U.S. patent application number 10/269010 was filed with the patent office on 2003-05-22 for identifying anti-tumor targets or agents by lipid raft immunization and proteomics.
Invention is credited to Green, Jennifer McPhate, Tso, J. Yun.
Application Number | 20030096285 10/269010 |
Document ID | / |
Family ID | 26986685 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030096285 |
Kind Code |
A1 |
Tso, J. Yun ; et
al. |
May 22, 2003 |
Identifying anti-tumor targets or agents by lipid raft immunization
and proteomics
Abstract
The present invention is directed to a method for identifying
anti-tumor targets by examining lipid rafts. It provides for a
method for identifying anti-tumor targets by lipid raft proteomics
and a method for identifying anti-tumor agents by lipid rafts
immunization. It also provides for hybridomas produced by the
method of identifying anti-tumor agents by lipid raft immunization,
and antibodies produced by the hybridomas. It also provides for the
anti-tumor targets or agents identified by the methods in the
present invention.
Inventors: |
Tso, J. Yun; (Menlo Park,
CA) ; Green, Jennifer McPhate; (Belmont, CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
26986685 |
Appl. No.: |
10/269010 |
Filed: |
October 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60329178 |
Oct 11, 2001 |
|
|
|
60331965 |
Nov 21, 2001 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
424/155.1; 435/344; 435/7.23; 506/9; 530/388.8; 536/23.2 |
Current CPC
Class: |
C07K 16/40 20130101;
C07K 2317/73 20130101; C07K 16/2833 20130101; A61K 2039/505
20130101; C07K 16/30 20130101; C07K 16/3069 20130101; G01N 2405/00
20130101; G01N 33/57492 20130101; C07K 16/3015 20130101 |
Class at
Publication: |
435/6 ; 435/7.23;
530/388.8; 536/23.2; 435/344; 424/155.1 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; A61K 039/395; C12N 005/06; C07K 016/30 |
Claims
1. A method for identifying a tumor target comprising examining a
lipid raft, wherein said lipid raft is derived from a tumor cell or
a normal cell.
2. The method according to claim 1, wherein said examining
comprises: a. isolating lipid rafts from a tumor cell and a normal
cells; b. comparing the lipid raft protein expressions of said
tumor cell and said normal cell; c. isolating a protein that is
differentially expressed in said tumor cell.
3. The method according to claim 2, further comprising: d.
identifying partial or full amino acid sequence of said molecule,
or partial or full nucleic acid sequence encoding said isolated
protein.
4. The method of identifying an anti-tumor agent comprising
selecting an inhibitor of said molecule according to claim 2.
5. A method for identifying anti-tumor targets comprising: a.
isolating lipid rafts; b. separating the lipid rafts by mean of
electrophoresis, so that individual protein bands are separated
from each other; c. comparing the protein expressions of said lipid
rafts from cancer cells and from normal cells. d. isolating a
protein band that is differentially expressed in cancer cells; and
e. identifying partial or full amino acid sequence of the protein,
or partial or full nucleic acid sequence encoding the protein.
6. The method according to claim 3, wherein said tumor target is a
prostate tumor target, wherein said tumor cell is a prostate tumor
cell and said normal cell is a normal prostate cell.
7. A method of identifying anti-prostate tumor agents comprising
selecting an inhibitor of said prostate tumor target according to
claim 6.
8. A method for generating an antibody against a tumor target
associated with a type of tumor cells, comprising: a. isolating
lipid rafts from said type of tumor cells; and b. immunizing an
animal host by said isolated lipid rafts.
9. The method according to claim 8 further comprising: c. producing
hybridomas from the immunized animal host, wherein said hybridomas
produce monoclonal antibodies; d. selecting said monoclonal
antibodies; and e. purifying said selected monoclonal
antibodies.
10. A method according to claim 9, wherein said selecting comprises
selecting monoclonal antibodies that bind to said type of tumor
cells but not to normal cells.
11. A method according to claim 10, wherein said selecting further
comprises selecting monoclonal antibodies that induces apoptosis of
said type of tumor cells.
12. A method according to claim 9, where said selecting comprising
selecting monoclonal antibodies that inhibit cell proliferation of
said type of tumor cell.
13. A method according to claim 10, wherein said type of tumor
cells are prostate tumor cells, said normal cells are non-cancerous
prostate cells.
14. A method according to claim 11, wherein said type of tumor
cells are leukemia cells, said normal cells are T cells.
15. A method of identifying a tumor target comprising identifying
an antigen that binds to the selected antibodies according to claim
9, wherein said identifying comprises identifying a partial or full
amino acid or nucleic acid of said antigen.
16. A hybridoma produced by the method according to claim 13.
17. A hybridoma produced by the method according to claim 14.
18. An antibody generated by the hybridoma according to claim
16.
19. An antibody generated by the hybridoma according to claim
17.
20. An antigen that binds to the antibody according to claim 18 or
19.
21. An isolated lipid raft derived from a prostate tumor cell,
wherein said isolated lipid raft comprises a polypeptide that is
differentially expressed in a prostate tumor cell.
22. The isolated lipid raft according to claim 21, wherein said
isolated lipid raft is clustered with other lipid rafts derived
from said prostate tumor cell.
23. The isolated lipid raft according to claim 21, wherein said
isolated lipid raft is a detergent resistant membrane (DRM).
24. The isolated lipid raft according to claim 21, wherein said
polypeptide is selected from the group consisting of PMSA, CD10,
Trop-1, ATP synthase, NCAM2, and CD222.
25. A monoclonal antibody that binds to the isolated lipid raft
according to claim 21.
26. The monoclonal antibody according to claim 25, wherein said
monoclonal antibody binds to or neutralizes PMSA.
27. The monoclonal antibody according to claim 25, wherein said
monoclonal antibody binds to or neutralizes CD10.
28. The monoclonal antibody according to claim 27, wherein said
monoclonal antibody specifically binds to prostate cancer cells but
not binds to other types of cancer cells.
29. The monoclonal antibody according to claim 25, wherein said
monoclonal antibody binds to or neutralizes Trop-1.
30. The monoclonal antibody according to claim 29, wherein said
monoclonal antibody reduces the colony formation of prostate cancer
cells by more than 60%.
31. The monoclonal antibody according to claim 25, wherein said
monoclonal antibody binds to or neutralizes ATP synthase.
32. The monoclonal antibody according to claim 25, wherein said
monoclonal antibody binds to or neutralizes NCAM2.
33. The monoclonal antibody according to claim 25, wherein said
monoclonal antibody binds to or neutralizes CD222.
34. An isolated lipid raft derived from a leukemia cell, wherein
said isolated lipid raft comprising a polypeptide that is
differentially expressed in said leukemia cell compared to a normal
T cell.
35. The isolated lipid raft according to claim 34, wherein said
leukemia cell is a KG-b 1 cell.
36. A monoclonal antibody that binds to the isolated lipid raft and
the polypeptide according to claim 35.
37. The antibody according to claim 36 wherein said antibody
induces apoptosis of said leukemia cell.
38. The isolated lipid raft according to claim 35, wherein said
polypeptide is HLA-DR antigen.
39. The antibody according to claim 38, wherein said antibody
induces apoptosis of said tumor cells by more than 80%.
40. A method of treating prostate cancer comprising administering
into a subject in need of such a treating a pharmaceutically
effective amount of the antibodies according to claim 25, 26, 28,
30, 32, or 33.
41. A pharmaceutical composition comprising the pharmaceutical
carrier and the antibodies according to claim 25, 26, 28, 30, 32,
or 33.
Description
[0001] This application claims the benefit of priority of the U.S.
provisional application U.S. Ser. No. 60/329,178 filed Oct. 11,
2001 and the U.S. provisional application U.S. Ser. No. 60/331,965,
filed Nov. 21, 2001, each of which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] This invention concerns methods for identifying anti-tumor
targets or agents by lipid raft proteomics or by lipid raft
immunization.
BACKGROUND OF THE INVENTION
[0003] Lipid rafts are regions on the plasma membrane that have a
different composition of lipids than the surrounding plasma
membrane. They are enriched in signaling molecules and can change
their size and composition in response to intra- or extracellular
stimuli (Simons, K., et al., Nature Reviews/Molecular Cell Biology:
Vol. 1 pp 31-39 (2000)). This action favors specific
protein-protein interactions, resulting in the activation of
signaling cascades. The most important role of rafts at the cell
surface is their function in signal transduction. It has been shown
that growth factor receptors and sensor molecules migrate to lipid
rafts after ligand binding or cross-linking. It is known that
growth factor receptors are closely related to tumor formation.
Therefore lipid rafts are a good source of tumor-associated
antigens. Accordingly, the current invention uses lipid rafts
instead of entire cell membranes in the search for the
cancer-related molecules (tumor targest).
SUMMARY OF THE INVENTION
[0004] The present invention provides a method for identifying a
tumor target comprising examining a lipid raft, wherein said lipid
raft is derived from a tumor cell.
[0005] Preferably, said examining comprises: isolating lipid rafts
from a tumor cell and a normal cell; comparing the lipid raft
protein expressions of said tumor cell and said normal cell;
isolating a molecule that is differentially expressed in said tumor
cell. More preferably, said method further comprises: identifying
partial or full amino acid sequence of said molecule, or partial or
full nucleic acid sequence encoding said molecule.
[0006] Preferably, said tumor target is a prostate tumor target,
wherein said tumor cell is a prostate tumor cell and said normal
cell is a normal prostate cell.
[0007] The present invention provides a method for generating an
antibody against a tumor target associated with a type of tumor
cells, comprising: isolating lipid rafts from said type of tumor
cells; and immunizing an animal host by said lipid rafts. producing
hybridomas from the immunized animal host selecting said monoclonal
antibodies; and purifying said selected antibodies
[0008] The present invention provide for an isolated lipid raft
derived from a prostate cancer cell, wherein said isolated lipid
raft comprising a polypeptide that is differentially expressed in a
prostate tumor cell. Preferably, said polypeptide is selected from
the group consisting of PMSA, CD10, Trop-1, ATP synthase, NCAM2,
and CD222.
[0009] The present invention provides an isolated monoclonal
antibody that binds to the isolated lipid raft, wherein said
monoclonal antibody binds to or neutralizes PMSA, CD10, Trop-1, ATP
synthase, NCAM2, or CD222.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1. Comparison of lipid rafts prepared from normal
prostate cells and three prostate carcinoma cell lines, DU 145,
LNCaP, and PC-3. Equal amounts of protein from lipid raft
preparations were separated by SDS-PAGE and then visualized by
silver staining. Differentially expressed proteins are marked.
[0011] FIG. 2. Comparison of lipid rafts prepared from LNCaP and
normal prostate cells by 2-dimensional electrophoresis. Equal
amounts of protein from lipid raft preparations were separated by
2-dimensional electrophoresis and then visualized by silver
staining. Protein spots that are present in the LNCaP sample, but
not the normal prostate sample are denoted with arrows. Protein
spots that have been identified by peptide mass profiling are
labeled with numbers (see Table 1).
[0012] FIG. 3. ATP synthase is present in lipid rafts preparations
from prostate cancer cell lines, but not from normal prostate
cells. Equal amounts of lipid raft proteins prepared from normal
prostate cells and three prostate carcinoma cell lines (LNCaP, DU
145, and PC-3) were separated by SDS-PAGE and then
electrotransferred onto a PVDF membrane. Western blotting was
performed using an antibody specific for ATP synthase followed by
HRP-conjugated goat anti-mouse IgG. The membrane was developed
using enhanced chemiluminescence.
[0013] FIG. 4. ATP synthase is present in rafts prepared from many
different cancer cell lines. Equal amounts of lipid raft proteins
prepared from various cancer cell lines (BeWo, Colo205, HT-29,
JEG-3, KG-1, LS 180, MCF-7, LNCaP, NCI-H292, PANC-1, RT-4, and
THP-1) were separated by SDS-PAGE and then electrotransferred onto
a PVDF membrane. Western blotting was performed as described in
FIG. 3.
[0014] FIG. 5. Flow cytometric analysis of ATP synthase cell
surface expression. LNCaP cells were resuspended in 100 .mu.L
PBS/5% fetal calf serum with 1 .mu.g anti-ATP synthase (middle) or
anti-Trop-1 (EpCAM) (right). Cells were also stained with a
negative control antibody (left). Cells were washed and bound
antibody was detected with PE-conjugated goat anti-mouse IgG. Cells
were then analyzed by flow cytometer.
[0015] FIG. 6. Flow cytometric analysis of ATP synthase expression
in the AML cell line THP-1. THP-1 cells were resuspended in 100
.mu.L PBS/5% fetal calf serum with 1 .mu.g anti-ATP synthase
.alpha. subunit (middle) or .beta. subunit (right). Cells were also
stained with a negative control antibody (left). Cells were washed
and bound antibody was detected with PE-conjugated goat anti-mouse
IgG. Cells were then analyzed by flow cytometer.
