U.S. patent application number 14/705638 was filed with the patent office on 2015-12-31 for method of diagnosing neoplastic conditions.
The applicant listed for this patent is MEDVET SCIENCE PTY LTD. Invention is credited to Michael Paul Brown.
Application Number | 20150377890 14/705638 |
Document ID | / |
Family ID | 32909159 |
Filed Date | 2015-12-31 |
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United States Patent
Application |
20150377890 |
Kind Code |
A1 |
Brown; Michael Paul |
December 31, 2015 |
METHOD OF DIAGNOSING NEOPLASTIC CONDITIONS
Abstract
The present invention relates generally to a method for
detecting an aberrant cell, and more particularly an apoptotic
cell, in a subject or in a biological sample from said subject, and
agents useful for same. The presence of the aberrant cell or group
of aberrant cells provides an indication of a particular disease or
condition or a propensity for development of a disease or
condition. More particularly, the present invention contemplates a
method for detecting an apoptotic cell by detecting the presence of
extranuclear nuclear molecules, in particular La, or a relative
increase in extranuclear nuclear molecule levels. The present
invention further provides a method for diagnosing or monitoring
conditions characterised by aberrant, unwanted or otherwise
inappropriate cellular apoptosis in a subject or in a biological
sample from said subject by screening for up-regulation of
extranuclear nuclear molecule levels in a cell or group of cells.
The present invention provides diagnostic agents useful for
detecting these molecules. Such diagnostic agents include
immunointeractive molecules, such as antibodies.
Inventors: |
Brown; Michael Paul; (St.
Georges, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDVET SCIENCE PTY LTD |
Stepney |
|
AU |
|
|
Family ID: |
32909159 |
Appl. No.: |
14/705638 |
Filed: |
May 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10546552 |
Oct 16, 2006 |
9063155 |
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PCT/AU04/00223 |
Feb 23, 2004 |
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14705638 |
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Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
A61P 25/00 20180101;
G01N 33/5017 20130101; A61P 25/16 20180101; A61P 35/02 20180101;
A61K 47/6843 20170801; A61K 51/1018 20130101; A61P 43/00 20180101;
A61P 35/00 20180101; G01N 33/6875 20130101; C07K 16/18 20130101;
A61P 35/04 20180101; A61P 37/02 20180101; A61P 31/12 20180101; A61P
9/10 20180101; A61P 37/06 20180101; G01N 2510/00 20130101; A61P
25/28 20180101; A61P 31/18 20180101; A61P 29/00 20180101; G01N
33/57488 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2003 |
AU |
2003900777 |
Mar 6, 2003 |
AU |
2003901126 |
Claims
1. A method for diagnosing or monitoring a condition characterised
by aberrant, unwanted or otherwise inappropriate cellular apoptosis
in a subject, said method comprising contacting cells or cell
extracts from said subject or a biological sample from said subject
with a nuclear molecule-binding effective amount of an interactive
molecule directed to said nuclear molecule or an antigenic
determinant or epitope thereof and then quantitatively or
qualitatively detecting nuclear molecule-immunointeractive molecule
complex formation wherein the non-nuclear localisation of said
complex is indicative of cellular apoptosis.
2. The method according to claim 1, wherein said nuclear molecule
is Ro52, Ro60, La-SS/B, gelsolin, .alpha.-fodrin, fibrillarin, U1
small nuclear ribonuclear protein (U1 snRNP), heteronuclear
ribonucleoproteins (hnRNP), lamin B, Poly(ADP-Ribose) Polymerase
(PARP), Proliferating Cell Nuclear Antigen (PCNA), SC-35 splicing
factor, Smith (Sm) antigen.
3. The method according to claim 2, wherein said nuclear molecule
is La.
4. The method according to claim 1, wherein said non-nuclear
localisation is cytoplasmic or associated with apoptotic
bodies.
5. The method according to claim 2, wherein said interactive
molecule is a immunointeractive molecule.
6. The method according to claim 5, wherein said immunointeractive
molecule is an antibody.
7. The method according to claim 6, wherein said antibody is a
monoclonal antibody.
8. The method according to claim 1, wherein said apoptotic cell is
an apoptotic cardiac cell, neural cell or lymphoid cell.
9. The method according to claim 1, wherein said condition is
infarction of cardiac muscle or brain tissue. autoimmune and other
inflammatory diseases, viral diseases such as AIDS, neurogenerative
diseases such as Alzheimer's disease or Parkinson's disease, acute
solid organ or bone marrow transplant rejection, chemotherapy- or
radiation-induced tissue damage (`mucositis`) or neoplasms such as
tumours.
10. The method according to claim 9, wherein said condition is a
central nervous system tumours, retinoblastoma, neuroblastoma or
other paediatric tumours, head and neck cancers, breast and
prostrate cancers, lung cancer, kidney cancers, oesophagogastric
cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias,
colorectal cancer, cervical and anal cancers, uterine and other
reproductive tract cancers, urinary tract cancers, germ cell
tumours, ovarian cancer, carcinomas of unknown primary, human
immunodeficiency associated malignancies, lymphomas, leukemias,
malignant melanomas, sarcomas, endocrine tumours, mesothelioma and
other pleural tumours, neuroendocrine tumours and carcinoid
tumours.
11. The method according to claim 10, wherein said head and neck
cancer is a squamous cell cancer, said lung cancer is a small or
non-small lung cell cancer, said kidney cancer is a renal cell
adenocarcinoma, said pancreatic reoplasma is an adenocarinoma islet
cell tumour, said germ cell tumour is testicular cancer or ovarian
cancer, and said ovarian cancer is an ovarian epithelial cancer,
said human immunodeficiency associated malignancies is kaposis
sarcoma and said endocrine tumour is a tumour of the thyroid gland.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a method for
detecting an aberrant cell, and more particularly an apoptotic
cell, in a subject or in a biological sample from said subject, and
agents useful for same. The presence of the aberrant cell or group
of aberrant cells provides an indication of a particular disease or
condition or a propensity for development of a disease or
condition. More particularly, the present invention contemplates a
method for detecting an apoptotic cell by detecting the presence of
extranuclear nuclear molecules, in particular La, or a relative
increase in extranuclear nuclear molecule levels. The present
invention further provides a method for diagnosing or monitoring
conditions characterised by aberrant, unwanted or otherwise
inappropriate cellular apoptosis in a subject or in a biological
sample from said subject by screening for up-regulation of
extranuclear nuclear molecule levels in a cell or group of cells.
The present invention provides diagnostic agents useful for
detecting these molecules. Such diagnostic agents include
immunointeractive molecules, such as antibodies.
[0002] The present invention still further relates to a means for
therapeutic targeting either in vitro or in vivo. The present
invention still further provides antibodies and, in particular,
monoclonal antibodies, which interact specifically with epitopes
present on the subject molecule. The ability to target apoptotic
cells may be useful, inter alia, in a range of diagnostic,
immuno-therapeutic and immuno-prophylactic treatments characterised
by the presence of apoptotic cells.
BACKGROUND OF THE INVENTION
[0003] Bibliographic details of the publications referred to by
author in this specification are collected alphabetically at the
end of the description.
[0004] The reference to any prior art in this specification is not,
and should not be taken as, an acknowledgment or any form of
suggestion that that prior art forms part of the common general
knowledge in Australia.
[0005] Malignant tumours, or cancers, grow in an uncontrolled
manner, invade normal tissues, and often metastasize and grow at
sites distant from the tissue of origin. In general, cancers are
derived from one or only a few normal cells that have undergone a
poorly understood process called malignant transformation. Cancers
can arise from almost any tissue in the body. Those derived from
epithelial cells, called carcinomas, are the most common kinds of
cancers. Sarcomas are malignant tumours of mesenchymal tissues,
arising from cells such as fibroblasts, muscle cells, and fat
cells. Solid malignant tumours of lymphoid tissues are called
lymphomas, and marrow and blood-borne malignant tumours of
lymphocytes and other hematopoietic cells are called leukemias.
[0006] Cancer is one of the three leading causes of death in
industrialized nations. As treatments for infectious diseases and
the prevention of cardiovascular disease continues to improve, and
the average life expectancy increases, cancer is likely to become
the most common fatal disease in these countries. Therefore,
successfully treating cancer requires that all the malignant cells
be removed or destroyed without killing the patient. An ideal way
to achieve this would be to induce an immune response against the
tumour that would discriminate between the cells of the tumour and
their normal cellular counterparts. However, immunological
approaches to the treatment of cancer have been attempted for over
a century with unsustainable results.
[0007] Accordingly, current methods of treating cancer continue to
follow the long used protocol of surgical excision (if possible)
followed by radiotherapy and/or chemotherapy, if necessary. The
success rate of this rather crude form of treatment is extremely
variable but generally decreases significantly as the tumour
becomes more advanced and metastasises. Further, these treatments
are associated with severe side effects including disfigurement and
scarring from surgery (e.g. mastectomy or limb amputation), severe
nausea and vomiting, chemotherapy, and most significantly, the
damage to normal tissues such as the hair follicles, gut and bone
marrow which is induced as a result of the relatively non-specific
targeting mechanism of the toxic drugs which form part of most
cancer treatments.
[0008] Further, most anti-cancer treatments, which include
cytotoxic chemotherapeutic agents, signal transduction inhibitors,
radiotherapy, monoclonal antibodies and cytotoxic lymphocytes, kill
cancer cells by apoptosis. Although tumours may contain a
proportion of apoptotic cells and even areas of necrosis before
anti-cancer treatment is given, an increased number of apoptotic
cells and larger areas of necrosis are anticipated in tumours that
respond to the anti-cancer treatment. However, when cytotoxic
chemotherapeutic agents are used for the treatment of advanced
cancer, the degree of cell kill and thus the response of the tumour
to the first treatment is frequently difficult to assess.
Conventionally, patients receive a minimum of three cycles of
chemotherapy before a clinical and radiological assessment of
tumour response is made. Usually, only a minority of patients with
advanced cancer responds to cytotoxic drugs and so patients may
experience the side effects of treatment without obtaining benefit.
Hence, there is an unmet medical need for a diagnostic method that
would enable rapid, convenient and reliable detection of tumour
cell kill after the first cycle of treatment that would predict
treatment response, which in turn often predicts survival. For
example, the use of positron emission tomography with
fluoro-deoxyglucose (FDG-PET) in patients with oesophageal
adenocarcinoma, who received chemoradiotherapy before surgery,
differentiated treatment responders from non-responders with
>90% sensitivity and specificity and tended to predict those who
would subsequently undergo a curative resection of their tumours.
Knowing whether the tumour is responding early would spare the
majority of patients from ineffective and potentially toxic
treatment. Then, non-responding patients can be offered second line
treatments or clinical trials of investigational agents.
[0009] Accordingly, there is an urgent and ongoing need to develop
new methods of diagnosing and treating cancers in a targeted
manner. This notion of effective targeted killing of malignant
cells has been, to date, unattainable.
[0010] In work leading up to the present invention, it has been
surprisingly determined that nuclear molecules can in fact be
reliably and detectably screened for, utilising an interactive
molecule, such as an immunointeractive molecule, and further,
provides an accurate and reliable means of detecting apoptotic
cells in a highly specific manner either in vitro or in vivo. In
particular, the diagnosis and monitoring of tumours and metastases,
which are often characterised by the presence of a proportion of
apoptotic cells, has now been facilitated. Still further, the use
of the interactive molecules of the present invention has now been
determined to facilitate anti-tumour therapy in a highly targeted
and specific context.
SUMMARY OF THE INVENTION
[0011] One aspect of the present invention contemplates a method
for detecting an apoptotic cell in a subject or in a biological
sample from said subject, said method comprising contacting cells
or cell extracts from said subject or said biological sample with
an interactive molecule directed to a nuclear molecule or antigenic
portion thereof and screening for the interactive molecule-nuclear
molecule complex formation wherein the non-nuclear localisation of
said complex is indicative of an apoptotic cell.
[0012] Another aspect of the present invention contemplates a
method for detecting an apoptotic cell in a subject or in a
biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an immunointeractive molecule directed to a
nuclear molecule or antigenic portion thereof and screening for
immunointeractive molecule-nuclear molecule complex formation
wherein the non-nuclear localisation of said complex is indicative
of an apoptotic cell.
[0013] In yet another aspect there is provided a method for
detecting an apoptotic neoplastic cell in a subject or in a
biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an immunointeractive molecule directed to La
or antigenic portion thereof and screening for immunointeractive
molecule-La complex formation wherein the non-nuclear localisation
of said complex is indicative of an apoptotic neoplastic cell.
[0014] In still another aspect there is provided a method for
detecting an apoptotic neoplastic cell in a subject or in a
biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an antibody directed to La or antigenic
portion thereof and screening for antibody-La complex formation
wherein the non-nuclear localisation of said complex is indicative
of an apoptotic cell.
[0015] Another aspect of the present invention is directed to a
method for diagnosing or monitoring a condition characterised by
aberrant, unwanted or otherwise inappropriate cellular apoptosis in
a subject, said method comprising contacting cells or cell extracts
from said subject or a biological sample from said subject with a
nuclear molecule-binding effective amount of an interactive
molecule directed to said nuclear molecule or an antigenic
determinant or epitope thereof and quantitatively or qualitatively
detecting nuclear molecule-immunointeractive molecule complex
formation wherein the non-nuclear localisation of said complex is
indicative of cellular apoptosis.
[0016] In yet another aspect the present invention is directed to a
method for diagnosing or monitoring a neoplastic condition in a
subject, said method comprising contacting said cells or cell
extracts from said subject or a biological sample from said subject
with an La-binding effective amount of an immunointeractive
molecule directed to said La or an antigenic determinant or epitope
thereof and quantitatively or qualitatively detecting
La-immunointeractive molecule complex formation wherein the
non-nuclear localisation of said complex is indicative of cellular
apoptosis and said cellular apoptosis is indicative of said
neoplastic condition.
[0017] The present invention further contemplates an assay to
detect an apoptotic cell in a biological sample, said assay
including the steps of:-- [0018] (1) contacting an interactive
molecule directed to a nuclear molecule or an antigenic determinant
thereof with a biological sample suspected of containing said
nuclear molecule; and [0019] (2) subjecting the complex formed in
step (1) to a signal detection step wherein detecting non-nuclear
interactive molecule-nuclear molecule complex formation is
indicative of apoptotic cells.
[0020] Another aspect of the present invention contemplates a
method for detecting apoptotic cells in a human, said method
comprising introducing into said patient an interactive molecule
directed to a nuclear molecule or an antigenic determinant thereof
labelled with a reporter molecule, allowing dissemination of the
labelled interactive molecule throughout the circulatory system, or
to selected parts of the circulatory system and then subjecting
said patient to reporter molecule-detection means to identify the
location of the interactive molecule.
[0021] A further aspect of the present invention provides a method
of detecting, in a sample, La or fragment, variant or derivative
thereof comprising contacting the sample with an antibody or
fragment or derivative thereof and detecting the formation of a
complex comprising said antibody and La or fragment, variant or
derivative thereof wherein non-nuclear localisation of La is
indicative of apoptosis.
[0022] The present invention still further contemplates the use of
an interactive molecule directed to a nuclear molecule in the
manufacture of a quantitative or semi-quantitative diagnostic kit
to detect apoptotic cells in a biological sample from a patient.
The kit may come with instructions for use and may be automated or
semi-automated or in a form which is compatible with automated
machine or software.
[0023] Still yet another aspect of the present invention is
directed to a method of therapeutically and/or prophylactically
treating a condition in a subject, which condition is characterised
by cellular apoptosis, said method comprising administering to said
subject an effective amount of an interactive molecule directed to
a nuclear molecule or antigenic portion thereof, which interactive
molecule is linked, bound or otherwise associated with a
therapeutic or prophylactic effector mechanism, for a time and
under conditions sufficient to treat said condition.
[0024] The present invention more particularly provides a method of
therapeutically and/or prophylactically treating a neoplastic
condition in a subject, said method comprising administering to
said subject an effective amount of an immunointeractive molecule
directed to La or antigenic portion thereof, which
immunointeractive molecule is linked, bound or otherwise associated
with a therapeutic effector mechanism, for a time and under
conditions sufficient to inhibit, reduce or otherwise down-regulate
the growth of the neoplasm.
