U.S. patent application number 12/229203 was filed with the patent office on 2009-07-30 for cytotoxicity mediation of cells evidencing surface expression of trop-2.
This patent application is currently assigned to Arius Research Inc.. Invention is credited to Luis A. G. DaCruz, Alison L. Ferry, Helen P. Findlay, Susan E. Hahn, David S. F. Young.
Application Number | 20090191118 12/229203 |
Document ID | / |
Family ID | 38436895 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191118 |
Kind Code |
A1 |
Young; David S. F. ; et
al. |
July 30, 2009 |
Cytotoxicity mediation of cells evidencing surface expression of
TROP-2
Abstract
The present invention relates to a method for producing
cancerous disease modifying antibodies using a novel paradigm of
screening. By segregating the anti-cancer antibodies using cancer
cell cytotoxicity as an end point, the process makes possible the
production of anti-cancer antibodies for therapeutic and diagnostic
purposes. The antibodies can be used in aid of staging and
diagnosis of a cancer, and can be used to treat primary tumors and
tumor metastases. The anti-cancer antibodies can be conjugated to
toxins, enzymes, radioactive compounds, cytokines, interferons,
target or reporter moieties and hematogenous cells.
Inventors: |
Young; David S. F.;
(Toronto, CA) ; Findlay; Helen P.; (Toronto,
CA) ; Hahn; Susan E.; (Toronto, CA) ; DaCruz;
Luis A. G.; (Toronto, CA) ; Ferry; Alison L.;
(Thornhill, CA) |
Correspondence
Address: |
MCHALE & SLAVIN, P.A.
2855 PGA BLVD
PALM BEACH GARDENS
FL
33410
US
|
Assignee: |
Arius Research Inc.
|
Family ID: |
38436895 |
Appl. No.: |
12/229203 |
Filed: |
August 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11709676 |
Feb 22, 2007 |
7420040 |
|
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12229203 |
|
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60776466 |
Feb 24, 2006 |
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Current U.S.
Class: |
424/1.49 ;
424/133.1; 424/155.1; 424/178.1; 530/387.3; 530/388.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/6851 20170801; A61P 1/18 20180101; A61K 51/1045 20130101;
C07K 2317/565 20130101; A61P 15/00 20180101; A61P 13/08 20180101;
A61P 35/00 20180101; C07K 2317/56 20130101; C07K 2317/24 20130101;
C07K 16/30 20130101; A61P 1/04 20180101; G01N 33/57484
20130101 |
Class at
Publication: |
424/1.49 ;
530/387.3; 424/133.1; 424/178.1; 530/388.1; 424/155.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 51/10 20060101 A61K051/10; C07K 16/00 20060101
C07K016/00; A61P 35/00 20060101 A61P035/00 |
Claims
1. A humanized antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05 or an antigen binding fragment produced from said
humanized antibody.
2. A chimeric antibody of the isolated monoclonal antibody produced
by the hybridoma deposited with the IDAC as accession number
141205-05 or an antigen binding fragment produced from said
chimeric antibody.
3. A method for initiating antibody induced cytotoxicity of
cancerous cells in a tissue sample selected from a human
pancreatic, ovarian, prostate or colon tumor comprising: providing
a tissue sample from said human pancreatic, ovarian, prostate or
colon tumor providing the isolated monoclonal antibody produced by
the hybridoma deposited with the IDAC as accession number
141205-05, the humanized antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05, the chimeric antibody of the isolated
monoclonal antibody produced by the hybridoma deposited with the
IDAC as accession number 141205-05, or an antigen binding fragment
of any of the foregoing, characterized by an ability to
competitively inhibit binding of said isolated monoclonal antibody
to its target antigen; and contacting said isolated monoclonal
antibody, said humanized antibody, said chimeric antibody or said
antigen binding fragment thereof with said tissue sample; wherein
binding of said isolated monoclonal antibody, said humanized
antibody, said chimeric antibody or said antigen binding fragment
thereof with said tissue sample induces cytotoxicity.
4. An antigen binding fragment of the humanized antibody of claim
1.
5. An antigen binding fragment of the chimeric antibody of claim
2.
6. A method of reduction of a human pancreatic, ovarian, prostate
or colon tumor susceptible to antibody induced cytotoxicity in a
mammal, wherein said human pancreatic, ovarian, prostate or colon
tumor expresses at least one epitope of an antigen which
specifically binds to the isolated monoclonal antibody produced by
the hybridoma deposited with the IDAC as accession number 141205-05
or an antigen binding fragment thereof, which antigen binding
fragment is characterized by an ability to competitively inhibit
binding of said isolated monoclonal antibody to its target antigen,
comprising administering to said mammal said monoclonal antibody or
said antigen binding fragment thereof in an amount effective to
result in a reduction of said mammal's pancreatic, ovarian,
prostate or colon tumor burden.
7. The method of claim 6 wherein said isolated monoclonal antibody
is conjugated to a cytotoxic moiety.
8. The method of claim 7 wherein said cytotoxic moiety is a
radioactive isotope.
9. The method of claim 6 wherein said isolated monoclonal antibody
or antigen binding fragment thereof activates complement.
10. The method of claim 6 wherein said isolated monoclonal antibody
or antigen binding fragment thereof mediates antibody dependent
cellular cytotoxicity.
11. The method of claim 6 wherein said isolated monoclonal antibody
is a humanized antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05.
12. The method of claim 6 wherein said isolated monoclonal antibody
is a chimeric antibody of the isolated monoclonal antibody produced
by the hybridoma deposited with the IDAC as accession number
141205-05.
13. A monoclonal antibody which specifically binds to the same
epitope or epitopes as the isolated monoclonal antibody produced by
the hybridoma deposited with the IDAC as accession number
141205-05.
14. A method of reduction of a human pancreatic, ovarian, prostate
or colon tumor in a mammal, wherein said human tumor expresses at
least one epitope of an antigen which specifically binds to the
isolated monoclonal antibody produced by the hybridoma deposited
with the IDAC as accession number 141205-05 or an antigen binding
fragment thereof, which antigen binding fragment is characterized
by an ability to competitively inhibit binding of said isolated
monoclonal antibody to its target antigen, comprising administering
to said mammal said monoclonal antibody or antigen binding fragment
thereof in an amount effective to result in a reduction of said
mammal's pancreatic, ovarian, prostate or colon tumor burden.
15. The method of claim 14 wherein said isolated monoclonal
antibody is conjugated to a cytotoxic moiety.
16. The method of claim 15 wherein said cytotoxic moiety is a
radioactive isotope.
17. The method of claim 14 wherein said isolated monoclonal
antibody or antigen binding fragment thereof activates
complement.
18. The method of claim 14 wherein said isolated monoclonal
antibody or antigen binding fragment thereof mediates antibody
dependent cellular cytotoxicity.
19. The method of claim 14 wherein said isolated monoclonal
antibody is a humanized antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05.
20. The method of claim 14 wherein said isolated monoclonal
antibody is a chimeric antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05.
21. A method of reduction of a human pancreatic, ovarian, prostate
or colon tumor in a mammal, wherein said human pancreatic, ovarian,
prostate or colon tumor expresses at least one epitope of an
antigen which specifically binds to the isolated monoclonal
antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05 or an antigen binding fragment thereof,
which antigen binding fragment is characterized by an ability to
competitively inhibit binding of said isolated monoclonal antibody
to its target antigen, comprising administering to said mammal said
monoclonal antibody or antigen binding fragment thereof in
conjunction with at least one chemotherapeutic agent in an amount
effective to result in a reduction of said mammal's pancreatic,
ovarian, prostate or colon tumor burden.
22. The method of claim 21 wherein said isolated monoclonal
antibody is conjugated to a cytotoxic moiety.
23. The method of claim 22 wherein said cytotoxic moiety is a
radioactive isotope.
24. The method of claim. 21 wherein said isolated monoclonal
antibody or antigen binding fragment thereof activates
complement.
25. The method of claim 21 wherein said isolated monoclonal
antibody or antigen binding fragment thereof mediates antibody
dependent cellular cytotoxicity.
26. The method of claim 21 wherein said isolated monoclonal
antibody is a humanized antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05.
27. The method of claim 21 wherein said isolated monoclonal
antibody is a chimeric antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05.
28. A binding assay to determine a presence of cancerous cells in a
tissue sample selected from a human tumor, which is specifically
bound by the isolated monoclonal antibody produced by hybridoma
cell line AR47A6.4.2 having IDAC Accession No. 141205-05, the
humanized antibody of the isolated monoclonal antibody produced by
the hybridoma deposited with the IDAC as accession number 141205-05
or the chimeric antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05, comprising: providing a tissue sample from said
human tumor; providing at least one of said isolated monoclonal
antibody, said humanized antibody, said chimeric antibody or
antigen binding fragment thereof that recognizes the same epitope
or epitopes as those recognized by the isolated monoclonal antibody
produced by a hybridoma cell line AR47A6.4.2 having IDAC Accession
No. 141205-05; contacting at least one said provided antibodies or
antigen binding fragment thereof with said tissue sample; and
determining binding of said at least one provided antibody or
antigen binding fragment thereof with said tissue sample; whereby
the presence of said cancerous cells in said tissue sample is
indicated.
29. Use of monoclonal antibodies for reduction of human pancreatic,
ovarian, prostate or colon tumor burden, wherein said human
pancreatic, ovarian, prostate or colon tumor expresses at least one
epitope of an antigen which specifically binds to the isolated
monoclonal antibody produced by the hybridoma deposited with the
IDAC as accession number 141205-05 or an antigen binding fragment
thereof, which antigen binding fragment is characterized by an
ability to competitively inhibit binding of said isolated
monoclonal antibody to its target antigen, comprising administering
to said mammal said monoclonal antibody or antigen binding fragment
thereof in an amount effective to result in a reduction of said
mammal's human pancreatic, ovarian, prostate or colon tumor
burden.