[0016] FIG. 7. ATP synthase is localized to the cell surface of
LNCaP prostate carcinoma as visualized by immunofluorescence. LNCaP
cells, grown on glass coverslips, were stained with antibodies
specific for ATP synthase (top) or Trop-1 (EpCAM) (bottom). Bound
antibody was detected with Alexa 488-conjugated goat anti-mouse
IgG. Coverslips were mounted onto slides and examined with a Nikon
Optiphot 2 microscope and photographed. No staining is observed on
cells that have been stained with the secondary antibody alone
(data not shown).
[0017] FIG. 8. Anti-ATP synthase inhibits LNCaP cell proliferation.
LNCaP cells (20,000 cells/well) were plated into a 96 well tissue
culture plate. After cells were allowed to grow undisturbed for two
days, antibodies (5 .mu.g/ml anti-ATP synthase, anti-Trop-1 (EpCAM)
(323/A3), or anti-MHC class II (Mu1D10)) were added and incubated
with the cells for 24 hours. AlamarBlue reagent was added to assess
cell proliferation. Fluorescence was detected at .lambda.ex=530 nm,
.lambda.em=590 nm. Data are expressed as the mean+/-SEM of 4
replicates.
[0018] FIG. 9. Anti-ATP synthase inhibits LNCaP colony formation in
soft agar. LNCaP cells were plated in soft agar and treated with
anti-ATP synthase or anti-Trop-1 (EpCAM) (5 .mu.g/ml) for up to 20
days. Colonies were counted under an inverted phase-contrast
microscope and a group of 5 or more cells were counted as a
colony.
[0019] FIG. 10. Anti-ATP synthase induces apoptosis in THP-1 cells.
THP-1 cells were treated with anti-ATP synthase or anti-Trop-1
(EpCAM) (5 .mu.g/mL) for 24 hours. Cells were then harvested at the
indicated times after the induction of apoptosis and were stained
with FITC-conjugated annexin V and propidium iodide. Flow cytometry
was used to assess percentage of apoptosis (annexin V.sup.+ and
propidium iodide.sup.-/+ cells).
[0020] FIG. 11. Comparison of lipid rafts from various cancer cell
lines. Equal amounts of protein from lipid raft preparations were
separated by SDS-PAGE and then visualized by silver staining.
[0021] FIG. 12. Flow chart summarizing how the tumor-specific
hybridomas were obtained from the LNCaP lipid raft
immunization.
[0022] FIG. 13. Antigen grouping by immunoprecipitation. .sup.125I
labeled LNCaP lysate was incubated individually with 20 hybridoma
supernatants (see Table 2). Antibody-antigen complexes were
captured by Gamma Bind Plus Sepharose and analyzed by SDS-PAGE.
Lane 1, P1-42; Lane 2, P2-23; Lane 3, P3-53; Lane 4, P4-79; Lane 5,
P6-49; Lane 6, P9-65; Lane 7, P8-2; Lane 8, P8-11; Lane 9, P8-14;
Lane 10, P8-35; Lane 11, P8-74; Lane 12, P9-32; Lane 13, P9-64;
Lane 14, P10-2; Lane 15, P10-28, Lane 16, P10-29; Lane 17, P10-62;
Lane 18, P10-70; Lane 19, P10-82; and Lane 20, P12-22. Molecular
weight standards (MW) are in kD.
[0023] FIG. 14. Anti-NCAM2 inhibits LNCaP cell proliferation. LNCaP
cells (20,000 cells/well) were plated into a 96 well tissue culture
plate. After cells were allowed to grow undisturbed for two days, 4
different NCAM2-specific antibodies (5 .mu.g/ml) were added and
incubated with the cells for 24 hours. AlamarBlue reagent was added
to assess cell proliferation. Fluorescence was detected at
.lambda.ex=530 nm, .lambda.em=590 nm. Data are expressed as the
mean+/-SEM of 4 replicates.
[0024] FIG. 15. Anti-NCAM2 inhibits LNCaP colony formation in soft
agar. LNCaP cells were plated in soft agar and treated with 4
different NCAM2-specific antibodies (5 .mu.g/ml) for up to 20 days.
Colonies were counted under an inverted phase-contrast microscope
and a group of 10 or more cells were counted as a colony.
[0025] FIG. 16. Antigen grouping by immunoprecipitation. 125I
labeled LNCaP lysate (Lanes 1-10) or Panc-1 lysate (Lanes 11-16)
were incubated individually with 16 hybridoma supernatants (see
Tables 2 and 3) that showed broad specficity against many cancer
cell lines (see Table 3). Antibody-antigen complexes were captured
by Gamma Bind Plus Sepharose and analyzed by SDS-PAGE. Lane 1,
P12-27; Lane 2, P11-65; Lane 3, P8-83; Lane 4, P8-32; Lane 5,
P8-20; Lane 6, P7-69; Lane 7, P4-48; Lane 8, P3-28; Lane 9, P2-68;
Lane 10, P1-95; Lane 11, P11-93; Lane 12, P11-85; Lane 13, P11-57;
Lane 14, P11-49; Lane 15, P8-35, Lane 16, P8-11. Molecular weight
standards (MW) are in kD.
[0026] FIG. 17. Flow chart summarizing how the tumor-specific
hybridomas were obtained from the KG-1 lipid raft immunization.
[0027] FIG. 18. Antigen grouping by immunoprecipitation. (A).
.sup.125I labeled KG-1 lysate was incubated individually with 36
hybridoma supernatants (see Table 4). Antibody-antigen complexes
were captured by Gamma Bind Plus Sepharose and analyzed by
SDS-PAGE. Lane 1, K1-34; Lane 2, K1-47; Lane 3, K1-79; Lane 4,
K1-95; Lane 5, K1-97; Lane 6, K2-109; Lane 7, K2-124; Lane 8,
K2-127; Lane 9, K2-167; Lane 10, K5-37; Lane 11, K5-71; Lane 12,
K6-98; Lane 13, K6-103; Lane 14, K6-114; Lane 15, K6-121, Lane 16,
K6-149. Lane 17, K6-150; Lane 18, K6-175; Lane 19, K6-179; Lane 20,
K7-196; Lane 21, K7-270; Lane 22, K7-275; Lane 23, K8-335; Lane 24,
K8-343; Lane 25, K8-355; Lane 26, K8-364; Lane 27, K8-365; Lane 28,
K9-3; Lane 29, K9-64; Lane 30, K9-92; Lane 31, K11-230, Lane 32,
K11-272, Lane 33, K11-280, Lane 34, K11-282, Lane 35, K12-328, Lane
36, K12-360. Molecular weight standards (MW) are in kD. (B).
Biotinylated KG-1 lysate was incubated individually with 15
hybridoma supernatants that failed to identify the molecular weight
of their antigens in the experiment shown in panel (A).
Antibody-antigen complexes were captured by Gamma Bind Plus
Sepharose, analyzed by SDS-PAGE. Lane 1, K8-364; Lane 2, K8-365;
Lane 3, K9-92; Lane 4, K11-272; Lane 5, 11-280; Lane 6, K6-150;
Lane 7, K6-149; Lane 8, K2-109; Lane 9, K2-127; Lane 10, K5-71;
Lane 11, K6-103; Lane 12, K6-114; Lane 13, K6-121; Lane 14, K9-64;
Lane 15, K7-196. Molecular weight standards (MW) are in kD.
[0028] FIG. 19. Nucleotide sequence and deduced amino acid sequence
of the heavy chain variable region (VH) of K8-355 (anti-HLA-DR).
The signal peptide is in italic, the three complementarity
determining regions (CDRs) are underlined, and the first
NH2-terminal amino acid residue of the matured heavy chain (Mouse
IgG1) is in bold. SEQ ID NO: 1 is the amino acid sequence of the
heavy chain variable region (VH) of K8-355 (anti-HLA-DR) (signaling
peptide is not included). FIG. 20. Nucleotide sequence and deduced
amino acid sequence of the light chain variable region (VH) of
K8-355 (anti-HLA-DR). The signal peptide is in italic, the three
complementarity determining regions (CDRs) are underlined, and the
first NH2-terminal amino acid residue of the matured light chain
(mouse kappa) is in bold. SEQ ID NO: 2 is the amino acid sequence
of the light chain variable region (VL) of K8-355 (anti-HLA-DR)
(signaling peptide is not included). FIG. 21. K8-355 (anti-HLA-DR)
induces apoptosis in Raji and Daudi cells. Raji or Daudi cells were
treated with K8-355 (5 .mu.g/mL) for 24 hours. Cells were then
harvested at the indicated times after the induction of apoptosis
and were stained with FITC-conjugated annexin V and propidium
iodide. Flow cytometry was used to assess percentage of apoptosis
(annexin V.sup.+ and propidium iodide.sup.-/+ cells).
[0029] FIG. 22. Table 1 shows five identified proteins from LNCaP
lipid raft 2-D samples by peptide mass profiling.
[0030] FIG. 23. Table 2 shows the reactivity profiles of 20
anti-LNCaP lipid raft hybridomas that showed limited binding to
other cancer cell lines
[0031] FIG. 24. Table 3 shows the reactivity profiles of 1
anti-LNCaP lipid raft hybridomas that showed broad binding to other
cancer cell lines.
[0032] FIG. 25. Table 4 shows the reactivity profiles of 36
anti-KG-1 lipid raft hybridomas that showed apoptosis-inducing
activity.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] As used herein, the term "differentially expressed" means
that a protein or polypeptide is expressed at a higher level, for
instance, 20% more, preferably 50% more, more preferably, 80% more,
in a cell type that is cancerous (tumor cell), than that in said
cell type that is non-cancerous (normal cell).
[0034] The term "tumor target" and "anti-tumor target" are used
interchangeable herein and refers to any molecules in correlation
with the occurrence or existence of a tumor or a tumor cell.
Preferably, a tumor target is expressed at a higher level in a type
of tumor cell than that in the normal cells. The term "anti-tumor
agent" refers to any molecules that can inhibit tumor or cancer
cell growth. The anti-tumor agent may reduce the growth rate or the
size of tumor cells, or inhibit or prevent proliferation or
migration of tumor cells. It may inhibit the colony formation of
cancer cells due to the anchorage-independent growth. Preferably,
such an inhibition at the cellular level can reduce the tumor size,
deter or reverse the growth of a tumor, reduce the aggressiveness
of a tumor, or prevent or inhibit tumor metastasis. Preferably
anti-tumor agent is an inhibitor of a tumor targets. The inhibitor
can be an antibody against the tumor targets, or a molecule
inhibiting the activities of the tumor targets, or a molecule
down-regulating the expression of tumor targets, or a molecule
down-regulating the transcription of DNA encoding the tumor
targets, or an anti-sense nucleic acid sequence of partial or full
nucleic acid sequence encoding the tumor target. More preferably,
it is an antibody against a tumor target associated with a type of
tumor.
[0035] The term "lipid raft" refers to a lipid raft or a portion
thereof in a clustered state or a non-clustered state, including
"lipid raft", "clustered lipid rafts", and "DRM", each of which has
been described in detail in Simons, K., et al., Nature
Reviews/Molecular Cell Biology: Vol. 1 pp 31-39 (2000). In
particular, "lipid raft" contains a given set of proteins that can
change size and composition in response to intra- or extracellular
stimuli. This favors specific protein-protein interactions,
resulting in the activation of signally cascade. Sometimes, the
lipid rafts may be clustered together. It has been reported that
clustering is used both artificially and physiologically to trigger
signally cascades. DRMs (detergent-resistant membranes) are the
rafts that remain insoluble after treatment on ice with detergents.
They are believed to be non-native aggregated rafts.
[0036] The term "apoptosis", "apoptotic cell death" or "programmed
cell death" as used herein refers to any cell death that results
from the complex cascade of cellular events that occur at specific
stages of cellular differentiation and in response to specific
stimuli. Apoptotic cell death is characterized by condensation of
the cytoplasm and nucleus of dying cells.
[0037] The term "colony formation" refers to the number of colonies
formed due to the inhibition of the anchorage-independent cell
growth. Various methods known in the art can be used to measure the
colony formation such as counting the number of tumor cell colonies
formed.
[0038] The term "antibody" or "immunoglobulin" is intended to
encompass both polyclonal and monoclonal antibodies. The preferred
antibody is a monoclonal antibody reactive with the antigen. The
term "antibody" is also intended to encompass mixtures of more than
one antibody reactive with the antigen (e.g., a cocktail of
different types of monoclonal antibodies reactive with the
antigen). The term "antibody" is further intended to encompass
whole antibodies, biologically functional fragments thereof,
single-chain antibodies, and genetically altered antibodies such as
chimeric antibodies comprising portions from more than one species,
bifunctional antibodies, antibody conjugates, humanized and human
antibodies. Biologically functional antibody fragments, which can
also be used, are those peptide fragments derived from an antibody
that are sufficient for binding to the antigen.
