U.S. patent application number 14/617788 was filed with the patent office on 2015-11-05 for treatment and diagnosis of cancer.
The applicant listed for this patent is CORNELL RESEARCH FOUNDATION, INC.. Invention is credited to Neil H. Bander.
Application Number | 20150316553 14/617788 |
Document ID | / |
Family ID | 29424685 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150316553 |
Kind Code |
A1 |
Bander; Neil H. |
November 5, 2015 |
TREATMENT AND DIAGNOSIS OF CANCER
Abstract
Use of antibodies or binding portions thereof, probes, ligands,
or other biological agents which either recognize an extracellular
domain of prostate specific membrane antigen (PSMA) or bind to and
are internalized with PSMA. These biological agents can be labeled
and used for detection of cancerous tissues, particularly cancerous
tissues proximate to or containing vascular endothelial cells,
which express an extracellular domain of PSMA. The labeled
biological agents can also be used to detect normal, benign
hyperplastic, and cancerous prostate epithelial cells or portions
thereof. They also can be used alone or bound to a substance
effective to ablate or kill such cells as a therapy for prostate or
other cancers. Also disclosed are four hybridoma cells lines, each
of which produces a monoclonal antibody recognizing extracellular
domains of PSMA of normal, benign hyperplastic, and cancerous
prostate epithelial cells or portions thereof.
Inventors: |
Bander; Neil H.; (Chappaqua,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CORNELL RESEARCH FOUNDATION, INC. |
ITHACA |
NY |
US |
|
|
Family ID: |
29424685 |
Appl. No.: |
14/617788 |
Filed: |
February 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11939422 |
Nov 13, 2007 |
8951737 |
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14617788 |
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11481344 |
Jul 5, 2006 |
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11939422 |
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09929546 |
Aug 13, 2001 |
7163680 |
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11481344 |
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09357708 |
Jul 20, 1999 |
6770450 |
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09929546 |
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08895914 |
Jul 17, 1997 |
6136311 |
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09357708 |
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08838682 |
Apr 9, 1997 |
6107090 |
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08895914 |
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60022125 |
Jul 18, 1996 |
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60016976 |
May 6, 1996 |
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/6869 20170801; G01N 33/57434 20130101; G01N 2333/705
20130101; C07K 2317/77 20130101; A61K 47/6899 20170801; A61P 35/00
20180101; B82Y 5/00 20130101; C07K 16/3069 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Claims
1. (canceled)
2. A method of detecting normal, benign hyperplastic, or cancerous
prostate cells in a biological sample, comprising: a. providing an
antibody or antigen binding portion thereof which competes for
binding to prostate specific membrane antigen (PSMA) with a
monoclonal antibody selected from the group consisting of an E99, a
J415, a J533, and a J591 monoclonal antibody, wherein the antibody
or antigen binding portion thereof is bound to a label effective to
permit detection of normal, benign hyperplastic, or cancerous
prostate cells; b. contacting the biological sample with the
antibody or antigen binding portion thereof under conditions
effective to permit binding of the antibody or antigen binding
portion thereof to the normal, benign hyperplastic, or cancerous
prostate cells; and c. detecting the presence of the normal, benign
hyperplastic, or cancerous prostate cells by detecting the
label.
3. The method of claim 2, wherein the antibody is a monoclonal
antibody produced by a hybridoma having an ATCC Accession Number
selected from the group consisting of HB-12101, HB-12109, HB-12127,
and HB-12126.
4. The method of claim 2, wherein the antigen binding portion is
selected from the group consisting of a Fab fragment, a
F(ab').sub.2 fragment, and a Fc fragment.
5. The method of claim 2, wherein the label is selected from the
group consisting of a fluorescent label, a biologically-active
enzyme label, a radiolabel, a nuclear magnetic resonance active
label, a luminescent label, and a chromophore label.
6. The method of claim 2, wherein the antibody or antigen binds to
an extracellular domain of PSMA on the normal, benign hyperplastic,
or cancerous prostate cells.
7. The method of claim 2, wherein the normal, benign hyperplastic,
or cancerous prostate cells are live cells in the biological
sample.
8. The method of claim 2, wherein the biological sample is a bodily
fluid sample.
9. The method of claim 8, wherein the bodily fluid sample is
selected from the group consisting of a urine sample and a serum
sample.
Description
RELATIONSHIP TO PRIOR APPLICATIONS
[0001] This invention relates to method and apparatus for
fertigation with wastewater, more specifically to method and The
present application is a continuation of U.S. patent application
Ser. No. 11/939,422, filed Nov. 13, 2007, which is a continuation
of U.S. patent application Ser. No. 11/481,344, filed Jul. 5, 2006,
which is a continuation of U.S. patent application Ser. No.
09/929,546, filed Aug. 13, 2001, now U.S. Pat. No. 7,163,680, which
is a continuation of U.S. patent application Ser. No. 09/357,708,
filed Jul. 20, 1999, now U.S. Pat. No. 6,770,450, which is a
divisional of U.S. patent application Ser. No. 08/895,914, filed
Jul. 17, 1997, now U.S. Pat. No. 6,136,311, which is a
continuation-in-part of U.S. patent application Ser. No.
08/838,682, filed Apr. 9, 1997, now U.S. Pat. No. 6,107,090, which
claims the benefit of U.S. Provisional Patent Application Ser. No.
60/016,976, filed May 6, 1996, and of U.S. Provisional Patent
Application Ser. No. 60/022,125, filed Jul. 18, 1996, the entire
contents of all of which are incorporated by reference herein.
FIELD OF INVENTION
[0002] The present invention relates to the treatment and diagnosis
of cancer with biological agents.
BACKGROUND
[0003] In spite of improved treatments for certain forms of cancer,
it is still a leading cause of death in the United States. Since
the chance for complete remission of cancer is, in most cases,
greatly enhanced by early diagnosis, it is very desirable that
physicians be able to detect cancers before a substantial tumor
develops. However, the development of methods that permit rapid and
accurate detection of many forms of cancers continues to challenge
the medical community. One such illustrative form of cancer is
prostate cancer.
[0004] Prostate cancer is the most common cancer in men with an
estimated 317,000 cases in 1996 in the United States. It is the
second leading cause of death among men who die from neoplasia with
an estimated 40,000 deaths per year. Prompt detection and treatment
is needed to limit mortality caused by prostate cancer.
[0005] Detection of Prostate Cancer
[0006] When it metastasizes, prostatic cancer has a distinct
predilection for bone and lymph nodes. Saitoh et al., "Metastatic
Patterns of Prostatic Cancer. Correlation Between Sites And Number
Of Organs Involved," Cancer, 54:3078-3084 (1984). At the time of
clinical diagnosis, as many as 25% of patients have bone metastasis
demonstrable by radionuclide scans. Murphy, G. P., et al., "The
National Survey Of Prostate Cancer In The United States By The
American College Of Surgeons," J. Urol., 127:928-939 (1982).
Accurate clinical evaluation of nodal involvement has proven to be
difficult. Imaging techniques such as computed tomography ("CT") or
magnetic resonance ("MR") imaging are unable to distinguish
metastatic prostate cancer involvement of lymph nodes by criterion
other than size (i.e., >1 cm). Therefore, by definition, these
imaging modalities are inherently insensitive in the detection of
small volume (<1 cm) disease as well as non-specific in the
detection of larger volume adenopathy. A recent study assessed the
accuracy of MR in patients with clinically localized prostate
cancer. Rifkin et al., "Comparison Of Magnetic Resonance Imaging
And Ultrasonography In Staging Early Prostate Cancer," N. Engel. J.
Med., 323:621-626 (1990). In this study, 194 patients underwent an
MR and 185 of these patients had a lymph node dissection. 23 (13%)
patients had pathologically involved lymph nodes. MR was suspicious
in only 1 of these 23 cases resulting in a sensitivity of 4%.
Similar results have also been noted with CT scans. Gasser et al.,
"MRI And Ultrasonography In Staging Prostate Cancer," N. Engl. J.
Med. (Correspondence), 324(7):49-495 (1991).
[0007] The elevation of serum acid phosphatase activity in patients
having metastasized prostate carcinoma was first reported by Gutman
et al., J. Clin. Invest 17:473 (1938). In cancer of the prostate,
prostatic acid phosphatase is released from the cancer tissue into
the blood stream with the result that the total serum acid
phosphatase level can be greatly increased above normal values.
Numerous studies of this enzyme and its relation to prostatic
cancer have been made since that time, e.g. Yam, Amer. J. Med.
56:604 (1974). However, the measurement of serum acid phosphatase
is elevated in about 65-90 percent of patients having carcinoma of
the prostate with bone metastasis; in about 30 percent of patients
without roentgenological evidence of bone metastasis; and in about
only 5-10 percent of patients lacking clinically demonstrable
metastasis.
[0008] Prior art attempts to develop a specific test for prostatic
acid phosphatase have met with only limited success, because
techniques which rely on enzyme activity on a so-called "specific"
substrate cannot take into account other biochemical and
immunochemical differences among the many acid phosphatases which
are unrelated to enzyme activity of prostate origin. In the case of
isoenzymes, i.e. genetically defined enzymes having the same
characteristic enzyme activity and a similar molecular structure
but differing in amino acid sequences and/or content and,
therefore, immunochemically distinguishable, it would appear
inherently impossible to distinguish different isoenzyme forms
merely by the choice of a particular substrate. It is, therefore,
not surprising that none of these prior art methods is highly
specific for the direct determination of prostatic acid phosphatase
activity; e.g. see Cancer 5:236 (1952); J. Lab. Clin. Med. 82:486
(1973); Clin. Chem. Acta. 44:21 (1973); and J. Physiol. Chem.
356:1775 (1975).
[0009] In addition to the aforementioned problems of
non-specificity which appear to be inherent in many of the prior
art reagents employed for the detection of prostate acid
phosphatase, there have been reports of elevated serum acid
phosphatase associated with other diseases, which further
complicates the problem of obtaining an accurate clinical diagnosis
of prostatic cancer. For example, Tuchman et al., Am. J. Med.
27:959 (1959) noted that serum acid phosphatase levels appear to be
elevated in patients with Gaucher's disease.
[0010] Due to the inherent difficulties in developing "specific"
substrate for prostate acid phosphatase, several researchers have
developed immunochemical methods for the detection of prostate acid
phosphatase. However, the previously reported immunochemical
methods have drawbacks of their own which have precluded their
widespread acceptance. For example, Shulman et al., Immunology
93:474 (1964) described an immuno-diffusion test for the detection
of human prostate acid phosphatase. Using antisera prepared from a
prostatic fluid antigen obtained by rectal massage from patients
with prostatic disease, no cross-reactivity precipitin line was
observed in the double diffusion technique against extracts of
normal kidney, testicle, liver, and lung. However, this method has
the disadvantages of limited sensitivity, even with the large
amounts of antigen employed, and of employing antisera which may
cross-react with other, antigenically unrelated serum protein
components present in prostatic fluid.
[0011] WO 79/00475 to Chu et. al. describes a method for the
detection of prostatic acid phosphatase isoenzyme patterns
associated with prostatic cancer which obviates many of the above
drawbacks. However, practical problems are posed by the need for a
source of cancerous prostate tissue from which the diagnostically
relevant prostatic acid phosphatase isoenzyme patterns associated
with prostatic cancer are extracted for the preparation of
antibodies thereto.
[0012] In recent years, considerable effort has been spent to
identify enzyme or antigen markers for various types of
malignancies with the view towards developing specific diagnostic
reagents. The ideal tumor marker would exhibit, among other
characteristics, tissue or cell-type specificity. Previous
investigators have demonstrated the occurrence of human prostate
tissue-specific antigens.
[0013] Treatment of Prostate Cancer
[0014] As described in W. J. Catalona, "Management of Cancer of the
Prostate," New Engl. J. Med., 331(15):996-1004 (1994), the
management of prostate cancer can be achieved by watchful waiting,
curative treatment, and palliation.
[0015] For men with a life expectancy of less than 10 years,
watchful waiting is appropriate where low-grade, low-stage prostate
cancer is discovered at the time of a partial prostatectomy for
benign hyperplasia. Such cancers rarely progress during the first
five years after detection. On the other hand, for younger men,
curative treatment is often more appropriate.
[0016] Where prostate cancer is localized and the patient's life
expectancy is 10 years or more, radical prostatectomy offers the
best chance for eradication of the disease. Historically, the
drawback of this procedure is that most cancers had spread beyond
the bounds of the operation by the time they were detected.
However, the use of prostate-specific antigen testing has permitted
early detection of prostate cancer. As a result, surgery is less
extensive with fewer complications. Patients with bulky, high-grade
tumors are less likely to be successfully treated by radical
prostatectomy.