30. The method of claim 29 wherein said isolated monoclonal
antibody is conjugated to a cytotoxic moiety.
31. The method of claim 30 wherein said cytotoxic moiety is a
radioactive isotope.
32. The method of claim 29 wherein said isolated monoclonal
antibody or antigen binding fragment thereof activates
complement.
33. The method of claim 29 wherein said isolated monoclonal
antibody or antigen binding fragment thereof mediates antibody
dependent cellular cytotoxicity.
34. The method of claim 29 wherein said isolated monoclonal
antibody is a humanized antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05.
35. The method of claim 29 wherein said isolated monoclonal
antibody is a chimeric antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05.
36. Use of monoclonal antibodies for reduction of human pancreatic,
ovarian, prostate or colon tumor burden, wherein said human
pancreatic, ovarian, prostate or colon tumor expresses at least one
epitope of an antigen which specifically binds to the isolated
monoclonal antibody produced by the hybridoma deposited with the
IDAC as accession number 141205-05 or an antigen binding fragment
thereof, which antigen binding fragment is characterized by an
ability to competitively inhibit binding of said isolated
monoclonal antibody to its target antigen, comprising administering
to said mammal said monoclonal antibody or antigen binding fragment
thereof; in conjunction with at least one chemotherapeutic agent in
an amount effective to result in a reduction of said mammal's human
pancreatic, ovarian, prostate or colon tumor burden.
37. The method of claim 36 wherein said isolated monoclonal
antibody is conjugated to a cytotoxic moiety.
38. The method of claim 37 wherein said cytotoxic moiety is a
radioactive isotope.
39. The method of claim 36 wherein said isolated monoclonal
antibody or antigen binding fragment thereof activates
complement.
40. The method of claim 36 wherein said isolated monoclonal
antibody or antigen binding fragment thereof mediates antibody
dependent cellular cytotoxicity.
41. The method of claim 36 wherein said isolated monoclonal
antibody is a humanized antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05.
42. The method of claim 36 wherein said isolated monoclonal
antibody is a chimeric antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the IDAC as accession
number 141205-05.
43. A composition effective for treating a human pancreatic,
ovarian, prostate or colon tumor comprising in combination: an
antibody or antigen binding fragment of any one of claims 1, 2, 4,
5, 13, 45 or 46; a conjugate of said antibody or an antigen binding
fragment thereof with a member selected from the group consisting
of cytotoxic moieties, enzymes, radioactive compounds, cytokines,
interferons, target or reporter moieties and hematogenous cells;
and a requisite amount of a pharmaceutically acceptable carrier;
wherein said composition is effective for treating said human
pancreatic, ovarian, prostate or colon tumor.
44. An assay kit for detecting the presence of a human cancerous
tumor, wherein said human cancerous tumor expresses at least one
epitope of an antigen which specifically binds to the isolated
monoclonal antibody produced by the hybridoma deposited with the
IDAC as accession number 141205-05 or an antigen binding fragment
thereof, which antigen binding fragment is characterized by an
ability to competitively inhibit binding of said isolated
monoclonal antibody to its target antigen, the kit comprising the
isolated monoclonal antibody produced by the hybridoma deposited
with the IDAC as accession number 141205-05 or an antigen binding
fragment thereof, and means for detecting whether the monoclonal
antibody, or an antigen binding fragment thereof, is bound to a
polypeptide whose presence, at a particular cut-off level, is
diagnostic of said presence of said human cancerous tumor.
45. An isolated monoclonal antibody or antigen binding fragment
thereof, which specifically binds to human TROP-2, in which the
isolated monoclonal antibody or antigen binding fragment thereof
reacts with the same epitope or epitopes of human TROP-2 as the
isolated monoclonal antibody produced by a hybridoma cell line
AR47A6.4.2 having IDAC Accession No. 141205-05; said isolated
monoclonal antibody or antigen binding fragment thereof being
characterized by an ability to competitively inhibit binding of
said isolated monoclonal antibody to its target human TROP-2
antigen.
46. An isolated monoclonal antibody or antigen binding fragment
thereof that recognizes the same epitope or epitopes as those
recognized by the isolated monoclonal antibody produced by the
hybridoma cell line AR47A6.4.2 having IDAC Accession No 141205-05;
said monoclonal antibody or antigen binding fragment thereof being
characterized by an ability to competitively inhibit binding of
said isolated monoclonal antibody to its target epitope or
epitopes.
47. A process for reduction of a human pancreatic, ovarian,
prostate or colon tumor which expresses at least one epitope of
human TROP-2 antigen which is specifically bound by the isolated
monoclonal antibody produced by hybridoma cell line AR47A6.4.2
having IDAC Accession No. 141205-05, comprising: administering to
an individual suffering from said human tumor, at least one
isolated monoclonal antibody or antigen binding fragment thereof
that binds the same epitope or epitopes as those bound by the
isolated monoclonal antibody produced by the hybridoma cell line
AR47A6.4.2 having IDAC Accession No. 141205-05; wherein binding of
said epitope or epitopes results in a reduction of pancreatic,
ovarian, prostate or colon tumor burden.
48. A process for reduction of a human pancreatic, ovarian,
prostate or colon tumor which expresses at least one epitope of
human TROP-2 antigen which is specifically bound by the isolated
monoclonal antibody produced by hybridoma cell line AR47A6.4.2
having IDAC Accession No. 141205-05, comprising: administering to
an individual suffering from said human tumor, at least one
isolated monoclonal antibody or antigen binding fragment thereof,
that binds the same epitope or epitopes as those bound by the
isolated monoclonal antibody produced by the hybridoma cell line
AR47A6.4.2 having IDAC Accession No. 141205-05; in conjunction with
at least one chemotherapeutic agent; wherein said administration
results in a reduction of tumor burden.
49. A binding assay to determine the presence of cells which
express TROP-2 which is specifically recognized by the isolated
monoclonal antibody produced by the hybridoma cell line AR47A6.4.2
having IDAC Accession No. 141205-05, or an antigen binding fragment
produced from said isolated monoclonal antibody comprising:
providing a cell sample; providing the isolated monoclonal antibody
produced by the hybridoma cell line AR47A6.4.2 having IDAC
Accession No. 141205-05 or said antigen binding fragment produced
from the isolated monoclonal antibody; contacting said isolated
monoclonal antibody or said antigen binding fragment with said cell
sample; and determining binding of said isolated monoclonal
antibody or antigen binding fragment thereof with said cell sample;
whereby the presence of cells which express an antigen of TROP-2
which is specifically bound by said isolated monoclonal antibody or
said antigen binding fragment is determined.
50. A method of extending survival and delaying disease progression
by treating a human pancreatic, ovarian, prostate or colon tumor in
a mammal, wherein said tumor expresses an antigen which
specifically binds to the isolated monoclonal antibody produced by
the hybridoma cell line AR47A6.4:2 having IDAC Accession No.
141205-05, or an antigen binding fragment produced from said
isolated monoclonal antibody comprising administering to said
mammal said monoclonal antibody in an amount effective to reduce
said mammal's tumor burden, whereby disease progression is delayed
and survival is extended.
51. A method of extending survival and delaying disease progression
by treating a human pancreatic, ovarian, prostate or colon tumor in
a mammal, wherein said tumor expresses TROP-2 which specifically
binds to the isolated monoclonal antibody produced by the hybridoma
cell line AR47A6.4.2 having IDAC Accession No. 141205-05, or a
TROP-2 binding fragment produced from said isolated monoclonal
antibody comprising administering to said mammal said monoclonal
antibody in an amount effective to reduce said mammal's tumor
burden, whereby disease progression is delayed and survival is
extended.
52. A composition effective for treating a human pancreatic,
ovarian, prostate or colon tumor comprising in combination: an
antibody or antigen binding fragment of any one of claims 1, 2, 4,
5, 13, 45 or 46; and a requisite amount of a pharmaceutically
acceptable carrier; wherein said composition is effective for
treating said human pancreatic, ovarian, prostate or colon
tumor.
53. A composition effective for treating a human pancreatic,
ovarian, prostate or colon tumor comprising in combination: a
conjugate of an antibody or antigen binding fragment of any one of
claims 1, 2, 4, 5, 13, 45 or 46 with a member selected from the
group consisting of cytotoxic moieties, enzymes, radioactive
compounds, cytokines, interferons, target or reporter moieties and
hematogenous cells; and a requisite amount of a pharmaceutically
acceptable carrier; wherein said composition is effective for
treating said human pancreatic, ovarian, prostate or colon tumor.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/709,676, filed on Feb. 22, 2007, which claims benefit
of the filing date of U.S. Provisional Patent Application No.
60/776,466, filed on Feb. 24, 2006, the contents of which are
herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the isolation and production of
cancerous disease modifying antibodies (CDMAB) and to the use of
these CDMAB alone or in combination with one or more
CDMAB/chemotherapeutic agents in therapeutic and diagnostic
processes. The invention further relates to binding assays which
utilize the CDMAB of the instant invention.
BACKGROUND OF THE INVENTION
[0003] TROP-2 is a cell surface glycoprotein expressed on most
carcinomas, as well as some normal human tissues. It was initially
defined as a molecule recognized by two murine monoclonal
antibodies raised to a human choriocarcinoma cell line BeWo that
recognized an antigen on human trophoblast cells (Faulk 1978). The
same molecule was independently discovered by other investigators
which led to multiple names describing the same antigen. Hence,
TROP-2 was also referred to as GA733-1 and epithelial
glycoprotein-1 (EGP-1) (Basu 1995, Fornaro 1995).
