U.S. patent application number 10/769612 was filed with the patent office on 2004-07-22 for compositions against cancer antigen liv-1 and uses thereof.
This patent application is currently assigned to Protein Design Labs, Inc.. Invention is credited to Culp, Patricia, Gish, Kurt C., Law, Debbie, Murray, Richard.
Application Number | 20040141983 10/769612 |
Document ID | / |
Family ID | 46300757 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040141983 |
Kind Code |
A1 |
Law, Debbie ; et
al. |
July 22, 2004 |
Compositions against cancer antigen LIV-1 and uses thereof
Abstract
Described herein are methods and compositions that can be used
for diagnosis and treatment of cancer.
Inventors: |
Law, Debbie; (San Francisco,
CA) ; Gish, Kurt C.; (Piedmont, CA) ; Murray,
Richard; (Cupertino, CA) ; Culp, Patricia;
(Fremont, CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Assignee: |
Protein Design Labs, Inc.
Fremont
CA
94555
|
Family ID: |
46300757 |
Appl. No.: |
10/769612 |
Filed: |
January 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10769612 |
Jan 29, 2004 |
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09642034 |
Aug 18, 2000 |
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09642034 |
Aug 18, 2000 |
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09525361 |
Mar 15, 2000 |
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09525361 |
Mar 15, 2000 |
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09453137 |
Dec 2, 1999 |
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09453137 |
Dec 2, 1999 |
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09450810 |
Nov 29, 1999 |
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09453137 |
Dec 2, 1999 |
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09268865 |
Mar 15, 1999 |
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60443712 |
Jan 29, 2003 |
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Current U.S.
Class: |
424/178.1 ;
530/391.1 |
Current CPC
Class: |
C07K 2317/73 20130101;
G01N 33/5011 20130101; C12Q 2600/106 20130101; C12Q 1/6886
20130101; A61P 35/00 20180101; C12Q 2600/158 20130101; G01N
33/57434 20130101; G01N 33/57415 20130101; A61P 43/00 20180101;
A61K 39/0011 20130101; A61K 49/0002 20130101; C07K 14/4748
20130101; A61K 49/0008 20130101; C07K 16/3015 20130101; A61K
2039/505 20130101; G01N 33/5748 20130101; C07K 16/3069 20130101;
A61K 39/00 20130101 |
Class at
Publication: |
424/178.1 ;
530/391.1 |
International
Class: |
C12Q 001/68; A61K
039/395; C07K 016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2000 |
WO |
PCT/US00/06952 |
Claims
What is claimed is:
1. An antibody that specifically binds a polypeptide or protein
encoded by SEQ ID NO:1.
2. The antibody of claim 1, wherein the antibody is conjugated to
an effector component.
3. The antibody of claim 2, wherein the effector component is
selected from the group consisting of a fluorescent label, a
radioisotope or a cytotoxic chemical.
4. The antibody of claim 3, wherein the cytotoxic chemical is
auristatin-E.
5. The antibody of claim 1, wherein the antibody is selected from
the group of Table 6.
6. The antibody of claim 1, wherein the antibody is an antibody
fragment.
7. The antibody of claim 1, wherein the antibody is a humanized
antibody.
8. The antibody of claim 1, wherein the antibody is a chimeric
antibody.
9. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the antibody of claim 1.
10. The pharmaceutical composition of claim 9, wherein the antibody
is further conjugated to an effector component.
11. The pharmaceutical composition of claim 10, wherein the
effector component is chosen from the group consisting of a
fluorescent label, a radioisotope or a cytotoxic chemical.
12. The pharmaceutical composition of claim 11, wherein the
cytotoxic chemical is auristatin-E.
13. The pharmaceutical composition of claim 9, wherein the antibody
is a humanized antibody.
14. The pharmaceutical composition of claim 9, wherein the antibody
is selected from the group of Table 6.
15. A method of detecting a prostate cancer cell or a breast cancer
cell in a biological sample from an individual, comprising: a.
obtaining a first prostate or breast tissue sample from an
individual and a normal tissue sample; b. contacting the first
prostate or breast tissue sample with an antibody of claim 1,
wherein the level of antibody binding to the first tissue sample
indicates the level of expression of SEQ ID NO:1; c. contacting the
normal tissue sample with an antibody of claim 1, wherein the level
of antibody binding to the normal tissue sample indicates the level
of expression of SEQ ID NO:1; and d. comparing the expression of
said SEQ ID NO:1 in the first prostate or breast tissue sample to
expression of said gene in the normal tissue sample; wherein a
higher level of protein expression in the first prostate or breast
tissue sample indicates that the first individual has prostate
cancer or breast cancer.
16. The method of claim 15, wherein the antibody is further
conjugated to a fluorescent label.
17. A method of treating an individual with prostate or breast
cancer, the method comprising administering an antibody that
specifically binds to a protein or polypeptide encoded by SEQ ID
NO:1.
18. The method of claim 17, wherein the antibody is a monoclonal
antibody.
19. The method of claim 17, wherein the antibody is an antibody
fragment.
20. The method of claim 17, wherein the antibody is a humanized
antibody.
21. The method of claim 17, wherein the antibody is conjugated to
an effector component.
22. The method of claim 21, wherein the effector component is
chosen from the group consisting of a fluorescent label, a
radioisotope or a cytotoxic chemical.
23. The method of claim 22, wherein the cytotoxic chemical is
auristatin-E.
24. A method for generating an immune response in an individual,
said method comprising administering to said individual the nucleic
acid of SEQ ID NO:1.
25. A method for generating an immune response in an individual,
said method comprising administering to said individual a protein
or polypeptide encoded by SEQ ID NO:1.
26. A double-stranded ribonucleic acid, comprising: a. a first
strand that is complementary to an mRNA encoded by SEQ ID NO:1, or
a fragment thereof; and b. a second strand that is complementary to
said first strand.
27. The ribonucleic acid of claim 26, wherein the double-stranded
ribonucleic acid is an siRNA.
28. The ribonucleic acid of claim 26, wherein the double-stranded
ribonucleic acid comprises SEQ ID NOS: 11 and 12.
29. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the ribonucleic acid of claim 26.
30. A method of treating an individual with prostate or breast
cancer, the method comprising administering a double-stranded
ribonucleic acid of claim 26.
31. The method of claim 30, wherein the double-stranded ribonucleic
acid is an siRNA.
32. The method of claim 30, wherein the double-stranded ribonucleic
acid comprises SEQ ID NOS: 11 and 12.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/443,712, filed Jan. 29, 2003; and is a CIP of
U.S. application Ser. No. 09/642,034, filed Aug. 18, 2000; which is
a CIP of U.S. application Ser. No. 09/525,361, filed Mar. 15, 2000;
which is a CIP of U.S. application Ser. No. 09/453,137, filed Dec.
2, 1999; which is a CIP of U.S. application Ser. No. 09/450,810,
filed Nov. 29, 1999, abandoned; and a CIP of U.S. application Ser.
No. 09/268,865, filed Mar. 15, 1999, all herein incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the identification and generation
of antibodies that specifically bind to LIV-1 proteins; and to the
use of such antibodies and compositions comprising them, in the
diagnosis, prognosis, and therapy of cancer.
BACKGROUND OF THE INVENTION
[0003] Zinc plays an essential role in cell growth, and is a
cofactor of over 300 enzymes, including enzymes important in
angiogenesis and cell remodeling. Vallee, B. L., Auld, D. S.,
Biochem. 29:5647-5659 (1990). Zinc associates with many
macromolecules in cells, including molecular components that act to
control growth, apoptosis, development and differentiation. Control
of intracellular zinc levels, therefore, may be important in
preventing the triggering of a variety of disease states, including
cancer.
[0004] LIV-1 is a member of the LZT (LIV-1-ZIP Zinc Transporters)
subfamily of zinc transporter proteins. Taylor, K. M. and
Nicholson, R. I., Biochim. Biophys. Acta 1611:16-30 (2003).
Computer analysis of the LIV-1 protein reveals a potential
metalloprotease motif,. fitting the consensus sequence for the
catalytic zinc-binding site motif of the zincin
metalloprotease.
[0005] The structure of LIV-1 implicates a role for the protein as
a zinc-influx transporter protein. Experiments with recombinant
LIV-1 localizes the protein to the plasma membrane, similarly
concentrated in lamellipodiae as membrane-type metalloproteases.
Taylor and Nicholson, supra. Computer analysis predicts six to
eight transmembrane domains, a long extracellular N terminus, a
short extracellular C terminus, as well as the consensus sequence
for the catalytic zinc-binding site of metalloproteases. LIV-1
distribution studies indicates primary expression in breast,
prostate, pituitary gland and brain tissue. Taylor and Nicholson,
supra.
[0006] The LIV-1 protein has also been implicated in certain
cancerous conditions, e.g. breast cancer and prostate cancer. The
detection of LIV-1 is associated with estrogen receptor-positive
breast cancer, McClelland, R. A., et al., Br. J. Cancer
77:1653-1656 (1998), and the metastatic spread of these cancers to
the regional lymph nodes. Manning, D. A. et al., Eur. J. Cancer
30A:675-678 (1994). Antibodies useful for diagnosis, prognosis, and
effective treatment of cancer, including metastatic cancer, would
be desirable. Accordingly, provided herein are compositions and
methods that can be used in diagnosis, prognosis, and therapy of
certain cancers.
SUMMARY OF THE INVENTION
[0007] The present invention provides anti-LIV-1 antibodies that
are useful for making conjugated antibodies for therapeutic
purposes. For example, the anti-LIV-1 antibodies of the invention
are useful as selective cytotoxic agents for LIV-1 expressing
cells. In some embodiments, the antibodies of the present invention
are therapeutically useful in persons diagnosed with cancer and
other proliferative conditions, including benign proliferative
conditions. In one aspect, the antibodies of the present invention
can be used to treat proliferative conditions of the prostate or
breast including, for example, prostate cancer or breast
cancer.
[0008] The present invention provides antibodies that competitively
inhibit binding of proteins encoded by vectors containing some or
all of the sequence associated with LIV-1 (Hs.79136). In some
embodiments the antibodies are further conjugated to an effector
component. The effector component can be a label (e.g., a
fluorescent label, an effector domain e.g. MicA) or can be a
cytotoxic moiety (e.g., a radioisotope or a cytotoxic chemical). An
exemplary cytotoxic chemical is auristatin-E. In other embodiments
the antibodies can be used alone to inhibit tumor cell growth.
[0009] The antibodies of the invention can be whole antibodies or
can be antibody fragments. In some embodiments the immunoglobulin
is a humanized antibody. An exemplary antibody of the invention is
defined by CDRs.
[0010] The invention further provides immunoassays using the
immunoglobulins of the invention. These methods involve detecting a
cancer cell in a biological sample from a patient by contacting the
biological sample with an antibody of the invention. The antibody
is typically conjugated to a label such as a fluorescent or other
label.
[0011] The invention also provides double-stranded ribonucleic
acids that bind to mRNA encoded by the LIV-1 nucleic acid of SEQ ID
NO:1. The double-stranded ribonucleic acids may cover the length of
the target mRNA, or may be short double-stranded ribonucleic acids
complementary to the target mRNA, e.g. siRNA.
[0012] The invention also provides pharmaceutical compositions
comprising a pharmaceutically acceptable excipient and the antibody
or double stranded ribonucleic acid of the invention. In these
embodiments, the antibody can be further conjugated to an effector
component. The effector component can be a label (e.g., a
fluorescent label) or can be a cytotoxic moiety (e.g., a
radioisotope or a cytotoxic chemical). An exemplary cytotoxic
chemical is auristatin-E. The antibodies in the pharmaceutical
compositions can be whole antibodies or antibody fragments. In some
embodiments the immunoglobulin is a humanized antibody.
[0013] The invention also provides methods of inhibiting
proliferation of a prostate cancer-associated or breast
cancer-associated cell. The method comprises contacting the cell
with an antibody or double-stranded ribonucleic acid of the
invention. In most embodiments, the cancer cell is in a patient,
typically a human. The patient may be undergoing a therapeutic
regimen to treat metastatic or benign prostate cancer or breast
cancer or may be suspected of having prostate cancer or breast
cancer.
DESCRIPTION OF THE TABLES AND FIGURES
[0014] Table 1 provides the cDNA (SEQ ID NO:1) and protein sequence
for LIV-1 (SEQ ID NO:2).
[0015] Table 2 provides DNA and peptide sequences for the LIV-1
antibody, #1.7A4 (SEQ ID NOS:3-6).