[0017] After surgery, if there are detectable serum
prostate-specific antigen concentrations, persistent cancer is
indicated. In many cases, prostate-specific antigen concentrations
can be reduced by radiation treatment. However, this concentration
often increases again within two years.
[0018] Radiation therapy has also been widely used as an
alternative to radical prostatectomy. Patients generally treated by
radiation therapy are those who are older and less healthy and
those with higher-grade, more clinically advanced tumors.
Particularly preferred procedures are external-beam therapy which
involves three dimensional, conformal radiation therapy where the
field of radiation is designed to conform to the volume of tissue
treated; interstitial-radiation therapy where seeds of radioactive
compounds are implanted using ultrasound guidance; and a
combination of external-beam therapy and interstitial-radiation
therapy.
[0019] For treatment of patients with locally advanced disease,
hormonal therapy before or following radical prostatectomy or
radiation therapy has been utilized. Hormonal therapy is the main
form of treating men with disseminated prostate cancer. Orchiectomy
reduces serum testosterone concentrations, while estrogen treatment
is similarly beneficial. Diethylstilbestrol from estrogen is
another useful hormonal therapy which has a disadvantage of causing
cardiovascular toxicity. When gonadotropin-releasing hormone
agonists are administered testosterone concentrations are
ultimately reduced. Flutamide and other nonsteroidal, anti-androgen
agents block binding of testosterone to its intracellular
receptors. As a result, it blocks the effect of testosterone,
increasing serum testosterone concentrations and allows patients to
remain potent--a significant problem after radical prostatectomy
and radiation treatments.
[0020] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. Its toxicity makes such therapy unsuitable for
elderly patients. In addition, prostate cancer is relatively
resistant to cytotoxic agents.
[0021] Use of Monoclonal Antibodies in Prostate Cancer Detection
and Treatment
[0022] Theoretically, radiolabeled monoclonal antibodies ("mAbs")
offer the potential to enhance both the sensitivity and specificity
of detecting prostatic cancer within lymph nodes and elsewhere.
While many mAbs have previously been prepared against prostate
related antigens, none of these mAbs were specifically generated
with an imaging objective in mind. Nevertheless, the clinical need
has led to evaluation of some of these mAbs as possible imaging
agents. Vihko et al., "Radioimaging of Prostatic Carcinoma With
Prostatic Acid Phosphatase--Specific Antibodies," Biotechnology in
Diagnostics, 131-134 (1985); Babaian et al., "Radioimmunological
Imaging of Metastatic Prostatic Cancer With 111-Indium-Labeled
Monoclonal Antibody PAY 276," J. Urol., 137:439-443 (1987); Leroy
et al., "Radioimmunodetection Of Lymph Node Invasion In Prostatic
Cancer. The Use Of Iodine 123 (123-I)-Labeled Monoclonal
Anti-Prostatic Acid Phosphatase (PAP) 227 A F (ab') 2 Antibody
Fragments In Vivo," Cancer, 64:1-5 (1989); Meyers et al.,
"Development Of Monoclonal Antibody Imaging Of Metastatic Prostatic
Carcinoma," The Prostate, 14:209-220 (1989).
[0023] In some cases, the monoclonal antibodies developed for
detection and/or treatment of prostate cancer recognize antigens
specific to malignant prostatic tissues. Such antibodies are thus
used to distinguish malignant prostatic tissue (for treatment or
detection) from benign prostatic tissue. See U.S. Pat. No.
4,970,299 to Bazinet et al. and U.S. Pat. No. 4,902,615 to Freeman
et al.
[0024] Other monoclonal antibodies react with surface antigens on
all prostate epithelial cells whether cancerous or benign. See U.S.
Pat. Nos. 4,446,122 and Re 33,405 to Chu et al., U.S. Pat. No.
4,863,851 to McEwan et al., and U.S. Pat. No. 5,055,404 to Ueda et
al. However, the antigens detected by these monoclonal antibodies
are present in the blood and, therefore, compete with antigens at
tumor sites for the monoclonal antibodies. This causes background
noise which makes the use of such antibodies inadequate for in vivo
imaging. In therapy, such antibodies, if bound to a cytotoxic
agent, could be harmful to other organs.
[0025] Horoszewicz et al., "Monoclonal Antibodies to a New
Antigenic Marker in Epithelial Prostatic Cells and Serum of
Prostatic Cancer Patients," Anticancer Research, 7:927-936 (1987)
("Horoszewicz") and U.S. Pat. No. 5,162,504 to Horoszewicz describe
an antibody, designated 7E11, which recognizes prostate specific
membrane antigen ("PSMA"). Israeli et al., "Molecular Cloning of a
Complementary DNA Encoding a Prostate-specific Membrane Antigen,"
Cancer Research, 53:227-230 (1993) ("Israeli") describes the
cloning and sequencing of PSMA and reports that PSMA is
prostate-specific and shows increased expression levels in
metastatic sites and in hormone-refractory states. Other studies
have indicated that PSMA is more strongly expressed in prostate
cancer cells relative to cells from the normal prostate or from a
prostate with benign hyperplasia. Furthermore, PSMA is not found in
serum (Troyer et al., "Detection and Characterization of the
Prostate-Specific Membrane Antigen (PSMA) in Tissue Extracts and
Body Fluids," Int. J. Cancer, 62:552-558 (1995)).
[0026] These characteristics make PSMA an attractive target for
antibody mediated targeting for imaging and therapy of prostate
cancer. Imaging studies using indium-labeled 7E11 have indicated
that the antibody localizes quite well to both the prostate and to
sites of metastasis. In addition, 7E11 appears to have clearly
improved sensitivity for detecting lesions compared to other
currently available imaging techniques, such as CT and MR imaging
or bone scan. Bander, "Current Status of Monoclonal Antibodies for
Imaging and Therapy of Prostate Cancer," Sem. In Oncology,
21:607-612 (1994).
[0027] However, the use of 7E11 and other known antibodies to PSMA
to mediate imaging and therapy has several disadvantages. First,
PSMA is an integral membrane protein known to have a short
intracellular tail and a long extracellular domain. Biochemical
characterization and mapping (Troyer et al., "Biochemical
Characterization and Mapping of the 7E11-05.3 Epitope of the
Prostate-specific Membrane Antigen," Urol. Oncol., 1:29-37 (1995))
have shown that the epitope or antigenic site to which the 7E11
antibody binds is present on the intracellular portion of the
molecule. Because antibody molecules do not, under normal
circumstances, cross the cell membrane unless they bind to the
extracellular portion of a molecule and become translocated
intracellularly, the 7E11 antibody does not have access to its
antigenic target site in an otherwise healthy, viable cell.
[0028] Consequently, imaging using 7E11 is limited to the detection
of dead cells within tumor deposits. Additionally, the therapeutic
use of the 7E11 antibody is limited, because only cells that are
already dead or tissue containing a large proportion of dead cells
can be effectively targeted.
[0029] Although the inadequacies and problems in the diagnosis and
treatment of one particular type of cancer are the focus of the
preceding discussion, prostate cancer is merely a representative
model. The diagnosis and treatment of numerous other cancers have
similar problems.
[0030] The present invention is directed to overcoming the
deficiencies of prior art antibodies in diagnosing and treating
prostate and other types of cancer.
BRIEF SUMMARY OF THE INVENTION
[0031] One aspect of the present invention relates to a method of
ablating or killing cancerous cells. The process involves providing
a biological agent which, when contacted with an extracellular
domain of prostate specific membrane antigen, recognizes the
extracellular domain of prostate specific membrane antigen. These
biological agents are contacted with vascular endothelial cells
proximate to the cancerous cells under conditions effective to
permit both binding of the biological agent to the vascular
endothelial cells proximate to the cancerous cells and killing or
ablating of the cancerous cells. The biological agent can be used
alone or can be bound to a substance effective to kill or ablate
the cancerous cells upon binding of the biological agent to
vascular endothelial cells that are proximate to the cancerous
cells.
[0032] In a particularly preferred embodiment of the method of
ablating or killing cancerous cells in accordance with the present
invention, the biological agent, when contacted with an
extracellular domain of prostate specific membrane antigen, binds
to and is internalized with the prostate specific membrane antigen
of such cells. Preferred biological agents for use in the method of
ablating or killing cancerous cells in accordance with the present
invention are antibodies or binding portions thereof, probes, or
ligands. The methods of the present invention are particularly
useful in killing or ablating renal, urothelial, colon, rectal,
lung, and breast cancerous cells and cancerous cells of metastatic
adenocarcinoma to the liver.
[0033] Another aspect of the present invention relates to a method
of detecting cancerous tissue in a biological sample. This method
involves providing a biological agent which, when contacted with an
extracellular domain of prostate specific membrane antigen, binds
to the extracellular domain of prostate specific membrane antigen.
The biological agent is bound to a label effective to permit
detection of vascular endothelial cells proximate to or within the
cancerous tissue upon binding of the biological agent to the
vascular endothelial cells proximate to or within the cancerous
tissue. The biological sample is contacted with the biological
agent having a label under conditions effective to permit binding
of the biological agent to the vascular endothelial cells proximate
to or within the cancerous tissue in the biological sample. The
presence of cancerous tissue in the biological sample is detected
by detection of the label.
[0034] In a particularly preferred embodiment of the method of
detecting cancerous tissue in accordance with the present
invention, the biological agent is one that, when contacted with an
extracellular domain of prostate specific membrane antigen, binds
to and is internalized with the prostate specific membrane antigen.
Preferred biological agents for use in the method of detecting
cancerous tissue in accordance with the present invention are
antibodies or binding portions thereof, probes, or ligands. The
method is especially useful in detecting renal, urothelial, colon,
rectal, lung, and breast cancerous tissue and cancerous tissue of
metastatic adenocarcinoma to the liver.
[0035] Still another aspect of the present invention relates to a
method of ablating or killing normal, benign hyperplastic, and
cancerous prostate epithelial cells. The process involves providing
a biological agent which recognizes an extracellular domain of
prostate specific membrane antigen. The biological agent can be
used alone or can be bound to a substance effective to kill the
cells upon binding of the biological agent to the cells. These
biological agents are then contacted with the cells under
conditions effective to permit both binding of the biological agent
to the extracellular domain of the prostate specific membrane
antigen and killing or ablating of the cells.
[0036] In a particularly preferred embodiment of the method of
ablating or killing normal, benign hyperplastic, and cancerous
prostate epithelial cells in accordance with the present invention,
the biological agent binds to and is internalized with the prostate
specific membrane antigen of such cells. Preferred biological
agents for use in the method of ablating or killing normal, benign
hyperplastic, and cancerous prostate epithelial cells in accordance
with the present invention are antibodies or binding portions
thereof, probes, or ligands.
[0037] Another aspect of the present invention relates to a method
of detecting normal, benign hyperplastic, and cancerous prostate
epithelial cells or portions thereof in a biological sample. This
method involves providing a biological agent which binds to an
extracellular domain of prostate specific membrane antigen. The
biological agent is bound to a label effective to permit detection
of the cells or portions thereof upon binding of the biological
agent to the cells or portions thereof. The biological sample is
contacted with the biological agent having a label under conditions
effective to permit binding of the biological agent to the
extracellular domain of the prostate specific membrane antigen of
any of the cells or portions thereof in the biological sample. The
presence of any cells or portions thereof in the biological sample
is detected by detection of the label.
[0038] In a particularly preferred embodiment of the method of
detecting normal, benign hyperplastic, and cancerous prostate
epithelial cells in accordance with the present invention, the
biological agent binds to and is internalized with the prostate
specific membrane antigen of such cells. Preferred biological
agents for use in the method of detecting normal, benign
hyperplastic, and cancerous prostate epithelial cells in accordance
with the present invention are antibodies or binding portions
thereof, probes, or ligands.
[0039] Another aspect of the present invention pertains to a
biological agent that recognizes an extracellular domain of
prostate specific membrane antigen. In a preferred embodiment, the
isolated biological agent binds to and is internalized with the
prostate specific membrane antigen. Preferred isolated biological
agents which recognize an extracellular domain of prostate specific
membrane antigen in accordance with the present invention are
isolated antibodies or binding portions thereof, probes, or
ligands. Hybridoma cell lines that produce monoclonal antibodies of
these types are also disclosed.
[0040] The biological agents of the present invention recognize the
extracellular domain of antigens of normal, benign hyperplastic,
and cancerous prostate epithelial cells. Unlike the 7E11 antibody,
which recognizes an epitope of prostate-associated antigens which
are exposed extracellularly only after cell lysis, the biological
agents of the present invention bind to antigenic epitopes which
are extracellularly exposed in living prostate cells. Using the
biological agents of the present invention, living, unfixed normal,
benign hyperplastic, and cancerous prostate epithelial cells can be
targeted, which makes treatment and diagnosis more effective. In a
preferred embodiment for treating prostate cancer, the biological
agents of the present invention also bind to and are internalized
with the prostate specific membrane antigen, which permits the
therapeutic use of intracellularly acting cytotoxic agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is an immuno-electron micrograph of gold-labeled
monoclonal antibody J591 on the surface of LNCaP cells after
incubation at 4.degree. C.