[0025] In a further aspect there is provided a method of
therapeutically treating a metastatic cancer in a subject, said
method comprising administering to said subject an effective amount
of an immunointeractive molecule directed to La or antigenic
portion thereof, which immunointeractive molecule is linked, bound
or otherwise associated with a therapeutic effector mechanism, for
a time and under conditions sufficient to inhibit, reduce or
otherwise down-regulate the growth of said metastatic cancer.
[0026] Another aspect of the present invention contemplates the use
of an anti-nuclear molecule interactive molecule conjugated to an
effector mechanism, in the manufacture of medicament for the
treatment of a condition in a subject, which condition is
characterised by cellular apoptosis, wherein said effector
mechanism treats said condition.
[0027] In yet another further aspect, the present invention
contemplates a pharmaceutical composition comprising the modulatory
agent as hereinbefore defined together with one or more
pharmaceutically acceptable carriers and/or diluents. Said agents
are referred to as the active ingredients.
[0028] Yet another aspect of the present invention relates to the
agent as hereinbefore defined, when used in the method of the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a graphical representation of the prostate cancer
cell line LNCaP which was cultured with or without serum for 24
hours before the cells were scraped off the tissue culture flask
for analysis by flow cytometry. Cells were stained with Propidium
Iodide (PI) and (A) Annexin V-FITC, (B) Normal human serum (NHS) or
affinity purified human La autoantibodies (hLa) and mouse
anti-human IgG-FITC, (C) Murine isotype control or murine anti-hLa
mAb clone SW3 and anti-mouse IgG-FITC, (D) Murine isotype control
or murine anti-hLa mAb clone 3B9 and anti-mouse IgG-FITC.
Histograms are shown for cells that were gated as PI intermediate
(i.e. late apoptotic cells). Blue line, NHS or murine isotype
control; Black line, Annexin V or anti-La staining for LNCaP cells
grown in serum; Red line, Annexin V or anti-La staining for
serum-starved LNCaP cells.
[0030] FIGS. 2A and 2B are a schematic representation of anti-La
functioning. Once anti-La antibodies diagnose chemotherapy-induced
apoptosis among cancer cells (FIG. 2A), they may subsequently
deliver other modalities of cancer treatment, which have non-cross
resistant mechanisms of action, to those viable cancer cells that
remain in the vicinity of the dead cells (FIG. 2B). FIG. 2A shows
that after the first chemotherapy, anti-La antibodies (yellow)
detect apoptotic cancer cells (dark grey), which live close to
viable cancer cells (light grey). FIG. 2B shows that anti-La
antibodies (purple), which are armed with a non-cross resistant
anti-cancer treatment, deliver bystander killing to the remaining
live cancer cells (red).
[0031] FIG. 3 is a graphical representation depicting that
apoptotic bodies progressively form in vitro and bind anti-La
antibody. Apoptosis was induced in the Jurkat human T cell leukemia
cell line using 0.5 .mu.M staurosporine (STS). Cells were stained
with FITC-labelled 3B9 and the nuclear impermeant nucleic acid
binding dye, propidium iodide (PI). The progressive formation of
apoptotic bodies with time is indicated by arrows. Quadrant cursors
are set for <3% staining with isotype control, Sal5.
[0032] FIG. 4 is a graphical representation depicting that anti-La
antibody binding to apoptotic Jurkat cells is associated with
increasing membrane permeability. Apoptosis was induced to Jurkat
cells using 0.5 .mu.M STS. Cells were stained FITC-labelled Sal5
(isotype CONTROL) or FITC-labelled 3B9 (anti-La antibody) or
FITC-labelled anti-.beta.-tubulin mAb (Tubulin) and PI.
[0033] FIG. 5 is a graphical representation of the time course of
anti-La antibody binding to apoptotic Jurkat cells. Apoptosis was
induced in Jurkat cells using 0.5 .mu.M STS. Cells were stained
with FITC-labelled Sal 5 (isotype CONTROL) or FITC-labelled 3B9
(anti-La antibody) or FITC-labelled anti-.beta.-tubulin mAb
(Tubulin) trypan blue. The percentage of total cells at each time
point that was positive for staining with each of these indicators
is shown on the ordinate axis. Data are plotted as simple lines (A)
or as fitted curves using a least squares method in Prism
v3.0(B).
[0034] FIG. 6 is a graphical representation depicting that
apoptotic bodies and anti-La antibody binding to apoptotic bodies
is stable in vitro. Apoptosis was induced in Jurkat cells using 0.5
.mu.M STS. Cells were stained with FITC-labelled 3B9 and propidium
iodide (PI) (upper row). The size and internal complexity of the
apoptotic bodies demonstrated using forward scatter (FSC) and side
scatter (SSC), respectively (lower row).
[0035] FIG. 7 is a graphical representation depicting that the
binding of annexin V, 7AAD and anti-La antibody to apoptotic cells
is interrelated and varies over time during apoptosis in vitro.
Apoptosis was induced in Jurkat cells using 0.5 .mu.M STS. Cells
were stained with FITC-labelled human annexin V (annV-FITC),
R-phycoerythrin-labelled 3B9 (PE-3B9) and 7AAD.
[0036] FIG. 8 is a graphical representation depicting that during
apoptosis in vitro, the time-dependent binding of annexin V, 7AAD
and anti-La antibody to apoptotic cells is interrelated. Apoptosis
was induced in Jurkat cells using 0.5 .mu.M STS. Cells were stained
with FITC-labelled human annexin V (annV-FITC),
R-phycoerthrin-labelled 3B9 (3B9) and 7AAD. Events in regions R1-3
(left hand panel) were analysed for size (forward scatter or FSC)
and internal complexity (side scatter or SSC) (middle panels) and
for staining with 7AAD and 3B9 (right hand panels).
[0037] FIG. 9 comprises both an image and a graphical
representation depicting that anti-La antibody binds to necrotic
Jurkat cells. Necrosis was induced in Jurkat cells by heating at
56.degree. C. for 1 hour. A. Cells were stained with
Alexa488-labelled 3B9 (anti-La antibody) (green) and the nuclear
impermeant DNA-binding dye, 7-amino-actinomycin D (7AAD) (red) or
B. Alexa488-labelled 3B9 (anti-La antibody) (green) and
R-phycoerythrin-labelled annexin V (red) and visualised by laser
scanning confocal microscopy. C. Cells were stained with
FITC-labelled Sal5 (isotype CONTROL) or FITC-labelled 3B9 (anti-La
antibody) and PI and analysed by flow cytometry.
[0038] FIG. 10 is a graphical representation depicting that anti-La
antibody preferentially binds late apoptotic cells and apoptotic
bodies. Apoptosis was induced in Jurkat cells using 0.5 .mu.M STS.
Cells were stained with FITC-labelled 3B9 and PI. At 20 h or 48 h
post-induction of apoptosis, 3B9.sup.+ subpopulations that stained
differentially with PI (1 and 2 in each left hand panel) were gated
for analysis of scatter characteristics. Scatter analysis (right
hand panels) shows that the 3B9.sup.+ PI.sup.intermediate events
that accumulate with time are smaller (lower size as measured by
forward scatter [FSC]) and less granular (reduced internal
complexity as measured by forward scatter [SSC]) (lower right hand
panel). Quadrant cursors are set for <3% staining with isotype
control, Sal5.
[0039] FIG. 11 is a graphical representation depicting that anti-La
antibody binding is caspase 3 dependent and associated with
apoptotic body formation. MCF-7 cells were transiently transfected
with plasmid DNA vectors that expressed enhanced green fluorescent
protein (EGFP) alone or pro-caspase 3 and EGFP. Apoptosis was
induced in the transiently transfected MCF-7 cells using 0.5 .mu.M
STS and the cells were stained with FITC-labelled 3B9 and 7AAD. The
cells shown in the scatter plots (left hand panels) had been gated
on green fluorescence. Subsequently, events in quadrants 1 and 2 of
each scatter plot (left hand panels) had been gated for analysis of
scatter characteristics. Scatter analysis (right hand panels) shows
that 3B9 binding is caspase 3 dependent and hence associated with
apoptotic body formation because apoptotic bodies were smaller
(lower size as measured by forward scatter [FSC]) and less granular
(reduced internal complexity as measured by forward scatter [SSC])
(lower right hand panels). Quadrant cursors are set for <3%
staining with isotype control, Sal5.
[0040] FIG. 12 comprises both an image and a graphical
representation depicting that anti-La antibody binding is caspase 3
dependent and associated with apoptotic body formation. MCF-7 cells
were stably transfected with either a vector control (B) or a
vector that expressed pro-caspase 3 (C and C'). MCF-7 transfectants
were rendered apoptotic by 24 h treatment with 1 .mu.M STS. A. For
flow cytometry, cells were stained with Alexa488-labelled 3B9 and
propidium iodide (PI). B, C and C'. For fluorescence microscopy,
cells were stained with Alexa488-labelled 3B9 (green) and the
nuclear dye DAPI (blue). Apoptotic bodies are indicated
(arrows).
[0041] FIG. 13 is an image of anti-La antibody loading the
cytoplasm of dead cells. Apoptosis was induced in Jurkat cells by
24 h treatment with 0.5 .mu.M STS. Cells were stained with the
nuclear impermeant dye TOPRO3 (blue), Alexa488-labelled 3B9 or
anti-La antibody, the isotype control Sal 5 or anti-PARP mAb
(green) and R-phycoerythrin (PE)-labelled human annexin V (red) and
viewed using confocal laser scanning microscopy. A. lower and A'.
higher magnifications are shown for Alexa488-labelled 3B9 staining;
B. negative isotype control staining for anti-La antibody using
Sal5; C. anti-PARP staining.
[0042] FIG. 14 is an image depicting that other monoclonal
antibodies directed against other nuclear and ribonuclear antigens
also bind apoptotic bodies. Apoptosis was induced in Jurkat cells
by a 24 h treatment with 0.5 .mu.M STS. Cells were stained with
Alexa488-labelled anti-.alpha.-fodrin mAb (green) and 7AAD (red)
and visualised by laser scanning confocal microscopy.
[0043] FIG. 15 is a graphical representation depicting that La/SS-B
expression is up-regulated after apoptosis of malignant Jurkat T
cells in comparison with apoptotic primary T cells. Ficoll-purified
peripheral blood mononuclear cells (PBMC) were cultured for 4d in
RPMI-1640 with 10% fetal calf serum and then treated with 1 .mu.M
STS in the final 24 h of culture. Similarly, PBMC were activated
with the T cell mitogen conconavalin A (PBMC-ConA) 10 .mu.g/mL for
4d before apoptosis was induced with 1 .mu.M STS in the final 24 h
of culture. Jurkat cells (Jurkat) were rendered apoptotic by 24 h
treatment with 0.5 .mu.M STS. Quadrant cursors are set for <3%
staining with isotype control, Sal5.
[0044] FIG. 16 is a graphical representation depicting that other
monoclonal antibodies directed again other nuclear and ribonuclear
antigens also bind apoptotic cells. Apoptosis was induced in Jurkat
cells using 0.5 .mu.M STS. Cells were stained with PI and various
FITC-labelled mAb: isotype control, Sal5, for anti-La/SS-B clone
3B9 (anti-La antibody), anti-.beta.-tubulin clone TUB2.1 FITC
conjugate (Sigma F 2043), Proliferating Cell Nuclear Antigen (PCNA)
Clone PC10 (Oncogene Cat#NA03), mouse anti-.alpha.-fodrin
(nonerythroid anti-spectrin) Chemicon MAB1622, anti-lamen B Clone
101-B7 (Oncogene cat#NA12) and anti-PARP clone C2-10 (Oncogene cat#
AM30). Quadrant cursors are set for <3% staining with
correspondence isotype control antibodies.
[0045] FIG. 17 is a graphical representative depicting that other
monoclonal antibodies directed against other nuclear and
ribonuclear antigens also bind apoptotic bodies. MCF-7 cells were
transiently transfected with plasmid DNA vectors that expressed
EGFP alone or pro-caspase 3 and EGFP. Apoptosis was induced in the
transiently transfected MCF-7 cells using 0.5 .mu.M STS and the
cells were stained with 7AAD and FITC-labelled isotype mAb, Sal5,
and mAb directed against La/SS-B (3B9), lamin B and Proliferating
Cell Nuclear Antigen (PCNA). Upper row of panels show
non-transfected MCF-7 cells (gated as EGFP-positive). The
appearances of the non-transfected MCF-7 cells are nearly identical
to those of MCF-7 cells that had been transfected with the DNA
vector that expressed EGFP alone (data not shown).
[0046] FIG. 18 is a graphical representation depicting that
apoptotic human cells are detected by human anti-La autoantibodies.
Jurkat cells that had been rendered apoptotic by treatment with 0.5
.mu.M STS were stained with human anti-La autoantibodies that had
been La-affinity purified (upper row of panels) or murine mAb 3B9
directed against human La (lower row of panels). PI.sup.+ cells
were gated and data are presented as histograms for each time point
after apoptosis induction. Negative control, human IgG (thick
line); human anti-hLa antibodies and 3B9 (thin line).
[0047] FIG. 19 is a graphical representation depicting that
apoptotic human cells were also detected by another anti-human
La/SS-B monoclonal antibody. Jurkat cells were either left
untreated in culture in vitro (Untreated) or treated with 0.5 .mu.M
STS for 17 h to induce apoptosis (Treated). Cells were stained with
PI and FITC-labelled anti-murine secondary antibody or mAb clone
SW3 or mAb clone 3B9.
[0048] FIG. 20 is a graphical representation depicting that anti-La
antibody binds primary apoptotic cells from rodent species. Murine
(A-C) or rat (D-F) thymocytes were cultured in vitro for 21-24 h
without supplements (A, D), or with the addition of 1 .mu.M
dexamethasone (B, E) or 0.5 .mu.M STS (C, F). Cells were stained
with FITC-labelled isotype mAb, Sal5 (CONTROL) or FITC-labelled 3B9
(anti-La antibody) and propidium iodide (PI).
[0049] FIG. 21 is a graphical representation depicting that anti-La
antibody binds apoptotic tumour cells from rodent species. The
murine thymic lymphoblastic cell line, EL-4 (A-E) or the rat
prostrate cancer cell line, AT-3.1 (F) were cultured in vitro (A-C,
F) for 24 h with 0.5 .mu.M STS (A, F), etoposide (B) or etoposide
and cyclophosphamide (C), or EL-4 tumour cells were recovered from
subcutaneous implants in syngeneic C57BL/6 mice, which had been
left untreated (D) or treated in vivo with cyclophosphamide and
etoposide for 48 h to induce tumour apoptosis (E). Cells were
stained with FITC-labelled isotype mAb, Sal5 (CONTROL) or
FITC-labelled 3B9 (anti-La antibody) and propidium iodide (PI).
[0050] FIG. 22 is a graphical representation depicting that anti-La
antibody binds a number of apoptotic human and monkey tumour cell
lines. The cell lines were treated with 0.5-1 .mu.M STS for 24 h to
induce apoptosis: A. Jurkat T cell leukemia; B. U2OS osteosarcoma
cells; C. HeLa cervical cancer cells; D. MG63 osteosarcoma cells;
E. COS-7 monkey kidney fibroblastic cells. Cells were stained with
FITC-labelled isotype mAb, Sal (CONTROL) or FITC-labelled 3B9
(anti-La antibody) and propidium iodide (PI).
[0051] FIG. 23 is a schematic depiction of the progression of
apoptosis through various stages in vitro. After an apoptotic
stimulus, apoptotic cells shrink and fragment into membrane bound
parcels known as apoptotic bodies that become increasingly leaky or
secondarily necrotic with time. Eventually the apoptotic bodies
disintegrate to oligonucleosomes and then free DNA.
[0052] FIG. 24 is a graphical depiction of the stage of apoptosis
is conventionally defined by staining with annexin V and nuclear
impermeant dyes such as 7AAD. Jurkat cells were rendered apoptotic
with 0.5 .mu.M staurosporine for 16 h and stained with annexin V
(AV) and 7-amino-actinomycin D (7AAD). 1, viable cells (AV.sup.-,
7AAD.sup.-); 2, early apoptotic cells (AV.sup.+, 7AAD.sup.-); 3,
late apoptotic cells (AV.sup.+, 7AAD.sup.+).