[0039] By "a pharmaceutically effective" amount of a drug or
pharmacologically active agent or pharmaceutical formulation is
meant a nontoxic but sufficient amount of the drug, agent or
formulation to provide the desired effect.
[0040] A "subject," "individual" or "patient" is used
interchangeably herein, which refers to a vertebrate, preferably a
mammal, more preferably a human.
[0041] The term "genetically altered antibodies" means antibodies
wherein the amino acid sequence has been varied from that of a
native antibody. Because of the relevance of recombinant DNA
techniques to this invention, one need not be confined to the
sequences of amino acids found in natural antibodies; antibodies
can be redesigned to obtain desired characteristics. The possible
variations are many and range from the changing of just one or a
few amino acids to the complete redesign of, for example, the
variable or constant region. Changes in the constant region will,
in general, be made in order to improve or alter characteristics,
such as complement fixation, interaction with membranes and other
effector functions. Changes in the variable region will be made in
order to improve the antigen binding characteristics.
[0042] The term "humanized antibody" or "humanized immunoglobulin"
refers to an immunoglobulin comprising a human framework, at least
one and preferably all complimentarity determining regions (CDRs)
from a non-human antibody, and in which any constant region present
is substantially identical to a human immunoglobulin constant
region, i.e., at least about 85-90%, preferably at least 95%
identical. Hence, all parts of a humanized immunoglobulin, except
possibly the CDRs, are substantially identical to corresponding
parts of one or more native human immunoglobulin sequences. See,
e.g.Queen et al., U.S. Pat. Nos. 5,5301,101; 5,585,089; 5,693,762;
and 6,180,370 (each of which is incorporated by reference in its
entirety).
[0043] The term "chimeric antibody" refers to an antibody in which
the constant region comes from an antibody of one species
(typically human) and the variable region comes from an antibody of
another species (typically rodent).
[0044] I. Lipid Rafts
[0045] The present invention provides for methods for identifying
anti-tumor agents (tumor targets) or anti-tumor targets by
examining lipid raft, preferably by lipid raft proteomics or lipid
raft immunization.
[0046] The methods of identifying anti-tumor targets or agents in
the present invention comprise examining lipid rafts. The methods
can be used in search for anti-tumor targets in various cancer
types, including but not limited to cancers of the brain, breast,
cervix, bladder, colon, head & neck, kidney, liver, lung,
non-small cell lung, lymphoid system, pancreas, prostate, ovary,
stomach, uterus, medulloblastoma, melanoma, mesothelioma sarcoma,
and other like cancers.
[0047] Preferably, the methods for identifying anti-tumor targets
or agents further comprises preparing and examining the lipid rafts
from a certain type of cells, wherein the type of cells can be of
normal cells or cancer cells from various tissues, including but
not limited to blood, brain, breast, cervix, bladder, colon, head
& neck, kidney, liver, lung, lymphoid system, non-small cell
lung, ovary, pancreas, prostate, stomach, uterus, Medulloblastoma,
melanoma, mesothelioma, sarcoma and other like tissues.
[0048] More preferably, the lipid rafts can be isolated from
established cancer cell lines and studied in search for anti-tumor
agents. The cell lines include, but are not limited to, LNCaP
(prostate cancer), DU 145 (prostate cancer), PC-3 (prostate
cancer), PANC-1 (pancreas cancer), RT4 (bladder cancer), HT-29
(colon cancer), NCI-H292 (lung cancer), T47D (breast cancer), Hep
G2 (liver cancer), and NIH:OVCAR-3 (ovary cancer), AML cells, and
other like tumor cell lines.
[0049] The lipid rafts can be isolated by the methods known in the
art, such as the method described in Green et al, J. Cell Biol.
146, 673-682 (1999). In particular, cells are lysed and add to a
sucrose solution to form a sucrose step-gradient. The gradients are
then centrifuged, and the lipid rafts float to a fraction of the
gradients. That fraction is then isolated and concentrated.
[0050] The isolated lipid rafts can then be used for the search of
tumor targets or anti-tumor agents by lipid raft proteomics or
lipid raft immunization.
[0051] II. Lipid Raft Proteomics
[0052] The method for identifying anti-tumor targets by lipid raft
proteomics comprises identifying proteins that are differentially
expressed in a type of cancer cells. In particular, protein
expressions of lipid rafts from tumor cells and normal cells are
compared. The molecules that are differentially expressed in the
lipid rafts of tumor cells will be isolated, analyzed, and
recognized as tumor targets. The anti-tumor agents can then be
identified by selecting the inhibitors of the tumor targets.
[0053] In particular, the present invention provides a method for
identifying a tumor target comprising examining a lipid raft,
wherein said lipid raft is derived from a tumor cell or a normal
cell. Typically, said examining comprises: isolating lipid rafts
from a tumor cell and a normal cells; comparing the lipid raft
protein expressions of said tumor cell and said normal cell;
isolating a protein that is differentially expressed in said tumor
cell. Preferably, said tumor target is a prostate tumor target,
said tumor cell is a prostate tumor cell and said normal cell is a
normal prostate cell. More preferably, the prostate cancer cells
are the cells of prostate cancer cell lines, including, but not
limited to, LNCaP, DU145, and PC-3. Preferably, the prostate tumor
target is ATP synthase
[0054] In addition, the method can further comprise: identifying
partial or full amino acid sequence of the isolated protein, or
partial or full nucleic acid sequence encoding said isolated
protein. The most commonly used method of identifying amino acid
sequence is N-terminal sequencing. The experimental procedure of
N-terminal amino acid sequencing is disclosed in the Examples of
the present application.
[0055] To compare the lipid raft proteins expressions, the proteins
contained in the isolated lipid rafts may need to be separated.
Various methods known in the art may be used to separate the
proteins of the lipid rafts, such as one-dimensional
electrophoresis or two-dimensional electrophoresis. The separated
proteins may then be visualized by any standard methods known in
the art, including, but not limited to, silver staining. The
experimental procedures of performing one or two-dimensional
protein electrophoresis and silver staining are disclosed in the
Examples of the present application.
[0056] Preferably, the method of identifying anti-tumor targets by
lipid raft proteomics comprises: isolating lipid rafts; separating
the lipid rafts by means of electrophoresis or liquid
chromatography, so that individual protein bands are separated from
each other. comparing the protein expressions of said lipid rafts
from cancer cells and from normal cells; isolating a protein band
that is differentially expressed in cancer cells; and; identifying
partial or full amino acid sequence of the protein, or partial or
full nucleic acid sequence encoding the protein.
[0057] Preferably, the present invention provides for a method for
identifying anti-tumor agents comprising selecting an inhibitor of
said isolated protein. The inhibitor can be an antibody against
said protein, or a molecule inhibiting the activities of said
protein, or a molecule down-regulating the expression of said
protein, or a molecule down-regulating the transcription of DNA
encoding said protein, or an anti-sense nucleic acid sequence of
partial or full nucleic acid sequence encoding said protein.
[0058] More preferably, the anti-tumor activity of said anti-tumor
agents may be further verified. Once a tumor-related protein (tumor
target) is identified, antibodies recognizing the protein can be
generated and a Western-blot analysis can be performed using the
antibodies to confirm that the protein is indeed differentially
expressed in the cancer cells. Anti-tumor agents may be created to
inhibit the expression or function of that cancer-related protein.
The Anti-tumor activity of the anti-tumor agent can be measured by
performing a variety of experiments, including but not limited to,
in vitro cell proliferation assay. The in vivo anti-tumor activity
of the anti-tumor agents can be further verified by testing the
efficacy using animal xenograft models. Cell adhesion and migration
assays can be performed to evaluate the inhibition of adhesion and
spreading of tumor cells by the anti-tumor agents. The experimental
details of cell proliferation assay, xenograft model, and cell
adhesion and migration assays are described in the Examples of the
present application.
[0059] Preferably, the method for lipid raft proteomics can be used
for the purpose of identifying anti-tumor targets or agents
exclusively for the treatment of a particular type or subtype of
cancer. Molecules (tumor targets) relating only to one type of
cancer or a subtype of cancer can be identified by comparing the
protein expressions of lipid rafts isolated from that type of
cancer cells (for example prostate cancer cells), from other cancer
cells, and from normal cells. The proteins only expressed in that
type of cancer cells (for example, prostate cancer cells), but not
to other type of cancers are further isolated and identified as the
tumor targets of certain type or subtype of cancer according to
methods provided in the present invention. The example of cancer
subtype includes, but is not limited to, androgen-dependent
prostate cancer and androgen-independent prostate cancer.
[0060] The present invention provides for an isolated protein,
wherein said isolated protein is expressed at higher rate in a cell
type that is cancerous than said cell type that is
non-cancerous.
[0061] The present invention provides for an inhibitor of said
isolated protein.
[0062] Preferably, the inhibitor is an antibody against said
isolated protein.
[0063] II. Lipid Raft Immunization
[0064] The present invention provides for a method of identifying
tumor targets or anti-tumor agents by lipid raft immunization.
Different from lipid raft proteomics, lipid raft immunization
produces monoclonal antibodies against lipid rafts derived from a
type of tumor cells. Such monoclonal antibodies can be directly
used as anti-tumor agents after the verification of their
anti-tumor activities. The antigens that bind to such monoclonal
antibodies are then identified. If the antigens are in correlation
with tumor cells, such as differentially expressed in certain type
of tumor cells, these antigens are then recognized as the tumor
targets for that type of tumor, and the monoclonal antibodies are
recognized as anti-tumor agents.
[0065] The present invention provide for a method for identifying
anti-tumor agents such as antibodies against a tumor target
associated with a type of tumor comprising isolating lipid rafts
from said type of tumor cells; immunizing an animal with the
isolated lipid rafts. Lipid raft preparation from cancer cells may
be injected into an appropriate host animal, such as cow, horse,
goat, rat, mouse, hamster, or macaque monkey, etc. The immunization
may be boosted by multiple sequential injections. A suggested
protocol includes, but is not limited to, injection of 50 .mu.g
lipid raft proteins on Day 7 and Day 14 after the initial
immunization. The experimental details of immunization are
described in the Examples of the present application.
[0066] Preferably, such a method further comprises: producing
hybridomas from the immunized animal host, wherein said hybridomas
produce monoclonal antibodies; selecting the hybridoma (monoclonal)
antibodies; and purifying and identifying the hybridoma
(monoclonal) antibodies.
[0067] In one embodiment of the present invention, after the
immunization, the animal may be sacrificed and the lymphocytes of
said animal may be elicited. The lymphocytes can produce or be
capable of producing antibodies that specifically bind to the
protein used for immunization. Lymphocytes then are fused with
myeloma cells using suitable fusing agents to form hybridomas
cells. Examples of myeloma cell lines include, but are not limited
to NS0. The hybridomas cells may be seeded and grow in suitable
culture medium in 96- well culture plate with a density of one
hybridoma cell per well. More preferably, nucleic acid encoding an
inhibitor of apoptosis may be delivered into the myeloma cells to
prevent the B-cell death induced by the production of
auto-antigens. Said nucleic acids include, but are not limited to,
anti-apoptosis genes, such as BCL-2. The experimental details of
creating hybridomas cells are described in the Examples of the
present invention.
[0068] Preferably, the anti-tumor agent may be identified by
selecting hybridoma antibodies based on their differential binding
reactivity to the type of cancer cells of interest. Hybridoma
antibodies that bind to the type of cancer cells but not normal
cells may be selected for further study. More preferably, the
selected hybridomas can be further screened against multiple other
types of cancer cells so that the antigen expression profiles of
hybridoma antibodies can be established. As a result, the selected
hybridomas may be categorized into two groups: one group of
hybridoma antibodies may show that their antigens are only
expressed in a limited number of types of cancer cells.
Accordingly, their antibodies can be employed for the treatment of
a particular type of cancer. The other group of hybridoma
antibodies may show that their antigens are expressed in multiple
cancer cells. As a result, their antibodies may be used more
broadly for the treatment of cancer. More experimental details are
disclosed in the Examples of the present application.
[0069] Alternatively, the anti-tumor agent may also be selected by
functional analysis. Apoptosis analysis may be performed to
identify the hybridoma antibodies that are capable of inducing the
cell-death of tumor cells. Thus, hybridomas inducing tumor death
can be directly selected without the binding reactivity assay.
Alternatively, the hybridomas can be screened based on the binding
reactivity and then selected based on the death-inducing activity
for tumor cells. In addition, the anti-tumor agents can be selected
based on its ability to inhibit tumor cell proliferation.
[0070] Preferably, the method of identifying anti-tumor agents by
lipid raft immunization comprises purifying and identifying the
hybridoma antibodies. In other words, the method comprises
purifying and identifying the antibodies produced by the hybridomas
and the antigens that bind to the antibody. The molecular weight of
the antigens can be determined by immunoprecipitation experiments.