[0004] The TROP-2 gene is an intronless gene that was thought to
have been formed through the retroposition of a homologous gene GA
733-2 (also known as epithelial glycoprotein-2, EpCAM and Trop-1)
via an RNA intermediate. The TROP-2 gene has been mapped to
chromosome 1p32 (Calabrese 2001). The protein component of TROP-2
has a molecular mass of approximately 35 kilodaltons. Its mass may
be increased by 11-13 kilodaltons with heterogeneous N-linked
glycosylation of its extracellular domain. There are many cysteine
residues in the extracellular domain which could form disulfide
bridge sites. TROP-2 is a substrate for protein kinase C, a
Ca.sup.2+ dependent protein kinase and the intracellular serine 303
residue has been shown to be phosphorylated (Basu 1995). It has
also been shown that crossing-linking of TROP-2 with anti-TROP-2
antibodies transduced a calcium signal as shown by a rise in
cytoplasmic Ca.sup.2+ (Ripani 1998). These data support signal
transduction as a physiological function of TROP-2, although to
date no physiological ligand has been identified. Recently an
association between TROP-2 expression and cancer has been shown as
TROP-2 was identified as a member of a group of genes reported to
be the most highly overexpressed in ovarian serous papillary
carcinoma compared to normal ovarian epithelium in a large-scale
gene expression analysis using cDNA microarray technology (Santin
2004).
[0005] The expression profile of TROP-2 has been elucidated through
immunohistochemistry (IHC) and flow cytometry studies using many
different TROP-2 antibodies. Anti-TROP-2 antibodies 162-25.3 and
162-46.2 were produced through immunization of mice with the human
choriocarcinoma cell line BeWo, and were investigated for their
reactivity to a series of tumor and lymphoid cell lines and
peripheral blood mononuclear cells. In this study both antibodies
appeared to be trophoblast specific, staining 3 of the 4
choriocarcinoma cell lines tested, while none of the other lymphoid
or tumor cell lines (representing fibrosarcoma, cervical sarcoma,
colon carcinoma, melanoma, neuroblastoma, erythroleukemia) were
stained in an indirect immunofluorescence FACS assay. In addition,
none of the normal peripheral blood cells were stained. The
antibodies were tested for staining of formalin-fixed
paraffin-embedded placenta tissue sections and frozen normal
sections of liver, kidney, spleen, thymus and lymph node tissues.
The placenta tissue sections were stained with both antibodies,
while there was no staining of the other normal tissues (Lipinski
1981). These two antibodies have strictly been reported for use in
in vitro diagnostic studies.
[0006] Anti-TROP-2 antibody MOv16 was generated through the
immunization of mice with a crude membrane preparation of poorly
differentiated ovarian carcinoma OvCa4343/83. MOv16 was tested for
reactivity to a series of frozen tissue sections of benign and
malignant ovarian tumors. MOv16 reacted with 31 of 54 malignant
ovarian tumors and 2 of 16 benign ovarian tumors. Of the 5 mucinous
ovarian tumors that were tested, MOv16 was completely unreactive.
MOv16 was also tested for reactivity to frozen sections of
non-ovarian malignant tumors where it was found to bind 117 of 189
breast carcinoma sections and 12 of 18 lung carcinoma sections.
MOv16 was completely unreactive on 16 non-epithelial tumors that
were tested (including liposarcomas, chondrosarcomas,
endotheliomas, histiocytomas and dysgerminomas). When tested on
frozen normal tissue sections, MOv-16 was reactive with breast,
pancreas, kidney and prostate sections. MOv16 reactivity was
reported to be negative on lung, spleen, skin, ovary, thyroid,
parotid gland, stomach, larynx, uterus and colon sections, though
the number of tissue sections that were used was not reported. The
authors noted that frozen tissue sections were used because MOv16
was unreactive to paraffin embedded tissues (Miotti 1987). This
antibody has also only been reported for use in in vitro diagnostic
studies.
[0007] Anti-TROP-2 antibody Rs7-3G11 (RS7) was generated through
the immunization of mice with a crude membrane preparation derived
from a surgically removed human primary squamous cell carcinoma of
the lung. IHC was used to examine the staining of RS7 on frozen
sections of human tumor and normal tissues. RS7 bound to 33 of the
40 sections representing tumors of the breast, colon, kidney, lung,
prostate and squamous cell cancer. Of the normal tissues RS7 bound
to 16 of 20 sections of breast, colon, kidney, liver, lung and
prostate tissues while none of the five sections of spleen tissue
were stained. In this study the authors noted that it appeared that
antigen density in tumors was higher than in normal epithelial
tissues (Stein 1990).
[0008] Additional studies of the tissue specificity of RS7 were
carried out on both tumor and normal tissues. RS7 was tested on a
panel of frozen tumor sections and bound to 65 of the 77 sections
representing tumors of the lung, stomach, kidney, bladder, colon,
breast, ovary, uterus and prostate. There was no binding to the 5
lymphomas tested. RS7 was tested on a panel of 85 frozen human
normal tissue sections composed of a total of 24 tissue types. 39
sections of 13 normal tissues (lung, bronchus, trachea, esophagus,
colon, liver, pancreas, kidney, bladder, skin, thyroid, breast and
prostate) were stained by RS7. The authors of this study noted that
in the tissues in which positive staining was observed, the
reactivity was generally restricted to epithelial cells, primarily
in ducts or glands. It was also noted that this study was limited
to frozen sections since it was observed that RS7 was not reactive
on formalin-fixed paraffin-embedded sections (Stein 1993).
[0009] Polyclonal anti-TROP-2 antibodies were prepared by
immunizing mice with a synthetic peptide corresponding to amino
acid positions between 169 and 182 of the cytoplasmic domain of
human TROP-2. The polyclonal antibodies were tested on a tissue
array slide that contained formalin-fixed human esophageal
hyperplasia and carcinoma tissues. Ten of the 55 carcinoma
specimens displayed heavy staining with the polyclonal antibodies,
while the mild hyperplasia tissue stained very weakly, indicating
expression levels may be related to malignant transformation
(Nakashima 2004).
[0010] Overall, IHC reactivity patterns obtained with different
anti-TROP-2 antibodies were consistent. Expression in cancer was
seen primarily in carcinomas, and most carcinomas were reactive. In
normal tissues, expression appeared to be limited to cells of
epithelial origin, and there was some evidence that staining of
carcinomas was stronger than staining of corresponding normal
epithelial tissues.
[0011] In addition to being used in IHC studies, antibody RS7 was
tested in in vivo models with initial experiments consisting of
tumor targeting studies in nude mouse xenograft models.
Radiolabeled RS7 injected i.v. was shown to accumulate specifically
in the tumor of mice bearing either Calu-3 (lung adenocarcinoma) or
GW-39 (colon carcinoma) tumors (Stein 1990). Further studies were
done to investigate the biodistribution of radiolabeled RS7 in a
xenograft system and to study the therapeutic potential of RS7 as
an immunoconjugate. In this study the therapeutic efficacy of
.sup.131I-labeled RS7 F(ab').sub.2 was investigated in nude mice
bearing Calu-3 human lung adenocarcinoma xenografts. Three weeks
following inoculation of the mice with Calu-3 cells, when the
tumors had reached a size of approximately 0.3-0.9 grams, groups of
6-7 mice were treated with a single dose i.v. of either 1.0 mCi
.sup.131I-RS7-F(ab').sub.2 or 1.5 mCi .sup.131I-RS7-F(ab').sub.2
and compared to a similar group of untreated control mice. The
single dose of 1.0 mCi .sup.131I-RS7-F(ab').sub.2 resulted in tumor
growth suppression for approximately 5 weeks, while the single dose
of 1.5 mCi .sup.131I-RS7-F(ab').sub.2 resulted in tumor regression,
and the mean tumor size did not exceed the pre-therapy size until
the eighth week after radioantibody injection. Mice receiving the
1.5 mCi .sup.131I-RS7-F(ab').sub.2 dose experienced a mean body
weight loss of 18.7 percent, indicating there was toxicity
associated with the treatment. In this study, effects of treatment
with naked RS7 or the F(ab').sub.2 fragment of RS7 were not tested
(Stein 1994a). Another study was done to test the efficacy of
.sup.131I-RS7 in a MDA-MB-468 breast cancer xenograft model. Groups
of ten mice bearing MDA-MB-468 tumors of approximately 0.1 cm.sup.3
were treated with a single dose i.v. of either 250 microcuries
.sup.131I-RS7 or 250 microcuries .sup.131I-Ag8 (an isotype matched
control antibody). Groups of six mice were treated with a single
dose i.v. of 30 micrograms of either unlabeled RS7 or Ag8. Complete
regression of the tumors (except for one animal that had a
transient reappearance of tumor) was seen in the animals treated
with .sup.131I-RS7, which lasted for the duration of the 11 week
observation period. Tumor regression was also seen in .sup.131I-Ag8
treated mice, though was only observed between 2 weeks and 5 weeks
with tumors either persisting or continuing to grow for the
remainder of the study. Tumor growth of mice that received
unlabeled RS7 or Ag8 was not inhibited and there did not appear to
be any differences in the mean tumor volume of RS7 treated mice
compared to the Ag8 treated mice. Two additional groups of 10 mice
bearing larger MDA-MB-468 tumors of approximately 0.2-0.3 cm.sup.3
were treated with a slightly higher single dose of either 275
microcuries .sup.131I-Rs7 or 275 microcuries .sup.131Ag8 and
compared to a similar group of untreated mice. Tumor volume was
measured weekly for 15 weeks. Although in this case there was a
significant difference in tumor growth between the .sup.131I-RS7
treated mice compared to the untreated mice, there was no
significant difference in the tumor growth of the .sup.131I-RS7
compared to the .sup.131I-Ag8 treated mice, indicating a portion of
the efficacy may have been due to non-specific effects of the
radiation. Unlabeled antibodies were not tested in mice containing
0.2-0.3 cm.sup.3 tumors (Shih 1995).