[0016] Table 3 provides a partial list of the variety of medical
conditions that LIV-1 may be implicated in.
[0017] Table 4 provides a list of cell lines that may be used to
validate anti-LIV-1 compositions in ovarian and bladder
systems.
[0018] Table 5 provides LIV-1 mutant (BCR4 M1) cDNA (5A) and
protein sequences (5B). Mutated residues are underlined.
[0019] Table 6 provides a list of antibodies generated against the
LIV-1 protein.
[0020] FIG. 1 shows a graph of the reduction in size of a prostate
tumor in vivo after Auristatin-E-conjugated LIV-1 antibody
treatment.
[0021] FIG. 2 shows a graph of the reduction in size of a breast
cancer tumor in vivo after Auristatin-E-conjugated LIV-1 antibody
treatment.
[0022] FIG. 3 shows fluorescence micrograph images of LIV-1
antibody stained tissue sections from breast cancer (left) and
other normal tissues.
[0023] FIG. 4A shows a bar graph of the effect of a LIV-1 RNAi
composition on MX-1 carcinoma cell growth in a clonogenic assay 14
days after addition of the LIV-1 siRNA.
[0024] FIG. 4B shows a bar graph of the effect of a LIV-1 RNAi
composition has on MX-1 carcinoma cell growth in a clonogenic assay
17 days after addition of the LIV-1 siRNA.
[0025] FIG. 5 shows a fluorescence microscope image of HCT116 cells
transfected without. (FIG. 5A) or with (FIG. 5B) a LIV-1 siRNA.
[0026] FIG. 6 shows a bar graph of the binding strength of various
LIV-1 antibodies on LIV-1 expressing cells (MX-1 breast carcinoma
cells).
[0027] FIG. 7 shows the inhibition of several LIV-1 antibodies on
epithelial ovarian carcinoma cell growth (CSOC), as compared to an
isotype IgG1 control.
[0028] FIG. 8 shows the inhibition of several LIV-1 antibodies on
mammary carcinoma cell growth (MX-1), as compared to an isotype
IgG1 control.
[0029] FIG. 9 shows the inhibition of several LIV-1 antibodies on
prostate carcinoma cell growth (LNCaP), as compared to an isotype
IgG1 control.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides novel reagents and methods
for treatment, diagnosis and prognosis for certain cancers using
antibodies and double-stranded ribonucleic acids against LIV-1. In
particular, the present invention provides anti-LIV-1 antibodies
that are particularly useful as selective cytotoxic agents for
LIV-1 expressing cells.
[0031] Epitope mapping of antibodies showing high affinity binding
can be carried out through competitive binding analyses. Using this
methodology, antibodies recognizing a number of individual epitopes
can be identified. The antibodies are then assessed for LIV-1
dependent cell death in vitro. Using these methods antibodies that
promote cell death can be identified.
[0032] Definitions
[0033] As used herein, "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies) and
heteroconjugate antibodies (e.g., bispecific antibodies). The term
"antibody" also includes antigen binding forms of antibodies,
including fragments with antigen-binding capability (e.g., Fab',
F(ab').sub.2, Fab, Fv and rIgG. See also, Pierce Catalog and
Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). See
also, e.g., Kuby, J., Immunology, 3.sup.rd Ed., W. H. Freeman &
Co., New York (1998). The term also refers to recombinant single
chain Fv fragments (scFv). The term antibody also includes bivalent
or bispecific molecules, diabodies, triabodies, and tetrabodies.
Bivalent and bispecific molecules are described in, e.g., Kostelny
et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992)
Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.
(1994) J. Immunol :5368, Zhu et al. (1997) Protein Sci 6:781, Hu et
al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res.
53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
[0034] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors,
see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al.,
Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech.
14:309-314 (1996), or by immunizing an animal with the antigen or
with DNA encoding the antigen.
[0035] Typically, an immunoglobulin has a heavy and light chain.
Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). Light
and heavy chain variable regions contain four "framework" regions
interrupted by three hypervariable regions, also called
"complementarity-determining regions" or "CDRs". The extent of the
framework regions and CDRs have been defined. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in three
dimensional space.
[0036] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found.
[0037] References to "V.sub.H" or a "V.sub.H" refer to the variable
region of an immunoglobulin heavy chain of an antibody, including
the heavy chain of an Fv, scFv, or Fab. References to "V.sub.L" or
a "V.sub.L" refer to the variable region of an immunoglobulin light
chain, including the light chain of an Fv, scFv, dsFv or Fab.
[0038] The phrase "single chain Fv" or "scFv" refers to an antibody
in which the variable domains of the heavy chain and of the light
chain of a traditional two chain antibody have been joined to form
one chain. Typically, a linker peptide is inserted between the two
chains to allow for proper folding and creation of an active
binding site.
[0039] A "chimeric antibody" is an immunoglobulin molecule in which
(a) the constant region, or a portion thereof, is altered, replaced
or exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0040] A "humanized antibody" is an immunoglobulin molecule which
contains minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient
antibody) in which residues from a complementary determining region
(CDR) of the recipient are replaced by residues from a CDR of a
non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some
instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies
may also comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. In
general, a humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin (Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
Humanization can be essentially performed following the method of
Winter and co-workers (Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species.
[0041] "Epitope" or "antigenic determinant" refers to a site on an
antigen to which an antibody binds. Epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
[0042] The term "LIV-1 protein" or "LIV-1 polynucleotide" refers to
nucleic acid and polypeptide polymorphic variants, alleles,
mutants, and interspecies homologues that: (1) have a nucleotide
sequence that has greater than about 60% nucleotide sequence
identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence
identity, preferably over a region of at least about 25, 50, 100,
200, 500, 1000, or more nucleotides, to a nucleotide sequence of
SEQ ID NO:1; (2) bind to antibodies, e.g., polyclonal antibodies,
raised against an immunogen comprising an amino acid sequence
encoded by a nucleotide sequence of SEQ ID NO:1, and conservatively
modified variants thereof; (3) specifically hybridize under
stringent hybridization conditions to a nucleic acid sequence, or
the complement thereof of SEQ ID NO:1 and conservatively modified
variants thereof or (4) have an amino acid sequence that has
greater than about 60% amino acid sequence identity, 65%, 70%, 75%,
80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% or greater amino sequence identity, preferably over a region of
at least about 25, 50, 100, 200, or more amino acids, to an amino
acid sequence of SEQ ID NO:2. A polynucleotide or polypeptide
sequence is typically from a mammal including, but not limited to,
primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig,
horse, sheep, or other mammal. A "LIV-1 polypeptide" and a "LIV-1
polynucleotide," include both naturally occurring or recombinant
forms.
[0043] A "full length" LIV-1 protein or nucleic acid refers to a
prostate cancer or breast cancer polypeptide or polynucleotide
sequence, or a variant thereof, that contains all of the elements
normally contained in one or more naturally occurring, wild type
LIV-1 polynucleotide or polypeptide sequences. For example, a full
length LIV-1 nucleic acid will typically comprise all of the exons
that encode for the full length, naturally occurring protein. The
"full length" may be prior to, or after, various stages of
post-translation processing or splicing, including alternative
splicing.
[0044] "Biological sample" as used herein is a sample of biological
tissue or fluid that contains nucleic acids or polypeptides, e.g.,
of a LIV-1 protein, polynucleotide or transcript. Such samples
include, but are not limited to, tissue isolated from primates,
e.g., humans, or rodents, e.g., mice, and rats. Biological samples
may also include sections of tissues such as biopsy and autopsy
samples, frozen sections taken for histologic purposes, blood,
plasma, serum, sputum, stool, tears, mucus, hair, skin, etc.
Biological samples also include explants and primary and/or
transformed cell cultures derived from patient tissues. A
biological sample is typically obtained from a eukaryotic organism,
most preferably a mammal such as a primate e.g., chimpanzee or
human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse;
rabbit; or a bird; reptile; or fish.
[0045] "Providing a biological sample" means to obtain a biological
sample for use in methods described in this invention. Most often,
this will be done by removing a sample of cells from an animal, but
can also be accomplished by using previously isolated cells (e.g.,
isolated by another person, at another time, and/or for another
purpose), or by performing the methods of the invention in vivo.
Archival tissues, having treatment or outcome history, will be
particularly useful.
[0046] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site or the
like). Such sequences are then said to be "substantially
identical." This definition also refers to, or may be applied to,
the compliment of a test sequence. The definition also includes
sequences that have deletions and/or additions, as well as those
that have substitutions, as well as naturally occurring, e.g.,
polymorphic or allelic variants, and man-made variants. As
described below, algorithms can account for gaps and the like.
Preferably, identity exists over a region that is at least about 25
amino acids or nucleotides in length, or more preferably over a
region that is 50-100 amino acids or nucleotides in length.
[0047] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default. program parameters can be
used, or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0048] A "comparison window", as used herein, includes reference to
a segment of one of the number of contiguous positions selected
from the group consisting typically of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0049] Examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity include the BLAST
and BLAST 2.0 algorithms, which are described in Altschul et al.,
Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol.
Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are used, with the
parameters described herein, to determine percent sequence identity
for the nucleic acids and proteins of the invention. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information. This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et
al., supra). These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are extended in both directions along each sequence for as far
as the cumulative alignment score can be increased. Cumulative
scores are calculated using, e.g., for nucleotide sequences, the
parameters M (reward score for a pair of matching residues;
always>0) and N (penalty score for mismatching residues;
always<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0050] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001. Log
values may be large negative numbers, e.g., 5, 10, 20, 30, 40, 40,
70, 90, 110, 150, 170, etc.
[0051] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, e.g.,
where the two peptides differ only by conservative substitutions.
Another indication that two nucleic acid sequences are
substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequences.
[0052] A "host cell" is a naturally occurring cell or a transformed
cell that contains an expression vector and supports the
replication or expression of the expression vector. Host cells may
be cultured cells, explants, cells in vivo, and the like. Host
cells may be prokaryotic cells such as E. coli, or eukaryotic cells
such as yeast, insect, amphibian, or mammalian cells such as CHO,
HeLa, and the like (see, e.g., the American Type Culture Collection
catalog).
[0053] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high performance liquid chromatography. A protein or nucleic acid
that is the predominant species present in a preparation is
substantially purified. In particular, an isolated nucleic acid is
separated from some open reading frames that naturally flank the
gene and encode proteins other than protein encoded by the gene.
The term "purified" in some embodiments denotes that a nucleic acid
or protein gives rise to essentially one band in an electrophoretic
gel. Preferably, it means that the nucleic acid or protein is at
least 85% pure, more preferably at least 95% pure, and most
preferably at least 99% pure. "Purify" or "purification" in other
embodiments means removing at least one contaminant from the
composition to be purified. In this sense, purification does not
require that the purified compound be homogenous,. e.g., 100%
pure.
[0054] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers, those containing modified
residues, and non-naturally occurring amino acid polymer.
[0055] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, e.g., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs may have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0056] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0057] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g.,
naturally contiguous, sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode most proteins. For instance, the codons GCA, GCC, GCG,
and GCU all encode the amino acid alanine. Thus, at every position
where an alanine is specified by a codon, the codon can be altered
to another of the corresponding codons described without altering
the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, often silent variations of a nucleic acid
which encodes a polypeptide is implicit in a described sequence
with respect to the expression product, but not with respect to
actual probe sequences.
[0058] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention. Typically conservative substitutions for
one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)
Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0059] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3rd ed., 1994) and Cantor &
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains.
Domains are portions of a polypeptide that often form a compact
unit of the polypeptide and are typically 25 to approximately 500
amino acids long. Typical domains are made up of sections of lesser
organization such as stretches of (-sheet and (-helices. "Tertiary
structure" refers to the complete three dimensional structure of a
polypeptide monomer. "Quaternary structure" refers to the three
dimensional structure formed, usually by the noncovalent
association of independent tertiary units. Anisotropic terms are
also known as energy terms.
[0060] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and proteins or other entities which can be made
detectable, e.g., by incorporating a radiolabel into the peptide or
used to detect antibodies specifically reactive with the peptide.
The radioisotope may be, for example, .sup.3H, .sup.14C, .sup.32P,
.sup.35S, or .sup.125I. In some cases, particularly using
antibodies against the proteins of the invention, the radioisotopes
are used as toxic moieties, as described below. The labels may be
incorporated into the LIV-1 nucleic acids, proteins and antibodies
at any position. Any method known in the art for conjugating the
antibody to the label may be employed, including those methods
described by Hunter et al., Nature, 144:945 (1962); David et al.,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407
(1982). The lifetime of radiolabeled peptides or radiolabeled
antibody compositions may extended by the addition of substances
that stablize the radiolabeled peptide or antibody and protect it
from degradation. Any substance or combination of substances that
stablize the radiolabeled peptide or antibody may be used including
those substances disclosed in U.S. Pat. No. 5,961,955.