The antigens and antibodies of the selected hybridomas can be
further purified by affinity chromatography and the antigen
identified by microsequencing or by mass spectrometry. The
experimental procedures of immunoprecipitation, affinity
chromatography, and microsequencing can be found in the Examples of
the present application. In addition, the anti-tumor agents can be
selected based on the ability to inhibit tumor cell
proliferation.
[0071] The antibody produced by hybridomas can be directly used as
an anti-tumor agent. The anti-tumor activity of the antibodies
produced by hybridomas can be verified by cell proliferation assay,
xenograft model, and cell adhesion and migration assay. The
experimental details are described in the Examples of the present
application.
[0072] The method of identifying anti-tumor targets by lipid raft
immunization comprises identifying the antigens that bind to the
antibodies produced by hybridomas. The identity of the antigen can
lead to the discovery of a group of potential anti-tumor agents.
The examples for those anti-tumor agents include, but are not
limited to, a molecule inhibiting the activities of said protein, a
molecule down-regulating the expression of said protein, the
molecule down-regulating the transcription of DNA encoding said
protein, or anti-sense nucleic acid sequence of partial or full
nucleic acid sequence encoding said protein.
[0073] Preferably, the present invention provides for a method of
identifying anti-tumor agents for the treatment of prostate cancer
by lipid raft immunization. More preferably, the method comprises:
immunizing an animal with lipid raft preparations from prostate
cancer cells; generating hybridomas; selecting monoclonal
antibodies that are prostate cancer-positive and normal
prostate-negative. The experimental details are described in the
Examples of the present application.
[0074] More preferably, the prostate cancer cells are the cells of
androgen-dependent prostate cancer (LNCaP), or androgen-independent
prostate cancer (DU 145).
[0075] Preferably, the present invention provides for a method of
identifying anti-tumor agents for the treatment of leukemia or
lymphoma. More preferably, the method comprises: immunizing an
animal with lipid raft preparations from leukemia or lymphoma
cells; generating hybridomas; selecting hybridoma antibodies that
are leukemia or lymphoma cell-positive and T-cell-negative;
screening the cell death-inducing activity of hybridoma antibodies;
obtaining hybridomas whose antibodies show specific killing of
tumor cells. The experimental details are described in the Examples
of the present application.
[0076] Preferably, the present invention provides for a hybridoma
produced by the method of identifying anti-tumor agents by lipid
raft immunization.
[0077] More preferably, the present invention provides for an
antibody generated by the hybridoma.
[0078] More preferably, the present invention provides for an
antigen that binds to the antibody generated by the hybridoma.
[0079] IV. Tumor Targets and Anti-tumor Agents and the Use
Thereof
[0080] The present invention provides for the tumor targets or the
anti-tumor agents identified by the method of identifying
anti-tumor targets by lipid raft proteomics and lipid raft
immunization.
[0081] The present invention provides an isolated lipid raft
derived form any cell, preferably from a tumor cell, more
preferably from a prostate cancer (tumor) cell, or a leukemia or
lymphoma cell. Preferably said isolated lipid raft is clustered
with other lipid rafts derived from said prostate cancer cell. More
preferably, said isolated lipid raft is a detergent resistant
membrane (DRM).
[0082] Said isolated lipid raft derived from a prostate tumor cell
may comprise a polypeptide that is differentially expressed in a
prostate tumor cell. Preferably, said polypeptide is selected from
the group consisting of PMSA, CD10 , Trop-1, ATP synthase, NCAM2,
and CD222.
[0083] The present invention provides a monoclonal antibody that
binds to an isolated lipid rafts, preferably an isolated lipid raft
derived from a tumor cell, more preferably, said isolated lipid
raft comprises a polypeptide that is differentially expressed in a
type of tumor cell Preferably, said monoclonal antibody is a
isolated monoclonal antibody.
[0084] Typically, the monoclonal antibody binds to both isolated
lipid raft and the polypeptide that is a component of the isolated
lipid raft and differentially expressed in the tumor cell where the
lipid raft is derived from. Preferably, the monoclonal antibody
binds to an exposed epitope of the polypeptide. The term "exposed
epitope" refers to an epitope of said polypeptide that is on the
surface of the lipid raft comprising said polypeptide, and not
concealed due to the association of the polypeptide with the lipid
raft. Thus, said antibody binds both to the lipid raft and said
polypeptide. Preferably, said polypeptide is differentially
expressed in prostate tumor cells, and is more preferably selected
from the group consisting of PMSA, CD10, Trop-1, ATP synthase,
NCAM2, and CD222. Accordingly, in addition to binding to said
isolated lipid raft, said monoclonal antibody binds to or
neutralizes PMSA, CD10, Trop-1, ATP synthase, NCAM2, or CD222.
[0085] More preferably, said anti-CD10 antibody specifically binds
to prostate cancer cells but not binds to other types of cancer
cells. Said anti-Trop-1 antibody reduces the colony formation of
prostate cancer cells by more than 40%, 50%, 60%, preferably by
about 65%.
[0086] The present invention provides an isolated lipid raft
derived from a leukemia cell, wherein said isolated lipid raft
comprising a polypeptide that is differentially expressed in said
leukemia cell compared to a normal T cell. Preferably, said
leukemia cell is a KG-1 cell.
[0087] The present invention provides a monoclonal antibody that
binds to the isolated lipid raft derived from leukemia cells as
well as the polypeptide that is differentially expressed in the
leukemia cells. Preferably, said antibody induces apoptosis of the
leukemia cell, preferably, by more than 50%, 60%, 70%, or 80%. More
preferably, said polypeptide is HLA-DR antigen. The monoclonal
antibody is preferably an isolated monoclonal antibody. In one
aspect of the invention, the antibody comprises a heavy chain
variable region of SEQ ID NO 1 and a light chain variable region of
SEQ ID NO 2.
[0088] The present invention provides a method of treating prostate
cancer comprising administering into a subject in need of such a
treating a pharmaceutically effective amount of the identified
anti-prostate tumor antibodies described herein.
[0089] The present invention also provides a pharmaceutical
composition comprising a pharmaceutical carrier and the antibodies
described herein.
[0090] Preferably, the anti-tumor agents, which are identified by
using the methods in the present invention, may be employed for the
treatment of disorders including, but not limited to, Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer,
lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,
genitourinary tract cancer, malignant hypercalcemia, cervical
cancer, endometrial cancer, adrenal cortical cancer, and leukemia,
and other like cancers.
[0091] In particular, the identified anti-prostate tumor agents can
be used for the treatment of prostate cancer. Said anti-prostate
tumor agents include the antibodies isolated by lipid raft
immunization of prostate cancer cells, such as the disclosed
antibodies that bind to both the isolated lipid rafts and the
identified prostate tumor targets including, but is not limited to,
antibodies against PMSA, CD10 , Trop-1, ATP synthase, NCAM2, or
CD222.
[0092] The identified anti-leukemia or lymphoma tumor antigens can
be used for the treatment of leukemia or lymphoma. Said anti-tumor
agents include the antibodies isolated by lipid raft immunization
of leukemia cells, such as the disclosed antibodies that bind to
both the isolated lipid rafts and the identified leukemia target,
such as HLA-DR.
[0093] Preferably, pharmaceutical compositions of the present
invention are useful for parenteral administration, i.e.,
subcutaneously, intramuscularly and particularly, intravenously.
The compositions for parenteral administration commonly comprise a
solution of the antibody or a cocktail thereof dissolved in an
acceptable carrier, preferably an aqueous carrier. A variety of
aqueous carriers can be used, e.g., water, buffered water, 0.4%
saline, 0.3% glycine and the like. These solutions are sterile and
generally free of particulate matter. 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, for
example sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate, histidine and arginine. The
concentration of the antibodies in these formulations can vary
widely, i.e., from less than about 0.01%, usually at least about
0.1% to as much as 5% by weight and are selected primarily based on
fluid volumes, and solubilities in accordance with the particular
mode of administration selected.
[0094] Thus, a typical pharmaceutical composition for injection
could be made up to contain 1 ml sterile buffered water, and 1-100
mg of an antibody. A typical composition for intravenous infusion
can be made up to contain 250 ml of sterile Ringer's solution, and
10 mg of the inhibitor. Actual methods for preparing parentally
administerable compositions are known or apparent to those skilled
in the art and are described in more detail in, for example,
Remington's Pharmaceutical Science (15th Ed., Mack Publishing
Company, Easton, Pa., 1980), which is incorporated herein by
reference.
[0095] The antibodies of this invention can be frozen or
lyophilized for storage and reconstituted in a suitable carrier
prior to use depending on the physical characteristics of the
inhibitors. This technique has been shown to be effective with
conventional antibodies and art-known lyophilization and
reconstitution techniques can be employed.
[0096] For the purpose of treatment of disease, the appropriate
dosage of antibodies will depend on the severity and course of
disease, the patient's clinical history and response, the toxicity
of the inhibitors, and the discretion of the attending physician.
The inhibitors are suitably administered to the patient at one time
or over a series of treatments. The initial candidate dosage may be
administered to a patient. The proper dosage and treatment regime
can be established by monitoring the progress of therapy using
conventional techniques known to the people skilled of the art.
[0097] The amount of active ingredients that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. It will be understood, however, that the specific
dose level for any particular patient will depend upon a variety of
factors, including the activity of the specific inhibitor employed,
the age, body weight, general health, sex, diet, time of
administration, route of administration, and rate of excretion,
drug combination and the severity of the particular disease
undergoing therapy, and can be determined by those skilled in the
art.
[0098] The compositions can be administered for prophylactic and/or
therapeutic treatments, comprising preventing, inhibiting, and
reversing cancer cell proliferation, or inducing the apoptosis of
the cancer cells. An amount adequate to accomplish the desired
effect without toxic effect is defined as a "pharmaceutically
effective amount" and will generally range from about 0.01 to about
100 mg of antibody per dose. Single or multiple administrations can
be carried out to achieve the desired therapeutic effect.
[0099] Antibodies disclosed herein are useful in diagnostic and
prognostic evaluation of diseases and disorders, particularly
cancers associated with the tumor target expression. At each stage
of disease, monoclonal antibodies against the identified tumor
target may be used to improve diagnostic accuracy and facilitate
treatment decisions. Labeled monoclonal antibodies can detect
abnormal cells at an early stage, because of their expression of
tumor antigens. Once cancer is diagnosed, accurate staging is
important in deciding on the most appropriate therapy. Later,
during follow-up of surgery, rising serum levels of tumor antigens
may indicate recurrence before it can be detected by conventional
methods.
[0100] Methods of diagnosis can be performed in vitro using a
cellular sample (e.g., blood sample, lymph node biopsy or tissue)
from a patient or can be performed by in vivo imaging.
[0101] Compositions comprising the antibodies of the present
invention can be used to detect the presence of a tumor target in a
type of cancer cells, for example, by radioimmunoassay, ELISA,
FACS, etc. One or more labeling moieties can be attached to the
humanized immunoglobulin. Exemplary labeling moieties include
radiopaque dyes, radiocontrast agents, fluorescent molecules,
spin-labeled molecules, enzymes, or other labeling moieties of
diagnostic value, particularly in radiologic or magnetic resonance
imaging techniques.
EXAMPLES
Example 1
[0102] This example describes the identification of anti-tumor
targets for the treatment of prostate cancer by using lipid raft
proteomics.
[0103] In the present invention, molecules critical to the
treatment of prostate cancer were sought by initially detecting
differential expression of proteins in normal cells and the
prostate tumor cell lines. Lipid rafts of each cell line were
isolated and studied subsequently.
[0104] Materials and Methods
[0105] a. Lipid Raft Preparation
[0106] Lipid rafts were prepared as described in Green et al, J.
Cell Biol. 146, 673-682 (1999). Briefly, cells (8.0.times.10.sup.6
cell/sample) were lysed in 0.1% vol/vol Brij-58, 20 mM Tris HCl, pH
8.2, 140 mM NaCl, 2 mM EDTA, 25 .mu.g/ml aprotinin, 25 .mu.g/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 10 minutes on
ice. Cells were homogenized using 10 strokes of a Dounce
homogenizer, then lysed 20 minutes more on ice. The resulting
lysate was adjusted to 40% wt/wt sucrose and applied onto a 60%
wt/wt sucrose cushion. A sucrose step-gradient consisting of 25%
wt/wt sucrose and 5% wt/wt sucrose were layered on top of the
lysate. Gradients were centrifuged 18 hours at 170,000.times.g at
4.degree. C. in a SW55 rotor. Fractions (0.2 ml) were taken from
the top of the gradient. Lipid rafts float to the interface of the
25% and 5% sucrose layers (Fractions 7 and 8). The amount of
protein in each fraction was determined using the BCA Protein Assay
Kit. Protein was concentrated by centrifugation at 2000.times.g in
Vivaspin 6 PES membrane columns (molecular weight cut off=10,000
kDa).