DETAILED DESCRIPTION OF THE INVENTION
[0053] The present invention is predicated, in part, on the
surprising determination that screening for nuclear molecule
expression outside the nucleus utilising an immunointeractive
molecule provides a highly specific and reliable means of detecting
the presence of apoptotic cells either in vitro or in vivo. This
finding is of particular significance since previous experimental
work directed to screening for (and thereby identifying) antigens
associated with apoptotic cells have not identified nuclear
molecules, in particular La, as candidate antigens. Rather, other
unrelated antigens such as phosphatidylserine have been repeatedly
identified. Unfortunately, these unrelated molecules exhibit
significant disadvantages, in particular the fact that transient
extracellular expression can occur due to non-apoptotic events,
such as mechanical or other disruption of the cell. Still further,
previously analysed nuclear molecules such as La have been
dismissed as markers of apoptotic cells, per se, on the basis that
cellular staining was thought to be the result of an active
process, by some apoptotic cells, of internalisation of cell
surface complexes of antibody-antigen. That is, membrane
permeability as a route of antibody entry to the cell had been
dismissed. Accordingly, no means of identifying apoptotic cells,
per se, as a class had been developed. However, there has now been
developed a method for the in vitro and in vivo detection of late
apoptotic cells and apoptotic bodies using anti-nuclear
immunointeractive molecules, in particular, anti-La/SS-B
immunointeractive molecules. The method is direct and does not
require additional steps such as permeabilisation to demonstrate
binding, as has been required prior to the advent of the present
invention. It had previously been understood that anti-nuclear
antibody binding was not related to apoptosis induction, that it
depended upon permeabilisation of the cell, that it was surface
membrane-related and depended upon mechanisms of binding other than
passive entry and specific binding to antigen contained within
apoptotic bodies.
[0054] The surprising identification of nuclear molecules such as
La as highly specific, detectable and exclusive markers of
apoptotic cells now permits the development of a range of agents
and methods directed to diagnosing and monitoring apoptotic
cellular populations and, in particular, tumours and their
metastases. Still further, it has been determined that the use of
immunointeractive molecules directed to these nuclear molecules
provides a highly specific means of targeting therapeutic and/or
prophylactic treatments to conditions characterised by the presence
of apoptotic cells. Of particular significance in the specific
context of tumour therapy has been the finding that targeting of an
anti-La immunointeractive molecules, for example, to apoptotic
cells within a tumour can be successfully utilised to provide a
means for achieving killing of bystander non-apoptotic tumour
cells, such as via the delivery of a toxic molecule.
[0055] Accordingly, one aspect of the present invention
contemplates a method for detecting an apoptotic cell in a subject
or in a biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an interactive molecule directed to a
nuclear molecule or antigenic portion thereof and screening for the
interactive molecule-nuclear molecule complex formation wherein the
non-nuclear localisation of said complex is indicative of an
apoptotic cell.
[0056] More particularly, the present invention contemplates a
method for detecting an apoptotic cell in a subject or in a
biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an immunointeractive molecule directed to a
nuclear molecule or antigenic portion thereof and screening for
immunointeractive molecule-nuclear molecule complex formation
wherein the non-nuclear localisation of said complex is indicative
of an apoptotic cell.
[0057] Reference to a "nuclear molecule" should be understood as a
reference to any proteinaceous or non-proteinaceous molecule which
is either permanently or transiently present prior to apoptosis, in
the nucleus of the cell which becomes the subject of apoptosis.
Preferably, said nuclear molecule is Ro52, Ro60, La/SS-B, gelsolin,
.alpha.-fodrin, fibrillarin, U1 small nuclear ribonuclear protein
(U1 snRNP), heteronuclear ribonucleoproteins (hnRNP), lamin B,
Poly(ADP-Ribose) Polymerase (PARP), Proliferating Cell Nuclear
Antigen (PCNA), SC-35 splicing factor, Smith (Sm) antigen. Even
more preferably said nuclear molecule is La.
[0058] Without limiting the present invention in any way, anti-La
antibodies specifically detect apoptosis. Prior understanding of
the non-nuclear localisation of La and its subsequent detection on
the exterior surface of apoptotic blebs was held to be dependent
upon caspase activation, which is a specific outcome of apoptosis.
However, although caspase activity is not detected in late
apoptotic cells (P. Smolewski et al. IJ Immunol Methods 2002) it
has been determined that nuclear molecules such as La can still be
detected. Late apoptotic cells are not detected readily by other
diagnostic methods.
[0059] Reference to "non-nuclear localisation" should be understood
as a reference to the subject nuclear molecule being localised to
any region of the cell, or part thereof, other than within the
intact nucleus. Preferably, the subject non-nuclear localisation is
such that the La is exposed to the extracellular environment,
herein referred to as "extracellular localisation", as occurs, for
example, where the nuclear molecule is translocated to the
cytoplasm of an apoptotic cell, the membrane of which cell has
become permeable or where the molecule is expressed within an
apoptotic body, which bodies form within the apoptosing cell and
are ultimately released to the extracellular environment upon
complete disintegration of the apoptosing cell.
[0060] Reference to an "apoptotic" cell should be understood as a
reference to a cell which is undergoing, or has undergone,
apoptosis. Without limiting the present invention to any one theory
or mode of action, apoptosis is an active process requiring
metabolic activity by the dying cell. Apoptosis is often
characterised by shrinkage of the cell, cleavage of the DNA into
fragments (which give a "laddering pattern" on gels) and by
condensation and margination of chromatin. Cellular apoptosis
occurs in a wide variety of contexts. Accordingly, identification
of the non-nuclear localisation of La, for example, together with
the nature and location of the cell type expressing this molecule
provides a means of monitoring and/or diagnosing a wide variety of
conditions including infarction of cardiac muscle (heart attack) or
brain (stroke), or autoimmune and other inflammatory diseases, or
viral diseases such as AIDS, or neurogenerative diseases such as
Alzheimer's disease or Parkinson's disease, or acute solid organ or
bone marrow transplant rejection, or chemotherapy- or
radiation-induced tissue damage (`mucositis`) or a neoplasm. In one
preferred embodiment, the subject apoptotic cell is an apoptotic
neoplastic cell.
[0061] According to this preferred embodiment, there is provided a
method for detecting an apoptotic neoplastic cell in a subject or
in a biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an immunointeractive molecule directed to La
or antigenic portion thereof and screening for immunointeractive
molecule-La complex formation wherein the non-nuclear localisation
of said complex is indicative of an apoptotic neoplastic cell.
[0062] Preferably, said non-nuclear localisation occurs within the
cytoplasm of the apoptotic cell or within the apoptotic bodies
formed by the apoptotic cell.
[0063] Reference to a "neoplastic cell" should be understood as a
reference to a cell exhibiting abnormal growth. The term "growth"
should be understood in its broadest sense and includes reference
to proliferation. In this regard, an example of abnormal cell
growth is the uncontrolled proliferation of a cell. The neoplastic
cell may be a benign cell or a malignant cell. The subject
neoplastic cell may be any cell type such as an epithelial cell or
a non-epithelial cell.
[0064] The common medical meaning of the term "neoplasia" refers to
"new cell growth" that results as a loss of responsiveness to
normal growth controls, e.g. to neoplastic cell growth. A
"hyperplasia" refers to cells undergoing an abnormally high rate of
growth. However, as used herein, the terms "neoplasia" and
"hyperplasia" can be used interchangeably, referring generally to
cells experiencing abnormal cell growth rates. Neoplasias and
hyperplasias include "tumours" which may be either benign,
pre-malignant or malignant. The term "neoplasm" should be
understood as a reference to a lesion, tumour or other encapsulated
or unencapsulated mass or other form of growth which comprises
neoplastic cells.
[0065] As used herein, the terms "hyperproliferative" and
"neoplastic" are used interchangeably and refer to those cells in
an abnormal state or condition characterized by rapid proliferation
or neoplasm. The terms are meant to include all types of cancerous
growths or oncogenic processes, metastatic tissues or malignantly
transformed cells, tissues or organs irrespective of
histopathologic type or state of invasiveness. "Pathologic
hyperproliferative" cells occur in disease states characterized by
malignant tumour growth.
[0066] The term "carcinoma" is recognized by those skilled in the
art and refers to malignancies of epithelial or endocrine tissues
including respiratory system carcinomas, gastrointestinal system
carcinomas, genitourinary system carcinomas, testicular carcinomas,
breast carcinomas, prostate carcinomas, endocrine system carcinomas
and melanomas. Exemplary carcinomas include those forming from
tissue of the breast. The term also includes carcinosarcomas, e.g.
which include malignant tumours composed of carcinomatous and
sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma
derived from glandular tissue or in which the tumour cells form
recognizable glandular structures.
[0067] The term "neoplasm" as used herein encompasses all the terms
discussed in the preceding three paragraphs.
[0068] Examples of neoplasms and neoplastic cells encompassed by
the present invention include, but are not limited central nervous
system tumours, retinoblastoma, neuroblastoma and other paediatric
tumours, head and neck cancers (eg. squamous cell cancers), breast
and prostate cancers, lung cancer (both small and non-small cell
lung cancer), kidney cancers (eg. renal cell adenocarcinoma),
oesophagogastric cancers, hepatocellular carcinoma,
pancreaticobiliary neoplasias (eg. adenocarcinomas and islet cell
tumours), colorectal cancer, cervical and anal cancers, uterine and
other reproductive tract cancers, urinary tract cancers (eg. of
ureter and bladder), germ cell tumours (eg. testicular germ cell
tumours or ovarian germ cell tumours), ovarian cancer (eg. ovarian
epithelial cancers), carcinomas of unknown primary, human
immunodeficiency associated malignancies (eg. Kaposi's sarcoma),
lymphomas, leukemias, malignant melanomas, sarcomas, endocrine
tumours (eg. of thyroid gland), mesothelioma and other pleural
tumours, neuroendocrine tumours and carcinoid tumours.
[0069] Reference herein to "La" includes reference to all forms of
La or their homologues, or orthologs or derivatives. Reference to
"La" should be understood to include reference to any isoforms
which arise from alternative splicing of La mRNA or mutants or
polymorphic variants of La. It should also be understood that "La"
is a molecule which is alternatively term SS-B.
[0070] The "interactive molecule" is any molecule having
specificity (not necessarily exclusive specificity, although this
is preferable) and binding affinity for La or its antigenic parts
or its homologues or derivatives. Examples of interactive molecules
include immunointeractive molecules and peptidomimetic agents.
Although the preferred immmunointeractive molecule is an
immunoglobulin molecule, the present invention extends to other
immunointeractive molecules such as antibody fragments, single
chain antibodies, deimmunized antibodies including humanized
antibodies and T-cell associated antigen-binding molecules (TABMs).
Most preferably, the immunointeractive molecule is an antibody such
as a polyclonal or monoclonal antibody. It should be understood
that the subject immunointeractive molecule may be linked, bound or
otherwise associated to any other proteinaceous or
non-proteinaceous molecule or cell. Most preferably, the antibody
is a monoclonal antibody.
[0071] The interactive molecule is "directed to" the nuclear
molecule, for example La, or, to the extent that the interactive
molecule is an immunointeractive molecule, to an antigenic
determinant or epitope. It should be understood that the molecule
may not necessarily exhibit complete exclusivity, although this is
preferable. For example, antibodies are known to sometimes
cross-react with other antigens. An antigenic determinant or
epitope includes that part of the molecule to which an immune
response can be directed. The antigenic determinant or epitope may
be a B-cell epitope or where appropriate a T-cell receptor binding
molecule. The term "antigenic part" includes an antigenic
determinant or epitope.
[0072] Preferably, the subject immunointeractive molecule is an
antibody.
[0073] Even more preferably, said antibody is a monoclonal
antibody.
[0074] According to this preferred embodiment, there is provided a
method for detecting an apoptotic neoplastic cell in a subject or
in a biological sample from said subject, said method comprising
contacting cells or cell extracts from said subject or said
biological sample with an antibody directed to La or antigenic
portion thereof and screening for antibody-La complex formation
wherein the non-nuclear localisation of said complex is indicative
of an apoptotic cell.
[0075] Preferably, said non-nuclear localisation occurs within the
cytoplasm of the apoptotic cell or within the apoptotic bodies
formed by the apoptotic cell.
[0076] Reference to a "biological sample" should be understood as a
reference to any sample of biological material derived from an
individual such as, but not limited to, mucus, stool, urine, blood,
serum, cell extract, biopsy specimens and fluid which has been
introduced into the body of an individual and subsequently removed
such as, for example, the saline solution extracted from the lung
following lung lavage or the solution retrieved from an enema wash.
The biological sample which is tested according to the method of
the present invention may be tested directly or may require some
form of treatment prior to testing. For example, a biopsy sample
may require homogenisation or sectioning prior to testing.
[0077] In accordance with the present invention, it is proposed
that apoptotic cells, including apoptotic malignant or
non-malignant neoplastic cells, express La extracellularly. The
quantitative or qualitative detection of levels of extracellular La
provides, therefore, an indicator that a cell is apoptotic and is
associated with a condition characterised by cellular
apoptosis.
[0078] The present invention therefore provides a method of
diagnosing or monitoring a condition characterised by aberrant,
unwanted or otherwise inappropriate cellular apoptosis. By
"aberrant, unwanted or otherwise inappropriate" is meant that the
subject apoptosis may be at an excessive level, inadequate level or
at a normal level, but which level in inappropriate or otherwise
unwanted. As detailed herein, there are a number of conditions
which are characterised by the presence of some degree of cellular
apoptosis, for example, infarction of cardiac muscle or brain
tissue or autoimmune and other inflammatory diseases, or viral
diseases such as AIDS, or neurogenerative diseases such as
Alzheimer's disease or Parkinson's disease, or acute solid organ or
bone marrow transplant rejection, or chemotherapy- or
radiation-induced tissue damage (`mucositis`) and neoplasms such as
tumours.
[0079] Although the preferred embodiments of the present invention
are directed to screening for the occurrence of apoptosis, this
being indicative of the onset of a particular disease condition,
there may also occur clinical situations where one is screening for
a drop in the level of cellular apoptosis or the absence of
apoptosis altogether. This latter situation may arise, for example,
where one is monitoring the progress of a therapeutic treatment
regime and a decrease in the level of apoptosis would indicate that
the disease under treatment is shifting into a remissive state. It
should also be understood that in some situations the absence of
cellular apoptosis events may be indicative of the development of a
disease condition. For example, in the course of normal thymocyte
development, a large portion of the thymocytes present in the
thymus undergo apoptosis during the course of the positive and
negative selection events which are necessary in order to develop
self/non-self discrimination. Accordingly, the absence of a normal
level of cellular apoptotic events in the thymus of a young child
may be indicative of the propensity of the child to developing
autoimmune conditions. It should therefore be understood that
although the present invention is likely to be largely applied in
the context of screening for the presence of specific cellular
apoptosis events in order to enable the diagnosis of a disease
condition, the method of the present invention can nevertheless be
applied to screening for the absence of apoptotic events, where
that would indicate the development of or a propensity to develop
certain disease conditions. The method of the present invention may
also be applied to screening for changes in the level of cellular
apoptosis in the context of monitoring the progress of a disease
condition or therapeutic or prophylactic treatment regime.
[0080] Accordingly, another aspect of the present invention is
directed to a method for diagnosing or monitoring a condition
characterised by aberrant, unwanted or otherwise inappropriate
cellular apoptosis in a subject, said method comprising contacting
cells or cell extracts from said subject or a biological sample
from said subject with a nuclear molecule-binding effective amount
of an interactive molecule directed to said nuclear molecule or an
antigenic determinant or epitope thereof and quantitatively or
qualitatively detecting nuclear molecule-immunointeractive molecule
complex formation wherein the non-nuclear localisation of said
complex is indicative of cellular apoptosis.
[0081] Preferably, said nuclear molecule is La.
[0082] Preferably, said interactive molecule is an
immunointeractive molecule and even more preferably an anti-La
antibody, such as an anti-La monoclonal antibody.