[0012] There have been numerous additional studies examining the
efficacy of RS7 as an immunoconjugate with an attempt to select the
optimal radiolabel for radioimmunotherapy (Stein 2001a, Stein
2001b, Stein 2003). A humanized version of RS7 has also been
generated, however it has only been tested in preclinical xenograft
models as a radioconjugate (Govindan 2004). These studies show
similar positive effects as the previously described studies with
RS7, however in one study, even when radiolabeled RS7 was delivered
at a previously determined maximum tolerable dose, toxicity
occurred leading to death in some mice (Stein 2001a). Although
effective treatment of xenograft tumors in mice was achieved with
radiolabeled RS7 in these studies, naked RS7 was not evaluated.
[0013] Immunizing mice with neuraminidase pre-treated H3922 human
breast carcinoma cells produced the anti-TROP-2 monoclonal antibody
BR110 (as disclosed in U.S. Pat. No. 5,850,854, refer to Prior
Patents section). By immunohistology, using human frozen tissue
specimens, BR110 was shown to react with a wide range of human
carcinoma specimens including those of the lung, colon, breast,
ovarian, kidney, esophagus, pancreas, skin, lung and tonsil. No
human normal tissue sections were tested. In vitro studies
demonstrated that BR110 had no ADCC or CDC activity on the human
carcinoma cell lines H3396 or H3922. In vitro studies analyzing the
cytotoxicity of BR110-immunotoxins was performed on the human
cancer cell lines H3619, H2987, MCF-7, H3396 and H2981. The
EC.sub.50 for the cell lines tested was 0.06, 0.001, 0.05, 0.09 and
>5 micrograms/mL respectively. No cytotoxicity data was
disclosed for the naked BR110 antibody. No in vivo data was
disclosed for the naked or immunoconjugated BR110.
[0014] A number of additional antibodies have been generated that
target TROP-2, such as MR54, MR6 and MR23 which were generated from
immunization of mice with the ovarian cancer cell line Colo 316
(Stein 1994b) and antibody T16 which was generated by immunization
of mice with the T24 bladder cancer cell line (Fradet 1984). The
use of these antibodies has been limited to biochemical
characterization of the TROP-2 antigen and cell line and tissue
expression studies. There have been no reports of anti-cancer
efficacy of these antibodies, either in vitro or in vivo. RS7 was
the only antibody that was tested for therapeutic efficacy in
preclinical cancer models, with its use being limited to a carrier
of radioisotope. There are no reports of any naked TROP-2
antibodies exhibiting therapeutic efficacy in preclinical cancer
models either in vitro or in vivo.
[0015] Monoclonal Antibodies as Cancer Therapy: Each individual who
presents with cancer is unique and has a cancer that is as
different from other cancers as that person's identity. Despite
this, current therapy treats all patients with the same type of
cancer, at the same stage, in the same way. At least 30 percent of
these patients will fail the first line therapy, thus leading to
further rounds of treatment and the increased probability of
treatment failure, metastases, and ultimately, death. A superior
approach to treatment would be the customization of therapy for the
particular individual. The only current therapy which lends itself
to customization is surgery. Chemotherapy and radiation treatment
cannot be tailored to the patient, and surgery by itself, in most
cases is inadequate for producing cures.
[0016] With the advent of monoclonal antibodies, the possibility of
developing methods for customized therapy became more realistic
since each antibody can be directed to a single epitope.
Furthermore, it is possible to produce a combination of antibodies
that are directed to the constellation of epitopes that uniquely
define a particular individual's tumor.
[0017] Having recognized that a significant difference between
cancerous and normal cells is that cancerous cells contain antigens
that are specific to transformed cells, the scientific community
has long held that monoclonal antibodies can be designed to
specifically target transformed cells by binding specifically to
these cancer antigens; thus giving rise to the belief that
monoclonal antibodies can serve as "Magic Bullets" to eliminate
cancer cells. However, it is now widely recognized that no single
monoclonal antibody can serve in all instances of cancer, and that
monoclonal antibodies can be deployed, as a class, as targeted
cancer treatments. Monoclonal antibodies isolated in accordance
with the teachings of the instantly disclosed invention have been
shown to modify the cancerous disease process in a manner which is
beneficial to the patient, for example by reducing the tumor
burden, and will variously be referred to herein as cancerous
disease modifying antibodies (CDMAB) or "anti-cancer"
antibodies.
[0018] At the present time, the cancer patient usually has few
options of treatment. The regimented approach to cancer therapy has
produced improvements in global survival and morbidity rates.
However, to the particular individual, these improved statistics do
not necessarily correlate with an improvement in their personal
situation.
[0019] Thus, if a methodology was put forth which enabled the
practitioner to treat each tumor independently of other patients in
the same cohort, this would permit the unique approach of tailoring
therapy to just that one person. Such a course of therapy would,
ideally, increase the rate of cures, and produce better outcomes,
thereby satisfying a long-felt need.
[0020] Historically, the use of polyclonal antibodies has been used
with limited success in the treatment of human cancers. Lymphomas
and leukemias have been treated with human plasma, but there were
few prolonged remission or responses. Furthermore, there was a lack
of reproducibility and there was no additional benefit compared to
chemotherapy. Solid tumors such as breast cancers, melanomas and
renal cell carcinomas have also been treated with human blood,
chimpanzee serum, human plasma and horse serum with correspondingly
unpredictable and ineffective results.
[0021] There have been many clinical trials of monoclonal
antibodies for solid tumors. In the 1980s there were at least four
clinical trials for human breast cancer which produced only one
responder from at least 47 patients using antibodies against
specific antigens or based on tissue selectivity. It was not until
1998 that there was a successful clinical trial using a humanized
anti-Her2/neu antibody (Herceptin.RTM.) in combination with
CISPLATIN. In this trial 37 patients were assessed for responses of
which about a quarter had a partial response rate and an additional
quarter had minor or stable disease progression. The median time to
progression among the responders was 8.4 months with median
response duration of 5.3 months.
[0022] Herceptin.RTM. was approved in 1998 for first line use in
combination with Taxol.RTM.. Clinical study results showed an
increase in the median time to disease progression for those who
received antibody therapy plus Taxol.RTM. (6.9 months) in
comparison to the group that received Taxol.RTM. alone (3.0
months). There was also a slight increase in median survival; 22
versus 18 months for the Herceptin.RTM. plus Taxol.RTM. treatment
arm versus the Taxol.RTM. treatment alone arm. In addition, there
was an increase in the number of both complete (8 versus 2 percent)
and partial responders (34 versus 15 percent) in the antibody plus
Taxol.RTM. combination group in comparison to Taxol.RTM. alone.
However, treatment with Herceptin.RTM. and Taxol.RTM. led to a
higher incidence of cardiotoxicity in comparison to Taxol.RTM.
treatment alone (13 versus 1 percent respectively). Also,
Herceptin.RTM. therapy was only effective for patients who over
express (as determined through immunohistochemistry (IHC) analysis)
the human epidermal growth factor receptor 2 (Her2/neu), a
receptor, which currently has no known function or biologically
important ligand; approximately 25 percent of patients who have
metastatic breast cancer. Therefore, there is still a large unmet
need for patients with breast cancer. Even those who can benefit
from Herceptin.RTM. treatment would still require chemotherapy and
consequently would still have to deal with, at least to some
degree, the side effects of this kind of treatment.
[0023] The clinical trials investigating colorectal cancer involve
antibodies against both glycoprotein and glycolipid targets.
Antibodies such as 17-1A, which has some specificity for
adenocarcinomas, has undergone Phase 2 clinical trials in over 60
patients with only 1 patient having a partial response. In other
trials, use of 17-1A produced only 1 complete response and 2 minor
responses among 52 patients in protocols using additional
cyclophosphamide. To date, Phase III clinical trials of 17-1A have
not demonstrated improved efficacy as adjuvant therapy for stage
III colon cancer. The use of a humanized murine monoclonal antibody
initially approved for imaging also did not produce tumor
regression.
[0024] Only recently have there been any positive results from
colorectal cancer clinical studies with the use of monoclonal
antibodies. In 2004, ERBITUX.RTM. was approved for the second line
treatment of patients with EGFR-expressing metastatic colorectal
cancer who are refractory to irinotecan-based chemotherapy. Results
from both a two-arm Phase II clinical study and a single arm study
showed that ERBITUX.RTM. in combination with irinotecan had a
response rate of 23 and 15 percent respectively with a median time
to disease progression of 4.1 and 6.5 months respectively. Results
from the same two-arm Phase II clinical study and another single
arm study showed that treatment with ERBITUX.RTM. alone resulted in
an 11 and 9 percent response rate respectively with a median time
to disease progression of 1.5 and 4.2 months respectively.
[0025] Consequently in both Switzerland and the United States,
ERBITUX.RTM. treatment in combination with irinotecan, and in the
United States, ERBITUX.RTM. treatment alone, has been approved as a
second line treatment of colon cancer patients who have failed
first line irinotecan therapy. Therefore, like Herceptin.RTM.,
treatment in Switzerland is only approved as a combination of
monoclonal antibody and chemotherapy. In addition, treatment in
both Switzerland and the US is only approved for patients as a
second line therapy. Also, in 2004, AVASTIN.RTM. was approved for
use in combination with intravenous 5-fluorouracil-based
chemotherapy as a first line treatment of metastatic colorectal
cancer. Phase III clinical study results demonstrated a
prolongation in the median survival of patients treated with
AVASTIN.RTM. plus 5-fluorouracil compared to patients treated with
5-fluorouracil alone (20 months versus 16 months respectively).
However, again like Herceptin.RTM. and ERBITUX.RTM., treatment is
only approved as a combination of monoclonal antibody and
chemotherapy.
[0026] There also continues to be poor results for lung, brain,
ovarian, pancreatic, prostate, and stomach cancer. The most
promising recent results for non-small cell lung cancer came from a
Phase II clinical trial where treatment involved a monoclonal
antibody (SGN-15; dox-BR96, anti-Sialyl-LeX) conjugated to the
cell-killing drug doxorubicin in combination with the
chemotherapeutic agent TAXOTERE.RTM.. TAXOTERE.RTM. is the only FDA
approved chemotherapy for the second line treatment of lung cancer.