[0042] FIG. 2 is an immuno-electron micrograph of LNCaP cells
treated with gold-labeled monoclonal antibody J591 after 5 minutes
incubation at 37.degree. C.
[0043] FIG. 3 is an immuno-electron micrograph of LNCaP cells
treated with gold-labeled monoclonal antibody J591 after 10 minutes
incubation at 37.degree. C.
[0044] FIG. 4 is an immuno-electron micrograph of LNCaP cells
treated with gold-labeled monoclonal antibody J591 after 15 minutes
incubation at 37.degree. C.
[0045] FIG. 5 is an immuno-electron micrograph of LNCaP cells
treated with gold-labeled monoclonal antibody J591 after 15 minutes
at 37.degree. C. showing J591 within endosomes.
[0046] FIG. 6 summarizes the sequencing strategy of the heavy chain
of monoclonal antibody J591.
[0047] FIG. 7 shows the nucleotide sequence of the heavy chain of
monoclonal antibody J591 (designated SEQ. ID. No. 1), the
nucleotide sequence of the corresponding reverse, non-coding strand
(designated SEQ. ID. No. 2), and the corresponding deduced amino
acid sequences (designated SEQ. ID. Nos. 3, 4, and 5).
[0048] FIG. 8 is a comparison of the heavy chain of monoclonal
antibody J591 (SEQ. ID. No. 8) with the consensus sequence for
Mouse Heavy Chains Subgroup IIA (SEQ. ID. No. 20), (resulting
comparison sequence SEQ. ID. No. 22).
[0049] FIG. 9 summarizes the sequencing strategy of the kappa light
chain of monoclonal antibody J591.
[0050] FIG. 10 shows the nucleotide sequences of the kappa light
chain of monoclonal antibody J591 (designated SEQ. ID. No. 9), the
nucleotide sequence of the corresponding reverse, non-coding strand
(designated SEQ. ID. No. 10), and the corresponding deduced amino
acid sequence (designated SEQ. ID. Nos. 11, 12, and 13).
[0051] FIG. 11 is a comparison of the kappa light chain of
monoclonal antibody J591 (SEQ. ID. No. 16) with the consensus
sequence for Mouse Kappa Chains Subgroup V (SEQ. ID. No. 21),
(resulting comparison sequence SEQ. ID. No. 23).
[0052] FIGS. 12A-12F are micrographs (250.times. magnification)
showing the immunohistochemical reactivity of mAb J591 to
neovasculature of various carcinomas.
DETAILED DESCRIPTION OF THE INVENTION
[0053] One aspect of the present invention relates to a method of
ablating or killing normal, benign hyperplastic, and cancerous
prostate epithelial cells. The process involves providing a
biological agent, such as an antibody or binding portion thereof,
probe, or ligand, which binds to an extracellular domain of
prostate specific membrane antigen of (i.e., a portion of prostate
specific membrane antigen which is external to) such cells. The
biological agent can be used alone or can be bound to a substance
effective to kill the cells upon binding of the biological agent to
the cells. These biological agents are then contacted with the
cells under conditions effective to permit both binding of the
biological agent to the extracellular domain of the prostate
specific membrane antigen and killing or ablating of the cells. In
its preferred form, such contacting is carried out in a living
mammal by administering the biological agent to the mammal under
conditions effective to permit both binding of the biological agent
to the extracellular domain of the prostate specific membrane
antigen and killing or ablating of the cells. Such administration
can be carried out orally or parenterally.
[0054] In a particularly preferred embodiment of the method of
ablating or killing normal, benign hyperplastic, and cancerous
prostate epithelial cells in accordance with the present invention,
the biological agent binds to and is internalized with the prostate
specific membrane antigen of such cells. Again, the biological
agent can be used alone. Alternatively, the biological agent can be
bound to a substance effective to kill the cells upon binding of
the biological agent to prostate specific membrane antigen and upon
internalization of the biological agent with the prostate specific
membrane antigen.
[0055] The mechanism by which the biological agent is internalized
with the prostate specific membrane antigen is not critical to the
practice of the present invention. For example, the biological
agent can induce internalization of the prostate specific membrane
antigen. Alternatively, internalization of the biological agent can
be the result of routine internalization of prostate specific
membrane antigen.
[0056] The above-described biological agents (i.e., biological
agents, such as an antibody or binding portion thereof, probe, or
ligand which, when contacted with an extracellular domain of
prostate specific membrane antigen, recognizes the extracellular
domain of prostate specific membrane antigen and, preferably, is
internalized therewith) can be used to ablate or kill cancerous
cells. In this aspect of the present invention, the biological
agent can be used alone or can be bound to a substance effective to
kill the cancerous cells upon binding of the biological agent to
vascular endothelial cells proximate thereto. These biological
agents are contacted with vascular endothelial cells proximate to
the cancerous cells. The contacting is carried out under conditions
that are effective to permit binding of the biological agent to the
vascular endothelial cells proximate to the cancerous cells and, in
addition, that are effective to kill or ablate the cancerous cells.
The mechanism by which the cancerous cells are killed or ablated is
not critical to the practice of the present invention. For example,
the cancerous cells can be killed or ablated directly by the
biological agent as a consequence of their proximity to the
vascular endothelial cells to which the biological agent binds.
Alternatively, the biological agent can kill, ablate, or otherwise
change the properties of the vascular endothelial cells to which it
binds so that blood flow to the cancerous cells proximate thereto
is stopped or otherwise reduced, thereby causing the cancerous
cells to be killed or ablated. Thus, the method of the present
invention is particularly useful for killing or ablating vascular
endothelial cells in cancerous tissue as well as the cancerous
cells contained in cancerous tissue.
[0057] In a particularly preferred embodiment of the method of
ablating or killing cancerous cells in accordance with the present
invention, the biological agent employed is one that, when
contacted with an extracellular domain of prostate specific
membrane antigen, binds to and is internalized with the
extracellular domain of prostate specific membrane antigen. The
methods of the present invention are particularly useful to kill or
ablate cancerous prostate epithelial cells as well as cancerous
cells other than cancerous prostate epithelial cells. Examples of
cancerous cells which are not cancerous prostate epithelial cells
are renal, urothelial, colon, rectal, lung, and breast cancerous
cells and cancerous cells of metastatic adenocarcinoma to the
liver. Although the method of the present invention can be used to
kill or ablate any cell which expresses an extracellular domain of
prostate specific membrane antigen or a portion thereof or whose
subsistence is dependent upon cells which express an extracellular
domain of prostate specific membrane antigen or a portion thereof,
the method of the present invention is particularly useful to kill
or ablate cancerous cells, because the vascular endothelial cells
supplying blood to cancerous tissues (e.g., tumors, collections of
cancerous cells, or other cancerous masses) express an
extracellular domain of prostate specific membrane antigen,
irrespective of the type of cancer involved. In contrast, vascular
endothelial cells supplying blood to normal tissues do not express
an extracellular domain of prostate specific membrane antigen.
[0058] Another aspect of the present invention relates to a method
of detecting normal, benign hyperplastic, and cancerous epithelial
cells or portions thereof in a biological sample. This method
involves providing a biological agent, such as an antibody or
binding portion thereof, probe, or ligand, which binds to an
extracellular domain of prostate specific membrane antigen of such
cells. The biological agent is bound to a label effective to permit
detection of the cells or portions (e.g., prostate specific
membrane antigen or fragments thereof liberated from such normal,
benign hyperplastic, and cancerous cells) thereof upon binding of
the biological agent to the cells or portions thereof. The
biological sample is contacted with the biological agent having a
label under conditions effective to permit binding of the
biological agent to the extracellular domain of the prostate
specific membrane antigen of any of the cells or portions thereof
in the biological sample. The presence of any cells or portions
thereof in the biological sample is detected by detection of the
label. In its preferred form, such contacting is carried out in a
living mammal and involves administering the biological agent to
the mammal under conditions effective to permit binding of the
biological agent to the prostate specific membrane antigen of any
of the cells or portions thereof in the biological sample. Again,
such administration can be carried out orally or parenterally.
[0059] The method of the present invention can be used to screen
patients for diseases associated with the presence of normal,
benign hyperplastic, and cancerous epithelial cells or portions
thereof. Alternatively, it can be used to identify the recurrence
of such diseases, particularly when the disease is localized in a
particular biological material of the patient. For example,
recurrence of prostatic disease in the prostatic fossa may be
encountered following radical prostatectomy. Using the method of
the present invention, this recurrence can be detected by
administering a short range radiolabeled antibody to the mammal and
then detecting the label rectally, such as with a transrectal
detector probe.
[0060] Alternatively, the contacting step can be carried out in a
sample of serum or urine or other body fluids, such as to detect
the presence of PSMA in the body fluid. When the contacting is
carried out in a serum or urine sample, it is preferred that the
biological agent recognize substantially no antigens circulating in
the blood other than PSMA. Since intact prostate cells do not
excrete or secrete PSMA into the extracellular environment,
detecting PSMA in serum, urine, or other body fluids generally
indicates that prostate cells are being lysed. Thus, the biological
agents and methods of the present invention can be used to
determine the effectiveness of a prostate cancer treatment protocol
by monitoring the level of PSMA in serum, urine or other body
fluids.
[0061] In a particularly preferred embodiment of the method of
detecting normal, benign hyperplastic, and cancerous prostate
epithelial cells in accordance with the present invention, the
biological agent, such as the antibody or binding portion thereof,
probe, or ligand, binds to and is internalized with the prostate
specific membrane antigen of such cells. Again, the biological
agent is bound to a label effective to permit detection of the
cells or portions thereof upon binding of the biological agent to
and internalization of the biological agent with the prostate
specific membrane antigen.
[0062] Another aspect of the present invention relates to a method
of detecting cancerous tissue in a biological sample. This method
involves providing the above-described biological agent (i.e., a
biological agent, such as an antibody or binding portion thereof,
probe, or ligand which, when contacted with an extracellular domain
of prostate specific membrane antigen, recognizes the extracellular
domain of prostate specific membrane antigen). The biological agent
is bound to a label that is effective to permit detection of
vascular endothelial cells proximate to or within the cancerous
tissue upon binding of the biological agent to vascular endothelial
cells proximate to or within the cancerous tissue. The biological
sample is then contacted with the biological agent having a label.
Contacting is carried out under conditions effective to permit
binding of the biological agent to the vascular endothelial cells
proximate to or within the cancerous tissue in the biological
sample. The presence of cancerous cells or portions thereof in the
biological sample is detected by detection of the label.
[0063] Rather than contacting the entire biological sample with the
biological agent, it is contemplated that a portion of the
biological sample can be used. For example, a tissue biopsy sample
can be contacted with the biological agent to determine the
presence of cancerous tissue in the tissue biopsy sample as well as
in the larger biological sample from which it is taken.
Alternatively, the biological agent can be contacted with a serum
or urine sample to acertain whether any vascular endothelial cells
expressing an extracellular domain of prostate specific membrane
antigen are present therein. Since vascular endothelial cells
expressing an extracellular domain of prostate specific membrane
antigen are found in the vasculature of cancerous tissues but not
in the vasculature of normal tissues, detection of the label in a
serum or urine sample indicates the presence of cancerous tissue in
the larger biological sample from which it is taken (e.g., a
patient).
[0064] In a particularly preferred embodiment of the method of
detecting cancerous tissues in accordance with the present
invention, the biological agent employed is one that, when
contacted with an extracellular domain of prostate specific
membrane antigen, binds to and is internalized with the prostate
specific membrane antigen. The methods of the present invention can
be used to detect cancerous prostate epithelial cells as well as
cancerous tissues containing cancerous cells other than cancerous
prostate epithelial cells. Examples of cancerous tissues containing
cancerous cells other than cancerous prostate epithelial cells
which can be detected with the methods of the present invention
include renal, urothelial, colon, rectal, lung, and breast
cancerous tissue and cancerous tissue of metastatic adenocarcinoma
to the liver.
[0065] As indicated above, biological agents suitable for either
killing, ablating, or detecting cancerous cells and normal, benign
hyperplastic, and cancerous prostate epithelial cells include
antibodies, such as monoclonal or polyclonal antibodies. In
addition, antibody fragments, half-antibodies, hybrid derivatives,
probes, and other molecular constructs may be utilized. These
biological agents, such as antibodies, binding portions thereof,
probes, or ligands, bind to extracellular domains of prostate
specific membrane antigens or portions thereof in normal, benign
hyperplastic, and cancerous prostate epithelial cells. As a result,
when practicing the methods of the present invention to kill,
ablate, or detect normal, benign hyperplastic, and cancerous
prostate epithelial cells, the biological agents bind to all such
cells, not only to cells which are fixed or cells whose
intracellular antigenic domains are otherwise exposed to the
extracellular environment. Consequently, binding of the biological
agents is concentrated in areas where there are prostate epithelial
cells, irrespective of whether these cells are fixed or unfixed,
viable or necrotic. Additionally or alternatively, these biological
agents, such as antibodies, binding portions thereof, probes, or
ligands, bind to and are internalized with prostate specific
membrane antigens or portions thereof in normal, benign
hyperplastic, and cancerous prostate epithelial cells.