[0061] An "effector" or "effector moiety" or "effector component"
is a molecule that is bound (or linked, or conjugated), either
covalently, through a linker or a chemical bond, or noncovalently,
through ionic, van der Waals, electrostatic, or hydrogen bonds, to
an antibody. The "effector" can be a variety of molecules
including, e.g., detection moieties including radioactive
compounds, fluorescent compounds, an enzyme or substrate, tags such
as epitope tags, a toxin; activatable moieties, a chemotherapeutic
agent; a chemoattractant, a lipase; an antibiotic; or a
radioisotope emitting "hard" e.g., beta radiation.
[0062] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, e.g., recombinant cells
express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all. By the term "recombinant nucleic acid" herein is meant nucleic
acid, originally formed in vitro, in general, by the manipulation
of nucleic acid, e.g., using polymerases and endonucleases, in a
form not normally found in nature. In this manner, operably linkage
of different sequences is achieved. Thus an isolated nucleic acid,
in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that are not normally joined, are both
considered recombinant for the purposes of this invention. It is
understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of
the host cell rather than in vitro manipulations; however, such
nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. Similarly, a "recombinant protein"
is a protein made using recombinant techniques, e.g., through the
expression of a recombinant nucleic acid as depicted above.
[0063] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not normally found in the same
relationship to each other in nature. For instance, the nucleic
acid is typically recombinantly produced, having two or more
sequences, e.g., from unrelated genes arranged to make a new
functional nucleic acid, e.g., a promoter from one source and a
coding region from another source. Similarly, a heterologous
protein will often refer to two or more subsequences that are not
found in the same relationship to each other in nature (e.g., a
fusion protein).
[0064] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element. A promoter also
optionally includes distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0065] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed
operably linked to a promoter.
[0066] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein, in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein sequences at least two times the
background and more typically more than 10 to 100 times
background.
[0067] Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies raised to a
particular protein, polymorphic variants, alleles, orthologs, and
conservatively modified variants, or splice variants, or portions
thereof, can be selected to obtain only those polyclonal antibodies
that are specifically immunoreactive with LIV-1 and not with other
proteins. This selection may be achieved by subtracting out
antibodies that cross-react with other molecules. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity).
[0068] "Tumor cell" refers to precancerous, cancerous, and normal
cells in a tumor.
[0069] "Cancer cells," "transformed" cells or "transformation" in
tissue culture, refers to spontaneous or induced phenotypic changes
that do not necessarily involve the uptake of new genetic material.
Although transformation can arise from infection with a
transforming virus and incorporation of new genomic DNA, or uptake
of exogenous DNA, it can also arise spontaneously or following
exposure to a carcinogen, thereby mutating an endogenous gene.
Transformation is associated with phenotypic changes, such as
immortalization of cells, aberrant growth control, nomnorphological
changes, and/or malignancy (see, Freshney, Culture of Animal Cells
a Manual of Basic Technique (3rd ed. 1994)).
[0070] Expression of LIV-1 Polypeptides from Nucleic Acids
[0071] Nucleic acids of the invention can be used to make a variety
of expression vectors to express LIV-1 polypeptides which can then
be used to raise antibodies of the invention, as described below.
Expression vectors and recombinant DNA technology are well known to
those of skill in the art and are used to express proteins. The
expression vectors may be either self-replicating extrachromosomal
vectors or vectors which integrate into a host genome. Generally,
these expression vectors include transcriptional and translational
regulatory nucleic acid operably linked to the nucleic acid
encoding the LIV-1 protein. The term "control sequences" refers to
DNA sequences used for the expression of an operably linked coding
sequence in a particular host organism. Control sequences that are
suitable for prokaryotes, e.g., include a promoter, optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and
enhancers.
[0072] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is typically
accomplished by ligation at convenient restriction sites. If such
sites do not exist, synthetic oligonucleotide adaptors or linkers
are used in accordance with conventional practice. Transcriptional
and translational regulatory nucleic acid will generally be
appropriate to the host cell used to express the LIV-1 protein.
Numerous types of appropriate expression vectors, and suitable
regulatory sequences are known in the art for a variety of host
cells.
[0073] In general, transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a one embodiment, the regulatory sequences include a
promoter and transcriptional start and stop sequences.
[0074] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0075] In addition, an expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, e.g. in mammalian or insect cells for expression and in
a prokaryotic host for cloning and amplification. Furthermore, for
integrating expression vectors, the expression vector contains at
least one sequence homologous to the host cell genome, and
preferably two homologous sequences which flank the expression
construct. The integrating vector may be directed to a specific
locus in the host cell by selecting the appropriate homologous
sequence for inclusion in the vector. Constructs for integrating
vectors are well known in the art (e.g., Fernandez & Hoeffler,
supra).
[0076] In addition, in another embodiment, the expression vector
contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0077] The LIV-1 proteins of the present invention are produced by
culturing a host cell transformed with an expression vector
containing nucleic acid encoding a LIV-1 protein, under the
appropriate conditions to induce or cause expression of the LIV-1
protein. Conditions appropriate for LIV-1 protein expression will
vary with the choice of the expression vector and the host cell,
and will be easily ascertained by one skilled in the art through
routine experimentation or optimization. For example, the use of
constitutive promoters in the expression vector will require
optimizing the growth and proliferation of the host cell, while the
use of an inducible promoter requires the appropriate growth
conditions for induction. In addition, in some embodiments, the
timing of the harvest is important. For example, the baculoviral
systems used in insect cell expression are lytic viruses, and thus
harvest time selection can be crucial for product yield.
[0078] Appropriate host cells include yeast, bacteria,
archaebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Saccharomyces
cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells,
C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, HUVEC
(human umbilical vein endothelial cells), THP1 cells (a macrophage
cell line) and various other human cells and cell lines.
[0079] In one embodiment, the LIV-1 proteins are expressed in
mammalian cells. Mammalian expression systems are also known in the
art, and include retroviral and adenoviral systems. One expression
vector system is a retroviral vector system such as is generally
described in PCT/US97/01019 and PCT/US97/01048, both of which are
hereby expressly incorporated by reference. Of particular use as
mammalian promoters are the promoters from mammalian viral genes,
since the viral genes are often highly expressed and have a broad
host range. Examples include the SV40 early promoter, mouse mammary
tumor virus LTR promoter, adenovirus major late promoter, herpes
simplex virus promoter, and the CMV promoter (see, e.g., Fernandez
& Hoeffler, supra). Typically, transcription termination and
polyadenylation sequences recognized by mammalian cells are
regulatory regions located 3' to the translation stop codon and
thus, together with the promoter elements, flank the coding
sequence. Examples of transcription terminator and polyadenlyation
signals include those derived from SV40.
[0080] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei.
[0081] In some embodiments, LIV-1 proteins are expressed in
bacterial systems. Bacterial expression systems are well known in
the art. Promoters from bacteriophage may also be used and are
known in the art. In addition, synthetic promoters and hybrid
promoters are also useful; e.g., the tac promoter is a hybrid of
the trp and lac promoter sequences. Furthermore, a bacterial
promoter can include naturally occurring promoters of non-bacterial
origin that have the ability to bind bacterial RNA polymerase and
initiate transcription. In addition to a functioning promoter
sequence, an efficient ribosome binding site is desirable. The
expression vector may also include a signal peptide sequence that
provides for secretion of the LIV-1 protein in bacteria. The
protein is either secreted into the growth media (gram-positive
bacteria) or into the periplasmic space, located between the inner
and outer membrane of the cell (gram-negative bacteria). The
bacterial expression vector may also include a selectable marker
gene to allow for the selection of bacterial strains that have been
transformed. Suitable selection genes include genes which render
the bacteria resistant to drugs such as ampicillin,
chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways. These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others. The bacterial
expression vectors are transformed into bacterial host cells using
techniques well known in the art, such as calcium chloride
treatment, electroporation, and others.
[0082] In one embodiment, LIV-1 polypeptides are produced in insect
cells. Expression vectors for the transformation of insect cells,
and in particular, baculovirus-based expression vectors, are well
known in the art.
[0083] LIV-1 polypeptides can also be produced in yeast cells.
Yeast expression systems are well known in the art, and include
expression vectors for Saccharomyces cerevisiae, Candida albicans
and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K.
lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces
pombe, and Yarrowia lipolytica.
[0084] The LIV-1 polypeptides may also be made as a fusion protein,
using techniques well known in the art. Thus, e.g., for the
creation of monoclonal antibodies, if the desired epitope is small,
the LIV-1 protein may be fused to a carrier protein to form an
immunogen. Alternatively, the LIV-1 protein may be made as a fusion
protein to increase expression, or for other reasons. For example,
when the LIV-1 protein is a LIV-1 peptide, the nucleic acid
encoding the peptide may be linked to other nucleic acid for
expression purposes.
[0085] The LIV-1 polypeptides are typically purified or isolated
after expression. LIV-1 proteins may be isolated or purified in a
variety of ways known to those skilled in the art depending on what
other components are present in the sample. Standard purification
methods include electrophoretic, molecular, immunological and
chromatographic techniques, including ion exchange, hydrophobic,
affinity, and reverse-phase HPLC chromatography, and
chromatofocusing. For example, the LIV-1 protein may be purified
using a standard anti-LIV-1 protein antibody column.
Ultrafiltration and diafiltration techniques, in conjunction with
protein concentration, are also useful. For general guidance in
suitable purification techniques, see Scopes, Protein Purification
(1982). The degree of purification necessary will vary depending on
the use of the LIV-1 protein. In some instances no purification
will be necessary.
[0086] One of skill will recognize that the expressed protein need
not have the wild-type LIV-1 sequence but may be derivative or
variant as compared to the wild-type sequence. These variants
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. These variants ordinarily are
prepared by site specific mutagenesis of nucleotides in the DNA
encoding the protein, using cassette or PCR mutagenesis or other
techniques well known in the art, to produce DNA encoding the
variant, and thereafter expressing the DNA in recombinant cell
culture as outlined above. However, variant protein fragments
having up to about 100-150 residues may be prepared by in vitro
synthesis using established techniques. Amino acid sequence
variants are characterized by the predetermined nature of the
variation, a feature that sets them apart from naturally occurring
allelic or interspecies variation of the LIV-1 protein amino acid
sequence. The variants typically exhibit the same qualitative
biological activity as the naturally occurring analogue, although
variants can also be selected which have modified characteristics
as will be more fully outlined below.
[0087] LIV-1 polypeptides of the present invention may also be
modified in a way to form chimeric molecules comprising a LIV-1
polypeptide fused to another, heterologous polypeptide or amino
acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of the LIV-1 polypeptide with a tag polypeptide
which provides an epitope to which an anti-tag antibody can
selectively bind. The epitope tag is generally placed at the amino-
or carboxyl-terminus of the LIV-1 polypeptide. The presence of such
epitope-tagged forms of a LIV-1 polypeptide can be detected using
an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the LIV-1 polypeptide to be readily purified by
affinity purification using an anti-tag antibody or another type of
affinity matrix that binds to the epitope tag. In an alternative
embodiment, the chimeric molecule may comprise a fusion of a LIV-1
polypeptide with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of the chimeric molecule, such
a fusion could be to the Fc region of an IgG molecule.
[0088] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; HIS6 and metal
chelation tags, the flu HA tag polypeptide and its antibody 12CA5
(Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag
and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et
al., Molecular and Cellular Biology 5:3610-3616 (1985)); and the
Herpes Simplex virus glycoprotein D (gD) tag and its antibody
(Paborsky et al., Protein Engineering 3(6):547-553 (1990)). Other
tag polypeptides include the FLAG-peptide (Hopp et al.,
BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide (Martin
et al., Science 255:192-194 (1992)); tubulin epitope peptide
(Skinner et al., J. Biol. Chem. 266:15163-15166 (1991)); and the T7
gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA 87:6393-6397 (1990)).
[0089] Antibodies to Cancer Proteins
[0090] Once the LIV-1 protein is produced, it is used to generate
antibodies, e.g., for immunotherapy or immunodiagnosis. In some
embodiments of the invention, the antibodies recognize the same
epitope as the CDRs shown in Table 2. The ability of a particular
antibody to recognize the same epitope as another antibody is
typically determined by the ability of one antibody to
competitively inhibit binding of the second antibody to the
antigen. Any of a number of competitive binding assays can be used
to measure competition between two antibodies to the same antigen.