Initial data indicate an improved overall survival compared to
TAXOTERE.RTM. alone. Out of the 62 patients who were recruited for
the study, two-thirds received SGN-15 in combination with
TAXOTERE.RTM. while the remaining one-third received TAXOTERE.RTM.
alone. For the patients receiving SGN-15 in combination with
TAXOTERE.RTM., median overall survival was 7.3 months in comparison
to 5.9 months for patients receiving TAXOTERE.RTM. alone. Overall
survival at 1 year and 18 months was 29 and 18 percent respectively
for patients receiving SNG-15 plus TAXOTERE.RTM. compared to 24 and
8 percent respectively for patients receiving TAXOTERE.RTM. alone.
Further clinical trials are planned.
[0027] Preclinically, there has been some limited success in the
use of monoclonal antibodies for melanoma. Very few of these
antibodies have reached clinical trials and to date none have been
approved or demonstrated favorable results in Phase III clinical
trials.
[0028] The discovery of new drugs to treat disease is hindered by
the lack of identification of relevant targets among the products
of 30,000 known genes that could contribute to disease
pathogenesis. In oncology research, potential drug targets are
often selected simply due to the fact that they are over-expressed
in tumor cells. Targets thus identified are then screened for
interaction with a multitude of compounds. In the case of potential
antibody therapies, these candidate compounds are usually derived
from traditional methods of monoclonal antibody generation
according to the fundamental principles laid down by Kohler and
Milstein (1975, Nature, 256, 495-497, Kohler and Milstein). Spleen
cells are collected from mice immunized with antigen (e.g. whole
cells, cell fractions, purified antigen) and fused with
immortalized hybridoma partners. The resulting hybridomas are
screened and selected for secretion of antibodies which bind most
avidly to the target. Many therapeutic and diagnostic antibodies
directed against cancer cells, including Herceptin.RTM. and
RITUXIMAB, have been produced using these methods and selected on
the basis of their affinity. The flaws in this strategy are
two-fold. Firstly, the choice of appropriate targets for
therapeutic or diagnostic antibody binding is limited by the
paucity of knowledge surrounding tissue specific carcinogenic
processes and the resulting simplistic methods, such as selection
by overexpression, by which these targets are identified. Secondly,
the assumption that the drug molecule that binds to the receptor
with the greatest affinity usually has the highest probability for
initiating or inhibiting a signal may not always be the case.
[0029] Despite some progress with the treatment of breast and colon
cancer, the identification and development of efficacious antibody
therapies, either as single agents or co-treatments, have been
inadequate for all types of cancer.
Prior Patents:
[0030] U.S. Pat. No. 5,750,102 discloses a process wherein cells
from a patient's tumor are transfected with MHC genes which may be
cloned from cells or tissue from the patient. These transfected
cells are then used to vaccinate the patient.
[0031] U.S. Pat. No. 4,861,581 discloses a process comprising the
steps of obtaining monoclonal antibodies that are specific to an
internal cellular component of neoplastic and normal cells of the
mammal but not to external components, labeling the monoclonal
antibody, contacting the labeled antibody with tissue of a mammal
that has received therapy to kill neoplastic cells, and determining
the effectiveness of therapy by measuring the binding of the
labeled antibody to the internal cellular component of the
degenerating neoplastic cells. In preparing antibodies directed to
human intracellular antigens, the patentee recognizes that
malignant cells represent a convenient source of such antigens.
[0032] U.S. Pat. No. 5,171,665 provides a novel antibody and method
for its production. Specifically, the patent teaches formation of a
monoclonal antibody which has the property of binding strongly to a
protein antigen associated with human tumors, e.g. those of the
colon and lung, while binding to normal cells to a much lesser
degree.
[0033] U.S. Pat. No. 5,484,596 provides a method of cancer therapy
comprising surgically removing tumor tissue from a human cancer
patient, treating the tumor tissue to obtain tumor cells,
irradiating the tumor cells to be viable but non-tumorigenic, and
using these cells to prepare a vaccine for the patient capable of
inhibiting recurrence of the primary tumor while simultaneously
inhibiting metastases. The patent teaches the development of
monoclonal antibodies which are reactive with surface antigens of
tumor cells. As set forth at col. 4, lines 45 et seq., the
patentees utilize autochthonous tumor cells in the development of
monoclonal antibodies expressing active specific immunotherapy in
human neoplasia.
[0034] U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen
characteristic of human carcinomas and not dependent upon the
epithelial tissue of origin.
[0035] U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies
which induce apoptosis in Her2 expressing cells, hybridoma cell
lines producing the antibodies, methods of treating cancer using
the antibodies and pharmaceutical compositions including said
antibodies.
[0036] U.S. Pat. No. 5,849,876 describes new hybridoma cell lines
for the production of monoclonal antibodies to mucin antigens
purified from tumor and non-tumor tissue sources.
[0037] U.S. Pat. No. 5,869,268 is drawn to a method for generating
a human lymphocyte producing an antibody specific to a desired
antigen, a method for producing a monoclonal antibody, as well as
monoclonal antibodies produced by the method. The patent is
particularly drawn to the production of an anti-HD human monoclonal
antibody useful for the diagnosis and treatment of cancers.
[0038] U.S. Pat. No. 5,869,045 relates to antibodies, antibody
fragments, antibody conjugates and single-chain immunotoxins
reactive with human carcinoma cells. The mechanism by which these
antibodies function is two-fold, in that the molecules are reactive
with cell membrane antigens present on the surface of human
carcinomas, and further in that the antibodies have the ability to
internalize within the carcinoma cells, subsequent to binding,
making them especially useful for forming antibody-drug and
antibody-toxin conjugates. In their unmodified form the antibodies
also manifest cytotoxic properties at specific concentrations.
[0039] U.S. Pat. No. 5,780,033 discloses the use of autoantibodies
for tumor therapy and prophylaxis. However, this antibody is an
antinuclear autoantibody from an aged mammal. In this case, the
autoantibody is said to be one type of natural antibody found in
the immune system. Because the autoantibody comes from "an aged
mammal", there is no requirement that the autoantibody actually
comes from the patient being treated. In addition the patent
discloses natural and monoclonal antinuclear autoantibody from an
aged mammal, and a hybridoma cell line producing a monoclonal
antinuclear autoantibody.
[0040] U.S. Pat. No. 5,850,854 discloses a specific antibody, BR110
directed against GA733-1. This patent discloses in vitro function
for BR110 as an immunotoxin conjugate. There was no in vitro
function as a naked antibody disclosed for this antibody. There was
also no in vivo function disclosed for this antibody.
[0041] U.S. Pat. No. 6,653,104 claims immunotoxin-conjugated
antibodies, including but not limited to RS7, directed against a
host of antigens, including but not limited to EGP-1. The
immunotoxin is limited to those possessing ribonucleolytic
activity. However, the examples disclose only a specific
immunotoxin-conjugated antibody, LL2, directed against CD22. There
was no in vitro or in vivo function for RS7 disclosed in this
application.
[0042] U.S. Application No. 20040001825A1 discloses a specific
antibody, RS7 directed against EGP-1. This application discloses in
vitro function for RS7 as a radiolabeled conjugate. There was no in
vitro function as a naked antibody disclosed for this antibody.
This application also discloses in vivo function for RS7 resulting
from radiolabeled and unlabeled conjugate administered
sequentially. However, this study was limited to one patient and it
is unknown whether any of the observed function was due to the
unlabeled antibody. There was no in vivo function for RS7 resulting
from the administration of the naked antibody.
SUMMARY OF THE INVENTION
[0043] This application utilizes methodology for producing patient
specific anti-cancer antibodies taught in the U.S. Pat. No.
6,180,357 patent for isolating hybridoma cell lines which encode
for cancerous disease modifying monoclonal antibodies. These
antibodies can be made specifically for one tumor and thus make
possible the customization of cancer therapy. Within the context of
this application, anti-cancer antibodies having either cell-killing
(cytotoxic) or cell-growth inhibiting (cytostatic) properties will
hereafter be referred to as cytotoxic. These antibodies can be used
in aid of staging and diagnosis of a cancer, and can be used to
treat tumor metastases. These antibodies can also be used for the
prevention of cancer by way of prophylactic treatment. Unlike
antibodies generated according to traditional drug discovery
paradigms, antibodies generated in this way may target molecules
and pathways not previously shown to be integral to the growth
and/or survival of malignant tissue. Furthermore, the binding
affinities of these antibodies are suited to requirements for
initiation of the cytotoxic events that may not be amenable to
stronger affinity interactions. Also, it is within the purview of
this invention to conjugate standard chemotherapeutic modalities,
e.g. radionuclides, with the CDMAB of the instant invention,
thereby focusing the use of said chemotherapeutics. The CDMAB can
also be conjugated to toxins, cytotoxic moieties, enzymes e.g.
biotin conjugated enzymes, cytokines, interferons, target or
reporter moieties or hematogenous cells, thereby forming an
antibody conjugate. The CDMAB can be used alone or in combination
with one or more CDMAB/chemotherapeutic agents.
[0044] The prospect of individualized anti-cancer treatment will
bring about a change in the way a patient is managed. A likely
clinical scenario is that a tumor sample is obtained at the time of
presentation, and banked. From this sample, the tumor can be typed
from a panel of pre-existing cancerous disease modifying
antibodies. The patient will be conventionally staged but the
available antibodies can be of use in further staging the patient.
The patient can be treated immediately with the existing
antibodies, and a panel of antibodies specific to the tumor can be
produced either using the methods outlined herein or through the
use of phage display libraries in conjunction with the screening
methods herein disclosed. All the antibodies generated will be
added to the library of anti-cancer antibodies since there is a
possibility that other tumors can bear some of the same epitopes as
the one that is being treated. The antibodies produced according to
this method may be useful to treat cancerous disease in any number
of patients who have cancers that bind to these antibodies.