[0066] Monoclonal antibody production may be effected by techniques
which are well-known in the art. Basically, the process involves
first obtaining immune cells (lymphocytes) from the spleen of a
mammal (e.g., mouse) which has been previously immunized with the
antigen of interest either in vivo or in vitro. The
antibody-secreting lymphocytes are then fused with (mouse) myeloma
cells or transformed cells, which are capable of replicating
indefinitely in cell culture, thereby producing an immortal,
immunoglobulin-secreting cell line. The resulting fused cells, or
hybridomas, are cultured, and the resulting colonies screened for
the production of the desired monoclonal antibodies. Colonies
producing such antibodies are cloned, and grown either in vivo or
in vitro to produce large quantities of antibody. A description of
the theoretical basis and practical methodology of fusing such
cells is set forth in Kohler and Milstein, Nature 256:495 (1975),
which is hereby incorporated by reference.
[0067] Mammalian lymphocytes are immunized by in vivo immunization
of the animal (e.g., a mouse) with the protein or polypeptide of
the present invention. Such immunizations are repeated as necessary
at intervals of up to several weeks to obtain a sufficient titer of
antibodies. Following the last antigen boost, the animals are
sacrificed and spleen cells removed.
[0068] Fusion with mammalian myeloma cells or other fusion partners
capable of replicating indefinitely in cell culture is effected by
standard and well-known techniques, for example, by using
polyethylene glycol ("PEG") or other fusing agents (See Milstein
and Kohler, Eur. J. Immunol. 6:511 (1976), which is hereby
incorporated by reference). This immortal cell line, which is
preferably murine, but may also be derived from cells of other
mammalian species, including but not limited to rats and humans, is
selected to be deficient in enzymes necessary for the utilization
of certain nutrients, to be capable of rapid growth, and to have
good fusion capability. Many such cell lines are known to those
skilled in the art, and others are regularly described.
[0069] Procedures for raising polyclonal antibodies are also well
known. Typically, such antibodies can be raised by administering
the protein or polypeptide of the present invention subcutaneously
to New Zealand white rabbits which have first been bled to obtain
pre-immune serum. The antigens can be injected at a total volume of
100 .quadrature.l per site at six different sites. Each injected
material will contain synthetic surfactant adjuvant pluronic
polyols, or pulverized acrylamide gel containing the protein or
polypeptide after SDS-polyacrylamide gel electrophoresis. The
rabbits are then bled two weeks after the first injection and
periodically boosted with the same antigen three times every six
weeks. A sample of serum is then collected 10 days after each
boost. Polyclonal antibodies are then recovered from the serum by
affinity chromatography using the corresponding antigen to capture
the antibody. Ultimately, the rabbits are euthenized with
pentobarbital 150 mg/Kg IV. This and other procedures for raising
polyclonal antibodies are disclosed in E. Harlow, et. al., editors,
Antibodies: A Laboratory Manual (1988), which is hereby
incorporated by reference.
[0070] In addition to utilizing whole antibodies, the processes of
the present invention encompass use of binding portions of such
antibodies. Such binding portions include Fab fragments, F(ab')2
fragments, and Fv fragments. These antibody fragments can be made
by conventional procedures, such as proteolytic fragmentation
procedures, as described in J. Goding, Monoclonal Antibodies:
Principles and Practice, pp. 98-118 (N.Y. Academic Press 1983),
which is hereby incorporated by reference.
[0071] Alternatively, the processes of the present invention can
utilize probes or ligands found either in nature or prepared
synthetically by recombinant DNA procedures or other biological or
molecular procedures. Suitable probes or ligands are molecules
which bind to the extracellular domains of prostate specific
membrane antigens identified by the monoclonal antibodies of the
present invention. Other suitable probes or ligands are molecules
which bind to and are internalized with prostate specific membrane
antigens. Such probes or ligands can be, for example, proteins,
peptides, lectins, or nucleic acid probes.
[0072] It is particularly preferred to use the monoclonal
antibodies identified below in Table 1.
TABLE-US-00001 TABLE 1 Monoclonal ATCC Designation for Antibody
Name Hybridoma Cell Line E99 HB-12101 J415 HB-12109 J533 HB-12127
J591 HB-12126
[0073] These antibodies can be used alone or as a component in a
mixture with other antibodies or other biological agents to treat
cancers or image cancerous tissues (particularly the vascular
endothelial cells therein) or prostate epithelial cells with
varying surface antigen characteristics.
[0074] Regardless of whether the biological agents are used for
treatment or diagnosis, they can be administered orally,
parenterally, subcutaneously, intravenously, intramuscularly,
intraperitoneally, by intranasal instillation, by intracavitary or
intravesical instillation, intraocularly, intraarterially,
intralesionally, or by application to mucous membranes, such as,
that of the nose, throat, and bronchial tubes. They may be
administered alone or with pharmaceutically or physiologically
acceptable carriers, excipients, or stabilizers, and can be in
solid or liquid form such as, tablets, capsules, powders,
solutions, suspensions, or emulsions.
[0075] The solid unit dosage forms can be of the conventional type.
The solid form can be a capsule, such as an ordinary gelatin type
containing the biological agent, such as an antibody or binding
portion thereof, of the present invention and a carrier, for
example, lubricants and inert fillers such as, lactose, sucrose, or
cornstarch. In another embodiment, these compounds are tableted
with conventional tablet bases such as lactose, sucrose, or
cornstarch in combination with binders like acacia, cornstarch, or
gelatin, disintegrating agents such as, cornstarch, potato starch,
or alginic acid, and a lubricant like stearic acid or magnesium
stearate.
[0076] The biological agent of the present invention may also be
administered in injectable dosages by solution or suspension of
these materials in a physiologically acceptable diluent with a
pharmaceutical carrier. Such carriers include sterile liquids such
as water and oils, with or without the addition of a surfactant and
other pharmaceutically and physiologically acceptable carrier,
including adjuvants, excipients or stabilizers. Illustrative oils
are those of petroleum, animal, vegetable, or synthetic origin, for
example, peanut oil, soybean oil, or mineral oil. In general,
water, saline, aqueous dextrose and related sugar solution, and
glycols such as, propylene glycol or polyethylene glycol, are
preferred liquid carriers, particularly for injectable
solutions.
[0077] For use as aerosols, the biological agent of the present
invention in solution or suspension may be packaged in a
pressurized aerosol container together with suitable propellants,
for example, hydrocarbon propellants like propane, butane, or
isobutane with conventional adjuvants. The materials of the present
invention also may be administered in a non-pressurized form such
as in a nebulizer or atomizer.
[0078] The biological agents may be utilized to detect cancerous
tissues (particularly the vascular endothelial cells therein) and
normal, benign hyperplastic, and cancerous prostate epithelial
cells in vivo. This is achieved by labeling the biological agent,
administering the labeled biological agent to a mammal, and then
imaging the mammal.
[0079] Examples of labels useful for diagnostic imaging in
accordance with the present invention are radiolabels such as 131I,
111In, 123I, 99mTc, 32P, 125I, 3H, 14C and 188Rh, fluorescent
labels such as fluorescein and rhodamine, nuclear magnetic
resonance active labels, positron emitting isotopes detectable by a
positron emission tomography ("PET") scanner, chemiluminescers such
as luciferin, and enzymatic markers such as peroxidase or
phosphatase. Short-range radiation emitters, such as isotopes
detectable by short-range detector probes, such as a transrectal
probe, can also be employed. These isotopes and transrectal
detector probes, when used in combination, are especially useful in
detecting prostatic fossa recurrences and pelvic nodal disease. The
biological agent can be labeled with such reagents using techniques
known in the art. For example, see Wensel and Meares,
Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. (1983),
which is hereby incorporated by reference, for techniques relating
to the radiolabeling of antibodies. See also, D. Colcher et al.,
"Use of Monoclonal Antibodies as Radiopharmaceuticals for the
Localization of Human Carcinoma Xenografts in Athymic Mice", Meth.
Enzymol. 121: 802-816 (1986), which is hereby incorporated by
reference.
[0080] A radiolabeled biological agent of this invention can be
used for in vitro diagnostic tests. The specific activity of a
tagged biological agent, such as a tagged antibody, binding portion
thereof, probe, or ligand, depends upon the half-life, the isotopic
purity of the radioactive label, and how the label is incorporated
into the biological agent. Table 2 lists several commonly-used
isotopes, their specific activities and half-lives. In immunoassay
tests, the higher the specific activity, in general, the better the
sensitivity.
TABLE-US-00002 TABLE 2 Specific Activity of Pure Isotope Isotope
(Curies/mole) Half-Life .sup. 14C 6.25 .times. 10.sup.1 5720 years
.sup. 3H 2.01 .times. 10.sup.4 12.5 years .sup. 35S 1.50 .times.
10.sup.6 87 days .sup.125I 2.18 .times. 10.sup.6 60 days .sup. 32P
3.16 .times. 10.sup.6 14.3 days .sup.131I 1.62 .times. 10.sup.7 8.1
days
[0081] Procedures for labeling biological agents with the
radioactive isotopes listed in Table 2 are generally known in the
art. Tritium labeling procedures are described in U.S. Pat. No.
4,302,438, which is hereby incorporated by reference. Iodinating,
tritium labeling, and 35S labeling procedures especially adapted
for murine monoclonal antibodies are described by Goding, J. W.
(supra, pp 124-126) and the references cited therein, which are
hereby incorporated by reference. Other procedures for iodinating
biological agents, such as antibodies, binding portions thereof,
probes, or ligands, are described by Hunter and Greenwood, Nature
144:945 (1962), David et al., Biochemistry 13:1014-1021 (1974), and
U.S. Pat. Nos. 3,867,517 and 4,376,110, which are hereby
incorporated by reference. Radiolabeling elements which are useful
in imaging include 123I, 131I, 111In, and 99m Tc, for example.
Procedures for iodinating biological agents are described by
Greenwood, F. et al., Biochem. J. 89:114-123 (1963); Marchalonis,
J., Biochem. J. 113:299-305 (1969); and Morrison, M. et al.,
Immunochemistry, 289-297 (1971), which are hereby incorporated by
reference. Procedures for 99m Tc-labeling are described by Rhodes,
B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The
Radioimmunochemical Detection of Cancer, New York: Masson 111-123
(1982) and the references cited therein, which are hereby
incorporated by reference. Procedures suitable for 111In-labeling
biological agents are described by Hnatowich, D. J. et al., J.
Immul. Methods, 65:147-157 (1983), Hnatowich, D. et al., J. Applied
Radiation, 35:554-557 (1984), and Buckley, R. G. et al., F.E.B.S.
166:202-204 (1984), which are hereby incorporated by reference.
[0082] In the case of a radiolabeled biological agent, the
biological agent is administered to the patient, is localized to
the tumor bearing the antigen with which the biological agent
reacts, and is detected or "imaged" in vivo using known techniques
such as radionuclear scanning using e.g., a gamma camera or
emission tomography. See e.g., A. R. Bradwell et al., "Developments
in Antibody Imaging", Monoclonal Antibodies for Cancer Detection
and Therapy, R. W. Baldwin et al., (eds.), pp. 65-85 (Academic
Press 1985), which is hereby incorporated by reference.
Alternatively, a positron emission transaxial tomography scanner,
such as designated Pet VI located at Brookhaven National
Laboratory, can be used where the radiolabel emits positrons (e.g.,
11C, 18F, 15O, and 13N).
[0083] Fluorophore and chromophore labeled biological agents can be
prepared from standard moieties known in the art. Since antibodies
and other proteins absorb light having wavelengths up to about 310
nm, the fluorescent moieties should be selected to have substantial
absorption at wavelengths above 310 nm and preferably above 400 nm.
A variety of suitable fluorescers and chromophores are described by
Stryer, Science, 162:526 (1968) and Brand, L. et al., Annual Review
of Biochemistry, 41:843-868 (1972), which are hereby incorporated
by reference. The biological agents can be labeled with fluorescent
chromophore groups by conventional procedures such as those
disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110,
which are hereby incorporated by reference.
[0084] One group of fluorescers having a number of the desirable
properties described above are the xanthene dyes, which include the
fluoresceins derived from 3,6-dihydroxy-9-henylxanthhydrol and
resamines and rhodamines derived from
3,6-diamino-9-phenylxanthydrol and lissanime rhodamine B. The
rhodamine and fluorescein derivatives of
9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group.