[0107] b. Electrophoresis and Western Blotting
[0108] Lipid raft proteins were separated by SDS-PAGE on a 4-20%
gradient gel and then electrotransferred onto a polyvinylidene
difluoride membrane (PVDF). The membrane was blocked for 1.5 hours
at room temperature in PBS with 5% milk. The membrane was then
incubated with 0.4 .mu.g/ml mouse anti-ATP synthase (Molecular
Probes, catalog # A-11144) in PBS with 1% BSA and 0.5% Tween-20 for
2 hours at room temperature. After extensive washing, the membrane
was incubated with HRP-conjugated goat antibodies specific for
mouse IgG for 1 hour at room temperature in PBS with 1% BSA and
0.5% Tween-20. After extensive washing, blot was developed using
enhanced chemiluminescence followed by fluorography.
[0109] C. N-terminal Sequencing
[0110] Proteins to be sequenced were separated by SDS-PAGE on a
4-20% gradient gel and then electrotransferred onto a PVDF
membrane. The membrane was stained for 2 minutes using colloidal
Coomassie and then destained in water. The resulting bands were
excised and subjected to N-terminal Edman sequencing as described
by Miller, Methods: A Companion to Methods in Enzymology 6, 315
(1994). Results were confirmed using matrix assisted laser
desorption ionization-time of flight (MALDI-TOF) peptide-mass
profiling.
[0111] d. MALDI-TOF Peptide-mass Profiling
[0112] Proteins to be analyzed were separated by SDS-PAGE on a
4-20% gradient gel. Alternatively, 2-dimensional electrophoresis
was used to further separate lipid raft proteins using an IPGphor
Isoelectric Focusing System according to the manufacturers protocol
(Amersham Pharmacia Biotech, Piscataway, N.J.). Proteins were
visualized by staining the resulting gel with 0.05% Coomassie Blue
R250, 50% methanol, 10% acetic acid in water followed by destaining
in 15% methanol, 10% acetic acid in water.
[0113] Protein band or spot of interest was excised with a razor
blade and equilibrated in 100 mM Tris HCl, pH 8.5 at room
temperature for 45 minutes. The solution was replaced with 150
.mu.L of 2 mM DTT in 100 mM Tris-HCl, pH 8.5. The samples were
incubated with agitation for 30 minutes at 60.degree. C. The
solution was replaced with 150 .mu.L of 20 mM iodoacetic acid in
100 mM Tris-HCl, pH 8.5. The samples were incubated in the dark at
37.degree. C. for 30 minutes. The solution was replaced with 150
.mu.L of equal parts 100 mM Tris-HCl, pH 8.5 and acetonitrile. The
tubes were shaken vigorously at 37.degree. C. for 45 minutes. This
step was repeated until the gel bands were clear. The solution was
removed and the gel slices were dried in a SpeedVac on low vacuum
strength for 15 minutes. The gel bands were re-swelled with
0.25-0.5 .mu.g of a concentrated endo-protease Lysine-C or trypsin
solution, then covered with 50-80 .mu.L of 100 mM Tris-HCl, pH 8.5,
10% acetonitrile and incubated with agitation for 18 hours at
37.degree. C. After digestion the samples were stored at
4-8.degree. C.
[0114] The digest solution was removed from the micro-centrifuge
tube, acidified with 10% trifluoroacetic acid (TFA) in water v/v to
1% TFA v/v, desalted and concentrated using a C18 Zip-Tip. The
micro-column eluate was combined 1:1 with 10 mg/ml
alpla-Cyano-4-hydroxycinamic acid in 60% acetonitrile and spotted
on a MALDI-TOF sample plate. A close external calibrant with the
approximate concentration of the sample was spotted adjacent to the
sample position on the MALDI plate. The calibrant was prepared by
diluting a pre-made calibration mix consisting of angiotensin II
fragment 1-7 and adrenocorticotropic hormone fragment 18-39 with
Zip-Tip eluant. The diluted calibration mixture was combined 1:1
with matrix solution. The sample and calibrant spots were dried
simultaneously at room temperature.
[0115] MALDI mass fingerprints were acquired on a Perceptive
Biosystems Voyager DE Pro MALDI-TOF in reflector mode with delayed
extraction and positive polarity. Approximately 100-300 shots from
a 20 KV laser were accumulated. During spectrum acquisition a
resolution calculator was employed to ensure accurate calibration.
After the spectra were acquired and calibrated, the monoisotopic
masses were automatically selected using the de-isotoping function
on Voyager Software. The calibrated monoisotopic peak lists were
exported into ProteinProspector MS-Fit version 3.2.1 and searched
against the largest non-redundant database available from the
National Center for Biotechnology Information (NCBInr).
[0116] e. Flow Cytometry Staining
[0117] Flow cytometry was used to screen hybridoma supernatants for
the presence of cell surface binding antibodies. The cells
(2.times.10.sup.5) were resuspended in 100 .mu.L ice cold PBS with
10 .mu.L tissue culture supernatant on ice for 1 hour. After
extensive washing, cells were incubated with
phycoerythrin-conjugated goat antibodies specific for mouse IgG for
30 minutes on ice. Cells were washed again and cell surface bound
antibody was detected using a Becton Dickenson FACScan.
Additionally, hybridoma supernatants were similarly screened on
many cancer cell lines or whole blood to test for specificity.
[0118] f. Immunofluorescence
[0119] LNCaP cells were grown on glass coverslips undisturbed for
two days. Cells were fixed with 3% paraformaldehyde in PBS for 15
minutes. After being washed with PBS, cells were incubated in 50 mM
NH.sub.4Cl in PBS for 10 minutes. After washing with PBS, cells
were subsequently incubated with 5% goat serum in PBS for 30
minutes followed by staining with 5 .mu.g/ml anti-ATP synthase or
anti-Trop-1 (EpCAM) in 2.5% goat serum in PBS for 1 hour at room
temperature. Cells were stained with anti-MHC class II (Kostelny et
al Int. J. Cancer 93, 556 (2001)) as a negative control. After
washing with PBS, bound antibody was detected by incubating cells
with Alexa 488-conjugated goat antibodies specific for mouse IgG in
2.5% goat serum in PBS for 30 minutes at room temperature. After
extensive washing, glass coverslips were mounted in a solution
containing Mowiol 4-88, glycerol, and 150 mM Tris HCl, pH 8.5.
Slides were placed at 4.degree. C. overnight before viewing. Cells
were analyzed by fluorescence microscopy on a Nikon Optiphot 2
microscope.
[0120] g. LNCaP Proliferation
[0121] LNCaP cells were plated at 20,000 cells/well into a 96 well
tissue culture plate. After cells were allowed to grow undisturbed
for two days, antibodies (5 .mu.g/ml anti-ATP synthase, anti-Trop-1
(EpCAM), or anti-MHC class II) were added and incubated with the
cells for 24 hours. Cell proliferation was measured using the
AlamarBlue vital dye indicator assay. AlamarBlue reagent was added
to each well and the plates were incubated for 3 to 4 hours at
37.degree. C. to allow for fluorescence development. Fluorescence
was detected at .lambda.ex=530 nm, .lambda.em=590 nm. Data are
expressed as the mean+/-SEM of 4 replicates.
[0122] h. Soft Agar Colony Formation Assay
[0123] For anchorage-independent cell growth, a soft agar colony
formation assay was performed in a six-well plate. Each well
contained 2 mL of 1% agar in complete medium as the bottom layer.
The top layer contained 2 mL 0.5% agar in complete medium,
1000-10000 LNCaP cells, and 5 .mu.g/mL mAb (anti-ATP synthase, or
anti-Trop-1). One mL complete medium was added and the cultures
were maintained at 37.degree. C. in a humidified 5% CO.sub.2
atmosphere for up to 20 days. One mL complete medium was added once
a week. Media was removed and the colonies were stained with 0.005%
crystal violet in PBS for 2 hours. The number of colonies was
determined by counting them under an inverted phase-contrast
microscope at 100.times., and a group of 10 or more cells were
counted as a colony.
[0124] Results and Discussion
[0125] Differential expression of ATP synthase in prostate tumor
cells
[0126] Lipid rafts were extracted from normal prostate cells and
three widely used prostate cancer cells--LNCaP, DU145, and PC-3.
The protein expression of each sample was compared using
one-dimensional electrophoresis together with silver staining.
Several protein bands in the one-dimensional electrophoresis gel
appeared only in the prostate cancer cell lines, indicating that
they are candidate proteins related to the prostate cancer. One of
the candidate proteins with molecular weight of approximately 50 kD
(denoted by an arrow with a "*" in FIG. 1) was selected for
N-terminal sequencing. The sequencing result indicated that it was
the .beta.-subunit of ATP synthase.
[0127] The differential protein expression was also confirmed by
two-dimensional electrophoresis and then visualized by silver
staining. Protein spots that are present in the LNCaP sample, but
not the normal prostate sample are denoted with arrows (16 spots
identified), as shown in FIG. 2. Many of these lipid raft proteins
were excised and subjected to MALDI-TOF peptide mass profiling
analysis. The identities of 5 of them are shown in Table 1. The
locations of these proteins on the 2-D gel are labeled with numbers
as shown in FIG. 2. We found 2 subunits of ATP synthase (.alpha.
and .beta. subunits), 2 voltage-dependent anion channel/porin
proteins, adenine nucleotide translocator, and prohibitin. All of
these 5 identified proteins are mitochondria proteins but somehow
are associated with lipid rafts of LNCaP cells. It is not known how
many of these lipid raft-associated proteins are exposed to the
outer surface of the cell membrane and therefore are accessible to
antibodies. As several anti-ATP synthase antibodies are
commercially available, we used one of them to confirm that some
ATP synthase molecules are located in lipid rafts and they are
accessible to antibodies.
[0128] Western blot analysis of lipid rafts using
anti-.alpha.-subunit ATP synthase antibody further confirmed the
correlation between the ATP synthase and prostate cancer. As shown
in FIG. 3, positive staining appeared in all three prostate cancer
cell lines, but not the normal cells, indicating that ATP synthase
was present in lipid rafts of prostate cancer cells but not normal
cells. In addition, similar Western blot analysis of lipid rafts
from cancer cells of different origins indicates that some cancer
cell lines, such as the AML cell lines KG-1 and THP-1, the breast
cancer cell line MCF-7, the colon cancer cell line LS180, and the
bladder cancer cell line RT4 also express ATP synthase in lipid
rafts (FIG. 4).
[0129] Cell Surface Localization of ATP Synthase
[0130] To investigate the surface localization of the ATP synthase,
prostate tumor cell line LNCaP was analyzed by FACS staining and
immunofluorescence microscopy. As shown in FIG. 5, FACS staining by
anti ATP-synthase antibody in LNCaP tumor cells revealed about
25.9% of cells had ATP synthase cell surface expression, Staining
with an antibody against Trop-1 (EpCAM), a cell surface protein
expressed on cancer cells, showed surface localization in 92.8% of
the cells. Similar analysis of the AML cell line THP-1 by flow
cytometry also showed that these cells have ATP synthase on their
cell surface (FIG. 6). ATP synthase was also showed to be expressed
on the surface of the androgen-independent cell line DU 145 by a
similar analysis.
[0131] The surface localization of ATP synthase was further
evidenced by the immunofluorescence microscopy. As shown in FIG. 7,
immunostaining of LNCaP cells with anti-ATP synthase gave rise to
positive cell surface staining. The colocalization of Trop-1
(EpCAM) and ATP synthase indicated that ATP synthase was indeed
expressed on the surface of prostate tumor cells.
[0132] Moreover, FACS staining of LNCaP tumor cells growing in
Matrigel.RTM. demonstrated a much higher percentage of cells
exhibiting a positive surface staining of ATP synthase, confirming
that ATP synthase cell surface expression can be modulated by
cellular environment, and this may play important roles in the real
biological environment (data not shown).
[0133] Inhibition of prostate cancer cell proliferation in the
presence of anti-ATP synthase
[0134] As shown in FIG. 8, LNCaP tumor cell proliferation can be
reduced by antibodies specific for ATP synthase. This reduction in
cellular proliferation ranges from 12.5% to 27.8%. This substantial
inhibition of cell proliferation suggests a potential of anti-ATP
synthase antibody as an anti-tumor agent for treating prostate
cancer.
[0135] The above experiments demonstrate that ATP synthase is
closely related to the prostate cancer cells. It is localized on
the surface of prostate tumor cells, and may play a key role in the
biological activities of prostate cancer cells. Blocking the
activity of ATP synthase by antibodies inhibits the prostate tumor
cell growth, suggesting the possibility of clinical application of
ATP synthase inhibitors as anti-tumor agents for treating prostate
cancer.