[0083] Most preferably, said non-nuclear localisation is
extracellular localisation and still more preferably within the
cytoplasm of the apoptotic cell or within the apoptotic bodies
formed by the apoptotic cell.
[0084] In another preferred embodiment, said condition is
infarction of cardiac muscle or brain tissue, autoimmune and other
inflammatory diseases, viral diseases such as AIDS, neurogenerative
diseases such as Alzheimer's disease or Parkinson's disease, acute
solid organ or bone marrow transplant rejection, chemotherapy- or
radiation-induced tissue damage (`mucositis`) or neoplasms such as
tumours.
[0085] In a most preferred embodiment the present invention is
directed to a method for diagnosing or monitoring a neoplastic
condition in a subject, said method comprising contacting said
cells or cell extracts from said subject or a biological sample
from said subject with an La-binding effective amount of an
immunointeractive molecule directed to said La or an antigenic
determinant or epitope thereof and quantitatively or qualitatively
detecting La-immunointeractive molecule complex formation wherein
the non-nuclear localisation of said complex is indicative of
cellular apoptosis and said cellular apoptosis is indicative of
said neoplastic condition.
[0086] Preferably said neoplasm is central nervous system tumours,
retinoblastoma, neuroblastoma and other paediatric tumours, head
and neck cancers (eg. squamous cell cancers), breast and prostate
cancers, lung cancer (both small and non-small cell lung cancer),
kidney cancers (eg. renal cell adenocarcinoma), oesophagogastric
cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias
(eg. adenocarcinomas and islet cell tumours), colorectal cancer,
cervical and anal cancers, uterine and other reproductive tract
cancers, urinary tract cancers (eg. of ureter and bladder), germ
cell tumours (eg. testicular germ cell tumours or ovarian germ cell
tumours), ovarian cancer (eg. ovarian epithelial cancers),
carcinomas of unknown primary, human immunodeficiency associated
malignancies (eg. Kaposi's sarcoma), lymphomas, leukemias,
malignant melanomas, sarcomas, endocrine tumours (eg. of thyroid
gland), mesothelioma and other pleural tumours, neuroendocrine
tumours and carcinoid tumours.
[0087] Most preferably, said immunointeractive molecule is an
antibody, and still more preferably a monoclonal antibody.
[0088] Reference herein to a "subject" should be understood to
encompass humans, primates, livestock animals (eg. sheep, pigs,
cattle, horses, donkeys), laboratory test animals (eg. mice,
rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and
captive wild animals (eg. foxes, kangaroos, deer). Preferably, the
mammal is a human.
[0089] The use of antibodies and in particular monoclonal
antibodies, such as those hereinbefore mentioned, to detect nuclear
molecules such as La is a preferred method of the present
invention. Antibodies may be prepared by any of a number of means.
For the detection of human La, for example, human-human monoclonal
antibody hybridomas may be derived from B cells, which have
been-obtained from patients who make anti-La autoantibodies because
they have systemic autoimmune diseases such as systemic lupus
erythematosis (SLE) or Sjogren's syndrome (Ravirajan et al. Lupus I
(3):157-165, 1992). Antibodies are generally but not necessarily
derived from non-human animals such as primates, livestock animals
(e.g. sheep, cows, pigs, goats, horses), laboratory test animals
(e.g. mice, rats, guinea pigs, rabbits) and companion animals (e.g.
dogs, cats). Generally, antibody based assays are conducted in
vitro on cell or tissue biopsies. However, if an antibody is
suitably deimmunized or, in the case of human use, humanized, then
the antibody can be labelled with, for example, a nuclear tag,
administered to a patient and the site of nuclear label
accumulation determined by radiological techniques. The La antibody
is regarded, therefore, as a cellular apoptosis targeting agent.
Accordingly, the present invention extends to deimmunized forms of
the antibodies for use in cellular apoptosis imaging in human and
non-human patients. This is described further below.
[0090] The present invention provides, therefore, an antibody and
in particular a monoclonal antibody for use in immunological assays
for La or for cellular apoptosis imaging in vivo. Currently
available antibodies include SW3 and 3B9.
[0091] For the generation of antibodies to La, this molecule is
required to be extracted from a biological sample whether this be
from animal including human tissue or from cell culture if produced
by recombinant means. The La can be separated from the biological
sample by any suitable means. For example, the separation may take
advantage of any one or more of La's surface charge properties,
size, density, biological activity and its affinity for another
entity (e.g. another protein or chemical compound to which it binds
or otherwise associates). Thus, for example, separation of La from
the biological fluid may be achieved by any one or more of
ultra-centrifugation, ion-exchange chromatography (e.g. anion
exchange chromatography, cation exchange chromatography),
electrophoresis (e.g. polyacrylamide gel electrophoresis,
isoelectric focussing), size separation (e.g., gel filtration,
ultra-filtration) and affinity-mediated separation (e.g.
immunoaffinity separation including, but not limited to, magnetic
bead separation such as Dynabead.TM. separation,
immunochromatography, immuno-precipitation). Choice of the
separation technique(s) employed may depend on the biological
activity or physical properties of the La sought or from which
tissues it is obtained.
[0092] Preferably, the separation of La from the biological fluid
preserves conformational epitopes present on the protein and, thus,
suitably avoids techniques that cause denaturation of the enzyme.
Persons of skill in the art will recognize the importance of
maintaining or mimicking as close as possible physiological
conditions peculiar to La (e.g. the biological fluid from which it
is obtained) to ensure that the antigenic determinants or active
sites on La, which are exposed to the animal, are structurally
identical to that of the native protein. This ensures the raising
of appropriate antibodies in the immunised animal that would
recognize the native protein. In a preferred embodiment, La is
separated from the biological fluid using any one or more of
affinity separation, gel filtration and ultra-filtration.
[0093] Immunization and subsequent production of monoclonal
antibodies can be carried out using standard protocols as for
example described by Kohler and Milstein, Nature 256: 495-499,
1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519, 1976;
Coligan et al., Current Protocols in Immunology, John Wiley &
Sons, Inc., 1991-1997, or Toyama et al., "Monoclonal Antibody,
Experiment Manual", published by Kodansha Scientific, 1987.
Essentially, an animal is immunized with a La-containing biological
fluid or fraction thereof by standard methods to produce
antibody-producing cells, particularly antibody-producing somatic
cells (e.g. B lymphocytes). These cells can then be removed from
the immunized animal for immortalization.
[0094] Where a fragment of La is used to generate antibodies, it
may need to first be associated with a carrier. By "carrier" is
meant any substance of typically high molecular weight to which a
non- or poorly immunogenic substance (e.g. a hapten) is naturally
or artificially linked to enhance its immunogenicity.
[0095] Immortalization of antibody-producing cells may be carried
out using methods which are well-known in the art. For example, the
immortalization may be achieved by the transformation method using
Epstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121:
140, 1986). In a preferred embodiment, antibody-producing cells are
immortalized using the cell fusion method (described in Coligan et
al., 1991-1997, supra), which is widely employed for the production
of monoclonal antibodies. In this method, somatic
antibody-producing cells with the potential to produce antibodies,
particularly B cells, are fused with a myeloma cell line. These
somatic cells may be derived from the lymph nodes, spleens and
peripheral blood of humans with circulating La-reactive antibodies,
and primed animals, preferably rodent animals such as mice and
rats. Mice spleen cells are particularly useful. It would be
possible, however, to use rat, rabbit, sheep or goat cells, or
cells from other animal species instead.
[0096] Specialized myeloma cell lines have been developed from
lymphocytic tumours for use in hybridoma-producing fusion
procedures (Kohler and Milstein, 1976, supra; Shulman et al.,
Nature 276: 269-270, 1978; Volk et al., J. Virol. 42(1): 220-227,
1982). These cell lines have been developed for at least three
reasons. The first is to facilitate the selection of fused
hybridomas from unfused and similarly indefinitely self-propagating
myeloma cells. Usually, this is accomplished by using myelomas with
enzyme deficiencies that render them incapable of growing in
certain selective media that support the growth of hybridomas. The
second reason arises from the inherent ability of lymphocytic
tumour cells to produce their own antibodies. To eliminate the
production of tumour cell antibodies by the hybridomas, myeloma
cell lines incapable of producing endogenous light or heavy
immunoglobulin chains are used. A third reason for selection of
these cell lines is for their suitability and efficiency for
fusion.
[0097] Many myeloma cell lines may be used for the production of
fused cell hybrids, including, e.g. P3X63-Ag8, P3X63-AG8.653,
P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1. The P3X63-Ag8
and NS-1 cell lines have been described by Kohler and Milstein
(1976, supra). Shulman et al. (1978, supra) developed the
Sp2/0-Ag14 myeloma line. The S194/5.XXO.Bu.1 line was reported by
Trowbridge, J. Exp. Med. 148(1): 313-323, 1978.
[0098] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually involve mixing
somatic cells with myeloma cells in a 10:1 proportion (although the
proportion may vary from about 20:1 to about 1:1), respectively, in
the presence of an agent or agents (chemical, viral or electrical)
that promotes the fusion of cell membranes. Fusion methods have
been described (Kohler and Milstein, 1975, supra; 1976, supra;
Gefter et al., Somatic Cell Genet. 3: 231-236, 1977; Volk et al.,
1982, supra). The fusion-promoting agents used by those
investigators were Sendai virus and polyethylene glycol (PEG).
[0099] Because fusion procedures produce viable hybrids at very low
frequency (e.g. when spleens are used as a source of somatic cells,
only one hybrid is obtained for roughly every 1.times.10.sup.5
spleen cells), it is preferable to have a means of selecting the
fused cell hybrids from the remaining unfused cells, particularly
the unfused myeloma cells. A means of detecting the desired
antibody-producing hybridomas among other resulting fused cell
hybrids is also necessary. Generally, the selection of fused cell
hybrids is accomplished by culturing the cells in media that
support the growth of hybridomas but prevent the growth of the
unfused myeloma cells, which normally would go on dividing
indefinitely. The somatic cells used in the fusion do not maintain
long-term viability in in vitro culture and hence do not pose a
problem. In the example of the present invention, myeloma cells
lacking hypoxanthine phosphoribosyl transferase (HPRT-negative)
were used. Selection against these cells is made in
hypoxanthine/aminopterin/thymidine (HAT) medium, a medium in which
the fused cell hybrids survive due to the HPRT-positive genotype of
the spleen cells. The use of myeloma cells with different genetic
deficiencies (drug sensitivities, etc.) that can be selected
against in media supporting the growth of genotypically competent
hybrids is also possible.
[0100] Several weeks are required to selectively culture the fused
cell hybrids. Early in this time period, it is necessary to
identify those hybrids which produce the desired antibody, so that
they may subsequently be cloned and propagated. Generally, around
10% of the hybrids obtained produce the desired antibody, although
a range of from about 1 to about 30% is not uncommon. The detection
of antibody-producing hybrids can be achieved by any one of several
standard assay methods, including enzyme-linked immunoassay and
radioimmunoassay techniques as, for example, described in Kennet et
al. (eds) Monoclonal Antibodies and Hybridomas: A New Dimension in
Biological Analyses, pp. 376-384, Plenum Press, New York, 1980 and
by FACS analysis.
[0101] Once the desired fused cell hybrids have been selected and
cloned into individual antibody-producing cell lines, each cell
line may be propagated in either of two standard ways. A suspension
of the hybridoma cells can be injected into a histocompatible
animal. The injected animal will then develop tumours that secrete
the specific monoclonal antibody produced by the fused cell hybrid.
The body fluids of the animal, such as serum or ascites fluid, can
be tapped to provide monoclonal antibodies in high concentration.
Alternatively, the individual cell lines may be propagated in vitro
in laboratory culture vessels. The culture medium containing high
concentrations of a single specific monoclonal antibody can be
harvested by decantation, filtration or centrifugation, and
subsequently purified.
[0102] The cell lines are tested for their specificity to detect
the La by any suitable immunodetection means. For example, cell
lines can be aliquoted into a number of wells and incubated and the
supernatant from each well is analyzed by enzyme-linked
immunosorbent assay (ELISA), indirect fluorescent antibody
technique, or the like. The cell line(s) producing a monoclonal
antibody capable of recognizing the target La but which does not
recognize non-target epitopes are identified and then directly
cultured in vitro or injected into a histocompatible animal to form
tumours and to produce, collect and purify the required
antibodies.
[0103] These antibodies are La specific. This means that the
antibodies are capable of distinguishing La from other molecules.
More broad spectrum antibodies may be used provided that they do
not cross react with molecules in a normal cell.
[0104] In a preferred embodiment, the subject antibody is
anti-human La monoclonal antibodies, 8G3 and 9A5 (Bachmann et al.
Proc Natl Acad Sci USA 83 (20):7770-7774, 1986), anti-human La
monoclonal antibody (mAb), La1B5 (Mamula et al. J Immunol
143(9):2923-2928, 1989), anti-human La monoclonal antibodies
(Carmo-Fonseca et al. ExpCell Res 185(1):73-85, 1989), anti-human
and anti-bovine La monoclonal antibodies, SW1, SW3 and SW5 (Pruijn
et al. Eur J Biochem 232(2):611-619, 1995), anti-human and
anti-rodent La mAb, La4B6 (Troster et al. J Autoimmunity
8(6):825-842, 1995) or anti-human and anti-murine La mAb, 3B9 (Tran
et al. Arthritis Rheum 46(1):202-208, 2002) or derivative,
homologue, analogue, chemical equivalent, mutant or mimetic
thereof.
[0105] The present invention should also be understood to extend to
the immunointeractive molecule and the cell lines which express the
subject immunointeractive molecule, in particular a hybridoma which
expresses a monoclonal antibody.
[0106] Where the monoclonal antibody is destined for use in in vivo
imaging or treatment, it may need to be deimmunized with respect to
the host into which it will be introduced (e.g. a human). The
deimmunization process may take any of a number of forms including
the preparation of chimeric antibodies which have the same or
similar specificity as the monoclonal antibodies prepared according
to the present invention. Chimeric antibodies are antibodies whose
light and heavy chain genes have been constructed, typically by
genetic engineering, from immunoglobulin variable and constant
region genes belonging to different species. Thus, in accordance
with the present invention, once a hybridoma producing the desired
monoclonal antibody is obtained, techniques are used to produce
interspecific monoclonal antibodies wherein the binding region of
one species is combined with a non-binding region of the antibody
of another species (Liu et al., Proc. Natl. Acad. Sci. USA 84:
3439-3443, 1987). For example, complementary determining regions
(CDRs) from a non-human (e.g. murine) monoclonal antibody can be
grafted onto a human antibody, thereby "humanizing" the murine
antibody (European Patent Publication No. 0 239 400; Jones et al.,
Nature 321: 522-525, 1986; Verhoeyen et al., Science 239:
1534-1536, 1988; Richmann et al., Nature 332: 323-327, 1988). In
this case, the deimmunizing process is specific for humans. More
particularly, the CDRs can be grafted onto a human antibody
variable region with or without human constant regions. The
non-human antibody providing the CDRs is typically referred to as
the "donor" and the human antibody providing the framework is
typically referred to as the "acceptor". Constant regions need not
be present, but if they are, they must be substantially identical
to human immunoglobulin constant regions, i.e. at least about
85-90%, preferably about 95% or more identical. Hence, all parts of
a humanized antibody, except possibly the CDRs, are substantially
identical to corresponding parts of natural human immunoglobulin
sequences. Thus, a "humanized antibody" is an antibody comprising a
humanized light chain and a humanized heavy chain immunoglobulin. A
donor antibody is said to be "humanized", by the process of
"humanization", because the resultant humanized antibody is
expected to bind to the same antigen as the donor antibody that
provides the CDRs. Reference herein to "humanized" includes
reference to an antibody deimmunized to a particular host, in this
case, a human host.
[0107] It will be understood that the deimmunized antibodies may
have additional conservative amino acid substitutions which have
substantially no effect on antigen binding or other immunoglobulin
functions. Exemplary conservative substitutions may be made
according to Table 1.