[0045] In addition to anti-cancer antibodies, the patient can elect
to receive the currently recommended therapies as part of a
multi-modal regimen of treatment. The fact that the antibodies
isolated via the present methodology are relatively non-toxic to
non-cancerous cells allows for combinations of antibodies at high
doses to be used, either alone, or in conjunction with conventional
therapy. The high therapeutic index will also permit re-treatment
on a short time scale that should decrease the likelihood of
emergence of treatment resistant cells.
[0046] If the patient is refractory to the initial course of
therapy or metastases develop, the process of generating specific
antibodies to the tumor can be repeated for re-treatment.
Furthermore, the anti-cancer antibodies can be conjugated to red
blood cells obtained from that patient and re-infused for treatment
of metastases. There have been few effective treatments for
metastatic cancer and metastases usually portend a poor outcome
resulting in death. However, metastatic cancers are usually well
vascularized and the delivery of anti-cancer antibodies by red
blood cells can have the effect of concentrating the antibodies at
the site of the tumor. Even prior to metastases, most cancer cells
are dependent on the host's blood supply for their survival and an
anti-cancer antibody conjugated to red blood cells can be effective
against in situ tumors as well. Alternatively, the antibodies may
be conjugated to other hematogenous cells, e.g. lymphocytes,
macrophages, monocytes, natural killer cells, etc.
[0047] There are five classes of antibodies and each is associated
with a function that is conferred by its heavy chain. It is
generally thought that cancer cell killing by naked antibodies are
mediated either through antibody dependent cellular cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC). For example
murine IgM and IgG2a antibodies can activate human complement by
binding the C-1 component of the complement system thereby
activating the classical pathway of complement activation which can
lead to tumor lysis. For human antibodies the most effective
complement activating antibodies are generally IgM and IgG1. Murine
antibodies of the IgG2a and IgG3 isotype are effective at
recruiting cytotoxic cells that have Fc receptors which will lead
to cell killing by monocytes, macrophages, granulocytes and certain
lymphocytes. Human antibodies of both the IgG1 and IgG3 isotype
mediate ADCC.
[0048] The cytotoxicity mediated through the Fc region requires the
presence of effector cells, their corresponding receptors, or
proteins e.g. NK cells, T-cells and complement. In the absence of
these effector mechanisms, the Fc portion of an antibody is inert.
The Fc portion of an antibody may confer properties that affect the
pharmacokinetics of an antibody in vivo, but in vitro this is not
operative.
[0049] The cytotoxicity assays under which we test the antibodies
do not have any of the effector mechanisms present, and are carried
out in vitro. These assays do not have effector cells (NK,
Macrophages, or T-cells) or complement present. Since these assays
are completely defined by what is added together, each component
can be characterized. The assays used herein contain only target
cells, media and sera. The target cells do not have effector
functions since they are cancer cells or fibroblasts. Without
exogenous cells which have effector function properties there is no
cellular elements that have this function. The media does not
contain complement or any cells. The sera used to support the
growth of the target cells do not have complement activity as
disclosed by the vendors. Furthermore, in our own labs we have
verified the absence of complement activity in the sera used.
Therefore, our work evidences the fact that the effects of the
antibodies are due entirely to the effects of the antigen binding
which is mediated through the Fab. Effectively, the target cells
are seeing and interacting with only the Fab, since they do not
have receptors for the Fc. Although the hybridoma is secreting
complete immunoglobulin which was tested with the target cells, the
only part of the immunoglobulin that interacts with the cells are
the Fab, which act as antigen binding fragments.
[0050] With respect to the instantly claimed antibodies and antigen
binding fragments, the application, as filed, has demonstrated
cellular cytotoxicity as evidenced by the data in FIG. 1. As
pointed out above, and as herein confirmed via objective evidence,
this effect was entirely due to binding by the Fab to the tumor
cells.
[0051] Ample evidence exists in the art of antibodies mediating
cytotoxicity due to direct binding of the antibody to the target
antigen independent of effector mechanisms recruited by the Fc. The
best evidence for this is in vitro experiments which do not have
supplemental cells, or complement (to formally exclude those
mechanisms). These types of experiments have been carried out with
complete immunoglobulin, or with antigen binding fragments such as
F(ab)'2 fragments. In these types of experiments, antibodies or
antigen binding fragments can directly induce apoptosis of target
cells such as in the case of anti-Her2 and anti-EGFR antibodies,
both of which have been approved by the US FDA for marketing in
cancer therapy.
[0052] Another possible mechanism of antibody mediated cancer
killing may be through the use of antibodies that function to
catalyze the hydrolysis of various chemical bonds in the cell
membrane and its associated glycoproteins or glycolipids, so-called
catalytic antibodies.
[0053] There are three additional mechanisms of antibody-mediated
cancer cell killing. The first is the use of antibodies as a
vaccine to induce the body to produce an immune response against
the putative antigen that resides on the cancer cell. The second is
the use of antibodies to target growth receptors and interfere with
their function or to down regulate that receptor so that its
function is effectively lost. The third is the effect of such
antibodies on direct ligation of cell surface moieties that may
lead to direct cell death, such as ligation of death receptors such
as TRAIL R1 or TRAIL R2, or integrin molecules such as alpha V beta
3 and the like.
[0054] The clinical utility of a cancer drug is based on the
benefit of the drug under an acceptable risk profile to the
patient. In cancer therapy survival has generally been the most
sought after benefit, however there are a number of other
well-recognized benefits in addition to prolonging life. These
other benefits, where treatment does not adversely affect survival,
include symptom palliation, protection against adverse events,
prolongation in time to recurrence or disease-free survival, and
prolongation in time to progression. These criteria are generally
accepted and regulatory bodies such as the U.S. Food and Drug
Administration (F.D.A.) approve drugs that produce these benefits
(Hirschfeld et al. Critical Reviews in Oncology/Hematology
42:137-143 2002). In addition to these criteria it is well
recognized that there are other endpoints that may presage these
types of benefits. In part, the accelerated approval process
granted by the U.S. F.D.A. acknowledges that there are surrogates
that will likely predict patient benefit. As of year-end 2003,
there have been sixteen drugs approved under this process, and of
these, four have gone on to full approval, i.e., follow-up studies
have demonstrated direct patient benefit as predicted by surrogate
endpoints. One important endpoint for determining drug effects in
solid tumors is the assessment of tumor burden by measuring
response to treatment (Therasse et al. Journal of the National
Cancer Institute 92(3):205-216 2000). The clinical criteria (RECIST
criteria) for such evaluation have been promulgated by Response
Evaluation Criteria in Solid Tumors Working Group, a group of
international experts in cancer. Drugs with a demonstrated effect
on tumor burden, as shown by objective responses according to
RECIST criteria, in comparison to the appropriate control group
tend to, ultimately, produce direct patient benefit. In the
pre-clinical setting tumor burden is generally more straightforward
to assess and document. In that pre-clinical studies can be
translated to the clinical setting, drugs that produce prolonged
survival in pre-clinical models have the greatest anticipated
clinical utility. Analogous to producing positive responses to
clinical treatment, drugs that reduce tumor burden in the
pre-clinical setting may also have significant direct impact on the
disease. Although prolongation of survival is the most sought after
clinical outcome from cancer drug treatment, there are other
benefits that have clinical utility and it is clear that tumor
burden reduction, which may correlate to a delay in disease
progression, extended survival or both, can also lead to direct
benefits and have clinical impact (Eckhardt et al. Developmental
Therapeutics: Successes and Failures of Clinical Trial Designs of
Targeted Compounds; ASCO Educational Book, 39.sup.th Annual
Meeting, 2003, pages 209-219).
[0055] The present invention describes the development and use of
AR47A6.4.2 identified by, its effect, in a cytotoxic assay, in
non-established and established tumor growth in animal models and
in prolonging survival time in those suffering from cancerous
disease. This invention represents an advance in the field of
cancer treatment in that it describes, for the first time, reagents
that bind specifically to an epitope or epitopes present on the
target molecule, TROP-2, and that also have in vitro cytotoxic
properties, as a naked antibody, against malignant tumor cells but
not normal cells, and which also directly mediate, as a naked
antibody, inhibition of tumor growth and extension of survival in
in vivo models of human cancer. This is an advance in relation to
any other previously described anti-TROP-2 antibody, since none
have been shown to have similar properties. It also provides an
advance in the field since it clearly demonstrates, and for the
first time, the direct involvement of TROP-2 in events associated
with growth and development of certain types of tumors. It also
represents an advance in cancer therapy since it has the potential
to display similar anti-cancer properties in human patients. A
further advance is that inclusion of these antibodies in a library
of anti-cancer antibodies will enhance the possibility of targeting
tumors expressing different antigen markers by determination of the
appropriate combination of different anti-cancer antibodies, to
find the most effective in targeting and inhibiting growth and
development of the tumors.
[0056] In all, this invention teaches the use of the AR47A6.4.2
antigen as a target for a therapeutic agent, that when administered
can reduce the tumor burden of a cancer expressing the antigen in a
mammal, and can also lead to a prolonged survival of the treated
mammal. This invention also teaches the use of CDMAB (AR47A6.4.2),
and its derivatives, and antigen binding fragments thereof, and
cellular cytotoxicity inducing ligands thereof to target their
antigen to reduce the tumor burden of a cancer expressing the
antigen in a mammal, and lead to prolonged survival of the treated
mammal. Furthermore, this invention also teaches the use of
detecting the AR47A6.4.2 antigen in cancerous cells that can be
useful for the diagnosis, prediction of therapy, and prognosis of
mammals bearing tumors that express this antigen.
[0057] Accordingly, it is an objective of the invention to utilize
a method for producing cancerous disease modifying antibodies
(CDMAB) raised against cancerous cells derived from a particular
individual, or one or more particular cancer cell lines, which
CDMAB are cytotoxic with respect to cancer cells while
simultaneously being relatively non-toxic to non-cancerous cells,
in order to isolate hybridoma cell lines and the corresponding
isolated monoclonal antibodies and antigen binding fragments
thereof for which said hybridoma cell lines are encoded.