An exemplary assay is a BIACORE.RTM. (chemicals for use in
biological assays) assay. Briefly in these assays, binding sites
can be mapped in structural terms by testing the ability of
interactants, e.g. different antibodies, to inhibit the binding of
another. Injecting two consecutive antibody samples in sufficient
concentration can identify pairs of competing antibodies for the
same binding epitope. The antibody samples should have the
potential to reach a significant saturation with each injection.
The net binding of the second antibody injection is indicative for
binding epitope analysis. Two response levels can be used to
describe the boundaries of perfect competition versus non-competing
binding due to distinct epitopes. The relative amount of binding
response of the second antibody injection relative to the binding
of identical and distinct binding epitopes determines the degree of
epitope overlap.
[0091] Other conventional immunoassays known in the art can be used
in the present invention. For example, antibodies can be
differentiated by the epitope to which they bind using a sandwich
ELISA assay. This is carried out by using a capture antibody to
coat the surface of a well. A subsaturating concentration of
tagged-antigen is then added to the capture surface. This protein
will be bound to the antibody through a specific antibody:epitope
interaction. After washing a second antibody, which has been
covalently linked to a detectable moeity (e.g., HRP, with the
labeled antibody being defined as the detection antibody) is added
to the ELISA. If this antibody recognizes the same epitope as the
capture antibody it will be unable to bind to the target protein as
that particular epitope will no longer be available for binding. If
however this second antibody recognizes a different epitope on the
target protein it will be able to bind and this binding can be
detected by quantifying the level of activity (and hence antibody
bound) using a relevant substrate. The background is defined by
using a single antibody as both capture and detection antibody,
whereas the maximal signal can be established by capturing with an
antigen specific antibody and detecting with an antibody to the tag
on the antigen. By using the background and maximal signals as
references, antibodies can be assessed in a pair-wise manner to
determine epitope specificity.
[0092] A first antibody is considered to competitively inhibit
binding of a second antibody, if binding of the second antibody to
the antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the first antibody using any of the assays described above.
[0093] Methods of preparing polyclonal antibodies are known to the
skilled artisan (e.g., Coligan, supra; and Harlow & Lane,
supra). Polyclonal antibodies can be raised in a mammal, e.g., by
one or more injections of an immunizing agent and, if desired, an
adjuvant. Typically, the immunizing agent and/or adjuvant will be
injected in the mammal by multiple subcutaneous or intraperitoneal
injections. The immunizing agent may include a protein encoded by a
nucleic acid of the figures or fragment thereof or a fusion protein
thereof. It may be useful to conjugate the immunizing agent to a
protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited
to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin,
and soybean trypsin inhibitor. Examples of adjuvants which may be
employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0094] The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such
as those described by Kohler & Milstein, Nature 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. The immunizing agent
will typically include a polypeptide encoded by a nucleic acid of
Table 1, a fragment thereof, or a fusion protein thereof.
Generally, either peripheral blood lymphocytes ("PBLs") are used if
cells of human origin are desired, or spleen cells or lymph node
cells are used if non-human mammalian sources are desired. The
lymphocytes are then fused with an immortalized cell line using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (1986)). Immortalized cell lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent
the growth of HGPRT-deficient cells.
[0095] In some embodiments the antibodies to the LIV-1 proteins are
chimeric or humanized antibodies. As noted above, humanized forms
of antibodies are chimeric immunoglobulins in which residues from a
complementary determining region (CDR) of human antibody are
replaced by residues from a CDR of a non-human species such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity.
[0096] Human antibodies can be produced using various techniques
known in the art, including phage display libraries (Hoogenboom
& Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol.
Biol. 222:581 (1991)). The techniques of Cole et al. and Boemer et
al. are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
p. 77 (1985) and Boemer et al., J. Immunol. 147(1):86-95 (1991)).
Similarly, human antibodies can be made by introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, e.g., in U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
and in the following scientific publications: Marks et al.,
Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intem. Rev.
Immunol. 13:65-93 (1995).
[0097] In some embodiments, the antibody is a single chain Fv
(scFv). The V.sub.H and the V.sub.L regions of a scFv antibody
comprise a single chain which is folded to create an antigen
binding site similar to that found in two chain antibodies. Once
folded, noncovalent interactions stabilize the single chain
antibody. While the V.sub.H and V.sub.L regions of some antibody
embodiments can be directly joined together, one of skill will
appreciate that the regions may be separated by a peptide linker
consisting of one or more amino acids. Peptide linkers and their
use are well-known in the art. See, e.g., Huston et al., Proc.
Nat'l Acad. Sci. USA 8:5879 (1988); Bird et al., Science 242:4236
(1988); Glockshuber et al., Biochemistry 29:1362 (1990); U.S. Pat.
No. 4,946,778, U.S. Pat. No. 5,132,405 and Stemmer et al.,
Biotechniques 14:256-265 (1993). Generally the peptide linker will
have no specific biological activity other than to join the regions
or to preserve some minimum distance or other spatial relationship
between the V.sub.H and V.sub.L. However, the constituent amino
acids of the peptide linker may be selected to influence some
property of the molecule such as the folding, net charge, or
hydrophobicity. Single chain Fv (scFv) antibodies optionally
include a peptide linker of no more than 50 amino acids, generally
no more than 40 amino acids, preferably no more than 30 amino
acids, and more preferably no more than 20 amino acids in length.
In some embodiments, the peptide linker is a concatamer of the
sequence Gly-Gly-Gly-Gly-Ser, preferably 2, 3, 4, 5, or 6 such
sequences. However, it is to be appreciated that some amino acid
substitutions within the linker can be made. For example, a valine
can be substituted for a glycine.
[0098] Methods of making scFv antibodies have been described. See,
Huse et al., supra; Ward et al. supra; and Vaughan et al., supra.
In brief, mRNA from B-cells from an immunized animal is isolated
and cDNA is prepared. The cDNA is amplified using primers specific
for the variable regions of heavy and light chains of
immunoglobulins. The PCR products are purified and the nucleic acid
sequences are joined. If a linker peptide is desired, nucleic acid
sequences that encode the peptide are inserted between the heavy
and light chain nucleic acid sequences. The nucleic acid which
encodes the scFv is inserted into a vector and expressed in the
appropriate host cell. The scFv that specifically bind to the
desired antigen are typically found by panning of a phage display
library. Panning can be performed by any of several methods.
Panning can conveniently be performed using cells expressing the
desired antigen on their surface or using a solid surface coated
with the desired antigen. Conveniently, the surface can be a
magnetic bead. The unbound phage are washed off the solid surface
and the bound phage are eluted.
[0099] Finding the antibody with the highest affinity is dictated
by the efficiency of the selection process and depends on the
number of clones that can be screened and the stringency with which
it is done. Typically, higher stringency corresponds to more
selective panning. If the conditions are too stringent, however,
the phage will not bind. After one round of panning, the phage that
binds to LIV-1 coated plates or to cells expressing LIV-1 on their
surface are expanded in E. coli and subjected to another round of
panning. In this way, an enrichment of many fold occurs in 3 rounds
of panning. Thus, even when enrichment in each round is low,
multiple rounds of panning will lead to the isolation of rare phage
and the genetic material contained within which encodes the scFv
with the highest affinity or one which is better expressed on
phage.
[0100] Regardless of the method of panning chosen, the physical
link between genotype and phenotype provided by phage display makes
it possible to test every member of a cDNA library for binding to
antigen, even with large libraries of clones.
[0101] In one embodiment, the antibodies are bispecific antibodies.
Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens or that have binding specificities for two
epitopes on the same antigen. In one embodiment, one of the binding
specificities is for the LIV-1 protein, the other one is for
another cancer antigen. Alternatively, tetramer-type technology may
create multivalent reagents.
[0102] In some embodiments, the antibodies to LIV-1 protein are
capable of reducing or eliminating cells expressing LIV-1 (e.g.,
prostate cancer or breast cancer cells). Generally, at least a 25%
decrease in activity, growth, size or the like is preferred, with
at least about 50% being particularly preferred and about a 95-100%
decrease being especially preferred.
[0103] By immunotherapy is meant treatment of prostate cancer or
breast cancer with an antibody raised against LIV-1 proteins. As
used herein, immunotherapy can be passive or active. Passive
immunotherapy as defined herein is the passive transfer of antibody
to a recipient (patient). Active immunization is the induction of
antibody and/or T-cell responses in a recipient (patient).
Induction of an immune response is the result of providing the
recipient with an antigen (e.g., LIV-1 or DNA encoding it) to which
antibodies are raised. As appreciated by one of ordinary skill in
the art, the antigen may be provided by injecting a polypeptide
against which antibodies are desired to be raised into a recipient,
or contacting the recipient with a nucleic acid capable of
expressing the antigen and under conditions for expression of the
antigen, leading to an immune response.
[0104] In some embodiments, the antibody is conjugated to an
effector moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. In one
aspect the therapeutic moiety is a small molecule that modulates
the activity of the LIV-1 protein. In another aspect the
therapeutic moiety modulates the activity of molecules associated
with or in close proximity to the LIV-1 protein.
[0105] In other embodiments, the therapeutic moiety is a cytotoxic
agent. In this method, targeting the cytotoxic agent to prostate
cancer or breast cancer tissue or cells, results in a reduction in
the number of afflicted cells, thereby reducing symptoms associated
with prostate cancer or breast cancer. Cytotoxic agents are
numerous and varied and include, but are not limited to, cytotoxic
drugs or toxins or active fragments of such toxins. Suitable toxins
and their corresponding fragments include diphtheria A chain,
exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin,
phenomycin, enomycin, auristatin-E and the like. Cytotoxic agents
also include radiochemicals made by conjugating radioisotopes to
antibodies raised against prostate cancer or breast cancer
proteins, or binding of a radionuclide to a chelating agent that
has been covalently attached to the antibody. Targeting the
therapeutic moiety to transmembrane prostate cancer or breast
cancer proteins not only serves to increase the local concentration
of therapeutic moiety in the prostate cancer or breast cancer
afflicted area, but also serves to reduce deleterious side effects
that may be associated with the therapeutic moiety.
[0106] Binding Affinity of Antibodies of the Invention
[0107] Binding affinity for a target antigen is typically measured
or determined by standard antibody-antigen assays, such as
BIACORE.RTM. competitive assays, saturation assays, or immunoassays
such as ELISA or RIA.
[0108] Such assays can be used to determine the dissociation
constant of the antibody. The phrase "dissociation constant" refers
to the affinity of an antibody for an antigen. Specificity of
binding between an antibody and an antigen exists if the
dissociation constant (K.sub.D=1/K, where K is the affinity
constant) of the antibody is<1 .mu.M, preferably<100 nM, and
most preferably<0.1 nM. Antibody molecules will typically have a
K.sub.D in the lower ranges. K.sub.D=[Ab-Ag]/[Ab][Ag] where [Ab] is
the concentration at equilibrium of the antibody, [Ag] is the
concentration at equilibrium of the antigen and [Ab-Ag] is the
concentration at equilibrium of the antibody-antigen complex.
Typically, the binding interactions between antigen and antibody
include reversible noncovalent associations such as electrostatic
attraction, Van der Waals forces and hydrogen bonds.
[0109] The antibodies of the invention specifically bind to LIV-1
proteins. By "specifically bind" herein is meant that the
antibodies bind to the protein with a K.sub.D of at least about 0.1
mM, more usually at least about 1 .mu.M, preferably at least about
0.1 .mu.M or better, and most preferably, 0.01 .mu.M or better.
[0110] Immunoassays
[0111] The antibodies of the invention can be used to detect LIV-1
or LIV-1 expressing cells using any of a number of well recognized
immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241;
4,376,110; 4,517,288; and 4,837,168). For a review of the general
immunoassays, see also Methods in Cell Biology, Vol. 37, Asai, ed.
Academic Press, Inc. New York (1993); Basic and Clinical Immunology
7th Edition, Stites & Terr, eds. (1991).
[0112] Thus, the present invention provides methods of detecting
cells that express LIV-1. In one method, a biopsy is performed on
the subject and the collected tissue is tested in vitro. The tissue
or cells from the tissue is then contacted, with an anti-LIV-1
antibody of the invention. Any immune complexes which result
indicate the presence of a LIV-1 protein in the biopsied sample. To
facilitate such detection, the antibody can be radiolabeled or
coupled to an effector molecule which is a detectable label, such
as a radiolabel. In another method, the cells can be detected in
vivo using typical imaging systems. Then, the localization of the
label is determined by any of the known methods for detecting the
label. A conventional method for visualizing diagnostic imaging can
be used. For example, paramagnetic isotopes can be used for MRI.