[0136] Inhibition of prostate cancer cell colony formation in soft
agar in the presence of anti-ATP synthase
[0137] Transformed cancer cells are resistant to
anchorage-independent growth inhibition and are able to growth in
soft agar without attaching to cell matrix. Formation of colonies
(three-dimensional growth under tissue culture growth conditions)
of cancer cells in soft agar is often correlated to the
aggressiveness of the tumor in vivo. To assess whether anti-ATP
synthase has any anti-cancer activity, we used it inhibit LNCaP
colony formation in vitro. As shown in FIG. 9, LNCaP colony
formation can be reduced by an antibody specific for ATP synthase
(anti-.alpha. subunit) at 5 .mu.g/ml. This reduction in colony
formation by anti-ATP synthase could be as high as 95%, whereas the
action of an anti-Trop-1 antibody (Ep-CAM) antibody was less
impressive, at about 68%. This substantial inhibition of colony
formation suggests a potential of anti-ATP synthase antibody as an
anti-tumor agent for treating prostate cancer.
[0138] AML cell death induction in the presence of anti-ATP
synthase
[0139] The anti-cancer activity of anti-ATP synthase can also be
demonstrated in the AML cell line THP-1, which expresses ATP
synthase on the cell surface. Incubation of THP-1 cells with
anti-ATP synthase at 5 .mu.g/ml for 24 hours led to substantial
cell death as assayed by flow cytometry (FIG. 10). Compared to PBS
or an irrelevant antibody control, the specific killing induced by
anti-ATP synthase was 25%. This anti-AML activity suggests a
potential of anti-ATP synthase antibody as anti-leukemia or
lymphoma agent to treat hematological malignancies.
[0140] The above experiments demonstrate that ATP synthase
expression is closely related to certain cancer cells. It is
localized on the surface of prostate and AML cancer cells, and may
play a key role in the biological activities of prostate and AML
cancer cells. Blocking the activity of ATP synthase by antibodies
inhibits the prostate cancer cell growth and colony formation, and
induces cell death in AML cancer cells, suggesting the possibility
of clinical application of ATP synthase inhibitors as anti-tumor
agents for treating prostate cancer and AML.
Example 2
[0141] This example describes the identification of anti-tumor
targets in various cancer cells by using lipid raft proteomics.
[0142] Lipid raft protein expressions were compared among various
cancer cell lines. Lipid rafts were extracted from normal
trophoblast cells (BeWo and JEG-3) and various cancer cells,
including colon cancer cells (HT-29, LS180, and Colo205), breast
cancer cells (MCF-7), prostate cancer cells (LNCaP), pancreas
cancer cells (PANC-1), and lung cancer cells (NCI-H292). The
protein expression of each sample was compared using
one-dimensional electrophoresis together with silver staining. As
shown in FIG. 11, many protein bands were shared among the various
cell lines, while many proteins are differentially expressed. In
addition, there are approximately 120 protein bands per lane,
confirming that lipid rafts are not too complex to be studied. The
differential protein expressions were also confirmed by
two-dimensional electrophoresis and Western blot analysis as
described in Example 1. The protein bands differentially expressed
in cancer cells were isolated, sequenced, and identified as
candidates for tumor related molecules as described in Example
1.
Example 3
[0143] This example describes identifying the anti-prostate tumor
agent by the immunization of lipid rafts from LNCaP cell lines.
[0144] Materials and Methods
[0145] a. Lipid Raft Preparation
[0146] See Example 1.
[0147] b. Lipid Raft Immunization
[0148] Lipid raft proteins (approximately 5 .mu.g) were mixed
together with 50 .mu.L Ribi.RTM., and then injected into the foot
pad of a BALB/c mouse. Mice were boosted with 50 .mu.L of lipid
raft proteins in Ribi.RTM. on day 7 and day 14. Three days after
the last boost, the mice were sacrificed and the hind leg lymph
node was harvested. The lymph node was washed in pre-warmed DMEM
and then ground using a Dounce homogenizer. After 5 gentle strokes,
the cell suspension was removed into the plastic tube. This process
was repeated four more times, each time adding 5 ml of fresh DMEM.
The lymphocytes were pooled and washed 3 times in DMEM. The
lymphocytes were mixed with the appropriate number of pre-washed
fusion partner NS0/BCL-2 (NS0 transfectant expressing the mouse
BCL-2 cDNA) to yield ratio of 2-3 lymphocytes for every 1 NS0. The
mixture was pelleted and warmed at 37.degree. C. for 1 min.
Pre-warmed 50% PEG was slowly added onto the pellet and then cells
were centrifuged at 300.times.g for 3 minutes at room temperature.
Five mL DMEM was added and then 10 mL DMEM with 10%FBS and 1%P/S
was added. The cells were then centrifuged 5 minutes at 300.times.g
at room temperature. The pellet was resuspended in HAT selection
medium (DMEM with 20% fetal bovine serum, 2 mM glutamine, 0.1 mM
non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM sodium
hypoxanthine, 16 .mu.M thymidine, 20 .mu.M aminopterin,
2.times.Origen cloning factor, 10 mM HEPES, 50 .mu.M
beta-mercaptoethanol, 0.2 units/mL penicillin, 0.2 .mu.g/mL
streptomycin) to yield 0.25.times.10.sup.6 lymphocytes/mL. Cells
were aliquoted into ten 96-well flat bottom plates at 200 .mu.L per
well-for the selection of hybridomas.
[0149] c. Flow Cytometry Screening
[0150] Flow cytometry was used to screen hybridoma supernatants for
the presence of cell surface binding antibodies. The cells
(2.times.10.sup.5) were resuspended in 100 .mu.L ice cold PBS with
10 .mu.L tissue culture supernatant on ice for 1 hour. After
extensive washing, cells were incubated with
phycoerythrin-conjugated goat antibodies specific for mouse IgG for
30 minutes on ice. Cells were washed again and cell surface bound
antibody was detected using a Becton Dickenson FACScan.
Additionally, hybridoma supernatants were similarly screened on
many cancer cell lines or whole blood to test for specificity.
[0151] d. Affinity Purification of Antigen
[0152] Cells (5.times.10.sup.8) were lysed in 1% vol/vol NP-40,
0.5% wt/vol deoxycholate, 20 mM Tris HCl, pH 8.2, 150 mM NaCl, 1 mM
EDTA, 25 .mu.g/ml aprotinin, 25 .mu.g/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride for 1 hour on ice with frequent
mixing. Lysate was centrifuged for 20 minutes at 300.times.g to
remove nuclei and debris. Antigens were purified by standard
hybridoma affinity chromatography techniques as described in Hill
et al, J. Immunol. 152, 2890-2898 (1994).
[0153] e. Antigen Grouping
[0154] Cells (2.times.10.sup.7) were cell surface iodinated as
described (Landolfi and Cook, Mol. Immunol. 23, 297-309 (1986)).
Cells were then lysed in 1% NP-40, 0.5% deoxycholate, 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 .mu.g/ml aprotinin, 10
.mu.g/ml leupeptin, and 1 mM PMSF for 1 hour on ice. Cell lysate
was centrifuged at 14,000.times.g for 5 minutes to remove nuclei
and debris. Cell lysate was pre-cleared with rotation by incubation
with Gamma Bind Plus Sepharose beads for 2 hours at 4.degree. C.
The beads were spun down and the cell lysate was then aliquoted
into Eppendorf tubes containing Gamma Bind Plus Sepharose beads
that had been pre-incubated with hybridoma supernatant. The tubes
were rotated overnight at 4.degree. C. After extensive washing,
bound antigen was eluted from the beads by boiling in the presence
of 5% wt/vol SDS, 125 mM Tris-HCl, pH 6.8, and 4% vol/vol
.beta.-mercaptoethanol, and 50% vol/vol glycerol. Proteins were
then subjected to SDS-PAGE. After electrophoresis, the gel was
fixed for 30 minutes with 60% H.sub.2O/30% methanol/10% acetic
acid. The gel was then washed for 30 minutes with water then dried
down. The dried gel was put on film (Kodak.RTM. Biomax MS.RTM. film
with appropriate Biomax MS.RTM. screen) overnight
[0155] f. Soft Agar Colony Formation Assay
[0156] For anchorage-independent cell growth, a soft agar colony
formation assay was performed in a six-well plate. Each well
contained 2 mL of 1% agar in complete medium as the bottom layer.
The top layer contained 2 mL 0.5% agar in complete medium,
1000-10000 LNCaP cells, and 5 .mu.g/mL mAb (anti-ATP synthase,
anti-NCAM2, or anti-Trop-1). One mL complete medium was added and
the cultures were maintained at 37.degree. C. in a humidified 5%
CO.sub.2 atmosphere for up to 20 days. One mL complete medium was
added once a week. Media was removed and the colonies were stained
with 0.005% crystal violet in PBS for 2 hours. The number of
colonies was determined by counting them under an inverted
phase-contrast microscope at 100.times., and a group of 10 or more
cells were counted as a colony.
[0157] g. LNCaP Proliferation
[0158] LNCaP cells were plated at 20,000 cells/well into a 96 well
tissue culture plate. After cells were allowed to grow undisturbed
for two days, antibodies (5 .mu.g/ml anti-ATP synthase, anti-NCAM2,
anti-Trop- 1, or anti-MHC class II) were added and incubated with
the cells for 24 hours. Cell proliferation was measured using the
AlamarBlue vital dye indicator assay. AlamarBlue reagent was added
to each well and the plates were incubated for 3 to 4 hours at
37.degree. C. to allow for fluorescence development. Fluorescence
was detected at .lambda.ex=530 nm, .lambda.em=590 nm. Data is
expressed as the mean+/-SEM of 4 replicates.
[0159] Results and Discussions p One BALB/c mouse was immunized
with a lipid raft preparation from the prostate cancer cell line
LNCaP. After two boosts, lymphocytes were isolated from the mouse
lymph nodes and fused with myeloma NS0 cells to generate
hybridomas. A total of about 700 hybridomas were generated and
supernatant from each hybridoma was screened by flow cytometry for
binding to LNCaP. About 203 supernatants tested positive, and they
were further tested for binding to normal prostate cells.
Thirty-four of these supernatants tested negative for binding to
normal prostate cells. Antibodies from these 34 hybridomas were
further tested against multiple cancer lines by flow cytometry to
determine whether they are LNCaP specific, prostate cancer
specific, or pan-cancer specific. The cancer cell lines we used
include: DU 145 (prostatic), PC-3 (prostatic), PANC-1 (pancreatic),
RT4 (bladder), HT-29 (colorectal), NCI-H292 (lung), T-47D (breast),
and NIH:OVCAR-3 (ovarian). In addition, a primary, non-transformed
HUVEC line (human umbilical vein endothelial cells) was used to
ensure that these antibodies do not cross-react with normal
endothelial cells. The 34 LNCaP reacting hybridomas can be divided
into two main groups based on their antigen expression profiles:
antibodies from 20 of these hybridomas (Table 2) showed that their
antigens are expressed only in a limited number (1-3) of cancer
cell lines and 14 (Table 3) showed that their antigens are
expressed in multiple lines. A flow chart describing how these
tumor-associated, LNCaP lipid raft-derived antigens were identified
by the hybridoma technology is shown in FIG. 12.
[0160] We performed an immunoprecipitation experiment to determine
the molecular weight of the antigens that showed limited expression
profiles. Of the twenty antibodies used, three predominant antigens
of MW 98 (recognized by 3 antibodies), 100 (recognized by 6
antibodies), and 120 kD (recognized by 4 antibodies) were
identified (FIG. 13, see also the last column of Table 2). Seven
antibodies were not able to immunoprecipitate antigen for molecular
weight determination. The antigen grouping results indicate the
hybridomas of Table 1 covered a minimum of four to a maximum of ten
antigens. Similarly antigen grouping by immunoprecipitation may
also apply to those antigens showing broad expression profiles
(antigens as defined by hybridomas of Table 3) to reduce the number
of tumor-associated antigens that need to be characterized.
[0161] Lipid Raft Tumor-associated Antigen NCAM2
[0162] The identity of the antigen defined by the hybridoma P3-53
had been determined. The monoclonal antibody produced by hybridoma
P3-53 (Table 2) immunoprecipitated a major protein band of 120 kD
and a minor band of 110 kD (see FIG. 13, Lane 3). The P3-53 antigen
is expressed only in the prostate cell line LNCaP and breast cell
line T-47D. The monoclonal antibody from P3-53 was conjugated to
CNBr-activated Sepharose to generate an affinity column (20 mg
conjugated to a 2 ml column). LNCaP whole cell lysate was prepared
from 2.times.10.sup.8 cells as described in MATERIAL AND METHODS
and passed onto the P3-53 affinity column. After extensive washing,
the retained protein was eluted with low pH buffer. About 5 .mu.g
of the P3-53 antigen was purified. SDS-PAGE analysis followed by
silver staining revealed the P3-53 antigen consisted of a major
protein band of 120 kD and a minor band of 110 kD. The purified
antigen was subjected to microsequencing analysis and the result
showed that it had a NH.sub.2-terminal sequence of
X-L-QV-T-I-S-L-S-K, where X was probably "L", but might also be
"G". This sequence was searched against the entire NCBInr database
using Protein Prospector. Only one human protein with the
NH.sub.2-terminal sequence of L-L-Q-V-T-I-S-L-S-K matched the
determined P3-53 antigen sequence. The human protein is called
neural cell adhesion molecule 2 (NCAM2, NCBI protein accession
number 4758764, see also Paoloni-Giacobino et al, Genomics 43,
43-51 (1997)), which is a homologue of a murine protein called Rb-8
neural adhesion molecule (RNCAM, NCBI protein accession number
3334269, see also Alenius, M. and Bohm, S., J. Biol. Chem. 272,
26083-26083 (1997) and Yoshihara et al. J. NeuroSci. 17: 5830-5842
(1997). The identification of the antigen recognized by P3-53 was
confirmed to be NCAM2 by MALDI-TOF peptide-mass profiling as
described in the MATERIALS AND METHODS in Example 1.