[0058] It is an additional objective of the invention to teach
cancerous disease modifying antibodies, ligands and antigen binding
fragments thereof.
[0059] It is a further objective of the instant invention to
produce cancerous disease modifying antibodies whose cytotoxicity
is mediated through antibody dependent cellular toxicity.
[0060] It is yet an additional objective of the instant invention
to produce cancerous disease modifying antibodies whose
cytotoxicity is mediated through complement dependent cellular
toxicity.
[0061] It is still a further objective of the instant invention to
produce cancerous disease modifying antibodies whose cytotoxicity
is a function of their ability to catalyze hydrolysis of cellular
chemical bonds.
[0062] A still further objective of the instant invention is to
produce cancerous disease modifying antibodies which are useful for
in a binding assay for diagnosis, prognosis, and monitoring of
cancer.
[0063] Other objects and advantages of this invention will become
apparent from the following description wherein are set forth, by
way of illustration and example, certain embodiments of this
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0064] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0065] FIG. 1 compares the percentage cytotoxicity and binding
levels of the hybridoma supernatants against cell lines OCC-1,
OVCAR-3 and CCD-27sk.
[0066] FIG. 2 tabulates binding of AR47A6.4.2 and the anti-EGFR
antibody control to cancer and normal cell lines. The data is
presented as the mean fluorescence intensity as a fold increase
above isotype control.
[0067] FIG. 3 includes representative FACS histograms of AR47A6.4.2
and anti-EGFR antibodies directed against several cancer and
non-cancer cell lines.
[0068] FIG. 4 demonstrates the effect of AR47A6.4.2 on tumor growth
in a prophylactic BxPC-3 pancreatic cancer model. The vertical
lines indicate the period during which the antibody was
administered. Data points represent the mean+/-SEM.
[0069] FIG. 5 demonstrates the effect of AR47A6.4.2 on body weight
in a prophylactic BxPC-3 pancreatic cancer model. Data points
represent the mean+/-SEM.
[0070] FIG. 6 demonstrates the effect of AR47A6.4.2 on tumor growth
in an established BxPC-3 pancreatic cancer model. The vertical
lines indicate the period during which the antibody was
administered. Data points represent the mean+/-SEM.
[0071] FIG. 7 demonstrates the effect of AR47A6.4.2 on body weight
in an established BxPC-3 pancreatic cancer model. Data points
represent the mean+/-SEM.
[0072] FIG. 8 demonstrates the effect of AR47A6.4.2 on tumor growth
in a prophylactic PL45 pancreatic cancer model. The vertical lines
indicate the period during which the antibody was administered.
Data points represent the mean+/-SEM.
[0073] FIG. 9 demonstrates the effect of AR47A6.4.2 on survival in
a prophylactic PL45 pancreatic cancer model.
[0074] FIG. 10 demonstrates the effect of AR47A6.4.2 on body weight
in a prophylactic PL45 pancreatic cancer model. Data points
represent the mean+/-SEM.
[0075] FIG. 11 demonstrates the effect of AR47A6.4.2 on tumor
growth in a prophylactic PC-3 prostate cancer model. The vertical
lines indicate the period during which the antibody was
administered. Data points represent the mean+/-SEM.
[0076] FIG. 12 demonstrates the effect of AR47A6.4.2 on survival in
a prophylactic PC-3 prostate cancer model.
[0077] FIG. 13 demonstrates the effect of AR47A6.4.2 on body weight
in a prophylactic PC-3 prostate cancer model. Data points represent
the mean+/-SEM.
[0078] FIG. 14 demonstrates the effect of AR47A6.4.2 on tumor
growth in a prophylactic MCF-7 breast cancer model. The vertical
lines indicate the period during which the antibody was
administered. Data points represent the median+/-SEM.
[0079] FIG. 15 demonstrates the effect of AR47A6.4.2 on body weight
in a prophylactic MCF-7 breast cancer model. Data points represent
the mean+/-SEM.
[0080] FIG. 16 demonstrates the effect of AR47A6.4.2 on survival in
a prophylactic MCF-7 breast cancer model.
[0081] FIG. 17 demonstrates the effect of AR47A6.4.2 on tumor
growth in a prophylactic Colo 205 colon cancer model. The vertical
lines indicate the period during which the antibody was
administered. Data points represent the median+/-SEM.
[0082] FIG. 18 demonstrates the effect of AR47A6.4.2 on body weight
in a prophylactic Colo 205 colon cancer model. Data points
represent the mean+/-SEM.
[0083] FIG. 19. Western blot of samples from the total membrane
fraction of MDA-MB-231 cells (lane 1) and from whole cell lysates
of PC-3 (lane 2) and CCD-27sk (lane 3) cell lines, probed with
AR47A6.4.2. Molecular weight markers are indicated on the left.
[0084] FIG. 20. Western blot of an immunocomplex prepared by
immunoprecipitation with AR47A6.4.2 (lane 1) and with an isotype
control (lane 2), from the total membrane fraction of the
MDA-MB-231 cell line. Antibody-conjugated Protein G-Sepharose beads
not incubated with total membrane fraction of MDA-MB-231 were also
used as negative controls (lanes 3 and 4). Three replicate blots
were probed either with the antibodies AR47A6.4.2, IgG2a isotype
control or without primary antibody. Molecular weight markers are
indicated on the left.
[0085] FIG. 21. Western blot of immunocomplexes prepared by
immunoprecipitation with AR47A6.4.2 (lanes 2 and 3) or with an
isotype control (lanes 1 and 4), from the total membrane fraction
of the MDA-MB-231 cell line. Replicate aliquots were incubated in
the presence (lanes 3 and 4) and absence (buffer only, lanes 1 and
2) of a mixture of glycosidase enzymes. Molecular weight markers
are indicated on the left.
[0086] FIG. 22. Alignment of images from Western blotting (center
panel) and colloidal staining (left panel) of an immunocomplex
prepared by a large-scale immunoprecipitation with AR47A6.4.2 (lane
1) and with an isotype control antibody (lane 2), from the total
membrane fraction of the MDA-MB-231 cell line.
AR47A6.4.2-conjugated protein G Sepharose beads only (not incubated
with MDA-MB-231 cells) were also used as a negative control (lane
3). Molecular weight markers are indicated on the left.
[0087] FIG. 23. Images of the Colloidal Blue stained gel prior to
and after coring to isolate protein samples, to be analyzed by Mass
Spectrometry after trypsin digestion. Lane description is the same
as in FIG. 20. Molecular weight markers are indicated on the
left.
[0088] FIG. 24. Mass spectrogram obtained after trypsin digestion
of the samples obtained from coring the Colloidal Blue-stained gel.
Below the mass spectograms is the Profound search result
summary.
[0089] FIG. 25. MASCOT search summary result from the MS/MS
analysis of one of the unique peptides obtained after trypsin
digestion of the AR47A6.4.2 immunoprecipitate. SEQ ID NO:9 is
shown.
[0090] FIG. 26. Western blot of immunocomplexes obtained, with
AR47A6.4.2 (lane 1) and with an isotype control antibody (lane 3),
from the total membrane fraction of the MDA-MB-231 cell line.
AR47A6.4.2-conjugated protein G Sepharose beads only (`mock IP`
were also used as a negative control (lane 2)). Replicate blots
were probed with the AR47A6.4.2, anti-human TROP-2 and isotype
control. Molecular weight markers are indicated on the left.
[0091] FIG. 27. Western blot of purified human recombinant proteins
TROP-2 (lane 1) and CD63 large extracellular domain (lane 2).
Replicate blots were probed with either AR47A6.4.2, anti-human
TROP-2, 1A245.6 or isotype control. Molecular weight markers are
indicated on the left.
[0092] FIG. 28. MDA-MB-231 membrane proteins (MB-231) and
recombinant human TROP-2 (rhTROP-2) glycosylated (G) and
deglycosylated (D) under denaturing and non-denaturing conditions,
probed with AR47A6.4.2.
[0093] FIG. 29. MDA-MB-231 membrane proteins (MB-231) and
recombinant human TROP-2 (rhTROP-2) glycosylated (G) and
deglycosylated (D) under denaturing and non-denaturing conditions,
probed with anti-human TROP-2.
[0094] FIG. 30. MDA-MB-231 membrane proteins (MB-231) and
recombinant human TROP-2 (rhTROP-2) glycosylated (G) and
deglycosylated (D) under denaturing and non-denaturing conditions,
probed with IgG isotype control.
[0095] FIG. 31. Western blot of recombinant human TROP-2 probed
with different primary antibody solutions. Lanes 3 to 7 were probed
with biotinylated AR52A301.5 mixed with 0.5 microgram/mL, 5
microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000
microgram/mL of non-biotinylated AR52A301.5 respectively. Lanes 9
to 13 were probed with biotinylated AR52A301.5 mixed with 0.5
microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and
1000 microgram/mL of non-biotinylated AR47A6.4.2 respectively.
Lanes 15 to 19 were probed with biotinylated AR52A301.5 mixed with
0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL
and 1000 microgram/mL of non-biotinylated 8A3B.6 respectively.
Lanes 8 and 14 were incubated with negative control solution and
lane 8 was not incubated in secondary solution. Lanes 1, 2 and 20
were incubated with TBST only.
[0096] FIG. 32. Western blot of recombinant human TROP-2 probed
with different primary antibody solutions. Lanes 3 to 7 were probed
with biotinylated AR47A6.4.2 mixed with 0.5 microgram/mL, 5
microgram/mL, 50 microgram/mL, 500 microgram/mL and 1000
microgram/mL of non-biotinylated AR52A301.5 respectively. Lanes 9
to 13 were probed with biotinylated AR47A6.4.2 mixed with 0.5
microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL and
1000 microgram/mL of non-biotinylated AR47A6.4.2 respectively.