Internalization of the antibody may be important to extend the life
within the organism beyond that provided by extracellular binding,
which will be susceptible to clearance by the extracellular
enzymatic environment coupled with circulatory clearance.
[0113] LIV-1 proteins can also be detected using standard
immunoassay methods and the antibodies of the invention. Standard
methods include, for example, radioimmunoassay, sandwich
immunoassays (including ELISA), immunofluorescence assays, Western
blot, affinity chromatography (affinity ligand bound to a solid
phase), and in situ detection with labeled antibodies.
[0114] Suppression of Endogenous LIV-1 Gene Expression Through the
Use of RNAi
[0115] In many species, introduction of double-stranded RNA (dsRNA)
which may alternatively be referred to herein as small interfering
RNA (siRNA), induces potent and specific gene silencing, a
phenomena called RNA interference or RNAi. siRNA, in particular, is
capable of rendering genes nonfunctional in a sequence specific
manner. This phenomenon has been extensively documented in the
nematode C. elegans (Fire, A., et al, Nature, 391, 806-811, 1998),
but is widespread in other organisms, ranging from trypanasomes to
mouse. Recent experiments demonstrate the inhibition of gene
expression in human somatic cells, including the embryonic kidney
cell line 293 and the epithelial carcinoma cell line HeLa. Caplen,
N. J., et al., P.N.A.S. 98:9742-9747 (2001). Depending on the
organism being discussed, RNA interference has been referred to as
"co-suppression", "post-transcriptional gene silencing", "sense
suppression" and "quelling".
[0116] RNAi is attractive as a biotechnological tool because it
provides a means for knocking out the activity of specific genes.
It is particularly useful for knocking out gene expression in
species that were not previously considered to be amenable to
genetic analysis or manipulation.
[0117] In designing RNAi experiments there are several factors that
need to be considered such as the nature of the dsRNA, the
durability of the silencing effect, and the choice of delivery
system. See Elbashir, S. M. et al., EMBO J 20:6877-6888 (2001).
[0118] To produce an RNAi effect, the dsRNA, or siRNA that is
introduced into the organism should contain exonic sequences.
Furthermore, the RNAi process is homology dependent, so the
sequences must be carefully selected so as to maximize gene
specificity, while minimizing the possibility of cross-interference
between homologous, but not gene-specific sequences. Preferably the
dsRNA exhibits greater than 90% or even 100% identity between the
sequence of the dsRNA and the gene to be inhibited. Sequences less
than about 80% identical to the target gene are substantially less
effective. Thus, the greater homology between the dsRNA and the
gene whose expression is to be inhibited, the less likely
expression of unrelated genes will be affected.
[0119] In addition, the size of the dsRNA is important. dsRNA may
be greater than 500 base pairs in length, however, smaller
fragments can also produce an RNAi effect. In particular, fragments
that are short enough to avoid activation of nonsequence specific
dsRNA responses (e.g. interferon responses) are effective in
silencing gene responses. See Elbashir, S. M., et al., Nature
411:494-498 (2001).
[0120] Introduction of dsRNA can be achieved by any method known in
the art, including for example, microinjection, liposome
transfection or electroporation. A variety of mechanisms by which
dsRNA may inhibit gene expression have been proposed, but evidence
in support of any specific mechanism is lacking (Fire, A., 1999;
Caplen, N. J. et al., 2001).
[0121] Administration of Pharmaceutical and Vaccine
Compositions
[0122] The antibodies, nucleic acids and polypeptides of the
invention can be formulated in pharmaceutical compositions. Thus,
the invention also provide methods and compositions for
administering a therapeutically effective dose of an anti-LIV-1
antibody, a LIV-1 nucleic acid, or a LIV-1 polypeptide or protein.
The exact dose will depend on the purpose of the treatment, and
will be ascertainable by one skilled in the art using known
techniques (e.g., Ansel et al., Pharmaceutical Dosage Forms and
Drug Delivery; Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,
1992), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981;
Lloyd, The Art, Science and Technology of Pharmaceutical
Compounding (1999); and Pickar, Dosage Calculations (1999)). As is
known in the art, adjustments for prostate cancer or breast cancer
degradation, systemic versus localized delivery, and rate of new
protease synthesis, as well as the age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0123] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals. Thus
the methods are applicable to both human therapy and veterinary
applications. In one embodiment the patient is a mammal, preferably
a primate. In other embodiments the patient is human.
[0124] The administration of the antibodies, nucleic acids and
polypeptides of the present invention can be done in a variety of
ways as discussed above, including, but not limited to, orally,
subcutaneously, intravenously, intranasally, transdermally,
intraperitoneally, intramuscularly, intrapulmonary, vaginally,
rectally, or intraocularly.
[0125] The pharmaceutical compositions of the present invention
comprise an antibody, nucleic acid or polypeptide of the invention
in a form suitable for administration to a patient. In one
embodiment, the pharmaceutical compositions are in a water soluble
form, such as being present as pharmaceutically acceptable salts,
which is meant to include both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those
salts that retain the biological effectiveness of the free bases
and that are not biologically or otherwise undesirable, formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, propionic acid, glycolic acid,
pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, p-toluenesulfonic acid, salicylic acid and the like.
"Pharmaceutically acceptable base addition salts" include those
derived from inorganic bases such as sodium, potassium, lithium,
ammonium, calcium, magnesium, iron, zinc, copper, manganese,
aluminum salts and the like. Particularly useful are the ammonium,
potassium, sodium, calcium, and magnesium salts. Salts derived from
pharmaceutically acceptable organic non-toxic bases include salts
of primary, secondary, and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines and
basic ion exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine.
[0126] The pharmaceutical compositions may also include one or more
of the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol.
[0127] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include, but are not limited to, powder, tablets,
pills, capsules and lozenges. It is recognized that compositions of
the invention when administered orally, should be protected from
digestion. This is typically accomplished either by complexing the
molecules with a composition to render them resistant to acidic and
enzymatic hydrolysis, or by packaging the molecules in an
appropriately resistant carrier, such as a liposome or a protection
barrier. Means of protecting agents from digestion are well known
in the art.
[0128] The compositions for administration will commonly comprise
an antibody, polypeptide or nucleic acid of the invention dissolved
in a pharmaceutically acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used, e.g., buffered
saline and the like. These solutions are sterile and generally free
of undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, e.g., sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
active agent in these formulations can vary widely, and will be
selected primarily based on fluid volumes, viscosities, body weight
and the like in accordance with the particular mode of
administration selected and the patient's needs (e.g., Remington's
Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman,
The Pharmacologial Basis of Therapeutics (Hardman et al., eds.,
1996)).
[0129] Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Substantially higher dosages are possible in
topical administration. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art, e.g., Remington's Pharmaceutical Science and
Goodman and Gillman, The Pharmacologial Basis of Therapeutics,
supra.
[0130] The compositions containing antibodies, polypeptides or
nucleic acids of the invention can be administered for therapeutic
or prophylactic treatments. In therapeutic applications,
compositions are administered to a patient suffering from a disease
(e.g., a cancer) in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the agents of this invention to
effectively treat the patient. An amount of modulator that is
capable of preventing or slowing the development of cancer in a
mammal is referred to as a "prophylactically effective dose." The
particular dose required for a prophylactic treatment will depend
upon the medical condition and history of the mammal, the
particular cancer being prevented, as well as other factors such as
age, weight, gender, administration route, efficiency, etc. Such
prophylactic treatments may be used, e.g., in a mammal who has
previously had cancer to prevent a recurrence of the cancer, or in
a mammal who is suspected of having a significant likelihood of
developing cancer.
[0131] It will be appreciated that the present prostate cancer or
breast cancer protein-modulating compounds can be administered
alone or in combination with additional prostate cancer or breast
cancer modulating compounds or with other therapeutic agent, e.g.,
other anti-cancer agents or treatments.
[0132] Kits for Use in Diagnostic and/or Prognostic
Applications
[0133] For use in diagnostic, research, and therapeutic
applications suggested above, kits are also provided by the
invention. In the diagnostic and research applications such kits
may include any or all of the following: assay reagents, buffers,
and LIV-1-specific antibodies of the invention. A therapeutic
product may include sterile saline or another pharmaceutically
acceptable emulsion and suspension base.
[0134] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
methods of this invention. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
EXAMPLES
Example 1
Antibodies to the Target Protein LIV-1, Inhibit Prostate Tumor Cell
Growth in Vivo
[0135] The following example illustrates that LIV-1 antibodies are
effective at reducing tumor volume in vivo. Animal studies were
conducted using male SCID mice implanted with a prostate cancer
cell line, LNCaP. The LNCaP cell line expresses the antigen
recognized by LIV-1 antibodies. The protein and nucleic acid
sequences of the anti-LIV-1 #1.7A4 antibodies which were effective
in these experiments, are provided as SEQ ID NOS: 3, 4, 5, and 6
(Table 2).
[0136] Tumors were allowed to grow until they reached a size of
between 50-100 mm.sup.3. At that time, animals were randomized into
groups and subjected to treatment with either a.) an
Auristatin-E-conjugated isotype control antibody, or b.) the
Auristatin-E-conjugated LIV-1 antibody, 1.7A4.
[0137] Antibodies were administered at a dose of 10 mg/kg for a
total of 10 doses given intra-peritoneally every four days. Tumor
size was measured twice weekly for 38 days. At the conclusion of
the study, only tumors in the group treated with
Auristatin-E-conjugated LIV-1 antibodies showed regression. See
FIG. 1.
[0138] Thus, these experiments showed that treatment with the
Auristatin-E-conjugated LIV-1 antibody results in a significant
tumor volume reduction. Therefore the Auristatin-E-conjugated LIV-1
antibodies function as anti-cancer therapeutics for the treatment
of patients bearing LIV-1 expressing tumors.
Example 2
Antibodies to the Target Protein LIV-1, Inhibit Breast Tumor Cell
Growth in Vivo
[0139] The following example illustrates that LIV-1 antibodies are
effective at reducing tumor volume in vivo. Animal studies were
conducted using female SCID mice implanted with estrogen pellets.
The fat pads of the mice were implanted with a breast cancer cell
line, MCF7. The MCF7 cell line expresses the antigen recognized by
LIV-1 antibodies. The protein and nucleic acid sequences of the
anti-LIV-1#1.7A4 antibodies which were one of the
auristatin-E-conjugated-LIV-1 antibodies effective in these
experiments, are provided as SEQ ID NOS: 3, 4, 5, and 6 (Table
2).
[0140] Tumors were allowed to grow until they reached a size of
between 50-100 mm.sup.3. At that time, animals were randomized into
groups and subjected to treatment with either a.) vehicle, b.) an
Auristatin-E-conjugated isotype control antibody, or c.) one of two
Auristatin-E-conjugated LIV-1 antibodies, 1.7A4 or 1.1F10.
[0141] Antibodies were administered at a dose of 5 mg/kg for a
total of 5 doses given intra-peritoneally every four days. Tumor
size was measure twice weekly for 30 days. At the conclusion of the
study, only tumors in the group treated with
Auristatin-E-conjugated LIV-1 antibodies showed regression. See
FIG. 2.
[0142] Thus, these experiments showed that treatment with the
Auristatin-E-conjugated LIV-1 antibody results in a
significant-tumor volume reduction. Therefore the
Auristatin-E-conjugated LIV-1 antibodies function as anti-cancer
therapeutics for the treatment of patients bearing LIV-1 expressing
tumors.
Example 3
Immunohistochemistry Analysis Using LIV-1 Antibodies
[0143] Tissue microarrays of primary breast cancer samples were
obtained from Clinomics Biosciences, Inc. (Pittsfield, Mass.).
Normal body tissues specimens were collected from samples harvested
at the time of cadaveric organ donation from 6 individuals (3
males, 3 females obtained from Zoion, Hawthorne, N.Y.). IHC on
formalin-fixed paraffin embedded tissues was performed using
standard methods: heat induced antigen retrieval was performed in
Dako Target Retrieval Solution for 15 minutes in a pressure cooker.