TABLE-US-00001 TABLE 1 ORIGINAL EXEMPLARY RESIDUE SUBSTITUTIONS Ala
Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro
His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu,
Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Tip, Phe Val Ile,
Leu
[0108] Exemplary methods which may be employed to produce
deimmunized antibodies according to the present invention are
described, for example, in references Richmann et al., 1988, supra;
European Patent Publication No. 0 239 400; Chou et al. (U.S. Pat.
No. 6,056,957); Queen et al. (U.S. Pat. No. 6,180,370); Morgan et
al. (U.S. Pat. No. 6,180,377).
[0109] Thus, in one embodiment, the present invention contemplates
a deimmunized antibody molecule having specificity for an epitope
recognized by a monoclonal antibody to La wherein at least one of
the CDRs of the variable domain of said deimmunized antibody is
derived from the said monoclonal antibody to La and the remaining
immunoglobulin-derived parts of the deimmunized antibody molecule
are derived from an immunoglobulin or an analogue thereof from the
host for which the antibody is to be deimmunized.
[0110] This aspect of the present invention involves manipulation
of the framework region of a non-human antibody.
[0111] The present invention extends to mutants, analogues and
derivatives of the subject antibodies but which still retain
specificity for La.
[0112] The terms "mutant" or "derivatives" includes one or more
amino acid substitutions, additions and/or deletions.
[0113] As used herein, the term "CDR" includes CDR structural loops
which covers the three light chain and the three heavy chain
regions in the variable portion of an antibody framework region
which bridge .beta. strands on the binding portion of the molecule.
These loops have characteristic canonical structures (Chothia et
al., J. Mol. Biol. 196: 901, 1987; Chothia et al., J. Mol. Biol.
227: 799, 1992).
[0114] By "framework region" is meant region of an immunoglobulin
light or heavy chain variable region, which is interrupted by three
hypervariable regions, also called CDRs. The extent of the
framework region and CDRs have been precisely defined (see, for
example, Kabat et al., "Sequences of Proteins of Immunological
Interest", U.S. Department of Health and Human Services, 1983). The
sequences of the framework regions of different light or heavy
chains are relatively conserved within a species. As used herein, a
"human framework region" is a framework region that is
substantially identical (about 85% or more, usually 90-95% or more)
to the framework region of a naturally occurring human
immunoglobulin. The framework region of an antibody, that is the
combined framework regions of the constituent light and heavy
chains, serves to position and align the CDRs. The CDRs are
primarily responsible for binding to an epitope of La.
[0115] As used herein, the term "heavy chain variable region" means
a polypeptide which is from about 110 to 125 amino acid residues in
length, the amino acid sequence of which corresponds to that of a
heavy chain of a monoclonal antibody of the invention, starting
from the amino-terminal (N-terminal) amino acid residue of the
heavy chain. Likewise, the term "light chain variable region" means
a polypeptide which is from about 95 to 130 amino acid residues in
length, the amino acid sequence of which corresponds to that of a
light chain of a monoclonal antibody of the invention, starting
from the N-terminal amino acid residue of the light chain.
Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino
acids) are encoded by a variable region gene at the
NH.sub.2-terminus (about 110 amino acids) and a .eta. or .lamda.
constant region gene at the COOH-terminus. Full-length
immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes, e.g. y
(encoding about 330 amino acids).
[0116] The term "immunoglobulin" or "antibody" is used herein to
refer to a protein consisting of one or more polypeptides
substantially encoded by immunoglobulin genes. The recognized
immunoglobulin genes include the .kappa., .lamda., .alpha.,
.epsilon. (IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4), .delta.,
.epsilon. and .mu. constant region genes, as well as the myriad
immunoglobulin variable region genes. One form of immunoglobulin
constitutes the basic structural unit of an antibody. This form is
a tetramer and consists of two identical pairs of immunoglobulin
chains, each pair having one light and one heavy chain. In each
pair, the light and heavy chain variable regions are together
responsible for binding to an antigen, and the constant regions are
responsible for the antibody effector functions. In addition to
antibodies, immunoglobulins may exist in a variety of other forms
including, for example, Fv, Fab, Fab' and (Fab').sub.2.
[0117] The invention also contemplates the use and generation of
fragments of monoclonal antibodies produced by the method of the
present invention including, for example, Fv, Fab, Fab' and
F(ab').sub.2 fragments. Such fragments may be prepared by standard
methods as for example described by Coligan et al. (1991-1997,
supra).
[0118] The present invention also contemplates synthetic or
recombinant antigen-binding molecules with the same or similar
specificity as the monoclonal antibodies of the invention.
Antigen-binding molecules of this type may comprise a synthetic
stabilised Fv fragment. Exemplary fragments of this type include
single chain Fv fragments (sFv, frequently termed scFv) in which a
peptide linker is used to bridge the N terminus or C terminus of a
V.sub.H domain with the C terminus or N-terminus, respectively, of
a V.sub.L domain. ScFv lack all constant parts of whole antibodies
and are not able to activate complement. Suitable peptide linkers
for joining the V.sub.H and V.sub.L domains are those which allow
the V.sub.H and V.sub.L domains to fold into a single polypeptide
chain having an antigen binding site with a three dimensional
structure similar to that of the antigen binding site of a whole
antibody from which the Fv fragment is derived. Linkers having the
desired properties may be obtained by the method disclosed in U.S.
Pat. No. 4,946,778. However, in some cases a linker is absent.
ScFvs may be prepared, for example, in accordance with methods
outlined in Krebber et al. (Krebber et al., J. Immunol. Methods
201(1): 35-55, 1997). Alternatively, they may be prepared by
methods described in U.S. Pat. No. 5,091,513, European Patent No
239,400 or the articles by Winter and Milstein (Winter and
Milstein, Nature 349: 293, 1991) and Pluckthun et al. (Pluckthun et
al., In Antibody engineering: A practical approach 203-252,
1996).
[0119] Alternatively, the synthetic stabilized Fv fragment
comprises a disulphide stabilized Fv (dsFv) in which cysteine
residues are introduced into the V.sub.H and V.sub.L domains such
that in the fully folded Fv molecule the two residues will form a
disulphide bond therebetween. Suitable methods of producing dsFv
are described, for example, in (Glockshuber et al., Biochem. 29:
1363-1367, 1990; Reiter et al., Biochem. 33: 5451-5459, 1994;
Reiter et al., Cancer Res. 54: 2714-2718, 1994; Reiter et al., J.
Biol. Chem. 269: 18327-18331, 1994; Webber et al., Mol. Immunol.
32: 249-258, 1995).
[0120] Also contemplated as synthetic or recombinant
antigen-binding molecules are single variable region domains
(termed dAbs) as, for example, disclosed in (Ward et al., Nature
341: 544-546, 1989; Hamers-Casterman et al., Nature 363: 446-448,
1993; Davies & Riechmann, FEBS Lett. 339: 285-290, 1994).
[0121] Alternatively, the synthetic or recombinant antigen-binding
molecule may comprise a "minibody". In this regard, minibodies are
small versions of whole antibodies, which encode in a single chain
the essential elements of a whole antibody. Suitably, the minibody
is comprised of the V.sub.H and V.sub.L domains of a native
antibody fused to the hinge region and CH3 domain of the
immunoglobulin molecule as, for example, disclosed in U.S. Pat. No.
5,837,821.
[0122] In an alternate embodiment, the synthetic or recombinant
antigen binding molecule may comprise non-immunoglobulin derived,
protein frameworks. For example, reference may be made to (Ku &
Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) which
discloses a four-helix bundle protein cytochrome b562 having two
loops randomized to create CDRs, which have been selected for
antigen binding.
[0123] The synthetic or recombinant antigen-binding molecule may be
multivalent (i.e. having more than one antigen binding site). Such
multivalent molecules may be specific for one or more antigens.
Multivalent molecules of this type may be prepared by dimerization
of two antibody fragments through a cysteinyl-containing peptide
as, for example disclosed by (Adams et al., Cancer Res. 53:
4026-4034, 1993; Cumber et al., J. Immunol. 149: 120-126,
1992).
[0124] Alternatively, dimerization may be facilitated by fusion of
the antibody fragments to amphiphilic helices that naturally
dimerize (Plunckthun, Biochem. 31: 1579-1584, 1992) or by use of
domains (such as leucine zippers jun and fos) that preferentially
heterodimerize (Kostelny et al., J. Immunol. 148: 1547-1553,
1992).
[0125] The present invention further encompasses chemical analogues
of amino acids in the subject antibodies. The use of chemical
analogues of amino acids is useful inter alia to stabilize the
molecules such as if required to be administered to a subject. The
analogues of the amino acids contemplated herein include, but are
not limited to, modifications of side chains, incorporation of
unnatural amino acids and/or their derivatives during peptide,
polypeptide or protein synthesis and the use of crosslinkers and
other methods which impose conformational constraints on the
proteinaceous molecule or their analogues.
[0126] Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with
2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride;
and pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4.
[0127] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0128] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivatisation, for example, to a corresponding amide.
[0129] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH.
[0130] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine
residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0131] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0132] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid,
4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid,
t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids. A list of unnatural amino acid,
contemplated herein is shown in Table 2.
TABLE-US-00002 TABLE 2 Non-conventional amino acid Code
.alpha.-aminobutyric acid Abu .alpha.-amino-.alpha.-methylbutyrate
Mgabu aminocyclopropane-carboxylate Cpro aminoisobutyric acid Aib
aminonorbornyl-carboxylate Norb cyclohexylalanine Chexa
cyclopentylalanine Cpen D-alanine Dal D-arginine Darg D-aspartic
acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamic acid Dglu
D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-lysine Dlys
D-methionine Dmet D-ornithine Dorn D-phenylalanine Dphe D-proline
Dpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp D-tyrosine
Dtyr D-valine Dval D-.alpha.-methylalanine Dmala
D-.alpha.-methylarginine Dmarg D-.alpha.-methylasparagine Dmasn
D-.alpha.-methylaspartate Dmasp D-.alpha.-methylcysteine Dmcys
D-.alpha.-methylglutamine Dmgln D-.alpha.-methylhistidine Dmhis
D-.alpha.-methylisoleucine Dmile D-.alpha.-methylleucine Dmleu
D-.alpha.-methyllysine Dmlys D-.alpha.-methylmethionine Dmmet
D-.alpha.-methylornithine Dmorn D-.alpha.-methylphenylalanine Dmphe
D-.alpha.-methylproline Dmpro D-.alpha.-methylserine Dmser
D-.alpha.-methylthreonine Dmthr D-.alpha.-methyltryptophan Dmtrp
D-.alpha.-methyltyrosine Dmty D-.alpha.-methylvaline Dmval
D-N-methylalanine Dnmala D-N-methylarginine Dnmarg
D-N-methylasparagine Dnmasn D-N-methylaspartate Dnmasp
D-N-methylcysteine Dnmcys D-N-methylglutamine Dnmgln
D-N-methylglutamate Dnmglu D-N-methylhistidine Dnmhis
D-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu
D-N-methyllysine Dnmlys N-methylcyclohexylalanine Nmchexa
D-N-methylornithine Dnmorn N-methylglycine Nala
N-methylaminoisobutyrate Nmaib N-(1-methylpropyl)glycine Nile
N-(2-methylpropyl)glycine Nleu D-N-methyltryptophan Dnmtrp
D-N-methyltyrosine Dnmtyr D-N-methylvaline Dnmval
.gamma.-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine
Etg L-homophenylalanine Hphe L-.alpha.-methylarginine Marg
L-.alpha.-methylaspartate Masp L-.alpha.-methylcysteine Mcys
L-.alpha.-methylglutamine Mgln L-.alpha.-methylhistidine Mhis
L-.alpha.-methylisoleucine Mile L-.alpha.-methylleucine Mleu
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorvaline Mnva
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylserine Mser
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methylvaline Mval
N-(N-(2,2-diphenylethyl) Nnbhm carbamylmethyl)glycine
1-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane
L-N-methylalanine Nmala L-N-methylarginine Nmarg
L-N-methylasparagine Nmasn L-N-methylaspartic acid Nmasp
L-N-methylcysteine Nmcys L-N-methylglutamine Nmgln
L-N-methylglutamic acid Nmglu L-Nmethylhistidine Nmhis
L-N-methylisolleucine Nmile L-N-methylleucine Nmleu
L-N-methyllysine Nmlys L-N-methylmethionine Nmmet
L-N-methylnorleucine Nmnle L-N-methylnorvaline Nmnva
L-N-methylornithine Nmorn L-N-methylphenylalanine Nmphe
L-N-methylproline Nmpro L-N-methylserine Nmser L-N-methylthreonine
Nmthr L-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr
L-N-methylvaline Nmval L-N-methylethylglycine Nmetg
L-N-methyl-t-butylglycine Nmtbug L-norleucine Nle L-norvaline Nva
.alpha.-methyl-aminoisobutyrate Maib
.alpha.-methyl-.gamma.-aminobutyrate Mgabu
.alpha.-methylcyclohexylalanine Mchexa
.alpha.-methylcylcopentylalanine Mcpen
.alpha.-methyl-.alpha.-napthylalanine Manap
.alpha.-methylpenicillamine Mpen N-(4-aminobutyl)glycine Nglu
N-(2-aminoethyl)glycine Naeg N-(3-aminopropyl)glycine Norn
N-amino-.alpha.-methylbutyrate Nmaabu .alpha.-napthylalanine Anap
N-benzylglycine Nphe N-(2-carbamylethyl)glycine Ngln
N-(carbamylmethyl)glycine Nasn N-(2-carboxyethyl)glycine Nglu
N-(carboxymethyl)glycine Nasp N-cyclobutylglycine Ncbut
N-cycloheptylglycine Nchep N-cyclohexylglycine Nchex
N-cyclodecylglycine Ncdec N-cylcododecylglycine Ncdod
N-cyclooctylglycine Ncoct N-cyclopropylglycine Ncpro
N-cycloundecylglycine Ncund N-(2,2-diphenylethyl)glycine Nbhm
N-(3,3-diphenylpropyl)glycine Nbhe N-(3-guanidinopropyl)glycine
Narg N-(1-hydroxyethyl)glycine Nthr N-(hydroxyethyl))glycine Nser
N-(imidazolylethyl))glycine Nhis N-(3-indolylyethyl)glycine Nhtrp
N-methyl-.gamma.-aminobutyrate Nmgabu D-N-methylmethionine Dnmmet
N-methylcyclopentylalanine Nmcpen D-N-methylphenylalanine Dnmphe
D-N-methylproline Dnmpro D-N-methylserine Dnmser
D-N-methylthreonine Dnmthr N-(1-methylethyl)glycine Nval
N-methyla-napthylalanine Nmanap N-methylpenicillamine Nmpen
N-(p-hydroxyphenyl)glycine Nhtyr N-(thiomethyl)glycine Ncys
penicillamine Pen L-.alpha.-methylalanine Mala
L-.alpha.-methylasparagine Masn L-.alpha.-methyl-t-butylglycine
Mtbug L-methylethylglycine Metg L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhomophenylalanine Mhphe
N-(2-methylthioethyl)glycine Nmet L-.alpha.-methyllysine Mlys
L-.alpha.-methylnorleucine Mnle L-.alpha.-methylornithine Morn
L-.alpha.-methylproline Mpro L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltyrosine Mtyr L-N-methylhomophenylalanine Nmhphe
N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine
[0133] Crosslinkers can be used, for example, to stabilize 3D
conformations, using homo-bifunctional crosslinkers such as the
bifunctional imido esters having (CH.sub.2).sub.n spacer groups
with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and
hetero-bifunctional reagents which usually contain an
amino-reactive moiety such as N-hydroxysuccinimide and another
group specific-reactive moiety such as maleimido or dithio moiety
(SH) or carbodiimide (COOH).
[0134] The present invention further contemplates an assay to
detect an apoptotic cell in a biological sample, said assay
including the steps of:-- [0135] (3) contacting an interactive
molecule directed to a nuclear molecule or an antigenic determinant
thereof with a biological sample suspected of containing said
nuclear molecule; and [0136] (4) subjecting the complex formed in
step (1) to a signal detection step wherein detecting non-nuclear
interactive molecule-nuclear molecule complex formation is
indicative of apoptotic cells.
[0137] Preferably, said nuclear molecule is La.