Fluorescein compounds having reactive coupling groups such as amino
and isothiocyanate groups such as fluorescein isothiocyanate and
fluorescamine are readily available. Another group of fluorescent
compounds are the naphthylamines, having an amino group in the
.alpha. or .beta. position.
[0085] Biological agents can be labeled with fluorchromes or
chromophores by the procedures described by Goding, J. (supra, pp
208-249). The biological agents can be labeled with an indicating
group containing the NMR-active 19F atom, or a plurality of such
atoms inasmuch as (i) substantially all of naturally abundant
fluorine atoms are the 19F isotope and, thus, substantially all
fluorine-containing compounds are NMR-active; (ii) many chemically
active polyfluorinated compounds such as trifluoracetic anhydride
are commercially available at relatively low cost, and (iii) many
fluorinated compounds have been found medically acceptable for use
in humans such as the perfluorinated polyethers utilized to carry
oxygen as hemoglobin replacements. After permitting such time for
incubation, a whole body NMR determination is carried out using an
apparatus such as one of those described by Pykett, Scientific
American, 246:78-88 (1982), which is hereby incorporated by
reference, to locate and image cancerous tissues (particularly the
vascular endothelial cells therein) and prostate epithelial
cells.
[0086] In cases where it is important to distinguish between
regions containing live and dead prostate epithelial cells or to
distinguish between live and dead prostate epithelial cells, the
antibodies of the present invention (or other biological agents of
the present invention), labeled as described above, can be
coadministered along with an antibody or other biological agent
which recognizes only living or only dead prostate epithelial cells
labeled with a label which can be distinguished from the label used
to label the subject antibody. By monitoring the concentration of
the two labels at various locations or times, spatial and temporal
concentration variations of living and dead normal, benign
hyperplastic, and cancerous prostate epithelial cells can be
ascertained. In particular, this method can be carried out using
the labeled antibodies of the present invention, which recognize
both living and dead epithelial prostate cells, and labeled 7E11
antibodies, which recognize only dead epithelial prostate cells.
The biological agents can also be utilized to kill or ablate
cancerous cells and normal, benign hyperplastic, and cancerous
prostate epithelial cells in vivo. This involves using the
biological agents by themselves or with a cytotoxic drug to which
the biological agents of the present invention (i.e., biological
agents recognizing normal, benign hyperplastic, and cancerous
prostate epithelial cells) are bound. This involves administering
the biological agents bonded to a cytotoxic drug to a mammal
requiring such treatment. In the case of normal, benign
hyperplastic, and cancerous prostate epithelial cells, since the
biological agents recognize prostate epithelial cells, any such
cells to which the biological agents bind are destroyed. Although
such administration may destroy normal prostate epithelial cells,
this is not problematic, because the prostate is not required for
life or survival. Although the prostate may indirectly contribute
to fertility, this is not likely to be a practical consideration in
patients receiving the treatment of the present invention. In the
case of cancerous tissues, since the biological agents recognize
vascular endothelial cells that are proximate to cancerous cells,
binding of the biological agent/cytotoxic drug complex to these
vascular endothelial cells destroys them, thereby cutting off the
blood flow to the proximate cancerous cells and, thus, killing or
ablating these cancerous cells. Alternatively, the biological
agents, by virtue of their binding to vascular endothelial cells
that are proximate to cancerous cells, are localized proximate to
the cancerous cells. Thus, by use of suitable biological agents
(including those containing substances effective to kill cells
nondiscriminatingly but only over a short range), cells in
cancerous tissue (including cancerous cells) can be selectively
killed or ablated.
[0087] The biological agents of the present invention may be used
to deliver a variety of cytotoxic drugs including therapeutic
drugs, a compound emitting radiation, molecules of plants, fungal,
or bacterial origin, biological proteins, and mixtures thereof. The
cytotoxic drugs can be intracellularly acting cytotoxic drugs, such
as short-range radiation emitters, including, for example,
short-range, high-energy a-emitters.
[0088] Enzymatically active toxins and fragments thereof are
exemplified by diphtheria toxin A fragment, nonbinding active
fragments of diphtheria toxin, exotoxin A (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
.alpha.-sacrin, certain Aleurites fordii proteins, certain Dianthin
proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S),
Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis
inhibitor, gelonin, mitogillin, restrictocin, phenomycin, and
enomycin, for example. Procedures for preparing enzymatically
active polypeptides of the immunotoxins are described in W084/03508
and W085/03508, which are hereby incorporated by reference. Certain
cytotoxic moieties are derived from adriamycin, chlorambucil,
daunomycin, methotrexate, neocarzinostatin, and platinum, for
example.
[0089] Procedures for conjugating the biological agents with the
cytotoxic agents have been previously described. Procedures for
conjugating chlorambucil with antibodies are described by Flechner,
I,. European Journal of Cancer, 9:741-745 (1973); Ghose, T. et al.,
British Medical Journal, 3:495-499 (1972); and Szekerke, M., et
al., Neoplasma, 19:211-215 (1972), which are hereby incorporated by
reference. Procedures for conjugating daunomycin and adriamycin to
antibodies are described by Hurwitz, E. et al., Cancer Research,
35:1175-1181 (1975) and Arnon, R. et al. Cancer Surveys, 1:429-449
(1982), which are hereby incorporated by reference. Procedures for
preparing antibody-ricin conjugates are described in U.S. Pat. No.
4,414,148 and by Osawa, T., et al. Cancer Surveys, 1:373-388 (1982)
and the references cited therein, which are hereby incorporated by
reference. Coupling procedures as also described in EP 86309516.2,
which is hereby incorporated by reference.
[0090] In a particularly preferred embodiment of the present
invention, especially well-suited for killing or ablating normal,
benign hyperplastic, and cancerous prostate epithelial cells, a
first biological agent is conjugated with a prodrug which is
activated only when in close proximity with a prodrug activator.
The prodrug activator is conjugated with a second biological agent
according to the present invention, preferably one which binds to a
non-competing site on the prostate specific membrane antigen
molecule. Whether two biological agents bind to competing or
non-competing binding sites can be determined by conventional
competitive binding assays. For example, monoclonal antibodies
J591, J533, and E99 bind to competing binding sites on the prostate
specific membrane antigen molecule. Monoclonal antibody J415, on
the other hand, binds to a binding site which is non-competing with
the site to which J591, J533, and E99 bind. Thus, for example, the
first biological agent can be one of J591, J533, and E99, and the
second biological agent can be J415. Alternatively, the first
biological agent can be J415, and the second biological agent can
be one of J591, J533, and E99. Drug-prodrug pairs suitable for use
in the practice of the present invention are described in Blakely
et al., "ZD2767, an Improved System for Antibody-directed Enzyme
Prodrug Therapy That Results in Tumor Regressions in Colorectal
Tumor Xenografts," Cancer Research, 56:3287-3292 (1996), which is
hereby incorporated by reference.
[0091] Alternatively, the biological agent can be coupled to high
energy radiation emitters, for example, a radioisotope, such as
1311, a .gamma.-emitter, which, when localized at the tumor site,
results in a killing of several cell diameters. See, e.g., S. E.
Order, "Analysis, Results, and Future Prospective of the
Therapeutic Use of Radiolabeled Antibody in Cancer Therapy",
Monoclonal Antibodies for Cancer Detection and Therapy, R. W.
Baldwin et al. (eds.), pp 303-316 (Academic Press 1985), which is
hereby incorporated by reference. Other suitable radioisotopes
include .alpha.-emitters, such as 212Bi, 213Bi, and 211At, and
.beta.-emitters, such as 186Re and 90Y. Radiotherapy is expected to
be particularly effective, because prostate epithelial cells and
vascular endothelial cells within cancers are relatively
radiosensitive.
[0092] Where the biological agents are used alone to kill or ablate
cancerous cells or prostate epithelial cells, such killing or
ablation can be effected by initiating endogenous host immune
functions, such as complement-mediated or antibody-dependent
cellular cytotoxicity.
[0093] The biological agent of the present invention can be used
and sold together with equipment, as a kit, to detect the
particular label.
[0094] Biological agents of the present invention can be used in
conjunction with other therapeutic treatment modalities. Such other
treatments include surgery, radiation, cryosurgery, thermotherapy,
hormone treatment, chemotherapy, vaccines, and other
immunotherapies.
[0095] Also encompassed by the present invention is a method of
killing or ablating which involves using the biological agents for
prophylaxis. For example, these materials can be used to prevent or
delay development or progression of prostate or other cancers. Use
of the therapeutic methods of the present invention to treat
prostate and other cancers has a number of benefits. Since the
biological agents according to the present invention only target
cancerous cells (such as cells of cancerous tissues containing
vascular endothelial cells) and prostate epithelial cells, other
tissue is spared. As a result, treatment with such biological
agents is safer, particularly for elderly patients. Treatment
according to the present invention is expected to be particularly
effective, because it directs high levels of biological agents,
such as antibodies or binding portions thereof, probes, or ligands,
to the bone marrow and lymph nodes where prostate cancer metastases
and metastases of many other cancers predominate. Moreover, the
methods of the present invention are particularly well-suited for
treating prostate cancer, because tumor sites for prostate cancer
tend to be small in size and, therefore, easily destroyed by
cytotoxic agents. Treatment in accordance with the present
invention can be effectively monitored with clinical parameters,
such as, in the case of prostate cancer, serum prostate specific
antigen and/or pathological features of a patient's cancer,
including stage, Gleason score, extracapsular, seminal, vesicle or
perineural invasion, positive margins, involved lymph nodes, etc.
Alternatively, these parameters can be used to indicate when such
treatment should be employed.
[0096] Because the biological agents of the present invention bind
to living prostate cells, therapeutic methods for treating prostate
cancer using these biological agents are much more effective than
those which target lysed prostate cells. For the same reasons,
diagnostic and imaging methods which determine the location of
living normal, benign hyperplastic, or cancerous prostate
epithelial cells (as well as vascular endothelial cells within
cancers) are much improved by employing the biological agents of
the present invention. In addition, the ability to differentiate
between living and dead prostate cells can be advantageous,
especially to monitor the effectiveness of a particular treatment
regimen.
[0097] Hybridomas E99, J415, J533, and J591 have been deposited
pursuant to, and in satisfaction of, the requirements of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure with the
American Type Culture Collection ("A.T.C.C.") at 12301 Parklawn
Drive, Rockville, Md. 20852. Hybridoma E99 was deposited on May 2,
1996, and received A.T.C.C. Designation Number HB-12101. Hybridoma
J415 was deposited on May 30, 1996, and received A.T.C.C.
Designation Number HB-12109. Hybridomas J533 and J591 were
deposited on Jun. 6, 1996, and received A.T.C.C. Designation
Numbers HB-12127 and HB-12126, respectively.
[0098] The present invention is further illustrated by the
following examples.
EXAMPLES
Example 1
Human Tissues
[0099] Fresh specimens of benign and malignant tissues were
obtained from the Department of Pathology of New York Hospital
Cornell University Medical Center ("NYH-CUMC"),
Example 2
Tissue Culture
[0100] Cultured cell lines of human cancers were obtained from the
Laboratory of Urological Oncology of NYH-CUMC. The prostate cancer
cell lines PC-3 (Mickey, D. D., et al., "Characterization Of A
Human Prostate Adenocarcinoma Cell Line (DU145) As A Monolayer
Culture And As A Solid
[0101] Tumor In Athymic Mice," Prog. Clin. Biol. Res., 37:67-84
(1980), which is hereby incorporated by reference), DU-145 (Mickey,
D. D., et al., "Characterization Of A Human Prostate Adenocarcinoma
Cell Line (DU145) As A Monolayer Culture And As A Solid Tumor In
Athymic Mice," Prog. Clin. Biol. Res., 37:67-84 (1980), which is
hereby incorporated by reference), and LNCaP (Horoszewicz, J. S.,
et al., "LNCaP Model Of Human Prostatic Carcinoma," Cancer Res.,
43:1809-1818 (1983), which is hereby incorporated by reference)
were obtained from the American Type Culture Collection (Rockville,
Md.). Hybridomas were initially cloned in RPMI-1640 medium
supplemented with 10% FCS, 0.1 mM nonessential amino acids, 2 mM
L-glutamine, 100 units/ml of penicillin, 100 ug/ml of streptomycin
and HAT medium (GIBCO, Grand Island, N.Y.). Subclones were cultured
in the same medium without aminopterin.