Samples were then incubated with 1.1F10 anti-LIV-1 antibodies or
control mouse IgG1 [TIB 191, a mouse anti-trinitrophenol mAb
(hybridoma clone 1B76.11)] for 30 minutes. Antibody binding was
detected using biotinylated secondary antibody [Goat-anti-mouse IgG
(3 mg/ml, 30 minutes; Jackson ImmunoResearch)], and developed using
the Vectastain Elite ABC Kit (Vector Laboratories) and stable DAB
(diaminobenzidine and H2O2; Research Genetics). Staining was
performed using the DAKO Autostainer at room temperature.
[0144] Analysis of primary breast cancer specimens showed that
LIV-1-specific staining was restricted to the cytoplasm and
membranes of the breast cancer epithelium, as compared to tissue
specimens from pancreas, liver, skeletal muscle, adrenal gland,
heart, spleen, cerebellum, lung, duodenum, kidney, myometrium and
placenta, which showed no significant staining. See FIG. 3. The
breast cancer cohort (n=133) displayed weak to strong staining in
27% of the cases, demonstrating that a large fraction of breast
cancer patients exhibit expression of LIV-1. Analysis of a prostate
cancer cohort (87 cases) demonstrated significant LIV-1 expression
in 42% of the cases (data not shown). The staining in the prostate
cancer specimens was also restricted to the glandular epithelium.
These results demonstrate that LIV-1 protein is highly expressed in
both breast and prostate cancer specimens. Therefore, LIV-1 is an
attractive target for antibody-based therapy for both breast and
prostate cancers.
Example 4
LIV-1 RNAi Clonogenic Assay
[0145] An RNAi Clonogenic assay was performed to determine the
extent of LIV-1 siRNA targeted inhibition of carcinoma cell
proliferation. MX-1 breast carcinoma cells were transfected with
siRNAs using Lipofectamine 2000 (Invitrogen) according to the
manufacturer's instructions except that cells were transfected in
suspension as follows: MX-1 cells were diluted to 500 cells/ml. 0.5
ml was placed into each well of a 24 well plate containing 100 ul
of a mixture of Lipofectamine 2000 (Invitrogen) and siRNAs in
Minimal Essential Medium without phenol red (Invitrogen), with a
final siRNA concentration of 10 nM. Each siRNA was assayed in four
replicate wells. Cells were incubated at 37 C for 14-17 days,
during which time the media was changed twice a week. The extent of
cell proliferation was assessed by adding alamar blue to a final
concentration of 12 ug/ml for 4-8 hours. Fluorescence was measured
by excitation at 544 nm and emission at 590 nmn.
[0146] siRNAs were purchased from Dharmacon as duplexes with 3'dTdT
overhangs. siRNA sequences used are as follows:
1 H2R-1 (negative control) sense (SEQ ID NO:7):
5'-CAGACACGGCCACGUGUGAdTdT-3' H2R-1 antisense (SEQ ID NO:8):
5'-UCACACGUGGCCGUGUCUGdTdT-3' HKSP-1 (positive control) sense (SEQ
ID NO:9): 5'-GCUAGCGCCCAUUCAAUAGdTdT-3' HKSP-1 antisense (SEQ ID
NO:10): 5'-CUAUUGAAUGGGCGCUAGCdTdT-3' BCR4-53 sense (SEQ ID NO:11):
5'-CAGCUUUUCUACCGCUAUGdTdT-3' BCR4-53 antisense (SEQ ID NO:12):
5'-CAUAGCGGUAGAAAAGCUGdTdT-3'
[0147] The results indicate that downmodulation of LIV-1 by siRNA
reduced carcinoma cell proliferation, as compared to controls. This
indicates that LIV-1 expression is essential for proliferation in
these carcinoma cells. As seen in FIGS. 4A & 4B, LIV-1 siRNA
(BCR4-53; SEQ ID NOS:11 and 12) decreased cellular proliferation as
compared to a non-specific siRNA control (H2R-1; SEQ ID NOS:7 and
8) in both 14- and 17-day cultures after addition of siRNA. The
inhibition of cellular proliferation by LIV-1 siRNA was also
comparable to a known inhibitor of carcinoma cell proliferation,
HKSP-1 (SEQ ID NOS:9 and 10). This experiment, therefore, validates
LIV-1 as a legitimate target for inhibiting cell proliferation.
Example 5
MMP Activity Assay
[0148] LIV-1 siRNA was also tested in a matrix metalloproteinase
(MMP) activity assay. Cancer cells exhibit a variety of MMP
activity, including MMP-2 and MMP-9 gelatinase activity. Increased
MMP activity has been directly implicated in tumor cell invasion
and angiogenesis because of their ability to degrade extracellular
matrix components (see Olson, M. W. et al., J. Biol. Chem.
272:29975 (1997); MacDougall, J. R. et al., Cancer Metastasis Rev.
14:351 (1995); Cockett, M. I. et al, Biochem. Soc. Symp. 63:295
(1998)). MMP-2 and MMP-9 activity can be measured by determining
the extent of gelatin degradation upon addition of carcinoma cells
onto a gelatin matrix.
[0149] HCT-116 colorectal adenocarcinoma cells (obtained from NCI)
were transfected with either BCR4-53 or H2R-1 siRNAs as described
in the RNAi clonogenic assay with the following changes: 2.5ml of
cells at 2.times.10.sup.4 cells/ml were plated into a single well
of a 6 well plate containing 0.5 ml Lipofectamine2000/siRNA mixture
for a final siRNA concentration of 10 nM.
[0150] Cells were harvested approximately 72 hours after
transfection by trypsinization, and counted using a hemacytometer.
1.times.10.sup.4 cells in 1 ml media were plated onto fluorescent
gelatin-coated glass coverslips in 24 well plates. Coverslips were
prepared by acid washing and coated with 10 ul 0.5 mg/ml gelatin
conjugated to Oregon Green (Molecular Probes). After drying, the
gelatin was fixed with 0.5% glutaraldehyde for 15 minutes on ice,
washed several times with H2O, and sterilized with 70% ethanol
before the cells were plated on top. The plates were incubated at
37 C to allow the cells to attach and degrade the gelatin.
[0151] Approximately 24 hours after plating, the cells were fixed
in Cytofix/Cytoperm (BD-Pharmingen). Total cellular LIV-1 protein
levels were then assessed with anti-LIV-1 antibody (1.7A4) at 10
ug/ml, followed by F(ab').sub.2 goat anti-mouse IgG conjugated to
Alexa Fluor 594 (Molecular Probes). Cell nuclei were also stained
with 10 uM Hoechst 33342. The coverslips were mounted onto glass
slides and subjected to fluorescence microscopy.
[0152] The results show (compare FIGS. 5A & 5B) that LIV-1
siRNA (SEQ ID NOS: 11 and 12) inhibited gelatin degradation by
HCT-116 colorectal adenocarcinoma cells. This indicates, that LIV-1
is important in the upregulation of MMP--expression and/or activity
in HCT-116 colorectal adenocarcinoma cells, and therefore validates
LIV-1's role as a mediator in the extracellular invasion, and
extracellular invasion and angiogenesis upregulation by carcinoma
cells.
Example 6
Additional Antibodies to LIV-1
[0153] Monoclonal antibodies were raised against an N-terminus
LIV-1 antigen and tested for binding on MX-1 cells by titration
through FACS analysis. Hybridomas were derived from a fusion of NSO
mouse myeloma cells and lymph nodes from mice immunized with
purified protein containing the N-terminal 329 aa of LIV-1 fused to
the human Fc domain. Hybridomas which expressed antibodies specific
for LIV-1 were subcloned, implanted into mice, and antibodies were
purified from ascites fluid.
[0154] Comparative binding of the various LIV-1 antibodies was
assessed by flow cytometry; MX-1 breast carcinoma cells were
incubated with a dilution series of LIV-1 and control antibody,
followed by incubation with goat anti-mouse IgG conjugated to FITC
(Caltag). Each dilution was done in triplicate.
[0155] The FACS results in FIG. 6 indicate that several LIV-1
antibodies show promising binding levels on MX-1 cells. In
particular, LIV-1 antibodies 14 (ATCC accession number PTA-5705),
19 (ATCC accession number PTA-5706 and 23 (ATCC accession number
PTA-5707).
Example 7
In Vitro Proliferation Assay with Toxin-Conjugated LIV-1
Antibodies
[0156] LIV-1 and control antibodies were conjugated to Auristatin-E
containing a valine-citrulline peptide linker and purified by HPLC.
Serial dilutions of two LIV-1 antibodies, 14a and 22a, were
compared to a previously tested LIV-1 antibody (see Examples 1 and
2, supra), 1.7A4, and an IgG isotype control antibody for their
ability to be internalized and kill LIV-1 expressing cells. 50 ul
cells were plated into 96 well plates: CSOC 882-2 ovarian carcinoma
cells at 650 cells/well, LNCaP prostate adenocarcinoma cells at
5000 cells/well, and MX-1 breast carcinoma cells at 650 cells/well.
24 hours after plating, 50 ul of the appropriate antibody dilution
was added to triplicate wells and incubated for an additional 72 or
96 hours at 37 C. The extent of cell proliferation was assessed by
adding alamar blue to a final concentration of 12 ug/ml and
incubating at 37 C for 2 hours. Fluorescence was measured by
excitation at 544 nm and emission at 590 nm. Fraction survival was
calculated by normalizing cell survival of antibody-exposed cells
to control cells growth in the absence of antibody.
[0157] The results in FIGS. 7, 8 and 9 indicate that
auristatin-E-conjugated 14a and 22a LIV-1 antibodies were more
effective in in vitro growth assays using a variety of carcinoma
cell types than the auristatin-E-conjugated 1.7a4 LIV-1 antibody.
For CSOC (FIG. 7), MX-1 (FIG. 8) and LNCaP cells (FIG. 9), both 14a
and 22a conjugated antibodies were more effective at tumor cell
growth suppression than 1.7A4 LIV-1 antibody and a control IgG1
antibody. Therefore, the LIV-1 14a and 22a antibodies are useful in
tumor cell growth suppression.
Example 8
Epitope Mapping of Anti-LIV-1 Antibodies Identifies Three Distinct
Epitopes
[0158] Epitope mapping of BCR4 antibodies was done with flow
cytometry using a competitive binding assay. MX-1 breast carcinoma
cells were incubated with one of three FITC-conjugated antibodies
in the presence or absence of a 10-fold molar excess of
unconjugated (or naked) antibodies. In some experiments, the naked
antibody was used at 5- or 20-fold molar excess with similar
results. The ability of each naked antibody to compete with a
FITC-conjugated antibody for binding was assessed. In all cases,
naked antibodies either did not compete at all (-) or competed as
well as the cognate naked antibody (+).
2 FITC-conjugated Abs Naked Abs 14 19 21 13 - + - 14 + - + 15 + ND
+ 16 + ND + 19 - + - 20 + ND + 21 + ND + 22 + ND + 23 + ND + 24 +
ND + 25 + ND + 1.7A4 - + - 1.1F10 - - - IGG1 - - - ISOTYPE ND: not
done
[0159] From this experiment we demonstrate that 9 of the antibodies
bind the same or overlapping epitopes. These antibodies are 14, 15,
16, 20, 21, 22, 23, 24, and 25. The epitope bound by these
antibodies is distinct from those of the previous antibodies we had
generated and thus represents a novel epitope for potential
therapeutic applications. The two new antibodies, 13 and 19,
represent an epitope binding group containing one of our previous
antibodies, 1.7A4. A third epitope binding group, represented by
1.1F10 from our previous set of antibodies, was not represented in
this new panel of antibodies.
Example 9
Mutant LIV-1 Protein
[0160] A mutant LIV-1 protein (BCR4 M1 cDNA (SEQ ID NO:13) and
protein sequence (SEQ ID NO:14) provided in Table 5) was generated
with a mutation in the putative MMP/Zn transporter domain. The goal
to generating this mutant was to further characterize the
biological activity of LIV-1.
[0161] This mutant was made using two rounds of PCR. Briefly two
fragments were generated from the wild type gene via PCR using
internal primers carrying the desired mutations.
[0162] The 5' end was generated using primers
3 1 (CTTTAATTAACACCGCCACCATGGCGAGGAAGTTATCTGTAATC) (SEQ ID NO:15)
and 2 (TAATGCAGCAGGCAACGCAGCACAGAACACAGCAACAGAAG). (SEQ ID
NO:16)
[0163] The 3' end was generated using primers
4 3 (TGCTGCGTTGCCTGCTGCATTAGGTGACTTTGCTGTTC) (SEQ ID NO:17) and 4
(GTCTCGAGGAAATTTATACGAAAC). (SEQ ID NO:18)
[0164] Since primers 2 and 3 contain overlapping sequences the two
gene fragments were gel purified and used as overlapping templates
in a second round of PCR using primers 1 and 4. The product of this
second round PCR were cloned into pCR4 topo blunt and selected
clones were sequenced. The desired mutant was selected and
subcloned into the NEF39 expression vector and transfected into
3T12 cells. Overexpressing cells were isolated using a CD4-based
screening procedure.