[0138] More preferably, said interactive molecule is an
immunointeractive molecule and even more preferably an anti-La
antibody, such as the monoclonal antibodies SW3 or 3B9.
[0139] Most preferably, said non-nuclear localisation is
extracellular localisation and, still more preferably, localisation
within the cytoplasm of the apoptotic cell or within the apoptotic
bodies formed by the apoptotic cells.
[0140] The signal detection step may include ELISA or any other
reporter molecule based assays. As part of this detection step, the
signal may first need to be amplified. It should be understood that
this assay may be performed in vivo or in vitro.
[0141] A deimmunized monoclonal antibody of the present invention
may also be useful for apoptosis imaging in vivo as well as for
targeting apoptotic cells in order to bring the apoptotic cells
into contact with bystander-cell growth retarding or bystander-cell
killing agents, i.e. cytostatic or cytocidal agents.
[0142] Anti-La antibodies are superior to currently known products,
such as Apomate.TM. (North American Scientific, Inc.), for the
detection of apoptotic cells in vivo. Apomate.TM. uses radiolabeled
annein V to bind apoptotic cells and, in particular, to ascertain
the responsiveness of cancers to chemotherapy. Annexin V binds to
phosphatidylserine that `flip-flops` to the outer plasma membrane
leaflet during the early part of apoptosis. However, the detection
of chemotherapy-induced tumour cell apoptosis by Apomate.TM. is
inconsistent because the timing of administration may be crucial
(Blankenberg et al. Clin Cancer Res 2002). Timing is less important
with the use of anti-La antibodies because the antibodies direct
apoptotic cells to macrophages, which accumulate at the site of
cancers. Moreover, annexin V inhibits macrophage-mediated
phagocytosis of apoptotic thymocytes in vitro, which is
circumvented by Fc receptor-mediated uptake of antibody-bound
erythrocytes, which have surface-exposed phosphatidylserine
(Callahan et al. Cell Death Different 7(7):645-653, 2000; Krahlung
et al. Cell Death Different 6(2):183-189, 1999). Hence, anti-La
antibodies opsonise apoptotic cells and facilitate their
phagocytosis by macrophages, in particular, so that macrophages are
targeted for diagnostic and/or therapeutic purposes, as hereinafter
described in more detail. Accordingly, one of the advantages of the
use of anti-La antibodies, as opposed to currently known diagnostic
methods such as those based on detecting Annexin V, is that the
opsonisation and uptake of apoptotic cells by macrophages results
in accumulation of the antibody in macrophages at the site of the
apoptotic cells, thereby aiding imaging.
[0143] With respect to imaging, a reporter molecule is attached to
the deimmunized monoclonal antibody and this is then introduced to
a host, such as a human. By detecting the reporter molecule,
cellular apoptotic clusters, such as those associated with tumours,
can be visualized. One particularly useful form of reporter
molecule is a nuclear tag. Some radioisotopes permit imaging by
positron emission tomography (PET) and some ligands facilitate
detection of target binding by magnetic resonance imaging
(MRI).
[0144] Accordingly, another aspect of the present invention
contemplates a method for detecting apoptotic cells in a human,
said method comprising introducing into said patient an interactive
molecule directed to a nuclear molecule or an antigenic determinant
thereof labelled with a reporter molecule, allowing dissemination
of the labelled interactive molecule throughout the circulatory
system, or to selected parts of the circulatory system and then
subjecting said patient to reporter molecule-detection means to
identify the location of the interactive molecule.
[0145] Preferably, said nuclear molecule is La.
[0146] More preferably, said interactive molecule is an
immunointeractive molecule and even more preferably an anti-La
antibody.
[0147] Most preferably, said non-nuclear localisation is
extracellular localisation and preferably, localisation within the
cytoplasm of the apoptotic cell or within the apoptotic bodies
formed by the apoptotic cells.
[0148] Preferably said apoptotic cells are characteristic of a
neoplasm.
[0149] Preferably said neoplastic cell is one which is
characteristic of central nervous system tumours, retinoblastoma,
neuroblastoma and other paediatric tumours, head and neck cancers
(eg. squamous cell cancers), breast and prostate cancers, lung
cancer (both small and non-small cell lung cancer), kidney cancers
(eg. renal cell adenocarcinoma), oesophagogastric cancers,
hepatocellular carcinoma, pancreaticobiliary neoplasias (eg.
adenocarcinomas and islet cell tumours), colorectal cancer,
cervical and anal cancers, uterine and other reproductive tract
cancers, urinary tract cancers (eg. of ureter and bladder), germ
cell tumours (eg. testicular germ cell tumours or ovarian germ cell
tumours), ovarian cancer (eg. ovarian epithelial cancers),
carcinomas of unknown primary, human immunodeficiency associated
malignancies (eg. Kaposi's sarcoma), lymphomas, leukemias,
malignant melanomas, sarcomas, endocrine tumours (eg. of thyroid
gland), mesothelioma and other pleural tumours, neuroendocrine
tumours and carcinoid tumours.
[0150] Immunologically based La detection protocols may take a
variety of forms. For example, a plurality of antibodies may be
immobilized in an array each with different specificities to
particular antigens or cancer cells including La. Cells from a
biopsy are then brought into contact with the antibody array and a
diagnosis may be made as to the type of neoplasm based on the cells
which are immobilized.
[0151] Other more conventional assays may also be conducted such as
by ELISA, Western blot analysis, immunoprecipitation analysis,
immunofluorescence analysis, immunochemistry analysis or FACS
analysis.
[0152] The present invention provides, therefore, a method of
detecting, in a sample, La or fragment, variant or derivative
thereof comprising contacting the sample with an antibody or
fragment or derivative thereof and detecting the formation of a
complex comprising said antibody and La or fragment, variant or
derivative thereof wherein non-nuclear localisation of La is
indicative of apoptosis.
[0153] Preferably, said non-nuclear localisation is extracellular
localisation and, still more preferably, localisation within the
cytoplasm of the apoptotic cell or within the apoptotic bodies
formed by the apoptotic cell.
[0154] As discussed above, any suitable technique for determining
formation of the complex may be used. For example, an antibody
according to the invention, having a reporter molecule associated
therewith, may be utilized in immunoassays. Such immunoassays
include but are not limited to radioimmunoassays (RIAs),
enzyme-linked immunosorbent assays (ELISAs) and
immunochromatographic techniques (ICTs), Western blotting which are
well known to those of skill in the art. For example, reference may
be made to "Current Protocols in Immunology", 1994 which discloses
a variety of immunoassays which may be used in accordance with the
present invention. Immunoassays may include competitive assays. It
will be understood that the present invention encompasses
qualitative and quantitative immunoassays.
[0155] Suitable immunoassay techniques are described, for example,
in U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include
both single-site and two-site assays of the non-competitive types,
as well as the traditional competitive binding assays. These assays
also include direct binding of a labelled antigen-binding molecule
to a target antigen. The antigen in this case is La or a fragment
thereof.
[0156] Two-site assays are particularly favoured for use in the
present invention. A number of variations of these assays exist,
all of which are intended to be encompassed by the present
invention. Briefly, in a typical forward assay, an unlabelled
antigen-binding molecule such as an unlabelled antibody is
immobilized on a solid substrate and the sample to be tested
brought into contact with the bound molecule. After a suitable
period of incubation, for a period of time sufficient to allow
formation of an antibody-antigen complex, another antigen-binding
molecule, suitably a second antibody specific to the antigen,
labelled with a reporter molecule capable of producing a detectable
signal is then added and incubated, allowing time sufficient for
the formation of another complex of antibody-antigen-labelled
antibody. Any unreacted material is washed away and the presence of
the antigen is determined by observation of a signal produced by
the reporter molecule. The results may be either qualitative, by
simple observation of the visible signal, or may be quantitated by
comparing with a control sample containing known amounts of
antigen. Variations on the forward assay include a simultaneous
assay, in which both sample and labelled antibody are added
simultaneously to the bound antibody. These techniques are well
known to those skilled in the art, including minor variations as
will be readily apparent.
[0157] In the typical forward assay, a first antibody having
specificity for the antigen or antigenic parts thereof is either
covalently or passively bound to a solid surface. The solid surface
is typically glass or a polymer, the most commonly used polymers
being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene. The solid supports may be in the form of
tubes, beads, discs of microplates, or any other surface suitable
for conducting an immunoassay.
[0158] The binding processes are well known in the art and
generally consist of cross-linking covalently binding or physically
adsorbing, the polymer-antibody complex is washed in preparation
for the test sample. An aliquot of the sample to be tested is then
added to the solid phase complex and incubated for a period of time
sufficient and under suitable conditions to allow binding of any
antigen present to the antibody. Following the incubation period,
the antigen-antibody complex is washed and dried and incubated with
a second antibody specific for a portion of the antigen. The second
antibody has generally a reporter molecule associated therewith
that is used to indicate the binding of the second antibody to the
antigen. The amount of labelled antibody that binds, as determined
by the associated reporter molecule, is proportional to the amount
of antigen bound to the immobilized first antibody.
[0159] An alternative method involves immobilizing the antigen in
the biological sample and then exposing the immobilized antigen to
specific antibody that may or may not be labelled with a reporter
molecule. Depending on the amount of target and the strength of the
reporter molecule signal, a bound antigen may be detectable by
direct labelling with the antibody. Alternatively, a second
labelled antibody, specific to the first antibody is exposed to the
target-first antibody complex to form a target-first
antibody-second antibody tertiary complex. The complex is detected
by the signal emitted by the reporter molecule.
[0160] From the foregoing, it will be appreciated that the reporter
molecule associated with the antigen-binding molecule may include
the following:-- [0161] (a) direct attachment of the reporter
molecule to the antibody; [0162] (b) indirect attachment of the
reporter molecule to the antibody; i.e., attachment of the reporter
molecule to another assay reagent which subsequently binds to the
antibody; and [0163] (c) attachment to a subsequent reaction
product of the antibody.
[0164] The reporter molecule may be selected from a group including
a chromogen, a catalyst, an enzyme, a fluorochrome, a
chemiluminescent molecule, a paramagnetic ion, a lanthanide ion
such as Europium (Eu.sup.34), a radioisotope including other
nuclear tags, semiconductor quantum dots (Wu et al. Nature
Biotechnol 2002) and a direct visual label. Recombinant
antibody-like molecules may be made by fusion to partners such as
enhanced green fluorescent protein (EGFP).
[0165] In the case of a direct visual label, use may be made of a
colloidal metallic or non-metallic particle, a dye particle, an
enzyme or a substrate, an organic polymer, a latex particle, a
liposome, or other vesicle containing a signal producing substance
and the like.
[0166] A large number of enzymes suitable for use as reporter
molecules is disclosed in U.S. Pat. No. 4,366,241, U.S. Pat. No.
4,843,000, and U.S. Pat. No. 4,849,338. Suitable enzymes useful in
the present invention include alkaline phosphatase, horseradish
peroxidase, luciferase, .beta.-galactosidase, glucose oxidase,
lysozyme, malate dehydrogenase and the like. The enzymes may be
used alone or in combination with a second enzyme that is in
solution.
[0167] Suitable fluorochromes include, but are not limited to,
fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other
exemplary fluorochromes include those discussed by Dower et al.,
International Publication No. WO 93/06121. Reference also may be
made to the fluorochromes described in U.S. Pat. No. 5,573,909,
Singer et al., and U.S. Pat. No. 5,326,692 Brinkley et al.
Alternatively, reference may be made to the fluorochromes described
in U.S. Pat. Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896,
5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270
and 5,723,218.
[0168] In the case of an enzyme immunoassay, an enzyme is
conjugated to the second antibody, generally by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different conjugation techniques exist
which are readily available to the skilled artisan. The substrates
to be used with the specific enzymes are generally chosen for the
production of, upon hydrolysis by the corresponding enzyme, a
detectable colour change.
[0169] Examples of suitable enzymes include those described supra.
It is also possible to employ fluorogenic substrates, which yield a
fluorescent product rather than the chromogenic substrates noted
above. In all cases, the enzyme-labelled antibody is added to the
first antibody-antigen complex, allowed to bind, and then the
excess reagent washed away. A solution containing the appropriate
substrate is then added to the complex of
antibody-antigen-antibody. The substrate will react with the enzyme
linked to the second antibody, giving a qualitative visual signal,
which may be further quantitated, usually spectrophotometrically,
to give an indication of the amount of antigen which was present in
the sample.
[0170] Alternately, fluorescent compounds, such as fluorescein,
rhodamine and the lanthanide, europium (EU), may be chemically
coupled to antibodies without altering their binding capacity. When
activated by illumination with light of a particular wavelength,
the fluorochrome-labelled antibody adsorbs the light energy,
inducing a state to excitability in the molecule, followed by
emission of the light at a characteristic colour visually
detectable with a light microscope. The fluorescent-labelled
antibody is allowed to bind to the first antibody-antigen complex.
After washing off the unbound reagent, the remaining tertiary
complex is then exposed to light of an appropriate wavelength. The
fluorescence observed indicates the presence of the antigen of
interest. Immunofluorometric assays (IFMA) are well established in
the art and are particularly useful for the present method.
However, other reporter molecules, such as radioisotope,
chemiluminescent or bioluminescent molecules may also be
employed.
[0171] The method of the present invention is useful as a one off
test or as an on-going monitor of those individuals thought to be
at risk of development or a condition characterised by cellular
apoptosis (eg. neoplasm) or as a monitor of the effectiveness of
therapeutic or prophylactic treatment regimes directed to
inhibiting or otherwise slowing the progress of such a condition.
In these situation, mapping the modulation of La levels in any one
or more classes of biological samples is a valuable indicator of
the status of an individual or the effectiveness of a therapeutic
or prophylactic regime which is currently in use. Accordingly, the
method of the present invention should be understood to extend to
monitoring for increases or decreases in marker levels in an
individual relative to their normal level (as hereinbefore defined)
or relative to one or more earlier marker levels determined from a
biological sample of said individual.
[0172] The present invention further contemplates the use of an
interactive molecule directed to a nuclear molecule in the
manufacture of a quantitative or semi-quantitative diagnostic kit
to detect apoptotic cells in a biological sample from a patient.
The kit may come with instructions for use and may be automated or
semi-automated or in a form which is compatible with automated
machine or software.
[0173] Preferably, said nuclear molecule is La.
[0174] Preferably, said apoptotic cells are apoptotic neoplastic
cells.
[0175] Without limiting the applications for the kit in any way, it
is useful for the detection of apoptotic cells in diagnostic and
research applications. Currently, there are many reagents on the
market for the detection of apoptosis in vitro, which include
annexin V, mitochondrial permeability dyes, APO2.7 (a monoclonal
antibody that recognises a mitochondrial protein only during
apoptosis), DNA binding fluorochromes such as propidium iodide and
7-amino-actinomycin D (7-AAD), and fluorogenic caspase inhibitors.
However, these reagents are unable to distinguish between apoptotic
and necrotic cells (H. Lecoeur et al. J Immunol Methods 2002) and
more than one is required to specifically identify late apoptotic
cells (P. Smolewski et al. J. Immunol Methods 2002; Hamel et al.
Cytometry 25(2):173-181, 1996).
[0176] The generation of antibodies to La may, in accordance with
the present invention, be directed to the active or inactive forms
of the molecule.
[0177] In addition to the clear diagnostic benefits of the method
of the present invention, the ability to accurately target
apoptotic cells now provides a means of delivering therapeutic
and/or prophylactic treatments in a localised and highly targeted
manner. To date, such treatments (often referred to as "magic
bullets") have not been possible. In particular, in the context of
tumour therapy the notion of targeted treatments has not been
possible due to the fact that it has not been possible to identify
suitable tumour specific antigens against which an antibody could
be directed. However, the present invention overcomes these
shortcomings by directing the therapeutic or prophylactic treatment
to the apoptotic cells which comprise the subject tumour. By
selecting therapeutic or prophylactic effector mechanisms which can
be coupled to an anti-La immunointeractive molecule, but which
function on cells located proximally to the apoptotic cells, that
is the non-apoptotic tumour cells, effective killing of the tumour
can be achieved. The subject effector mechanism may take any
suitable form but will preferably deliver a toxic molecule or
otherwise kill the proximally located non-apoptotic tumour
cells.