Example 3
Preparation of Mouse Monoclonal Antibodies
[0102] Female BALB/c mice were immunized intraperitoneally with
LNCaP (6.times.106 cells) three times at 2 week intervals. A final
intraperitoneal booster immunization was administered with fresh
prostate epithelial cells which had been grown in vitro. Three days
later, spleen cells were fused with SP-2 mouse myeloma cells
utilizing standard techniques (Ueda, R., et al., "Cell Surface
Antigens Of Human Renal Cancer Defined By Mouse Monoclonal
Antibodies: Identification Of Tissue-Specific Kidney
Glycoproteins," Proc. Natl. Acad. Sci. USA, 78:5122-5126 (1981),
which is hereby incorporated by reference). Supernatants of the
resulting clones were screened by rosette and complement
cytotoxicity assays against viable LNCaP. Clones which were
positive by these assays were screened by immunochemistry vs normal
kidney, colon, and prostate. Clones which were
LNCap+/NmlKid-/colon-/prostate+ were selected and subcloned 3 times
by limiting dilution. The immunoglobulin class of cultured
supernatant from each clone was determined by immunodiffusion using
specified rabbit antisera (Calbiochem, San Diego, Calif.). mAbs
were purified using the MAPS-II kit (Bio-Rad, Richmond,
Calif.).
Example 4
Biotinylation of mAbs
[0103] Purified mAbs were dialyzed in 0.1 M NaHCO3 for 2 hours. One
ml of mAb at 1 mg/ml was mixed with 0.1 ml of biotinamidocaproate
N-hydroxysuccinamide ester (Sigma) in dimethylsulfoxide (1 mg/ml)
and stirred for 4 hours at room temperature. Unbound biotin was
removed by dialysis against phosphate buffered saline ("PBS").
Example 5
Immunohistochemical Staining of Prostate Tissues
[0104] Cryostat sections of prostate tissues were placed inside
rings of Falcon 3034 plate covers (Becton-Dickenson, Lincoln Park,
N.J.) previously coated with 0.45% gelatin solution as described in
Marusich, M. F., "A Rapid Method For Processing Very Large Numbers
Of Tissue Sections For Immunohistochemical Hybridoma Screening," J.
Immunol. Methods, 111:143-145 (1988), which is hereby incorporated
by reference. Plates were stored at -80.degree. C. Cryostat
sections were fixed with 2% paraformaldehyde in PBS for 10 min at
room temperature, and, after washing with PBS, endogenous
peroxidase activity was blocked by treatment with 0.3% hydrogen
peroxide in PBS for 10 min at room temperature. After sections were
incubated with 2% BSA in PBS for 20 min, mAbs were added for 60 min
at room temperature. Slides were extensively washed with PBS and
incubated with peroxidase-conjugated rabbit anti-mouse Ig (DAKO
Corp., Santa Barbara, Calif.) diluted 1:100 in 10% normal human
serum in PBS for 60 min at room temperature. After a
diaminobenzidine reaction, sections were counterstained with
hematoxylin.
Example 6
Serological Analysis
[0105] The anti-mouse immunoglobulin mixed hemadsorption assay was
performed as described in Ueda, R., et al., "Cell Surface Antigens
Of Human Renal Cancer Defined By Mouse Monoclonal Antibodies:
Identification of Tissue-Specific Kidney Glycoproteins," Proc.
Natl. Acad. Sci. USA, 78:5122-5126 (1981), which is hereby
incorporated by reference. To prepare the indicator cells,
anti-mouse Ig (DAKO Corp.) was conjugated to type O human RBC using
0.01% chromium chloride. Serological assays were performed on cells
previously plated in Terasaki plates (Nunc, Denmark). Antibodies
were incubated with target cells at room temperature for 1 hour.
Target cells were then washed, and indicator cells added for 1
hour.
Example 7
Immunoprecipitation
[0106] LNCaP cells (2.times.107) were biotinylated with biotin-NHSS
(at final concentration of 5 mM) for 30 minutes on ice. After
washing, the biotinylated cells were resuspended in 1 ml lysis
buffer (20 mM Tris/HCl pH 8.0, 1 mM EDTA, 1 mM PMSF, 1% triton
X-100) for 30 min on ice. The suspension was centrifuged at 1500
g.times.100 min at 4.degree. C., and the supernatant was
centrifuged at 12,000 rpm.times.15 min at 4.degree. C. The
resulting lysate was preabsorbed with rabbit or goat anti-mouse
IgG-coated pansorbin for 1 hour at 4.degree. C. The pre-absorbed
lysate was incubated with the mAb overnight at 4.degree. C. Rabbit
or goat anti-mouse Ig-coated agarose beads were added for 2 hours
at 4.degree. C. and then washed. The beads were resuspended in
Tris-base/NaCl, added to sample buffer with 2-mercaptoethanol, and
boiled for 5 min. After centrifuging, the supernatant was run on an
SDS-PAGE 12% gel. The gel was transferred to a nitrocellulose
membrane which was blocked and stained with
straptavidin-peroxidase. The membrane was developed with
diaminobenzidine ("DAB").
[0107] Sequential immunoprecipitation was similar except that the
lysate was initially pre-cleared with one mAb overnight at
4.degree. C. A second mAb was then used to immunoprecipitate the
pre-cleared lysate.
[0108] Approximately 2000 clones were screened, of which four
clones were selected as described in Example 3, above. After
subcloning, supernatants from the 4 hybridomas, E99, J415, J533,
and J591, were assayed by immunofluorescence against viable (i.e.
unfixed) LNCaP, immunoprecipitation, and sequential
immunoprecipitation to confirm reactivity to PSMA.
[0109] The immunofluorescence study using the LNCaP target cell
(described originally in Horoszewicz, which is hereby incorporated
by reference, to make the 7E11 antibody and the prototype cell line
for expression for PSMA) shows that E99 antibody binds to and
renders viable LNCaP cells immunofluorescent. This is in contrast
to the 7E11 antibody, which, as noted originally in Horoszewicz,
which is hereby incorporated by reference, gives only poor or no
binding to viable LNCaP cells but exhibits strong binding once the
cells are fixed (killed).
[0110] The reactivities of the four mAbs with normal human tissues
were examined immunohistochemically; these results are presented in
Table 3.
TABLE-US-00003 TABLE 3 Reactivity of mAbs with human normal tissues
by indirect immunoperosidase staining E99 J415 J533 J591 Tissues
(.gamma..sub.3) (.gamma..sub.3) (.gamma..sub.3) (.gamma..sub.3)
Prostate* Kidney Glomerulus Prox. Tubule .box-solid. .box-solid.
.box-solid. .box-solid. Ureter Bladder Testis Uterus Esophagus
Small Intestine Stomach Colon Spleen Thyroid Lung Pancreas Liver
*BPH 0-3.sup.+ 0-3.sup.+ 0-4.sup.+ 0-4.sup.+ *Prostate Cancer
0-3.sup.+ 0-3.sup.+ 0-4.sup.+ 0-4.sup.+ *LNCaP (scid) 3.sup.+
3.sup.+ 4.sup.+ 4.sup.+ *LuCaP (scid) 0-2.sup.+ 0-2.sup.+ 0-3.sup.+
0-3.sup.+ --positive; .box-solid.--weak, heterogeneous;
--negative
[0111] The above sequential Immunoprecipitation study showed that
7E11, E99, J415, J533, and J591 bind to the same molecule, i.e.
PSMA.
Example 8
Western Blot Analysis
[0112] To confirm that antibodies E99, J415, J533, and J591
precipitate an identical band to the 7E11 antibody (i.e., PSMA),
Western Blot analyses were performed. Seminal plasma (400
.quadrature.g/lane) or LNCaP lysate were loaded into lanes of 12%
SDS-PAGE gels. After electrophoresis, the gels are transferred to
nitrocellulose membranes. The membranes were blocked with 5% dry
milk/Tris-buffered saline-tween 20 ("TBST") for 60 min at room
temperature. After washing, the membranes were incubated with
primary mAb for 60 min at room temperature. After repeat washing,
the membranes were incubated with sheep anti-mouse-Ig-peroxidase
1/5000 in 5% dry milk/TBST for 60 min at room temperature. After
repeat washing, the membranes were developed using a
chemiluminescent tag designated "ECL" (Amersham Life Sciences,
International, Arlington Heights, Ill.) according to the
manufacturer's directions. The results of the Western Blot
experiment are presented in Table 4.
TABLE-US-00004 TABLE 4 Western blot data Sample 7E11 E99 J415 J533
J591 Prostatic 100 KD 100 KD 100 KD 100 KD 100 KD (seminal) band
band band band band fluid LNCaP 100 KD & 100 KD & 100 KD
& 100 KD & 100 KD & cell lysate 200 KD 200 KD 200 KD
200 KD 200 KD bands bands bands bands bands
Example 9
mAb Reactivity to External Domain of PSMA
[0113] To confirm cell surface (external) expression of the
detected PSMA, fresh, viable LNCaP cells were tested, without
fixation, in vitro, by immunofluorescence. LNCaP cells were washed
and incubated with mAb for 1 hour at room temperature and then with
a rabbit anti-mouse Ig-fluorescein (DAKO Corp., Santa Barbara,
Calif.). Wells were read with a fluorescent microscope. Negative
control consisted of an isotype-matched irrelevant mAb, while an
anti-class I MHC mAb served as a positive control.
[0114] Immunofluorescence and rosette assay results are presented
in Table 5.
TABLE-US-00005 TABLE 5 Comparison of 7E11 with new mAbs LNCaP 7E11
E99 J415 J533 J591 Viable Cells Immuno- neg 3+ 3+ 4+ 4+
fluorescence Rosette neg + + + + assay LNCaP-fixed +++ ++++ +++ ++
+++
Example 10
Competition Studies
[0115] A competition study was carried out to determine whether
J591, J533, E99, and J415 detected the same or different antigenic
sites (epitopes) of the prostate specific membrane antigen molecule
using the following procedure.
[0116] Plates were coated with LNCaP cell line lysate as a source
of prostate specific membrane antigen and washed to remove unbound
material. "Cold" (unlabeled) monoclonal antibody was incubated on
the plate for 1 hour at room temperature to allow binding to its
antigenic site. Subsequently, a second monoclonal antibody, labeled
either with biotin or 125I, was added for an additional hour.
Plates were washed to remove unbound material. The amount of the
second monoclonal antibody bound to the prostate specific membrane
antigen-coated plate was determined either by avidin-alkaline
phosphatase in an enzyme-linked immunoassay (in the case of
biotin-labeled second monoclonal antibody) or by physically
counting the well in a gamma counter (in the case of 125I-labeled
second monoclonal antibody). Controls consisted of using the same
monoclonal antibody both cold and labeled to define "100%
competition" or using monoclonal antibody to a totally different
molecule (e.g., monoclonal antibody I-56, which detects inhibin, a
prostate related protein different from prostate specific membrane
antigen) to define "0% competition".
[0117] The results indicated that J591, J533, and E99 each
interfere, compete, or block binding of one another but do not
block binding of J415 and vice versa. 7E11/CYT356, known to bind
PSMA at a different (intracellular) site, did not block any of
J591, J533, E99, or J415.
[0118] Having pairs of monoclonal antibodies which bind to
non-competing sites permits development of antibody sandwich assays
for detecting soluble antigens, such as solubilized prostate
specific membrane antigen or fragment thereof, in, for example,
body fluids. For example, the antigen (e.g., prostate specific
membrane antigen or a fragment thereof) could be "captured" from
body fluid with J591 and, in another step, detected by labeled
J415.
[0119] In another setting, e.g. treatment, one could increase
antibody binding by using a combination of non-competing monoclonal
antibodies. For example, assuming the non-competing sites are each
represented once on the prostate specific membrane antigen
molecule, adding a combination of J591 plus J415 would bind twice
as many monoclonal antibody molecules as either monoclonal antibody
alone. Binding two non-competing antigenic binding sites also can
result in greater antigen cross-linking and, perhaps, increased
internalization. Furthermore, since the two detected sites are
physically located on the same prostate specific membrane antigen
molecule, the binding of two monoclonal antibody molecules to that
single prostate specific membrane antigen molecule puts the two
monoclonal antibody molecules in close proximity to each other, a
setting which provides optimal drug-prodrug interaction. For
example, monoclonal antibody J591 can be conjugated with an
inactive pro-drug and J415 can be conjugated with a pro-drug
activator. Since prodrug and activator would be bound in close
proximity only at the site of prostate specific membrane
antigen-expressing cells (e.g., prostate cancer cells), prodrug
activation to the active form would occur only at those sites.
Example 11
Microscopy
[0120] Confocal microscopy and immuno-electron microscopy
demonstrated that E99, J591, J533, and J415 are bound to the cell
membrane at clathrin-coated pits and then rapidly internalize into
endosomes (cytoplasmic vesicles). FIGS. 1-4 are immuno-electron
micrographs which follow the interaction of gold-labeled monoclonal
antibody J591 with the cell surface as a function of time. In these
figures, the location of the monoclonal antibody is indicated by
the black dots.