[0165] The isolated clones expressed significantly higher levels of
mutant LIV-1 protein compared to clones isolated from experiments
in which wild-type LIV-1 protein were transfected into 3T12 cells
(data not shown). This result suggests that a functional activity,
possibly MMP and/or Zn transporter activity, limits the amount of
LIV-1 protein that can be expressed in a cell. It is known that
expressing high levels of certain oncogenes in cells is difficult,
because these oncogenes activate signaling pathways that under
normal tissue culture conditions lead to cell death. It is possible
that LIV-1 activity has a similar effect in cells and that
mutational inactivation abrogates this effect, allowing for higher
overexpression. This result suggests a functional role for LIV-1 in
regulating cell proliferation and/or cell survival.
Example 10
Use of LIV-1 Antibodies to Delay the Onset of Androgen-Independence
of Prostate Cancer and/or to Treat Androgen-Independent Disease
[0166] Prostate cancer is a hormone regulated disease that affects
men in the later years of life. Untreated prostate cancer
metastasizes to lymph nodes and bone in advanced cases. In such
cases current treatment consists of antagonizing the androgenic
growth-stimulus that feeds the tumor by chemical or surgical
hormone-ablation therapy (Galbraith and Duchesne. (1997) Eur. J.
Cancer 33:545-554). An unfortunate consequence of anti-androgen
treatment is the development of androgen-independent cancer.
Androgen regulated genes such as the gene encoding
prostate-specific antigen (PSA) are turned off with
hormone-ablation therapy, but reappear when the tumor becomes
androgen-independent (Akakura et al. (1993) Cancer 71:2782-2790).
There are no viable treatment regimens for androgen-independent
prostate cancer.
[0167] To study the progression of androgen-dependent prostate
cancer to androgen-independent prostate cancer, the human CWR22
prostate cancer xenograft model was propagated in nude mice (see
Pretlow, et al. (1993) J. Natl. Cancer Inst. 85:394-398). The CWR22
xenograft is androgen-dependent when grown in male nude mice.
Androgen-independent sub-lines can be derived by first establishing
androgen-dependent tumors in male mice. The mice are then castrated
to remove the primary source of growth stimulus (androgen),
resulting in tumor regression. Within 3-4 months, molecular events
prompt the tumors to relapse and start growing as
androgen-independent tumors. See, e.g., Nagabhushan, et al. (1996)
Cancer Res. 56:3042-3046; Amler, et al. (2000) Cancer Res.
60:6134-6141; and Bubendorf, et al. (1999) J. Natl. Cancer Inst.
91:1758-1764.
[0168] We have previously monitored the gene expression changes
that occur during the transition from androgen-dependence to
androgen-independence using the CWR22 xenograft model (see
WO02098358). Tumors were grown subcutaneously in male nude mice.
Tumors were harvested at different times after castration. The time
points ranged from 0 to 125 days post-castration. Castration
resulted in tumor regression. At day 120 and thereafter, the tumors
relapsed and started growing in the absence of androgen.
[0169] Gene expression profiling of the harvested tumors was
accomplished using the Eos Hu03 oligonucleotide microarray
(Affymetrix Eos Hu03) (Henshall et al. (2003) Cancer Res.
63:4196-4203). Our results identified several hundred genes that
exhibited significant gene expression changes associated with
androgen ablation therapy. Some genes were associated with the
androgen-dependent growth phase of the CWR22 tumors (pre-castration
and 1-5 days post-castration), some genes were associated with the
androgen-withdrawal phase (10-82 days post castration,
characterized by tumor regression and/or tumor growth stasis), and
some genes were associated with the androgen-independent growth of
CWR22 (greater than 120 days post castration). See WO02098358. From
these results, we determined that the gene encoding LIV-1 is not
androgen-regulated and exhibited high expression levels in
androgen-dependent tumors and in all tumors undergoing
androgen-withdrawal experiment, including tumors that grew in an
androgen-independent manner (data not shown).
[0170] Castrated CWR22 xenograft nude male mice would be used as a
model system for prevention of androgen-independent prostate cancer
growth. CWR22 tumor bearing mice would be treated, post
androgen-ablation therapy (castration), with anti-LIV-1 antibody
conjugated with Auristatin-E. Post-castration treatment with
anti-LIV-1 conjugated with Auristatin-E during the
androgen-withdrawal phase (10-82 days post castration) should
result in a delay in the onset of androgen-independent CWR22 tumor
growth.
[0171] To accomplish this, CWR22 tumors would be grown in male
immunodeficient mice for 2-3 weeks. The mice would then be
castrated to induce tumor regression and entry into the
androgen-withdrawal phase. Twenty days post-castration the tumors
would be treated with anti-LIV-1 conjugated with Auristatin-E as
described in Examples 1 and 2. A significant effect of
anti-LIV-1-Auristatin-E would manifest itself in a delay in the
onset of androgen-independence (e.g., 5 months or more post
castration). This would suggest that androgen-ablation therapy
patients with advanced stage prostate cancer would greatly benefit
from treatment with humanized anti-LIV-1 drug conjugates.
[0172] A non-significant effect of anti-LIV-1 ADC treatment would
be due to several potential factors: (a) CWR22 xenograft tumors may
be resistant to Auristatin E; (b) the tumor cells may not
efficiently internalize anti-LIV-1 ADC during the
androgen-withdrawal phase; or (c) LIV-1 protein expression may be
significantly decreased during the androgen-withdrawal phase.
Modifications in treatment are available to address these
issues.
[0173] As a model system for treating established
androgen-independent prostate cancer, CWR22 tumor bearing mice
would be treated at the time of onset of androgen-independence with
anti-LIV-1 drug conjugate. The objective would be to show that
post-castration treatment with anti-LIV-1 drug conjugates during
the emergence of androgen-independence (>120 days post
castration) would result in regression of androgen-independent
CWR22 tumors.
[0174] CWR22 tumors would be grown in male immunodeficient mice for
2-3 weeks. The mice would be then castrated to induce tumor
regression and entry into the androgen-withdrawal phase. Ten days
after the tumors start growing in an androgen-independent manner,
the tumors would be treated with anti-LIV-1 conjugated with
Auristatin-E as described in Examples 1 and 2. A significant effect
of anti-LIV-1-Auristatin-E would manifest itself in regression of
androgen-independent tumors. This would suggest that patients that
were treated with androgen-ablation therapy and that suffered
relapse in the form of androgen-independent tumor growth and
metastasis would greatly benefit from treatment with humanized
anti-LIV-1 drug conjugate.
5TABLE 1 DNA AND PROTEIN SEQUENCES OF LIV-1 (GENBANK ACCESSION
NM_012319) SEQ ID NO: 1 LIV-1 DNA SEQUENCE CTCGTGCCGA ATTCGGCACG
AGACCGCGTG TTCGCGCCTG GTAGAGATTT CTCGAAGACA CCAGTGGGCC CGTGTGGAAC
CAAACCTGCG CGCGTGGCCG GGCCGTGGGA CAACGAGGCC GCGGAGACGA AGGCGCAATG
GCGAGGAAGT TATCTGTAAT CTTGATCCTG ACCTTTGCCC TCTCTGTCAC AAATCCCCTT
CATGAACTAA AAGCAGCTGC TTTCCCCCAG ACCACTGAGA AAATTAGTCC GAATTGGGAA
TCTGGCATTA ATGTTGACTT GGCAATTTCC ACACGGCAAT ATCATCTACA ACAGCTTTTC
TACCGCTATG GAGAAAATAA TTCTTTGTCA GTTGAAGGGT TCAGAAAATT ACTTCAAAAT
ATAGGCATAG ATAAGATTAA AAGAATCCAT ATACACCATG ACCACGACCA TCACTCAGAC
CACGAGCATC ACTCAGACCA TGAGCGTCAC TCAGACCATG AGCATCACTC AGACCACGAG
CATCACTCTG ACCATAATCA TGCTGCTTCT GGTAAAAATA AGCGAAAAGC TCTTTGCCCA
GACCATGACT CAGATAGTTC AGGTAAAGAT CCTAGAAACA GCCAGGGGAA AGGAGCTCAC
CGACCAGAAC ATGCCAGTGG TAGAAGGAAT GTCAAGGACA GTGTTAGTGC TAGTGAAGTG
ACCTCAACTG TGTACAACAC TGTCTCTGAA GGAACTCACT TTCTAGAGAC AATAGAGACT
CCAAGACCTG GAAAACTCTT CCCCAAAGAT GTAAGCAGCT CCACTCCACC CAGTGTCACA
TCAAAGAGCC GGGTGAGCCG GCTGGCTGGT AGGAAAACAA ATGAATCTGT GAGTGAGCCC
CGAAAAGGCT TTATGTATTC CAGAAACACA AATGAAAATC CTCAGGAGTG TTTCAATGCA
TCAAAGCTAC TGACATCTCA TGGCATGGGC ATCCAGGTTC CGCTGAATGC AACAGAGTTC
AACTATCTCT GTCCAGCCAT CATCAACCAA ATTGATGCTA GATCTTGTCT GATTCATACA
AGTGAAAAGA AGGCTGAAAT CCCTCCAAAG ACCTATTCAT TACAAATAGC CTGGGTTGGT
GGTTTTATAG CCATTTCCAT CATCAGTTTC CTGTCTCTGC TGGGGGTTAT CTTAGTGCCT
CTCATGAATC GGGTGTTTTT CAAATTTCTC CTGAGTTTCC TTGTGGCACT GGCCGTTGGG
ACTTTGAGTG GTGATGCTTT TTTACACCTT CTTCCACATT CTCATGCAAG TCACCACCAT
AGTCATAGCC ATGAAGAACC AGCAATGGAA ATGAAAAGAG GACCACTTTT CAGTCATCTG
TCTTCTCAAA ACATAGAAGA AAGTGCCTAT TTTGATTCCA CGTGGAAGGG TCTAACAGCT
CTAGGAGGCC TGTATTTCAT GTTTCTTGTT GAACATGTCC TCACATTGAT CAAACAATTT
AAAGATAAGA AGAAAAAGAA TCAGAAGAAA CCTGAAAATG ATGATGATGT GGAGATTAAG
AAGCAGTTGT CCAAGTATGA ATCTCAACTT TCAACAAATG AGGAGAAAGT AGATACAGAT
GATCGAACTG AAGGCTATTT ACGAGCAGAC TCACAAGAGC CCTCCCACTT TGATTCTCAG
CAGCCTGCAG TCTTGGAAGA AGAAGAGGTC ATGATAGCTC ATGCTCATCC ACAGGAAGTC
TACAATGAAT ATGTACCCAG AGGGTGCAAG AATAAATGCC ATTCACATTT CCACGATACA
CTCGGCCAGT CAGACGATCT CATTCACCAC CATCATGACT ACCATCATAT TCTCCATCAT
CACCACCACC AAAACCACCA TCCTCACAGT CACAGCCAGC GCTACTCTCG GGAGGAGCTG
AAAGATGCCG GCGTCGCCAC TTTGGCCTGG ATGGTGATAA TGGGTGATGG CCTGCACAAT
TTCAGCGATG GCCTAGCAAT TGGTGCTGCT TTTACTGAAG GCTTATCAAG TGGTTTAAGT
ACTTCTGTTG CTGTGTTCTG TCATGAGTTG CCTCATGAAT TAGGTGACTT TGCTGTTCTA
CTAAAGGCTG GCATGACCGT TAAGCAGGCT GTCCTTTATA ATGCATTGTC AGCCATGCTG
GCGTATCTTG GAATGGCAAC AGGAATTTTC ATTGGTCATT ATGCTGAAAA TGTTTCTATG
TGGATATTTG CACTTACTGC TGGCTTATTC ATGTATGTTG CTCTGGTTGA TATGGTACCT
GAAATGCTGC ACAATGATGC TAGTGACCAT GGATGTAGCC GCTGGGGGTA TTTCTTTTTA
CAGAATGCTG GGATGCTTTT GGGTTTTGGA ATTATGTTAC TTATTTCCAT ATTTGAACAT
AAAATCGTGT TTCGTATAAA TTTCTAGTTA AGGTTTAAAT GCTAGAGTAG CTTAAAAAGT
TGTCATAGTT TCAGTAGGTC ATAGGGAGAT GAGTTTGTAT GCTGTACTAT GCAGCGTTTA
AAGTTAGTGG GTTTTGTGAT TTTTGTATTG AATATTGCTG TCTGTTACAA AGTCAGTTAA
AGGTACGTTT TAATATTTAA GTTATTCTAT CTTGGAGATA AAATCTGTAT GTGCAATTCA
CCGGTATTAC CAGTTTATTA TGTAAACAAG AGATTTGGCA TGACATGTTC TGTATGTTTC
AGGGAAAAAT GTCTTTAATG CTTTTTCAAG AACTAACACA GTTATTCCTA TACTGGATTT
TAGGTCTCTG AAGAACTGCT GGTG SEQ ID NO: 2 LIV-1 PROTEIN SEQUENCE
MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLA- ISTRQYHLQQLFY
RYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHS- DHERHSDHEHHSDHEHHS
DHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRP- EHASGRRNVKDSVSASEVTSTVY
NTVSEGTHFLETIETPRPGKLFPKDVSSSTPPSV- TSKSRVSRLAGRKTNESVSEPRKGFMYS
RNTNENPQECFNASKLLTSHGMGIQVPLN- ATEFNYLCPAIINQIDARSCLIHTSEKKAEIPP
KTYSLQIAWVGGFIAISIISFLSL- LGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLL
PHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHV
LTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPS
HFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHI
LHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEG- LSSGL
STSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFI- GHYAENVSMW
IFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLG- FGIMLLISIFEHKIV
FRINF
[0175]
6TABLE 2 ANTI-LIV-1 #1.7A4 DNA AND PEPTIDE SEQUENCES PROTEIN
SEQUENCES SEQ ID NO:3 Heavy chain variable domain; CDRs in bold and
underlined:
eiqlqqsgpelmkpgasvkisckastysftryfmhwvkqshgeslewigyidpfnggtgynqkfkgkat
ltvdkssstaymhlssltsedsavyycvtygsdyfdywgqgttltvss SEQ ID NO:4 Light
chain variable domain; CDRs in bold and underlined:
divmtqpqkfmstsvgdrvsvtckasqnvetdvvwyqqkpgqppkaliysasyrhsgvpdrftgsg-
sgt nftltistvqsedlaeyfcqqynnypftfgsgtkleiir DNA SEQUENCES SEQ ID
NO:5 Heavy chain variable domain:
GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGATGAAGCCTGGGGCTTCAGTGAAGATATC
TTGCAAGGCTTCTACTTACTCATTCACTAGGTACTTCATGCACTGGGTGAAGCAGAGCCATG
GAGAGAGCCTTGAGTGGATTGGATATATTGATCCTTTCAATGGTGGTACTGGCTACAATCAG
AAATTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATG- CATCT
CAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAACGTATGGC- TCCGACTACT
TTGACTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO:6 LIGHT CHAIN
VARIABLE DOMAIN:
GACATTGTGATGACCCAGCCACAAAAATTCATGTCCACGTCTGTAGGCGACAGGGTCAGTGT
CACCTGCAAGGCCAGTCAGAATGTGGAAACTGATGTAGTCTGGTATCAACAGAAACCTGGGC
AACCACCTAAAGCACTGATTTACTCGGCATCCTACCGGCACAGTGGAGTCCCTGATCGCTTC
ACAGGCAGTGGATCTGGGACAAATTTCACTCTCACCATCAGCACTGTACAGTCTGAA- GACTT
GGCAGAGTATTTCTGTCAGCAATATAACAACTATCCATTCACGTTCGGCTCG- GGGACAAAGT
TGGAAATAATACGG
[0176]
7TABLE 3 LISTS OF MEDICAL CONDITIONS LIV-1 has been found to be
over-expressed in cancers of the organs listed in the Table.