[0163] The sequence of RNCAM predicted molecules having an
extracellular region of 5 immunoglobulin C2-type domains followed
by two fibronectin type III domains. Alternative splicing of the
NCAM2 and RNCAM transcripts generate two isoforms: the long form
containing a transmembrane domain and the short form containing a
glycosylphosphatidylinositol-anchor attached to the membrane. The
expression of RNCAM is restricted to the olfactory neurons in the
brain and in the nasal vomeronasal organ. The transcript of RNCAM
is not detectable in lung, gut, liver, heart, testis and kidney.
The function of NCAM2 or RNCAM is not known, but the molecule may
play a role in selective axon projection. NCAM2 was also shown to
be a homophilic adhesion molecule (see Yoshihara et al. J.
NeuroSci. 17: 5830-5842 (1997)). Thus we found that certain
prostate and breast cancer cell lines express this protein marker
that is neural in origin. Accordingly, because NCAM2 is expressed
in a substantial percentage of prostate and breast cancer cells, an
antibody to NCAM2 provides an effective treatment for prostate or
breast cancer. Antibodies are better drugs than small molecules
against cancer cells expressing neural markers because they do not
cross the blood-brain barrier to potentially have toxic effects on
normal neurons.
[0164] Inhibition of Prostate Cancer Cell Proliferation by
Anti-NCAM2 Antibodies
[0165] As shown in Table 2 and FIG. 13, P3-53, P9-64, P10-28, and
P10-29 all immunoprecipitated an antigen with a molecular weight of
120/110. The P3-53 antigen was identified as NCAM2. We tested the
anti-cancer activity of 4 anti-NCAM2 antibodies in a proliferation
assay using the prostate cancer cell line LNCaP. The results are
shown in FIG. 14. One of the 4 antibodies, namely P9-64, had
significant inhibition activity against proliferation of LNCaP
cells. This reduction in cellular proliferation by P9-64 was about
39%. P10-28 and P10-29 had about 16 and 11% inhibition activity,
respectively, and P3-53 was not effective. This substantial
inhibition of cell proliferation by some anti-NCAM2 antibodies
suggests a potential of anti-NCAM2 antibody as an anti-tumor agent
for treating prostate cancer.
[0166] Inhibition of Prostate Cancer Cell Colony Formation by
Anti-NCAM2 Antibodies
[0167] Transformed cancer cells are resistant to
anchorage-independent growth inhibition and are able to growth in
soft agar without attaching to cell matrix. Formation of colonies
(three-dimensional growth under tissue culture growth conditions)
of cancer cells in soft agar is often correlated to the
aggressiveness of the tumor in vivo. To assess whether the four
anti-NCAM2 antibodies any anti-cancer activity, we used them
inhibit LNCaP colony formation in vitro. As shown in FIG. 15, LNCaP
cancer cell colony formation can be reduced by some anti-NCAM2
antibodies at 5 .mu.g/ml. As in the proliferation assay, the most
potent inhibitor is P9-64. It inhibited colony formation by 42%.
P10-28 and P10-29 inhibited about 22% and 36% respectively. The
inhibitory activity of P3-53 was again low, at about 10%
inhibition. The substantial inhibition of colony formation by some
anti-NCAM2 antibodies suggests a potential of anti-NCAM2 antibody
as an anti-tumor agent for treating prostate cancer.
[0168] The above experiments demonstrate that NCAM2 expression is
closely linked to some types of prostate cancer cells. It is
localized on the surface of cancer cells, and may play a key role
in the biological activities of prostate cancer cells. Blocking the
activity of NCAM2 by antibodies inhibits prostate cancer cell
growth, suggesting the possibility of clinical application of NCAM2
inhibitors as anti-tumor agents for treating prostate cancer.
[0169] Lipid Raft Tumor-associated Antigens PSMA and CD10
[0170] The identities of two more antigens defined by hybridomas
listed in Table 2 have also been determined. The monoclonal
antibody produced by hybridoma P12-22, representing 6 hybridomas
(Table 2), immunoprecipitated a protein band of 100 kD (see FIG.
13, lane 20). The P12-22 antigen is expressed in the prostate
cancer cell line LNCaP. The monoclonal antibody from P12-22 was
conjugated to CNBr-activated Sepharose to generate an affinity
column (20 mg conjugated to a 2 ml column). LNCaP whole cell lysate
was prepared from 2.times.10.sup.8 cells as described in MATERIAL
AND METHODS and passed onto the P12-22 affinity column. After
extensive washing, the retained protein was eluted with low pH
buffer. About 15 .mu.g of the P12-22 antigen was purified. SDS-PAGE
analysis followed by silver staining revealed the P12-12 antigen
had a molecular weight of 100 kD. The purified antigen was
subjected to microsequencing analysis and the result showed No
Edman degradation products were obtained, indicating the
NH.sub.2-terminus of this antigen might have been blocked. The
purified P12-22 antigen was then subjected to MALDI-TOF peptide
mass profiling as described in MATERIALS AND METHODS in Example 1.
This method identified the P12-22 antigen as prostate
specific-membrane antigen (PSMA) also known as folate hydrolase
(NCBI accession number 20561181), a well-known prostate cancer
associated antigen that is expressed in LNCaP, but not in PC-3 or
DU 145. This antigen is a type II transmembrane protein that is
highly expressed in prostate cancer. The NH.sub.2-terminus of this
protein is known to be blocked (see Israeli et al. Cancer Research
53, 227-230 (1993)).
[0171] The monoclonal antibody produced by hybridoma P10-82,
representing 3 hybridomas (Table 2), immunoprecipitated a protein
band of 98 kD (see FIG. 13, lane 19), which is expressed mostly in
the prostate cancer cell line LNCaP. The P10-82 antigen was
purified by the antibody affinity column affinity described above.
About 10 .mu.g of the antigen was obtained. Microsequencing
analysis of this protein yielded no Edman degradation products, but
MALDI-TOF peptide mass profiling identified the P10-82 antigen as
CD10, also known as neutral endopeptidase (NCBI accession number
14733461). This is also a type II transmembrane protein. Both PSMA
and CD10 were previously identified as prostate cancer-associated
antigens (see Gong et al. Cancer Metastasis Rev.18, 483-490 (1999)
for review of PSMA, and Krongrad et al. Urol. Res. 25,113-116
(1997) and Sumitomo et al. J. Clin. Invest. 106, 1399-1407 (2000)
for CD10), although they were not known to be lipid-raft
associated. The finding of these known cancer-associated antigens
by the lipid raft immunization approach provides a good validation
of the method.
[0172] Lipid Raft Tumor-associated Antigens Trop-1 (EpCAM) and
CD222
[0173] We also performed an immunoprecipitation experiment to
determine the molecular weight of the antigens that showed broad
expression profiles (Table 3). The molecular weights of 8 antigens
were determined and are summarized in FIG. 16 and Table 3. Unlike
the antigens in Table 2, the molecular weights of 8 antigens
determined in Table 3 appeared to be different from each other. No
immunoprecipitation result was obtained with the P8-20 antibody but
the antigen was identified as Trop-1 (EpCAM), which we knew as a
lipid raft associated tumor antigen (unpublished observation). The
identity of P8-20 antigen as Trop-1 was determined by staining of
Trop-1/mouse myeloma NS0 transfectant with all 14 antibodies listed
in Table 2 by flow cytometry. Only P8-20 antibody stained
positively with the Trop-1 transfectant.
[0174] By using affinity chromatography and MALDI-TOF, two more
antigens in Table 3 were also identified. The P7-69 antigen was
identified as PSMA, the tumor-associated antigen that we previously
identified in Table 2. The P11-65 antigen (>220 kD) was
identified as CD222, also known as mannose-6-phosphate receptor or
insulin-like growth facto II receptor (NCBI accession number
13642251). It is a ubiquitously expressed multifunctional type I
transmembrane protein. Its main functions include internalization
of insulin-like growth factor II and internalization and sorting of
lysosomal enzymes and other mannose-6-phophate-containing proteins
(Korenfied, Annu. Rev. Biochem. 61, 307-330 (1992)). The majority
of CD222 molecules (90-95%) are located intracellularly in normal
cells; only 5-10% is presented on the membrane surface. CD222 is
also a receptor of TGF-.beta. latency associated peptide (LAP), the
angiogenesis-inducing protein proliferin, plasminogen, and retinoic
acid. The data in Table 3 indicate the epitope on CD222 recognized
by P11-65 antibody is highly expressed in many cancer cell lines
but detectable only weakly in one of the three normal cell line
tested.
Example 4
[0175] This example describes generation and characterization of
anti-KG-1 lipid raft hybridomas.
[0176] Materials and Methods
[0177] a. Flow Cytometry Screening
[0178] Flow cytometry was used to screen hybridoma supernatants for
the presence of cell surface binding antibodies. The cells
(2.times.10.sup.5) were resuspended in 100 .mu.L ice cold PBS with
10 .mu.L tissue culture supernatant on ice for 1 hour. After
extensive washing, cells were incubated with
phycoerythrin-conjugated goat antibodies specific for mouse IgG for
30 minutes on ice. Cells were washed again and cell surface bound
antibody was detected using a Becton Dickenson FACScan.
Additionally, hybridoma supernatants were similarly screened on
whole blood to test for specificity. Normal blood cell populations
were purified as described from peripheral blood from volunteers
(Brown Methods in Cell Biol. 45, 147 (1994)) or purchased from the
Stanford Medical School Blood Center (Stanford, Calif.).
[0179] b. Apoptosis
[0180] Flow cytometry was used to screen hybridoma supernatants for
the ability to induce apoptosis of acute myelogenous leukemia
cancer cells. KG-1 cells or THP-1 cells (2.times.10.sup.5) were
resuspended in 100 .mu.L media with 100 .mu.L tissue culture
supernatant for 24 hours at 37.degree. C. Cells were centrifuged
and then incubated with FITC-conjugated annexin V and propidium
iodide in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM KCl, 1 mM
MgCl.sub.2, 1.8 mM CaCl.sub.2 for 15 minutes at room temperature.
After extensive washing, cells were analyzed using a Becton
Dickenson FACScan. Apoptotic events were considered to be annexin
V.sup.+ and PI.sup.-/+. The apoptosis-inducing activity of purified
anti-HLA-DR antibodies was similarly determined using KG-1 or THP-1
cells.
[0181] C. Flow Cytometry for Cell Line and Blood Cell
Reactivities
[0182] To determine blood cell reactivities of K8-355, peripheral
blood from normal donors was stained with FITC-conjugated K8-355,
L227, or L243 for 15 minutes on ice. Red blood cells were removed
by the addition of FACS lysing solution (Becton Dickinson catalog #
349202) for 10 minutes at room temperature. After washing, cell
surface bound antibody was detected using a Becton Dickenson
FACScan.
[0183] To determine if K8-355 is a pan-anti-MHC class II antibody,
CESS (myelomonocytic leukemia, American Type Culture Collection
(ATCC)), Daudi (Burkitts lymphoma, ATCC), KG-1 (acute myelocytic
leukemia, ATCC), Raji (Burkitt's lymphoma, ATCC), Ramos (Burkitt's
lymphoma, ATCC), RL (non-Hodgkin's lymphoma, ATCC), and THP-1
(acute myelocytic leukemia, ATCC) were analyzed by flow cytometry.
In addition, binding of K8-355 was assessed on 9 individual normal
donors as described above.