[0178] Further, the phagocytosis of apoptotic cells, which have
been opsonised with anti-La antibodies, will be facilitated by
antibodies such as human IgGI and IgG3. Without limiting the
present invention to any one theory or mode of action, anti-La
antibodies will become targeted to and accumulate in macrophages or
other phagocytic cells in vivo. Cells that undergo apoptosis are
first divided into membrane-bound parcels or apoptotic bodies,
which are subsequently disposed of by surrounding cells and, in
particular, by professional scavenger cells known as macrophages.
Anti-La antibodies recognize apoptotic cells specifically at a
particular stage in the apoptotic process both in vitro and in
vivo. Moreover, anti-La antibodies preferentially localize in vivo
to macrophages, which engulf the apoptotic cells. Macrophages
contribute to the healing of the tissue damage that occurs in heart
attach, stroke and organ transplant rejection. Cancers have a high
content of macrophages known as tumour associated macrophages,
which may either retard or promote the growth of the cancer. Hence,
anti-La antibodies serve as a vehicle for the delivery of
therapeutically active technologies to cancers.
[0179] Accordingly, another aspect of the present invention is
directed to a method of therapeutically and/or prophylactically
treating a condition in a subject, which condition is characterised
by cellular apoptosis, said method comprising administering to said
subject an effective amount of an interactive molecule directed to
a nuclear molecule or antigenic portion thereof, which interactive
molecule is linked, bound or otherwise associated with a
therapeutic or prophylactic effector mechanism, for a time and
under conditions sufficient to treat said condition.
[0180] Preferably, said nuclear molecule is La.
[0181] More preferably, said interactive molecule is an
immunointeractive molecule and even more preferably an anti-La
antibody.
[0182] In another preferred embodiment, said condition is
infarction of cardiac muscle or brain tissue, autoimmune and other
inflammatory diseases, viral diseases such as AIDS, neurogenerative
diseases such as Alzheimer's disease or Parkinson's disease, acute
solid organ or bone marrow transplant rejection, chemotherapy- or
radiation-induced tissue damage (`mucositis`) or neoplasms such as
tumours.
[0183] Examples of neoplasms and neoplastic cells encompassed by
the present invention include, but are not limited central nervous
system tumours, retinoblastoma, neuroblastoma and other paediatric
tumours, head and neck cancers (eg. squamous cell cancers), breast
and prostate cancers, lung cancer (both small and non-small cell
lung cancer), kidney cancers (eg. renal cell adenocarcinoma),
oesophagogastric cancers, hepatocellular carcinoma,
pancreaticobiliary neoplasias (eg. adenocarcinomas and islet cell
tumours), colorectal cancer, cervical and anal cancers, uterine and
other reproductive tract cancers, urinary tract cancers (eg. of
ureter and bladder), germ cell tumours (eg. testicular germ cell
tumours or ovarian germ cell tumours), ovarian cancer (eg. ovarian
epithelial cancers), carcinomas of unknown primary, human
immunodeficiency associated malignancies (eg. Kaposi's sarcoma),
lymphomas, leukemias, malignant melanomas, sarcomas, endocrine
tumours (eg. of thyroid gland), mesothelioma and other pleural
tumours, neuroendocrine tumours and carcinoid tumours.
[0184] Reference to "nuclear molecule", "La", "immunointeractive
molecule", "subject" and "apoptosis" should be understood to have
the same meaning as hereinbefore provided.
[0185] The present invention more particularly provides a method of
therapeutically and/or prophylactically treating a neoplastic
condition in a subject, said method comprising administering to
said subject an effective amount of an immunointeractive molecule
directed to La or antigenic portion thereof, which
immunointeractive molecule is linked, bound or otherwise associated
with a therapeutic effector mechanism, for a time and under
conditions sufficient to inhibit, reduce or otherwise down-regulate
the growth of the neoplasm.
[0186] Preferably said neoplastic condition is a malignant
tumour.
[0187] Reference to an "effector mechanism" should be understood as
a reference to any suitable mechanism which, when localised to the
site of apoptotic cells, either directly or indirectly treats the
condition in issue, for example, down-regulating the growth of
tumour cells. In the context of this preferred embodiment, the
effector mechanism is most likely a proteinaceous or
non-proteinaceous molecule or group of molecules which achieve this
outcome. Examples of effector mechanisms suitable for use in the
method of the present invention include, but are not limited to:
[0188] (i) Use of an antibody which has been linked to a cytokine,
chemokine or other factor, such as macrophage, dendritic cell
and/or T cell activators which act to induce or enhance one or more
aspects of an immune response, thereby augmenting bystander
killing. For example, the chemotactic peptide,
N-formyl-methionyl-leucyl-phenylalanine (FMLP) (Morikawa et al.
Cancer Immunol Immunother 27(1):1-6, 1988) and the novel bacterial
lipopeptide, JBT 2002 (Shinohara et al. J Immunother 23(3):321-331,
2000) are both activators of tumour associated macrophages. [0189]
(ii) Use of an antibody which has been conjugated to a toxin.
[0190] Reference to "toxin" should be understood as a reference to
any suitable proteinaceous or non-proteinaceous molecule which
achieves the object of providing a signal which reduces, prevents
or otherwise inhibits the proliferation, differentiation or
maintenance of the subject cell (herein referred to as
"down-regulating the growth" of said cell). The subject toxin may
act by a variety of means including providing its signal via direct
contact with a subject cell or emitting a molecule or particle,
such as radiation in the case of a radioactive isotope toxin, which
provides the signal to the subject cell. Preferably the toxin is a
radioisotope and even more preferably a radioisotope which is
highly toxic over a short range and exhibits a short half life
thereby minimizing the occurrence of inadvertent toxicity on
proximally located non-target cells. Most particularly, said
radioisotope is an alpha particle emitting radioisotope. However,
it should be understood that radioisotopes are not limited to alpha
particle emitting radioisotope and may include beta- and
gamma-emitting radioisotopes, depending on the clinical context.
Examples of alpha-emitting radioisotopes suitable for use in the
method of the present invention include, but are not limited to,
Tb-149 or Bi-213. It should be understood that the toxin which is
utilised in the method of the present invention may be in a
purified, partially purified or unpurified form. It may also form a
component of a larger molecule. The toxin may be naturally
occurring or it may be synthetically or recombinantly produced.
[0191] Other examples of molecules which should be understood to
fall within the scope of "toxin" include ricin, colicheamicin,
prodrugs (as antibody-directed prodrug converting enzyme therapy
[ADEPT]) and novel biotherapeutic agents, such as catalytic
antibodies.
[0192] It should be understood that the method of the present
invention may be performed either in vivo or in vitro. Examples of
in vitro applications include, but are not limited to, the in vitro
purging of biological samples comprising neoplastic cells. For
example, bone marrow and/or blood may be removed from a patient,
purged to kill tumour cells in accordance with the method of the
present invention and then returned to the patient. Such procedures
are currently performed, albeit in a significantly less targeted
manner, and avoid the significant risks associated with the
transplantation of MHC incompatible bone marrow.
[0193] Reference to an effector mechanism being "linked, bound or
otherwise associated" with an anti-La, for example, antibody or
other immunointeractive molecule should be understood as a
reference to any covalent or non-covalent interactive mechanism
which achieves linking of the two molecules. This includes, but is
not limited to the use of peptide bonds, ionic bonds, hydrogen
bonds, van Der Waals forces or any other interactive bonding
mechanism.
[0194] Reference to "growth" of a cell or neoplasm should be
understood as a reference to the proliferation, differentiation
and/or maintenance of viability of the subject cell, while
"down-regulating the growth" of a cell or neoplasm is a reference
to the process of cellular senescence or to reducing, preventing or
inhibiting the proliferation, differentiation and/or maintenance of
viability of the subject cell. In a preferred embodiment the
subject growth is proliferation and the subject down-regulation is
killing. In this regard, killing may be achieved either by
delivering a fatal hit to the cell or by delivering to the cell a
signal which induces the cell to apoptose.
[0195] Reference herein to "therapeutic" or "prophylactic"
"treatment" is to be considered in its broadest context. The term
"treatment" does not necessarily imply that a subject is treated
until total recovery. Similarly, "prophylaxis" does not necessarily
mean that the subject will not eventually contract a disease
condition. Accordingly, treatment and prophylaxis include
amelioration of the symptoms of a particular condition or
preventing or otherwise reducing the risk of developing a
particular condition. The term "prophylaxis" may be considered as
reducing the severity or onset of a particular condition.
"Treatment" may also reduce the severity of an existing
condition.
[0196] Without limiting the present invention to any one theory or
mode of action anti-cancer treatments usually kill by apoptosis but
in many cases of advanced cancer, some cancer cells are resistant
to the apoptosis that may be induced by a particular anti-cancer
treatment. These apoptosis-resistant tumour cells are the source of
clinical relapse of disease that ultimately kills most patients
with advanced cancers and a significant proportion of those
patients with earlier stage cancers. In those patients with
advanced cancers who could be shown to be responding to the initial
modality of treatment because tumour cell kill could be documented
in vivo, additional gains in survival and quality of life may be
made if another non-cross resistant treatment modality were also to
be employed. Therefore, diagnosis of responding cancer patients
using the method of the present invention can identify those
patients who could benefit from supplementary treatment with
therapeutic conjugates or hybrid fusion proteins, as detailed.
[0197] The use of an anti-La antibody may also be favoured in the
adjuvant clinical setting. Although early stage breast and colon
cancers can both be cured by surgery, the risk of overt and
incurable systemic relapse is heightened because, in those patients
whose primary tumour has certain high-risk features and/or whose
regional lymph nodes contain metastases, undetectable systemic
micrometastases may already exist. So the use of adjuvant
chemotherapy and/or adjuvant hormonal therapy (in the case of
breast cancer) cures an additional minor proportion of these
patients presumably because the systemic micrometastases are
cleared successfully.
[0198] Further, although dormant tumours remain small because they
lack a blood supply, the tumour cells within the lesion turnover at
a rapid rate with the rate of cell division balancing the rate of
apoptosis. Therefore, even dormant tumours will be suitable targets
for the present technology. Both in the case of clinically evident
metastases and micrometastases, apoptosis-resistant cancer cells
can be admixed with susceptible cancer cells. Bystander killing of
these surviving cancer cells can occur if a non-cross resistant
and/or synergistic means of tumour killing were delivered to nearby
cancer cells that had been rendered apoptotic by the first
treatment. Additional technologies that arm this technology with
bystander killing potential can improve its therapeutic
efficacy.
[0199] A most preferred embodiment of the present invention is
therefore directed to the treatment of a metastatic cancer.
[0200] According to this preferred embodiment, there is provided a
method of therapeutically treating a metastatic cancer in a
subject, said method comprising administering to said subject an
effective amount of an immunointeractive molecule directed to La or
antigenic portion thereof, which immunointeractive molecule is
linked, bound or otherwise associated with a therapeutic effector
mechanism, for a time and under conditions sufficient to inhibit,
reduce or otherwise down-regulate the growth of said metastatic
cancer.
[0201] As detailed hereinbefore, the present invention should also
be understood to extend to the down-regulation of growth of
neoplastic cells in an in vitro environment. For example,
neoplastic cells may be purged from an inoculum of bone marrow or
peripheral blood stem cells before an autologous transplant.
[0202] An "effective amount" means an amount necessary at least
partly to attain the desired response, or to delay the onset or
inhibit progression or halt altogether, the onset or progression of
a particular condition being treated. The amount varies depending
upon the health and physical condition of the individual to be
treated, the taxonomic group of individual to be treated, the
degree of protection desired, the formulation of the composition,
the assessment of the medical situation, and other relevant
factors. It is expected that the amount will fall in a relatively
broad range that can be determined through routine trials.
[0203] The present invention further contemplates a combination of
therapies, such as the administration of the antibody together with
subjection of the mammal to circulating cytotoxic agents or to
radiotherapy in the treatment of cancer.
[0204] Administration of the interactive molecule (herein referred
to as the "modulatory agent"), in the form of a pharmaceutical
composition, may be performed by any convenient means. The
modulatory agent of the pharmaceutical composition is contemplated
to exhibit therapeutic activity when administered in an amount
which depends on the particular case. The variation depends, for
example, on the human or animal and the modulatory agent chosen. A
broad range of doses may be applicable. Considering a patient, for
example, from about 0.1 mg to about 1 mg of modulatory agent may be
administered per kilogram of body weight per day. Dosage regimes
may be adjusted to provide the optimum therapeutic response. For
example, several divided doses may be administered continuously,
daily, weekly, monthly or other suitable time intervals or the dose
may be proportionally reduced as indicated by the exigencies of the
situation.
[0205] The modulatory agent may be administered in a convenient
manner such as by the oral, intravenous (where water soluble),
intraperitoneal, intramuscular, subcutaneous, intradermal or
suppository routes or implanting (e.g. using slow release
molecules). The modulatory agent may be administered in the form of
pharmaceutically acceptable nontoxic salts, such as acid addition
salts or metal complexes, e.g. with zinc, iron or the like (which
are considered as salts for purposes of this application).
Illustrative of such acid addition salts are hydrochloride,
hydrobromide, sulphate, phosphate, maleate, acetate, citrate,
benzoate, succinate, malate, ascorbate, tartrate and the like. If
the active ingredient is to be administered in tablet form, the
tablet may contain a binder such as tragacanth, corn starch or
gelatin; a disintegrating agent, such as alginic acid; and a
lubricant, such as magnesium stearate.
[0206] Routes of administration include, but are not limited to,
respiratorally, intratracheally, nasopharyngeally, intravenously,
intraperitoneally, subcutaneously, intracranially, intradermally,
intramuscularly, intraoccularly, intrathecally, intracereberally,
intranasally, infusion, orally, rectally, via IV drip patch and
implant.
[0207] In accordance with these methods, the agent defined in
accordance with the present invention may be coadministered with
one or more other compounds or molecules. By "coadministered" is
meant simultaneous administration in the same formulation or in two
different formulations via the same or different routes or
sequential administration by the same or different routes. For
example, the subject agent may be administered together with an
agonistic agent in order to enhance its effects. By "sequential"
administration is meant a time difference of from seconds, minutes,
hours or days between the administration of the two types of
molecules. These molecules may be administered in any order.
[0208] Another aspect of the present invention contemplates the use
of an anti-nuclear molecule interactive molecule conjugated to an
effector mechanism, in the manufacture of medicament for the
treatment of a condition in a subject, which condition is
characterised by cellular apoptosis, wherein said effector
mechanism treats said condition.
[0209] Preferably, said nuclear molecule is La.
[0210] Preferably, said interactive molecule is an
immunointeractive molecule and even more preferably an anti-La
antibody, such as a monoclonal antibody.
[0211] Most preferably, said non-nuclear localisation is
extracellular localisation, as hereinbefore defined.
[0212] In another preferred embodiment, said condition is
infarction of cardiac muscle or brain tissue, autoimmune and other
inflammatory diseases, viral diseases such as AIDS, neurogenerative
diseases such as Alzheimer's disease or Parkinson's disease, acute
solid organ or bone marrow transplant rejection, chemotherapy- or
radiation-induced tissue damage (`mucositis`) or neoplasms such as
tumours.
[0213] Examples of neoplasms and neoplastic cells encompassed by
the present invention include, but are not limited central nervous
system tumours, retinoblastoma, neuroblastoma and other paediatric
tumours, head and neck cancers (eg. squamous cell cancers), breast
and prostate cancers, lung cancer (both small and non-small cell
lung cancer), kidney cancers (eg. renal cell adenocarcinoma),
oesophagogastric cancers, hepatocellular carcinoma,
pancreaticobiliary neoplasias (eg. adenocarcinomas and islet cell
tumours), colorectal cancer, cervical and anal cancers, uterine and
other reproductive tract cancers, urinary tract cancers (eg. of
ureter and bladder), germ cell tumours (eg. testicular germ cell
tumours or ovarian germ cell tumours), ovarian cancer (eg. ovarian
epithelial cancers), carcinomas of unknown primary, human
immunodeficiency associated malignancies (eg. Kaposi's sarcoma),
lymphomas, leukemias, malignant melanomas, sarcomas, endocrine
tumours (eg. of thyroid gland), mesothelioma and other pleural
tumours, neuroendocrine tumours and carcinoid tumours.