[0121] Viable LNCaP cells were incubated with J591 for one hour at
4.degree. C. The cells were washed and then held at 37.degree. C.
for 0, 5, 10, or 15 minutes, after which time they were fixed and
processed for immuno-electron microscopy. FIG. 1 shows the cell
prior to 37.degree. C. incubation. J591 can be seen bound to the
cell along the external aspect of the cell membrane. In this
Figure, "M" denotes the cell's mitochondria, and "N" denotes its
nucleus. FIG. 2 shows the cell after incubation at 37.degree. C.
for 5 minutes. The arrow indicates formation of a clathrin-coated
pit. In FIG. 3, which shows the cell after a 10 minute 37.degree.
C. incubation, pinching off or endocytosis of the clathrin-coated
pit can be seen, as indicated by the arrow. FIG. 4 shows that,
after incubation at 37.degree. C. for 15 minutes, monoclonal
antibody J591 is contained in endocytic vesicles within the cell,
as indicated by the arrows. As can be seen in FIG. 5, after
incubation at 37.degree. C. for 15 minutes, monoclonal antibody
J591 is also contained within endosomes, as indicated by the
arrows.
Example 12
Sequencing of the Variable Region of Monoclonal Antibody J591
[0122] Total RNA was prepared from 107 murine hybridoma J591 cells.
A sample of the conditioned medium from these cells was tested for
binding to the specific antigen for J591 on prostate cells. The
conditioned medium was positive by both ELISA and Western Blot for
binding to the antigen.
[0123] VH and VK cDNA were prepared using reverse transcriptase and
mouse .kappa. constant region and mouse IgG constant region
primers. The first strand cDNAs were amplified by PCR using a
variety of mouse signal sequence primers (6 for VH and 7 for VK).
The amplified DNAs were gel-purified and cloned into the vector
pT7Blue.
[0124] The VH and VK clones obtained were screened for correct
inserts by PCR, and the DNA sequence of selected clones was
determined by the dideoxy chain termination method.
[0125] Excluding the primer region (as the sequence of this
depended on the sequence of the primer that was used), all the VH
clones obtained gave identical sequence. This sequence was obtained
from clones produced with three different 5' primers. One clone had
one base pair change within the signal sequence, and one clone
contained an aberrant PCR product. Using the sequencing strategy
shown in FIG. 6, the nucleotide sequence for the heavy chain was
obtained. It is designated SEQ. ID. No. 1 and is presented in FIG.
7, along with the nucleotide sequence of the corresponding reverse,
non-coding strand (designated SEQ. ID. No. 2). These sequences
include part of the signal sequence and part of the constant region
of the antibody. The corresponding deduced amino acid sequences of
J591 VH, designated SEQ. ID. No. 3, SEQ. ID. No. 4, and SEQ. ID.
No. 5, are also shown in FIG. 7. The coding strand of the J591
heavy chain's variable region (exclusive of signal sequence and
constant region components) has the following nucleotide sequence
(designated SEQ. ID. No. 6):
TABLE-US-00006 GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTC
AGTGAGGATATCCTGCAAGACTTCTGGATACACATTCACTGAATATACCA
TACACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAAC
ATCAATCCTAACAATGGTGGTACCACCTACAATCAGAAGTTCGAGGACAA
GGCCACATTGACTGTAGACAAGTCCTCCAGTACAGCCTACATGGAGCTCC
GCAGCCTAACATCTGAGGATTCTGCAGTCTATTATTGTGCAGCTGGTTGG
AACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
[0126] The reverse, non-coding strand of the J591 heavy chain's
variable region (exclusive of signal sequence and constant region
components) has the following nucleotide sequence (designated SEQ.
ID. No. 7):
TABLE-US-00007 TGAGGAGACTGTGAGAGTGGTGCCTTGGCCCCAGTAGTCAAAGTTCCAAC
CAGCTGCACAATAATAGACTGCAGAATCCTCAGATGTTAGGCTGCGGAGC
TCCATGTAGGCTGTACTGGAGGACTTGTCTACAGTCAATGTGGCCTTGTC
CTCGAACTTCTGATTGTAGGTGGTACCACCATTGTTAGGATTGATGTTTC
CAATCCACTCAAGGCTCTTTCCATGGCTCTGCTTCACCCAGTGTATGGTA
TATTCAGTGAATGTGTATCCAGAAGTCTTGCAGGATATCCTCACTGAAGT
CCCAGGCTTCACCAGTTCAGGTCCAGACTGTTGCAGCTGGACCTC
[0127] The protein sequence corresponding to the J591 heavy chain's
variable region (exclusive of signal sequence and constant region
components) has the following nucleotide sequence (designated SEQ.
ID. No. 8):
TABLE-US-00008 EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGN
INPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGW
NFDYWGQGTTLTVSS
[0128] The J591 VH is in Mouse Heavy Chains Subgroup HA (Kabat et
al., Sequences of Proteins of Immunological Interest, U.S.
Department of Health and Human Services (1991) ("Kabat"), which is
hereby incorporated by reference). The sequence of J591 VH is
compared to the consensus sequence for this subgroup in FIG. 8.
[0129] In contrast to the VH, more than one VK sequence was
obtained. Out of the 15 VK clones examined, four gave the sequence
of an aberrant mouse Ig.kappa. from the fusion partner (Carol et
al., Molecular Immunology, 25:991-995 (1988), which is hereby
incorporated by reference). These clones originated from two
specific 5' primers. No further work was done with these clones. Of
the remaining clones, ten gave identical nucleotide sequences, and
one clone, VK17, gave an alternative VK sequence. The ten identical
clones originated from three 5' primers (different from the two
that gave the aberrant sequence), one of which also produced VK17.
The sequencing strategy that was employed is shown in FIG. 9.
[0130] The nucleic acid sequence of J591 VK corresponding to the
ten identical clones (designated SEQ. ID. No. 9) is presented in
FIG. 10, along with the nucleic acid sequence of the corresponding
reverse, non-coding strand (designated SEQ. ID. No. 10) and the
deduced amino acid sequences, which are designated SEQ. ID. No. 11,
SEQ. ID. No. 12, and SEQ. ID. No. 13. These sequences include part
of the signal sequence and part of the constant region of the
antibody. The coding strand of the J591 light (kappa) chain's
variable region (exclusive of signal sequence and constant region
components) corresponding to the ten identical clones has the
following nucleotide sequence (designated SEQ. ID. No. 14):
TABLE-US-00009 AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGA
GAGGGTCACCTTGACCTGCAAGGCCAGTGAGAATGTGGTTACTTATGTTT
CCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGG
GCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATC
TGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTG
CAGATTATCACTGTGGACAGGGTTACAGCTATCCGTACACGTTCGGAGGG
GGGACCAAGCTGGAAATAAAA
[0131] The reverse, non-coding strand of the J591 light (kappa)
chain's variable region (exclusive of signal sequence and constant
region components) corresponding to the ten identical clones has
the following nucleotide sequence (designated SEQ. ID. No. 15):
TABLE-US-00010 TTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGATAGCTGTAAC
CCTGTCCACAGTGATAATCTGCAAGGTCTTCAGCCTGCACACTGCTGATG
GTCAGAGTGAAATCTGTTGCAGATCCACTGCCTGTGAAGCGATCGGGGAC
CCCAGTGTACCGGTTGGATGCCCCGTATATCAGCAGTTTAGGAGACTGCT
CTGGTTTCTGTTGATACCAGGAAACATAAGTAACCACATTCTCACTGGCC
TTGCAGGTCAAGGTGACCCTCTCTCCTACTGACATGGACATGGATTTGGG
AGATTGGGTCATTACAATGTT
[0132] The protein sequence corresponding to the J591 light (kappa)
chain's variable region (exclusive of signal sequence and constant
region components) corresponding to the ten identical clones has
the following nucleotide sequence (designated SEQ. ID. No. 16):
TABLE-US-00011 NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYG
ASNRYTG VPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFG GGTKLEIK
[0133] The coding strand of the J591 light (kappa) chain's variable
region (exclusive of signal sequence and constant region
components) corresponding to clone VK17 has the following
nucleotide sequence (designated SEQ. ID. No. 17):
TABLE-US-00012 GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGA
CAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAG
ACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGG
GCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATC
TGGGACAGACTTCACTCTCACCATTACTAATGTTCAGTCTGAAGACTTGG
CAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCT
GGGACCATGCTGGACCTGAAA
[0134] The reverse, non-coding strand of the J591 light (kappa)
chain's variable region (exclusive of signal sequence and constant
region components) corresponding to clone VK17 has the following
nucleotide sequence (designated SEQ. ID. No. 18):
TABLE-US-00013 TTTCAGGTCCAGCATGGTCCCAGCACCGAACGTGAGAGGATAGCTGTTAT
ATTGCTGACAGAAATAATCTGCCAAGTCTTCAGACTGAACATTAGTAATG
GTGAGAGTGAAGTCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGAC
TCCAGTGTGCCGAGTGGATGCCCAATAAATCAGTAGTTTAGGAGATTGTC
CTGGTTTCTGTTGATACCAGTCTACAGCAGTACCCACATCTTGACTGGCC
TTACAGATGATGCTGACCCTGTCTCCTACTGATGTGGACATGAATTTGTG
AGACTGGGTCATCACAATGTC
[0135] The protein sequence corresponding to the J591 light (kappa)
chain's variable region (exclusive of signal sequence and constant
region components) corresponding to clone VK17 has the following
nucleotide sequence (designated SEQ. ID. No. 19):
TABLE-US-00014 DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYW
ASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGA GTMLDLK
[0136] J591 VK is in the Mouse Kappa Chains Subgroup V (Kabat,
which is hereby incorporated by reference). The sequence of J591 VK
corresponding to the ten identical clones is compared to the
consensus sequence for the subgroup in FIG. 11.
[0137] Preferred J591's are those having heavy chain variable
region DNA coding strand sequences corresponding to SEQ. ID. No. 6
and non-coding strand (reverse) sequences corresponding to SEQ. ID.
No. 7. The heavy chain variable region of J591 preferably has an
amino acid sequence corresponding to SEQ. ID. No. 8. The light
chain variable region of J591 preferably has a DNA coding strand
sequence corresponding to SEQ. ID. No. 17, a DNA non-coding strand
(reverse) sequence corresponding to SEQ. ID. No. 18, and a amino
acid sequence corresponding to SEQ. ID. No. 19.
Example 13
Immunohistochemical Staining of Normal and Cancer Tissues
[0138] Cancer tissues from 23 carcinomas were pre-cooled in liquid
nitrogen, snap-frozen in OCT compound (Miles, Elkhart, Ind.) on dry
ice, and stored at -80.degree. C. Cryostat tissue sections (5
.mu.m) were fixed in cold acetone (4.degree. C.) for 10 minutes.
mAbs (5 .mu.g/ml or hybridoma supernatants) were incubated for 1
hour at room temperature. Antibody binding was detected using
rabbit anti-mouse Ig-peroxidase (Dako, Carpinteria, Calif.) as a
secondary antibody and DAB (Sigma, St. Louis, Mo.) as chromogen.
Isotype-matched irrelevant antibody was used as negative
control.
[0139] mAbs J591, J533, J415, and E99 reacted strongly with
vascular endothelia in all 23 carcinomas studied, including 9/9
renal, 5/5 urothelial, 6/6 colon, 1/1 lung, and 1/1 breast
carcinomas, and 1/1 metastatic adenocarcinoma to the liver FIGS.
12A-12F, respectively, show the immunohistochemical reactivity of
mAb J591 to neovasculature of renal, urothelial, colon, lung, and
breast carcinomas, and metastatic adenocarcinoma to the liver.
[0140] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose and variations can be made by those skilled
in the art without departing from the spirit and scope of the
invention which is defined by the following claims.
SEQUENCE LISTING
[0141] The specification incorporates herein by reference the
sequence listing filed with the instant application in compliance
with 37 C.F.R. .sctn..sctn.1.821-1.825.
Sequence CWU 1
1
231391DNAMus sp. 1tctcctgtca ggaactgcag gtgtcctctc tgaggtccag
ctgcaacagt ctggacctga 60actggtgaag cctgggactt cagtgaggat atcctgcaag
acttctggat acacattcac 120tgaatatacc atacactggg tgaagcagag
ccatggaaag agccttgagt ggattggaaa 180catcaatcct aacaatggtg
gtaccaccta caatcagaag ttcgaggaca aggccacatt 240gactgtagac
aagtcctcca gtacagccta catggagctc cgcagcctaa catctgagga
300ttctgcagtc tattattgtg cagctggttg gaactttgac tactggggcc
aaggcaccac 360tctcacagtc tcctcagcca aaacgacacc c 3912391DNAMus sp.