Therefore, targeting or inhibiting LIV-1, by any means known in the
art, including but limited to antibodies, may be a an effective
treatment for such diseases. bladder: carcinoma in situ, papillary
carcinomas, transitional cell carcinoma, squamous cell carcinoma
breast: ductal carcinoma in situ, lobular carcinoma in situ ovary:
ovarian carcinoma (e.g., epithelial (serous tumors, mucinous
tumors, endometrioid tumors), germ cell (e.g., teratomas,
choriocarcinomas, polyembryomas, embryomal carcinoma, endodermal
sinus tumor, dysgerminoma, gonadoblastoma), stromal carcinomas
(e.g., granulosal stromal cell tumors)), fallopian tube carcinoma,
peritoneal carcinoma, leiomyoma prostate: epithelial neoplasms
(e.g., adenocarcinoma, small cell tumors, transitional cell
carcinoma, carcinoma in situ, and basal cell carcinoma),
carcinosarcoma, non-epithelial neoplasms (e.g., mesenchymal and
lymphoma), germ cell tumors, prostatic intraepithelial neoplasia
(PIN), hormone independent prostate cancer, benign prostate
hyperplasia, prostatitis
[0177]
8TABLE 4 CELL LINES FOR VALIDATING LIV-1 ANTIBODIES FOR BLADDER AND
OVARIAN CANCERS: The following cell lines may be used to validate
the effectiveness of anti-LIV-1 antibodies in diseases involving
ovaries and bladder. Experiments similar to those described in the
Examples above, could be carried out. SW780 (bladder), OVCAR3
(ovarian), ES-2 (ovarian). It is understood that the examples
described above in no way serve to limit the true scope of this
invention, but rather are presented for illustrative purposes. All
publications, sequences of accession numbers, and patent
applications cited in this specification are herein incorporated by
reference as if each individual publication or patent application
were specifically and individually indicated to be incorporated by
reference. All UniGene cluster identification numbers and accession
numbers herein are for the GenBank sequence database and the
sequences of the accession numbers are hereby expressly
incorporated by reference. GenBank is known in the art, see, e.g.,
Benson, DA, et al., Nucleic Acids Research 26: 1-7 (1998).
Sequences are also available in other databases, e.g., European
Molecular Biology Laboratory (EMBL) and DNA Database of Japan
(DDBJ).
[0178]
9TABLE 5 Liv-1 Mutant BCR4M1 cDNA and Protein Sequences BCR4 M1
cDNA (SEQ ID NO:13)
ATGGCGAGGAAGTTATCTGTAATCTTGATCCTGACCTTTGCCCTCTCTGTCACAAATCCCCTTCATGA
ACTAAAAGCAGCTGCTTTCCCCCAGACCACTGAGAAAATTAGTCCGAATTGGGAATCTGGCA-
TTAATG TTGACTTGGCAATTTCCACACGGCAATATCATCTACAACAGCTTTTCTACC-
GCTATGGAGAAAATAAT TCTTTGTCAGTTGAAGGGTTCAGAAAATTACTTCAAAATA-
TAGGCATAGATAAGATTAAAAGAATCCA TATACACCATGACCACGACCATCACTCAG-
ACCACGAGCATCACTCAGACCATGAGCGTCACTCAGACC
ATGAGCATCACTCAGACCACGAGCATCACTCTGACCATGATCATCACTCTCACCATAATCATGCTGCT
TCTGGTAAAAATAAGCGAAAAGCTCTTTGCCCAGACCATGACTCAGATAGTTCAGGTAAAGA-
TCCTAG AAACAGCCAGGGGAAAGGAGCTCACCGACCAGAACATGCCAGTGGTAGAAG-
GAATGTCAAGGACAGTG TTAGTGCTAGTGAAGTGACCTCAACTGTGTACAACACTGT-
CTCTGAAGGAACTCACTTTCTAGAGACA ATAGAGACTCCAAGACCTGGAAAACTCTT-
CCCCAAAGATGTAAGCAGCTCCACTCCACCCAGTGTCAC
ATCAAAGAGCCGGGTGAGCCGGCTGGCTGGTAGGAAAACAAATGAATCTGTGAGTGAGCCCCGAAAAG
GCTTTATGTATTCCAGAAACACAAATGAAAATCCTCAGGAGTGTTTCAATGCATCAAAGCTA-
CTGACA TCTCATGGCATGGGCATCCAGGTTCCGCTGAATGCAACAGAGTTCAACTAT-
CTCTGTCCAGCCATCAT CAACCAAATTGATGCTAGATCTTGTCTGATTCATACAAGT-
GAAAAGAAGGCTGAAATCCCTCCAAAGA CCTATTCATTACAAATAGCCTGGGTTGGT-
GGTTTTATAGCCATTTCCATCATCAGTTTCCTGTCTCTG
CTGGGGGTTATCTTAGTGCCTCTCATGAATCGGGTGTTTTTCAAATTTCTCCTGAGTTTCCTTGTGGC
ACTGGCCGTTGGGACTTTGAGTGGTGATGCTTTTTTACACCTTCTTCCACATTCTCATGCAA-
GTCACC ACCATAGTCATAGCCATGAAGAACCAGCAATGGAAATGAAAAGAGGACCAC-
TTTTCAGTCATCTGTCT TCTCAAAACATAGAAGAAAGTGCCTATTTTGATTCCACGT-
GGAAGGGTCTAACAGCTCTAGGAGGCCT GTATTTCATGTTTCTTGTTGAACATGTCC-
TCACATTGATCAAACAATTTAAAGATAAGAAGAAAAAGA
ATCAGAAGAAACCTGAAAATGATGATGATGTGGAGATTAAGAAGCAGTTGTCCAAGTATGAATCTCAA
CTTTCAACAAATGAGGAGAAAGTAGATACAGATGATCGAACTGAAGGCTATTTACGAGCAGA-
CTCACA AGAGCCCTCCCACTTTGATTCTCAGCAGCCTGCAGTCTTGGAAGAAGAAGA-
GGTCATGATAGCTCATG CTCATCCACAGGAAGTCTACAATGAATATGTACCCAGAGG-
GTGCAAGAATAAATGCCATTCACATTTC CACGATACACTCGGCCAGTCAGACGATCT-
CATTCACCACCATCATGACTACCATCATATTCTCCATCA
TCACCACCACCAAAACCACCATCCTCACAGTCACAGCCAGCGCTACTCTCGGGAGGAGCTGAAAGATG
CCGGCGTCGCCACTTTGGCCTGGATGGTGATAATGGGTGATGGCCTGCACAATTTCAGCGAT-
GGCCTA GCAATTGGTGCTGCTTTTACTGAAGGCTTATCAAGTGGTTTAAGTACTTCT-
GTTGCTGTGTTCTGTGC TGCGTTGCCTGCTGCATTAGGTGACTTTGCTGTTCTACTA-
AAGGCTGGCATGACCGTTAAGCAGGCTG TCCTTTATAATGCATTGTCAGCCATGCTG-
GCGTATCTTGGAATGGCAACAGGAATTTTCATTGGTCAT
TATGCTGAAAATGTTTCTATGTGGATATTTGCACTTACTGCTGGCTTATTCATGTATGTTGCTCTGGT
TGATATGGTACCTGAAATGCTGCACAATGATGCTAGTGACCATGGATGTAGCCGCTGGGGGT-
ATTTCT TTTTACAGAATGCTGGGATGCTTTTGGGTTTTGGAATTATGTTACTTATTT-
CCATATTTGAACATAAA ATCGTGTTTCGTATAAATTTC BCR4 M1 protein sequence
(SEQ ID NO:14) MARKLSVILILTFALSVTNPLHELKA-
AAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENN
SLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSDHEHHSDHDHHSHHNHAA
SGKNKRKALCPDHDSDSSGKDPPNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEG-
THFLET IETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSR-
NTNENPQECFNASKLLT SHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKA-
EIPPKTYSLQIAWVGGFIAISIISFLSL LGVILVPLMNRVFFKFLLSFLVALAVGTL-
SGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLS
SQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQ
LSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKN-
KCHSHF HDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATL-
AWMVIMGDGLHNFSDGL AIGAAFTEGLSSGLSTSVAVFCAALPAALGDFAVLLKAGM-
TVKQAVLYNALSAMLAYLGMATGIFIGH YAENVSMWIFALTAGLFMYVALVDMVPEM-
LHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHK IVFRINF
[0179]
10TABLE 6 Liv-1 Antibodies Number Designation 1 1.1F10 2 1.7A4 3
BR2-10b 4 BR2-11a 5 BR2-13a 6 BR2-14a 7 BR2-15a 8 BR2-16a 9 BR2-17a
10 BR2-18a 11 BR2-19a 12 BR2-20a 13 BR2-21a 14 BR2-22a 15 BR2-23a
16 BR2-24a 17 BR2-25a
* * * * *