[0214] In yet another further aspect, the present invention
contemplates a pharmaceutical composition comprising the modulatory
agent as hereinbefore defined together with one or more
pharmaceutically acceptable carriers and/or diluents. Said agents
are referred to as the active ingredients.
[0215] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion or may be in the form of a cream
or other form suitable for topical application. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol and liquid polyethylene glycol,
and the like), suitable mixtures thereof, and vegetable oils. The
proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
superfactants. The preventions of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0216] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilisation. Generally,
dispersions are prepared by incorporating the various sterilised
active ingredient into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof.
[0217] When the active ingredients are suitably protected they may
be orally administered, for example, with an inert diluent or with
an assimilable edible carrier, or it may be enclosed in hard or
soft shell gelatin capsule, or it may be compressed into tablets,
or it may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compound may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 1% by weight of active compound. The
percentage of the compositions and preparations may, of course, be
varied and may conveniently be between about 5 to about 80% of the
weight of the unit. The amount of active compound in such
therapeutically useful compositions in such that a suitable dosage
will be obtained. Preferred compositions or preparations according
to the present invention are prepared so that an oral dosage unit
form contains between about 0.1 .mu.g and 2000 mg of active
compound.
[0218] The tablets, troches, pills, capsules and the like may also
contain the components as listed hereafter: a binder such as gum,
acacia, corn starch or gelatin; excipients such as dicalcium
phosphate; a disintegrating agent such as corn starch, potato
starch, alginic acid and the like; a lubricant such as magnesium
stearate; and a sweetening agent such as sucrose, lactose or
saccharin may be added or a flavouring agent such as peppermint,
oil of wintergreen, or cherry flavouring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills, or capsules may be coated with
shellac, sugar or both. A syrup or elixir may contain the active
compound, sucrose as a sweetening agent, methyl and propylparabens
as preservatives, a dye and flavouring such as cherry or orange
flavour. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compound(s) may be
incorporated into sustained-release preparations and
formulations.
[0219] Yet another aspect of the present invention relates to the
agent as hereinbefore defined, when used in the method of the
present invention.
[0220] The present invention is further defined by the following
non-limiting examples:
Example 1
[0221] Anti-La antibody used herein is mouse monoclonal antibody
hybridoma 3B9, which recognises both the human and mouse versions
of the small ribonucleoprotein antigen, La/SS-B. The antibody was
sourced from Professor Tom Gordon, Flinders Medical Centre,
Adelaide, South Australia, who had in turn obtained it from Dr. M.
Bachmann of the Oklahoma Medical Research Foundation, Oklahoma
City, Okla., USA. 3B9 was published by Dr. Gordon's group (Tran et
al. 2002a). The corresponding isotype control antibody hybridoma is
Sal5.
[0222] Six wild type (ST) C57BL/6 male mice (seven weeks old) or
two six month-old TRansgenic Adenocarcinoma of Mouse Prostate
(TRAMP) mice (column 1) were given two daily intravenous injections
of 400 .mu.L of normal human serum (NHS) or human serum that
contained La-reactive autoantibodies (La serum). Mice were left
intact or surgically castrated on the day that the first injection
was given (column 2). Mice were killed for analysis two days after
the second injection. High levels of hIgG cross-reactive with mouse
La were detected by ELISA in all mice injected with La serum. The
negative controls injected with NHS showed the same low background
level of binding as non-injected normal mouse serum (NMS) (column
3). Hen egg lysozyme (HEL) was used as a negative control in the
ELISA (column 4). Serum levels of hIgG were measured directly by
quantitative ELISA and were found to be much higher than in the
previously cited experiments (Tran et al, 2002b). Pregnant BALB/c
mice were given injections of La serum and serum hIgG ranged from
0.01 to 0.4 g/L, which produced good opsonization of fetal
apoptotic cardiomyocytes (column 5). The extent of prostate
epithelial apoptosis was measured by TUNEL assay. Numbers indicated
in the table represent TUNEL.sup.+ nuclei detected in the prostatic
epithelial layers that were available on whole sections. The
numerous TUNEL.sup.+ particles detected in the prostatic lumina of
nearly all animals were not counted. There was a tendency toward
increased apoptosis in the castrated mice in comparison to the
intact mice (column 6). Binding of hIgG to apoptotic prostatic
epithelial cells was detected in one of three mice, which was
castrated and given an injection of La serum (column 7). In this
case, numerous TUNEL.sup.+ cells coated with bright particulate
hIgG were detected in the prostatic lumen. Binding of hIgG to
TUNEL.sup.+ cells was seen also within the epithelium and on its
surface. Immunolabeling of serial (adjacent) sections suggested the
presence of both MHC-II.sup.+ and F4/80.sup.+ cells in the vicinity
of these immunocomplexes (data not shown). Significantly, no
binding of hIgG was detected in NHS-injected animals, or in La
serum injected but non-castrated animals. As an additional control,
ample background interstitial and intravascular hIgG was detected
in all animals injected with either La serum or NHS (prostate,
spleen, liver, salivary glands), which was in keeping with the
detectable serum levels of hIgG. As expected, numerous apoptotic
cells were detected in the spleen and thymus. In particular, the
spleen showed significant speckle staining of hIgG (blood-tissue
barrier being low in this tissue) without associated TUNEL staining
Apoptotic cells were barely detected in the salivary glands
following castration, consistent with the notion that
castration-induced apoptosis in glandular epithelium was
prostate-specific.
TABLE-US-00003 TABLE 3 Tabulated and Narrative Summary of
Experimental Results. Serum hIgG bound to TUNEL(+) recombinant
antigens nuclei in prostate Animal (ELISA OD.sub.405) Serum levels
epithelial layers hIgG bound to ID Treatment 6HismLa 6HisHEL of
hIgG (g/L) per section apoptotic cells WT Intact, La serum 1.850 0
3.2 2, 3 - WT Intact, La serum 1.684 0 2.5 1 - WT Castrated, La
serum 1.534 0 1.8 >30 + WT Castrated, La serum 1.685 0 2.7 8, 10
- WT Intact, NHS 0.007 0 1.5 N/A - WT Castrated, NHS 0.014 0 0.9 1,
3, 10 - TRAMP Castrated, La serum 1.588 0 2.4 >30 - TRAMP
Intact, La serum 1.389 0 1.6 5 - NMS N/A 0.016 0 N/A N/A N/A
Example 2
Anti-La Antibody Binds Apoptotic and Necrotic Cells
[0223] As apoptosis is induced and progresses in vitro, apoptotic
cells increasingly become leaky because of the loss of membrane
integrity, which is manifest as increasing avidity for the nucleic
acid and DNA binding dyes, propidium iodide (PI) and
7-amino-actinomycin D (7AAD), respectively. Binding of anti-La
antibody to apoptotic cells has a slower time course than PI
binding. However, the observed fluorescence intensity of anti-La
antibody staining indicates that it binds with similar avidity to
apoptotic cells irrespective of whether the PI staining is of high
or intermediate intensity (FIG. 3). As is shown below, the
PI.sup.intermediate subpopulation comprises apoptotic bodies. The
intermediate staining does not result from quenching artefacts
since it was present irrespective of changes in the concentration
of PI.
[0224] Accordingly, anti-La antibody binding to apoptotic cells is
a function of the loss of membrane integrity as apoptosis proceeds
in vitro. However, anti-La antibody binding to apoptotic cells is
not simply a matter of passive binding of mAb because the
fluorescence intensity of staining with Sal5, the isotype control
mAb, was one log-fold lower than 3B9 (anti-monoclonal antibody)
fluorescence intensity, which indicated that 3B9 bound specifically
to its target antigen, human La/SS-B (upper and middle rows of
panels, FIG. 4). While an anti-tubulin mAb, which recognises a
component of the cytoskeleton, also demonstrated a high level of
binding to apoptotic cells, it bound fewer PI.sup.intermediate
apoptotic bodies (lower row of panels, FIG. 2) than 3B9 (46% of
total PI events cf. 69%).
[0225] Some of the data shown in FIG. 4 are illustrated graphically
and compared at each time point with the percentage of cells
excluding trypan blue, which is another indicator of loss of cell
viability (FIG. 5). It is clear that the kinetics of anti-La
antibody binding to apoptotic cells parallels known markers of cell
permeability (tubulin) and loss of cell viability (trypan blue),
which supports the notion that the loss membrane integrity
associated with apoptosis permits anti-La antibody binding.
[0226] As FIG. 6 illustrates, apoptotic bodies derived from
apoptotic Jurkat cells were stable in size (lower row of panels,
FIG. 6) with comparable intensity of staining for 3B9 (upper row of
panels, FIG. 6) over a period that lasted for almost four days.
[0227] Further kinetic analysis of anti-La antibody binding to
apoptotic Jurkat cells was performed using annexin V to probe
phosphatidylserine exposure on the outer plasma membrane, which is
an early feature of apoptosis, and 7AAD to bind the DNA of leaky
cells, which is a feature of late apoptosis. This analysis reveals
again that anti-La antibody binding is commensurate with 7AAD
binding but occurs later than annexin V binding (FIG. 7). This
concept is displayed in another way in FIG. 8. anti-La antibody
binding is insignificant during early apoptosis when cells are
annexin V-positive but 7AAD-negative (R3 in left hand panel, FIG.
8) but anti-La antibody binding increases to high levels once the
cells become permeable to 7AAD (R1 in left hand panel, FIG. 8).
[0228] To emphasize that loss of cell membrane integrity is
required for anti-La antibody binding, primary necrotic cells also
demonstrate avid binding for anti-La antibody (FIG. 9). Note that
anti-La antibody staining does not co-localise with DNA (FIG. 9A)
or phosphatidylserine, which is exposed on necrotic cell membranes
(FIG. 9B).
Example 3
Anti-La Antibody Binding to Apoptotic Bodies is Caspase 3
Dependent
[0229] As mentioned in Example 2, more detailed flow cytometric
analysis indicated that anti-La antibody bound apoptotic bodies,
which are characterized by their smaller size and reduced internal
complexity because they contain varying proportions of
membrane-bound remnants of nuclear components and cellular
organelles (FIG. 10). Furthermore, it is shown that apoptotic body
formation is a requirement for anti-La antibody binding. MCF-7 is a
human breast cancer cell line that lacks the gene for pro-caspase
3.
[0230] Pro-caspase 3 is a pro-enzyme form of the crucial
executioner caspase, caspase 3, which catalyses the cleavage of
many functional and structural proteins in the dying cell. Caspase
3-mediated cleavage of these proteins contributes to the
morphological appearances of apoptotic body formation, which is the
splitting of the apoptotic cell into several (or more) smaller
membrane-bound parcels known as apoptotic bodies. Although MCF-7
cells do not demonstrate apoptotic body formation during cell
death, this phenotype can be rescued by the transfection of the
gene for pro-caspase 3 into MCF-7 cells.
[0231] As illustrated in FIG. 10, transient transfection of MCF-7
cells with the gene for pro-caspase 3 generates apoptotic bodies
and consequent binding of anti-La antibody. Hence, anti-La antibody
binding to apoptotic bodies is caspase 3 dependent. These apoptotic
bodies did not stain with 7AAD, which preferentially stains DNA
rather than RNA (lower left hand panel, FIG. 10). Moreover, when
evaluated by scatter criteria, apoptotic bodies were smaller in
size (lower right hand panel, FIG. 10).
[0232] Next, MCF-7 cells that had been stably transfected with the
gene for pro-caspase 3 were studied. As in FIG. 11, MCF-7 cells
that contained the control vector or the pro-caspase 3 gene were
rendered apoptotic and stained with 3B9 that had been labelled with
the green fluorochrome, Alexa488 and propidium iodide (FIG. 11A).
Again, caspase 3 activity was shown to be required for anti-La
antibody binding to apoptotic bodies. Fluorescence microscopy of
these cells demonstrated that pro-caspase 3 expressing MCF-7
transfectants had budded and partitioned distinctly 3B9.sup.+
apoptotic bodies (FIG. 11C). On the other hand, vector control
MCF-7 cells showed a less discrete pattern of 3B9 staining (FIG.
11C), which is consistent with the broad distribution of 3B9
staining observed by flow cytometry in vector control MCF-7 cells
(upper right hand panel, FIG. 11A). In contrast, as in FIG. 10,
caspase 3 activity conferred a restricted pattern of 3B9 staining
(lower right hand panel, FIG. 11A), which suggested that the
activity of caspase 3 had resulted in uniform partitioning of
La/SS-B antigen among apoptotic bodies.
[0233] As illustrated in FIG. 12, confocal laser scanning
microscopy of apoptotic Jurkat cells was used to show that anti-La
antibody staining neither overlapped with staining for apoptotic
cell membranes that had everted phosphatidylserine (as detected by
annexin V) nor staining for DNA (as detected by TOPRO3) (FIG. 13A).
Moreover, serial vertical-sections confirmed that anti-La antibody
staining occurred throughout the cytoplasmic region of dead cells.
This staining with 3B9 was specific because barely any staining was
observed using the isotype control mAb, Sal5 (FIG. 13B). PARP,
which is an abundant nuclear antigen (comprising approximately 2%
of nuclear protein) and which is also cleaved by caspase 3 during
apoptosis, adopted a similar pattern of staining to 3B9 when
detected with a specific mAb (FIG. 12C). Together, these data
suggested that anti-La antibody `loads` the cytoplasm of dead cells
(FIG. 13).
[0234] Other mAb specific for nuclear antigens such as Fodrin,
which is also cleaved by caspase 3 and thus contributes to
apoptotic body formation, stain apoptotic Jurkat cells with a
staining pattern that is similar to that exhibited by 3B9 (FIG.
14). Similar patterns of staining were also observed for mAb, which
are specific for PCNA and lamin B. These mAb demonstrated
widespread cytoplasmic staining that did not co-localise with DNA
as detected by 7AAD. Indeed, 7AAD tended to be restricted to
peripheral apoptotic blebs. In contrast, staining with
anti-.beta.-tubulin mAb was evident throughout apoptotic cells
where it co-localised to some extent with 7AAD staining.
[0235] Anti-La antibody also binds apoptotic primary T cells, which
comprise the majority of peripheral blood mononuclear cells (PMBC).
However, the fluorescence intensity of anti-La antibody binding to
primary T cells is approximately one half-log fold less than that
observed for malignant Jurkat T cells even if the PBMC had
previously been activated with the T cell mitogen, conconavalin A
(FIG. 15).
Example 4
Other Monoclonal Antibodies Directed Against Other Nuclear and
Ribonuclear Antigens Also Bind Apoptotic Cells
[0236] As observed in confocal scanning laser microscopy studies,
flow cytometry shows similar kinetics and patterns of binding of a
number of mAb, which are specific for nuclear and ribonuclear
antigens. Anti-tubulin mAb is included as a control for cytoplasmic
binding in permeable apoptotic cells (FIG. 16). Similarly, some of
these mAb specifically bind apoptotic bodies that form after MCF-7
cells have been transfected with the gene for pro-caspase 3 (FIG.
17).
Example 5
Other Antibodies Directed Against Human La/SS-B Also Detect
Apoptotic Cells
[0237] Binding to apoptotic cells of human anti-La autoantibodies
(FIG. 18) and another mAb, clone SW3, which is specific for human
La/SS-B (FIG. 19) was studied.
Example 6
Anti-La Antibody Binds Primary and Malignant Apoptotic Cells from
Human and Rodent Species
[0238] In addition to the binding of apoptotic primary human cells
(FIG. 15), anti-La antibody also binds apoptotic primary cells from
the thymi of mice and rats in which apoptosis was induced in vitro
with dexamethasone or staurosporine. Anti-La antibody specifically
binds PI.sup.+ thymocytes in response to apoptosis that was induced
with either stimulus (FIG. 20). A similar pattern of binding was
observed using a mAb directed against proliferating cell nuclear
antigen (PCNA). Anti-La antibody also binds apoptotic tumour cells
from rodent species (FIG. 21), which includes tumour cells that
have undergone apoptosis in vivo in response to cytotoxic drugs,
together with a number of apoptotic human and monkey tumour cell
lines (FIG. 22).
[0239] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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