2gggtgtcgtt ttggctgagg agactgtgag agtggtgcct tggccccagt agtcaaagtt
60ccaaccagct gcacaataat agactgcaga atcctcagat gttaggctgc ggagctccat
120gtaggctgta ctggaggact tgtctacagt caatgtggcc ttgtcctcga
acttctgatt 180gtaggtggta ccaccattgt taggattgat gtttccaatc
cactcaaggc tctttccatg 240gctctgcttc acccagtgta tggtatattc
agtgaatgtg tatccagaag tcttgcagga 300tatcctcact gaagtcccag
gcttcaccag ttcaggtcca gactgttgca gctggacctc 360agagaggaca
cctgcagttc ctagcaggag a 3913123PRTMus sp. 3Ser Pro Val Arg Asn Cys
Arg Cys Pro Leu Gly Pro Ala Ala Thr Val 1 5 10 15 Trp Thr Thr Gly
Glu Ala Trp Asp Phe Ser Glu Asp Ile Leu Gln Asp 20 25 30 Phe Trp
Ile His Ile His Ile Tyr His Thr Leu Gly Glu Ala Glu Pro 35 40 45
Trp Lys Glu Pro Val Asp Trp Lys His Gln Ser Gln Trp Trp Tyr His 50
55 60 Leu Gln Ser Glu Val Arg Gly Gln Gly His Ile Asp Cys Arg Gln
Val 65 70 75 80 Leu Gln Tyr Ser Leu His Gly Ala Pro Gln Pro Asn Ile
Gly Phe Cys 85 90 95 Ser Leu Leu Leu Cys Ser Trp Leu Glu Leu Leu
Leu Gly Pro Arg His 100 105 110 His Ser His Ser Leu Leu Ser Gln Asn
Asp Thr 115 120 4130PRTMus sp. 4Leu Leu Ser Gly Thr Ala Gly Val Leu
Ser Glu Val Gln Leu Gln Gln 1 5 10 15 Ser Gly Pro Glu Leu Val Lys
Pro Gly Thr Ser Val Arg Ile Ser Cys 20 25 30 Lys Thr Ser Gly Tyr
Thr Phe Thr Glu Tyr Thr Ile His Trp Val Lys 35 40 45 Gln Ser His
Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn 50 55 60 Asn
Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu Asp Lys Ala Thr Leu 65 70
75 80 Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser
Leu 85 90 95 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ala Gly
Trp Asn Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
Ser Ser Ala Lys Thr 115 120 125 Thr Pro 130 5125PRTMus sp. 5Leu Ser
Cys Gln Glu Leu Gln Val Ser Ser Leu Arg Ser Ser Cys Asn 1 5 10 15
Ser Leu Asp Leu Asn Trp Ser Leu Gly Leu Gln Gly Tyr Pro Ala Arg 20
25 30 Leu Leu Asp Thr His Ser Leu Asn Ile Pro Tyr Thr Gly Ser Arg
Ala 35 40 45 Met Glu Arg Ala Leu Ser Gly Leu Glu Thr Ser Ile Leu
Thr Met Val 50 55 60 Val Pro Pro Thr Ile Arg Ser Ser Arg Thr Arg
Pro His Leu Thr Ser 65 70 75 80 Pro Pro Val Gln Pro Thr Trp Ser Ser
Ala Ala His Leu Arg Ile Leu 85 90 95 Gln Ser Ile Ile Val Gln Leu
Val Gly Thr Leu Thr Thr Gly Ala Lys 100 105 110 Ala Pro Leu Ser Gln
Pro Ser Gln Pro Lys Arg His Pro 115 120 125 6345DNAMus sp.
6gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctgggacttc agtgaggata
60tcctgcaaga cttctggata cacattcact gaatatacca tacactgggt gaagcagagc
120catggaaaga gccttgagtg gattggaaac atcaatccta acaatggtgg
taccacctac 180aatcagaagt tcgaggacaa ggccacattg actgtagaca
agtcctccag tacagcctac 240atggagctcc gcagcctaac atctgaggat
tctgcagtct attattgtgc agctggttgg 300aactttgact actggggcca
aggcaccact ctcacagtct cctca 3457345DNAMus sp. 7tgaggagact
gtgagagtgg tgccttggcc ccagtagtca aagttccaac cagctgcaca 60ataatagact
gcagaatcct cagatgttag gctgcggagc tccatgtagg ctgtactgga
120ggacttgtct acagtcaatg tggccttgtc ctcgaacttc tgattgtagg
tggtaccacc 180attgttagga ttgatgtttc caatccactc aaggctcttt
ccatggctct gcttcaccca 240gtgtatggta tattcagtga atgtgtatcc
agaagtcttg caggatatcc tcactgaagt 300cccaggcttc accagttcag
gtccagactg ttgcagctgg acctc 3458115PRTMus sp. 8Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Arg
Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr
Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45 Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60 Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 9363DNAMus sp.
9ttatatggag ctgatgggaa cattgtaatg acccaatctc ccaaatccat gtccatgtca
60gtaggagaga gggtcacctt gacctgcaag gccagtgaga atgtggttac ttatgtttcc
120tggtatcaac agaaaccaga gcagtctcct aaactgctga tatacggggc
atccaaccgg 180tacactgggg tccccgatcg cttcacaggc agtggatctg
caacagattt cactctgacc 240atcagcagtg tgcaggctga agaccttgca
gattatcact gtggacaggg ttacagctat 300ccgtacacgt tcggaggggg
gaccaagctg gaaataaaac gggctgatgc tgcaccaact 360gta 36310363DNAMus
sp. 10tacagttggt gcagcatcag cccgttttat ttccagcttg gtcccccctc
cgaacgtgta 60cggatagctg taaccctgtc cacagtgata atctgcaagg tcttcagcct
gcacactgct 120gatggtcaga gtgaaatctg ttgcagatcc actgcctgtg
aagcgatcgg ggaccccagt 180gtaccggttg gatgccccgt atatcagcag
tttaggagac tgctctggtt tctgttgata 240ccaggaaaca taagtaacca
cattctcact ggccttgcag gtcaaggtga ccctctctcc 300tactgacatg
gacatggatt tgggagattg ggtcattaca atgttcccat cagctccata 360taa
36311121PRTMus sp. 11Leu Tyr Gly Ala Asp Gly Asn Ile Val Met Thr
Gln Ser Pro Lys Ser 1 5 10 15 Met Ser Met Ser Val Gly Glu Arg Val
Thr Leu Thr Cys Lys Ala Ser 20 25 30 Glu Asn Val Val Thr Tyr Val
Ser Trp Tyr Gln Gln Lys Pro Glu Gln 35 40 45 Ser Pro Lys Leu Leu
Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val 50 55 60 Pro Asp Arg
Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile
Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln 85 90
95 Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val 115 120 12114PRTMus
sp. 12Tyr Met Glu Leu Met Gly Thr Leu Pro Asn Leu Pro Asn Pro Cys
Pro 1 5 10 15 Cys Gln Glu Arg Gly Ser Pro Pro Ala Arg Pro Val Arg
Met Trp Leu 20 25 30 Leu Met Phe Pro Gly Ile Asn Arg Asn Gln Ser
Ser Leu Leu Asn Cys 35 40 45 Tyr Thr Gly His Pro Thr Gly Thr Leu
Gly Ser Pro Ile Ala Ser Gln 50 55 60 Ala Val Asp Leu Gln Gln Ile
Ser Leu Pro Ser Ala Val Cys Arg Leu 65 70 75 80 Lys Thr Leu Gln Ile
Ile Thr Val Asp Arg Val Thr Ala Ile Arg Thr 85 90 95 Arg Ser Glu
Gly Gly Pro Ser Trp Lys Asn Gly Leu Met Leu His Gln 100 105 110 Leu
Tyr 13116PRTMus sp. 13Ile Ile Trp Ser Trp Glu His Cys Asn Asp Pro
Ile Ser Gln Ile His 1 5 10 15 Val His Val Ser Arg Arg Glu Gly His
Leu Asp Leu Gln Gly Gln Glu 20 25 30 Cys Gly Tyr Leu Cys Phe Leu
Val Ser Thr Glu Thr Arg Ala Val Ser 35 40 45 Thr Ala Asp Ile Arg
Gly Ile Gln Pro Val His Trp Gly Pro Arg Ser 50 55 60 Leu His Arg
Gln Trp Ile Cys Asn Arg Phe His Ser Asp His Gln Gln 65 70 75 80 Cys
Ala Gly Arg Pro Cys Arg Leu Ser Leu Trp Thr Gly Leu Gln Leu 85 90
95 Ser Val His Val Arg Arg Gly Asp Gln Ala Gly Asn Lys Thr Gly Cys
100 105 110 Cys Thr Asn Cys 115 14321DNAMus sp. 14aacattgtaa
tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc 60ttgacctgca
aggccagtga gaatgtggtt acttatgttt cctggtatca acagaaacca
120gagcagtctc ctaaactgct gatatacggg gcatccaacc ggtacactgg
ggtccccgat 180cgcttcacag gcagtggatc tgcaacagat ttcactctga
ccatcagcag tgtgcaggct 240gaagaccttg cagattatca ctgtggacag
ggttacagct atccgtacac gttcggaggg 300gggaccaagc tggaaataaa a
32115321DNAMus sp. 15ttttatttcc agcttggtcc cccctccgaa cgtgtacgga
tagctgtaac cctgtccaca 60gtgataatct gcaaggtctt cagcctgcac actgctgatg
gtcagagtga aatctgttgc 120agatccactg cctgtgaagc gatcggggac
cccagtgtac cggttggatg ccccgtatat 180cagcagttta ggagactgct
ctggtttctg ttgataccag gaaacataag taaccacatt 240ctcactggcc
ttgcaggtca aggtgaccct ctctcctact gacatggaca tggatttggg
300agattgggtc attacaatgt t 32116107PRTMus sp. 16Asn Ile Val Met Thr
Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val
Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr
Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 17321DNAMus sp. 17gacattgtga tgacccagtc tcacaaattc
atgtccacat cagtaggaga cagggtcagc 60atcatctgta aggccagtca agatgtgggt
actgctgtag actggtatca acagaaacca 120ggacaatctc ctaaactact
gatttattgg gcatccactc ggcacactgg agtccctgat 180cgcttcacag
gcagtggatc tgggacagac ttcactctca ccattactaa tgttcagtct
240gaagacttgg cagattattt ctgtcagcaa tataacagct atcctctcac
gttcggtgct 300gggaccatgc tggacctgaa a 32118321DNAMus sp.
18tttcaggtcc agcatggtcc cagcaccgaa cgtgagagga tagctgttat attgctgaca
60gaaataatct gccaagtctt cagactgaac attagtaatg gtgagagtga agtctgtccc
120agatccactg cctgtgaagc gatcagggac tccagtgtgc cgagtggatg
cccaataaat 180cagtagttta ggagattgtc ctggtttctg ttgataccag
tctacagcag tacccacatc 240ttgactggcc ttacagatga tgctgaccct
gtctcctact gatgtggaca tgaatttgtg 300agactgggtc atcacaatgt c
32119107PRTMus sp. 19Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Ile Cys Lys Ala
Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr
Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu
Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys 100 105 20125PRTMus
sp. 20Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30 Tyr Met Asn Asn Trp Val Lys Gln Ser Pro Gly
Lys Ser Leu Glu Trp 35 40 45 Ile Gly Asp Ile Asn Pro Gly Asn Gly
Gly Thr Ser Tyr Asn Gln Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95 Cys Ala Arg
Gly Tyr Tyr Ser Ser Ser Tyr Met Ala Tyr Tyr Ala Phe 100 105 110 Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
21109PRTMus sp. 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Asp Ile Ser Asn 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Gly Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Ala Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser
Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu 65 70 75 80 Gln Glu
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95
Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
2296PRTArtificial Sequencecomparison sequence 22Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Val Ile Ser
Cys Lys Ser Gly Tyr Thr Phe Thr Tyr Trp Val Lys Gln 20 25 30 Ser
Gly Lys Ser Leu Glu Trp Ile Gly Ile Asn Pro Asn Gly Gly Thr 35 40
45 Tyr Asn Gln Lys Phe Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
50 55 60 Thr Ala Tyr Met Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr 65 70 75 80 Cys Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Thr Val Ser Ser 85 90 95 2367PRTArtificial Sequencecomparison
sequence 23Ile Met Thr Gln Ser Pro Ser Ser Ser Gly Arg Val Thr Thr
Cys Ala 1 5 10 15 Ser Tyr Trp Tyr Gln Gln Lys Pro Ser Pro Lys Leu
Leu Ile Tyr Ala 20 25 30 Ser Gly Val Pro Arg Phe Gly Ser Gly Ser
Thr Asp Leu Thr Ile Ser 35 40 45 Glu Asp Ala Tyr Cys Gln Gly Pro
Thr Phe Gly Gly Gly Thr Lys Leu 50 55 60 Glu Ile Lys 65
* * * * *