[0184] e. Antibody Variable Region Sequences Determination
[0185] Total RNA was extracted from approximately 10.sup.7
hybridoma cells using TRIzol reagent (Life Technologies,
Gaithersburg, Md.) and poly(A).sup.+ RNA was isolated with the
PolyATract mRNA Isolation System (Promega, Madison, Wis.) according
to the suppliers' protocols. Double-stranded cDNA was synthesized
using the SMART.TM.RACE cDNA Amplification Kit (Clontech, Palo
Alto, Calif.) following the supplier's protocol. The variable
region cDNAs for the light and heavy chains were amplified by
polymerase chain reaction (PCR) using 3' primers that anneal
respectively to the mouse kappa and gamma chain constant regions,
and a 5' universal primer provided in the SMART.TM.RACE cDNA
Amplification Kit. The 5' universal primer for VL has the
sequence:
[0186] 5' GAT GGA TAC AGT TGG TGC AGC-3', and that for VH has the
sequence:
[0187] 5'-GCC AGT GGA TAG ACA GAT GG-3'.
[0188] For VL PCR, the 3' primer has the sequence:
[0189] 5"-TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC-3'
[0190] with residues 17-46 hybridizing to the mouse Ck region. For
VH PCR, the 340 primers have the degenerate sequences:
1 5'-TATAGAGCTCAAGCTTCCAGTGGATAGAC(ACT)GATGGGG(GC)TG
T(CT)GTTTTGGC-3'
[0191] with residues 17-50 hybridizing to mouse gamma chain CH1.
The VL and VH cDNAs were subcloned into pCR4Blunt-TOPO vector
(Invitrogen, Carlsbad, Calif.) for sequence determination. DNA
sequencing was carried out by PCR cycle sequencing reactions with
fluorescent dideoxy chain terminators (Applied Biosystems, Foster
City, Calif.) according to the manufacturer's instructions. The
sequencing reactions were analyzed on a Model 377 DNA Sequencer
(Applied Biosystems).
[0192] f. Affinity Purification of Antigen
[0193] Cells (5.times.10.sup.8) were lysed in 1% vol/vol NP-40,
0.5% wt/vol deoxycholate, 20 mM Tris HCl, pH 8.2, 150 mM NaCl, 1 mM
EDTA, 25 .mu.g/ml aprotinin, 25 .mu.g/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride for 1 hour on ice with frequent
mixing. Lysate was centrifuged for 20 minutes at 300.times.g to
remove nuclei and debris. Antigens were purified by standard
hybridoma affinity chromatography techniques as described in Hill
et al, J. Immunol. 152, 2890-2898 (1994).
[0194] g. N-terminal Sequencing
[0195] Proteins to be sequenced were separated by SDS-PAGE on a
4-20% gradient gel and then electrotransferred onto a PVDF
membrane. The membrane was stained for 2 minutes using colloidal
Coomassie and then destained in water. The resulting bands were
excised and subjected to N-terminal Edman sequencing as described
by Miller, Methods: A Companion to Methods in Enzymology 6, 315
(1994).
[0196] h. Antigen Grouping
[0197] Cells (2.times.10.sup.7) were cell surface iodinated as
described (Landolfi and Cook, Mol. Immunol. 23, 297-309 (1986)).
Alternately, cells were cell surface biotinylated with EZ-Link
Sulfo-NHS-LC-Biotin per manufacturers protocol (Pierce cat #
21335). Cells were then lysed in 1% NP-40, 0.5% deoxycholate, 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 .mu.g/ml aprotinin, 10
.mu.g/ml leupeptin, and 1 mM PMSF for 1 hour on ice. Cell lysate
was centrifuged at 14,000.times.g for 5 minutes to remove nuclei
and debris. Cell lysate was pre-cleared with rotation by incubation
with Gamma Bind Plus Sepharose beads for 2 hours at 4.degree. C.
The beads were spun down and the cell lysate was then aliquoted
into Eppendorf tubes containing Gamma Bind Plus Sepharose beads
that had been pre-incubated with hybridoma supernatant. The tubes
were rotated overnight at 4.degree. C. After extensive washing,
bound antigen was eluted from the beads by boiling in the presence
of 5% wt/vol SDS, 125 mM Tris-HCl, pH 6.8, and 4% vol/vol
.beta.-mercaptoethanol, and 50% vol/vol glycerol. Proteins were
then subjected to SDS-PAGE. After electrophoresis, the gel was
fixed for 30 minutes with 60% H.sub.2O/30% methanol/10% acetic
acid. The gel was then washed for 30 minutes with water then dried
down. The dried gel was put on film (Kodak.RTM. Biomax MS.RTM. film
with appropriate Biomax MS.RTM. screen) overnight. For cell surface
biotinylated samples, after electrophoresis proteins were
electrotransferred onto a PVDF membrane. The membrane was blocked
for 1.5 hours at room temperature in Superblock (Pierce cat
#37515). The membrane was then incubated with HRP-conjugated avidin
in PBS with 1% BSA and 0.5% Tween-20 for 1 hour at room
temperature. After extensive washing, the membrane was developed
using enhanced chemiluminescence followed by fluorography.
[0198] Results and Discussions
[0199] One BALB/c mouse was immunized with a lipid raft preparation
from the acute myclogenous leukemia cell line KG-1. After two
boosts, lymphocytes were isolated from the mouse lymph nodes and
fused with myeloma NS0 cells to generate hybridomas according to
the methods described in Example 3. A total of about 1142
hybridomas were generated and supernatant from each hybridoma was
screened by flow cytometry for binding to KG-1. About 392
hybridomas tested positive. As the normal counter part of acute
myelogenous leukemic cells are difficult to purify to screen for
differentially expressed antigens, we opted to identify those
monoclonal antibodies that have a more restricted expression in
blood cells and have anti-cancer activity against KG-1. The 392
KG-1-positive hybridomas were screened against a human T cell line
Jurkat by flow cytometry to eliminate those that were reactive to T
cells. Antibodies from one hundred and four hybridomas that were
Jurkat-negative were identified and their supernatants were tested
for KG-1 cell death-inducing activity. Antibodies from 36 selected
hybridomas that had apoptosis-inducing activity in an overnight
assay were retested in various assays for further characterization.
These additional assays included one for the antibody's
apoptosis-inducing activity in another AML cell line (THP-1) and
several for the antibody's binding activity to T cells, red blood
cells, platelets, granulocytes, stem cells (CD34+), lymphocytes and
monocytes by flow cytometry. A flow chart of how these 36 anti-KG-1
hybridomas were selected is shown in FIG. 17. The reactivity
profiles of these 36 anti-KG-1 hybridomas were summarized in Table
4.
[0200] Antigen grouping by immunoprecipitation as demonstrated in
Example 3 was used to reduce the number of tumor-associated
antigens that need to be characterized. The molecular weights of 24
antigens were determined (FIG. 18) and summarized in Table 4.
Fifteen antibodies immunoprecipitated an antigen consisting of two
chains of 32 kD and 28 kD. The molecular weights of these two
chains resemble those of major histocompatibility complex class II
antigen HLA-DR, a predominant antigen in blood cells. We also know
by previous experience that some anti-HLA-DR antibodies have potent
apoptosis-inducing activity against lymphoma and leukemic cells
(Kostelny et al. Int. J. Cancer 93, 556-565 (2001)). Using antibody
from K8-355 as a representative of this group, we made an affinity
column to isolate the antigen from KG-1 cells and confirmed its
identity to be HLA-DR by MALDI-TOF peptide profiling.
[0201] Data indicate that the antibody K8-355 (murine IgG1/kappa)
has a pan-HLA-DR reactivity; it binds to all cells that express
HLA-DR in spite of the polymorphic nature of the molecules. The
variable regions of the antibody K8-355 have also been determined
(FIGS. 19 and 20). In addition to having apoptosis-inducing
activity against AML cells such as KG-1 and THP-1, K8-355 also has
apoptosis-inducing against the B cell lines Raji and Daudi (FIG.
21). These data indicate that pan-HLA-DR antibodies may have
clinical application against leukemia and lymphoma.
Example 5
[0202] This example describes determining the identity of lipid
raft tumor-associated antigens.
[0203] Monoclonal antibody that binds to each tumor-associated
antigen is to be purified from hybridoma spent medium by protein-G
affinity chromatography. The purified monoclonal antibody is then
covalently linked to CNBr-activated Sepharose resin to generate an
affinity column for a particular antigen. LNCaP or KG-b 1 cell
lysate is to be prepared as in the immunoprecipitation experiment
and passed onto the affinity column. Antigen retained in affinity
column is eluted and subjected to protein sequence determination by
the Edman degradation method. The determined N-terminal sequence is
used to search for gene product identity against the Human Genome
data bank. Alternatively, the eluted antigen can be subjected to
MALDI-TOF peptide-mass profiling and the derived fingerprints be
used to search for protein identity against the Human Genome data
bank.
Example 6
[0204] This example describes inhibition of tumor adhesion and
spreading by cell adhesion and migration assay
[0205] Antibody inhibition of adhesion and spreading is evaluated.
Tissue culture 12-well plates were coated 2 hours at room
temperature with components of the extracellular matrix, i.e.
vitronectin (VN), fibronectin (FN), collagen type I, type III and
type IV, laminin (LA), or hyaluronic acid (HA) in Hanks buffered
salt solution (HBSS). Plates are blocked for 2 hours with 1% BSA in
PBS. Cells are plated in HBSS with 1 mM CaCl.sub.2 and 1 mM
MgCl.sub.2 in the presence or absence of antibody. Cells are
allowed to spread for 30 minutes to 2 hours at 37.degree. C. prior
to photography.
[0206] Inhibition of cancer cell migratory activity of anti-tumor
agents is evaluated in a matrigel assay. Membranes with a pore size
of 8 .mu.m were coated with 50 .mu.l matrigel. The membranes were
inserted into 24 well plates that contain medium without
supplements. Cancer cells are resuspended in medium with 10% FCS in
the presence or absence of antibodies and then seeded on the
matrigel coated transwell plates. Plates are incubated for 48 hours
at 37.degree. C. Thereafter, cells at the bottom of the chamber are
counted using an inverted microscope.
Example 7
[0207] This example describes using xenograft models to test the
efficacy of the anti-tumor agents.
[0208] For solid tumor models, such as LNCaP xenograft, six to ten
week old male nude NCR nu/nu mice are inoculated subcutaneously in
the mid-scapular region with 5.times.10.sup.6 androgen-dependent
LNCaP cells. Cells that are injected are reconstituted with
basement membrane in the form of Matrigel as described (Sato et al
Cancer Res. 5, 1584-1589 (1997)). To maintain serum testosterone
levels, male mice are implanted with 12.5 mg sustained release
testosterone pellets subcutaneously prior to receiving the tumor
cell inoculation. Antibodies against ATP synthase, or specific for
LNCaP lipid raft antigens are given intraperitoneally on day 2 and
4. Tumors are measured every three to four days with vernier
calipers. Tumor volumes are calculated by the formula
.pi./6.times.(larger diameter).times.(smaller diameter).sup.2. For
the androgen-independent prostate cancer xenograft studies, DU 145
or PC-3 cells are used. For the hematological tumor model, such as
KG-1 xenograft, a similar approach may also be tried (Dao, et al.,
Curr. Opin. Mol. Ther. 1=553-7, 1999)
Example 8
[0209] This example describes selection of anti-lipid raft
antibodies for cancer therapy based on their antigen expression
profiles and anti-cancer activities in vitro.
[0210] For solid tumors, monoclonal antibodies against the
identified antigens are used to stain by immunohistochemistry
normal or neoplastic human tissues to establish the expression
profiles of the tumor associated antigens. Valuable
tumor-associated tumor antigens should have low or no expression in
normal tissues and high expression in cancer cells. To be a good
targets for antibody therapy, tumor associated antigens should be
differentially expressed in substantial percentage (20% and above)
of certain cancer type. Valuable antibodies against these antigens
may have anti-cancer activities in vitro. These activities include
inhibition of cell proliferation, induction of apoptosis and
inhibition of cell migration.
[0211] For hematological malignancies, monoclonal antibodies
against the identified antigens are used to stain by flow cytometry
patient's leukemic cell as well as normal human blood and bone
marrow cells. Antigens that are expressed in hematopoietic stem
cells (within the CD34-positive population), T cells, platelets, or
granulocytes should be excluded because triggering or killing of
these cells by antibodies will cause severe toxicity in humans.
Antigens of interest may be expressed in B cells, macrophages or
monocytes but not in other normal tissues. The ideal
tumor-associated antigens are the ones that can be triggered to
induce cell death in leukemic cells.
[0212] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
[0213] All publications, patents, patent applications, and web
sites are herein incorporated by reference in their entirety to the
same extent as if each individual patent, patent application, or
web site was specifically and individually indicated to be
incorporated by reference in its entirety.
Sequence CWU 1
1
2 1 119 PRT Mouse 1 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys
Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Lys
Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala
Pro Gly Lys Val Leu Arg Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr
Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe
Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln
Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys 85 90 95
Ala Thr Thr Thr Leu Ile Thr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Thr Leu Thr Val Ser Ser 115 2 107 PRT Mouse 2 Asp Ile
Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ser Ser Gln Asp Ile Ser Lys Tyr 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln
Gln Gly Asp Thr Val Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105
* * * * *