Lanes 15 to 19 were probed with biotinylated AR47A6.4.2 mixed with
0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500 microgram/mL
and 1000 microgram/mL of non-biotinylated 1B7.11 respectively.
Lanes 8 and 14 were incubated with negative control solution and
lane 8 was not incubated in secondary solution. Lanes 1, 2 and 20
were incubated with TBST only.
[0097] FIG. 33 tabulates an IHC comparison of AR47A6.4.2 versus
positive and negative controls on a normal human tissue micro
array.
[0098] FIG. 34. Representative micrographs showing the binding
pattern on spleen tissue obtained with AR47A6.4.2 (A) or the
isotype control antibody (B) and on brain tissue obtained with
AR47A6.4.2 (C) or the isotype control antibody (D) from a normal
human tissue microarray. Magnification is 200.times..
[0099] FIG. 35 tabulates an IHC comparison of AR47A6.4.2 on various
human tumor and normal tissue sections from different tissue micro
arrays.
[0100] FIG. 36. Representative micrographs showing the binding
pattern on breast tumor tissue obtained with AR47A6.4.2 (A) or the
isotype control antibody (B) and on prostate tumor tissue obtained
with AR47A6.4.2 (C) or the isotype control antibody (D) and on
pancreatic tumor tissue obtained with AR47A6.4.2 (E) or the isotype
control antibody (F) from various human tumor tissue microarrays.
Magnification is 400.times. for the breast and pancreatic tumor
tissue and 200.times. for the prostate tumor tissue.
[0101] FIG. 37 tabulates an IHC comparison of AR47A6.4.2 on various
human and other species normal tissue sections from different
normal species tissue micro arrays.
[0102] FIG. 38. Representative micrographs showing the binding
pattern on normal human kidney tissue obtained with AR47A6.4.2 (A)
or the isotype control antibody (B) and on normal cynomolgus kidney
tissue obtained with AR47A6.4.2 (C) or the isotype control antibody
(D) and on normal rhesus tissue obtained with AR47A6.4.2 (E) or the
isotype control antibody (F) from various multi-species tissue
microarrays. Magnification is 200.times..
[0103] FIG. 39. Sequences of all oligonucleotide primers (SEQ ID
NOS:10-47) used in the murine sequence determination of
AR47A6.4.2.
[0104] FIG. 40. Agarose gel of the RT/PCR amplification of
AR47A6.4.2 V.sub.H and V.sub.L regions.
[0105] FIG. 41. Agarose gel of the PCR colony screen of AR47A6.4.2
V.sub.H-C and V.sub.H-E.
[0106] FIG. 42. Agarose gel of the PCR colony screen of AR47A6.4.2
V.sub.LA and V.sub.LG.
[0107] FIG. 43. AR47A6.4.2 V.sub.L amino acid sequence (SEQ ID
NO:8).
[0108] FIG. 44. AR47A6.4.2 V.sub.H amino acid sequence (SEQ ID
NO:7).
DETAILED DESCRIPTION OF THE INVENTION
[0109] In general, the following words or phrases have the
indicated definition when used in the summary, description,
examples, and claims.
[0110] The term "antibody" is used in the broadest sense and
specifically covers, for example, single monoclonal antibodies
(including agonist, antagonist, and neutralizing antibodies,
de-immunized, murine, chimeric or humanized antibodies), antibody
compositions with polyepitopic specificity, single-chain
antibodies, diabodies, triabodies, immunoconjugates and antibody
fragments (see below).
[0111] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma (murine or human) method first described
by Kohler et al., Nature, 256:495 (1975), or may be made by
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example.
[0112] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding or variable
region thereof. Examples of antibody fragments include less than
full length antibodies, Fab, Fab', F(ab').sub.2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules;
single-chain antibodies, single domain antibody molecules, fusion
proteins, recombinant proteins and multispecific antibodies formed
from antibody fragment(s).
[0113] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (C.sub.L) and heavy chain constant domains, C.sub.H1,
C.sub.H2 and C.sub.H3. The constant domains may be native sequence
constant domains (e.g. human native sequence constant domains) or
amino acid sequence variant thereof. Preferably, the intact
antibody has one or more effector functions.
[0114] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five-major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0115] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding;
complement dependent cytotoxicity; Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g. B cell receptor;
BCR), etc.
[0116] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0117] "Effector cells" are leukocytes which express one or more
FcRs and perform effector functions. Preferably, the cells express
at least Fc.gamma.RIII and perform ADCC effector function. Examples
of human leukocytes which mediate ADCC include peripheral blood
mononuclear cells (PBMC), natural killer (NK) cells, monocytes,
cytotoxic T cells and neutrophils; with PBMCs and NK cells being
preferred. The effector cells may be isolated from a native source
thereof, e.g. from blood or PBMCs as described herein.
[0118] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma. RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term
"FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., Eur. J.
Immunol. 24:2429 (1994)).
[0119] "Complement dependent cytotoxicity" or "CDC" refers to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (C1q) to a
molecule (e.g. an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0120] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody dependent cellular cytotoxicity (ADCC).
[0121] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 2632 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined. Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0122] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site. The Fab fragment also contains the
constant domain of the light chain and the first constant domain
(CH I) of the heavy chain. Fab' fragments differ from Fab fragments
by the addition of a few residues at the carboxy terminus of the
heavy chain CH1 domain including one or more cysteines from the
antibody hinge region. Fab'-SH is the designation herein for Fab'
in which the cysteine residue(s) of the constant domains bear at
least one free thiol group. F(ab').sub.2 antibody fragments
originally were produced as pairs of Fab' fragments which have
hinge cysteines between them. Other chemical couplings of antibody
fragments are also known.
[0123] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0124] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0125] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a variable
heavy domain (V.sub.H) connected to a variable light domain
(V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using
a linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0126] The term "triabodies" or "trivalent trimers" refers to the
combination of three single chain antibodies. Triabodies are
constructed with the amino acid terminus of a V.sub.L or V.sub.H
domain, i.e., without any linker sequence. A triabody has three Fv
heads with the polypeptides arranged in a cyclic, head-to-tail
fashion. A possible conformation of the triabody is planar with the
three binding sites located in a plane at an angle of 120 degrees
from one another. Triabodies can be monospecific, bispecific or
trispecific.
[0127] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0128] An antibody "which binds" an antigen of interest, e.g.
TROP-2 antigen, is one capable of binding that antigen with
sufficient affinity such that the antibody is useful as a
therapeutic or diagnostic agent in targeting a cell expressing the
antigen. Where the antibody is one which binds TROP-2, it will
usually preferentially bind TROP-2 as opposed to other receptors,
and does not include incidental binding such as non-specific Fc
contact, or binding to post-translational modifications common to
other antigens and may be one which does not significantly
cross-react with other proteins. Methods, for the detection of an
antibody that binds an antigen of interest, are well known in the
art and can include but are not limited to assays such as FACS,
cell ELISA and Western blot.
[0129] As used herein, the expressions "cell", "cell line", and
"cell culture" are used interchangeably, and all such designations
include progeny. It is also understood that all progeny may not be
precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included. It will be clear from the context
where distinct designations are intended.
[0130] "Treatment or treating" refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented. Hence, the mammal to be
treated herein may have been diagnosed as having the disorder or
may be predisposed or susceptible to the disorder.
[0131] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth or death. Examples of cancer include,
but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and
leukemia or lymphoid malignancies. More particular examples of such
cancers include squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney or renal cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,
anal carcinoma, penile carcinoma, as well as head and neck
cancer.
[0132] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlomaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
carnomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(TAXOTERE.RTM., Aventis, Rhone-Poulenc Rorer, Antony, France);
chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C;
mitoxantrone; vincristine; vinorelbine; navelbine; novantrone;
teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoic acid; esperamicins; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above. Also
included in this definition are anti-hormonal agents that act to
regulate or inhibit hormone action on tumors such as anti-estrogens
including for example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0133] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, mice, SCID or nude mice
or strains of mice, domestic and farm animals, and zoo, sports, or
pet animals, such as sheep, dogs, horses, cats, cows, etc.
Preferably, the mammal herein is human.
[0134] "Oligonucleotides" are short-length, single- or
double-stranded polydeoxynucleotides that are chemically
synthesized by known methods (such as phosphotriester, phosphite,
or phosphoramidite chemistry, using solid phase techniques such as
described in EP 266,032, published 4 May 1988, or via
deoxynucleoside H-phosphonate intermediates as described by
Froehler et al., Nucl. Acids Res., 14:5399-5407, 1986. They are
then purified on polyacrylamide gels.
[0135] In accordance with the present invention, "humanized" and/or
"chimeric" forms of non-human (e.g. murine) immunoglobulins refer
to antibodies which contain specific chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
which results in the decrease of a human anti-mouse antibody
(HAMA), human anti-chimeric antibody (HACA) or a human anti-human
antibody (HAHA) response, compared to the original antibody, and
contain the requisite portions (e.g. CDR(s), antigen binding
region(s), variable domain(s) and so on) derived from said
non-human immunoglobulin, necessary to reproduce the desired
effect, while simultaneously retaining binding characteristics
which are comparable to said non-human immunoglobulin. For the most
part, humanized antibodies are human immunoglobulins (recipient
antibody) in which residues from the complementarity determining
regions (CDRs) of the recipient antibody are replaced by residues
from the CDRs of a non-human species (donor antibody) such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework region (FR) residues of
the human immunoglobulin are replaced by corresponding non-human FR
residues. Furthermore, the humanized antibody may comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or FR sequences. These modifications are made to
further refine and optimize antibody performance. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR residues are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
[0136] "De-immunized" antibodies are immunoglobulins that are
non-immunogenic, or less immunogenic, to a given species.
De-immunization can be achieved through structural alterations to
the antibody. Any de-immunization technique known to those skilled
in the art can be employed. One suitable technique for
de-immunizing antibodies is described, for example, in WO 00/34317
published Jun. 15, 2000.
[0137] An antibody which induces "apoptosis" is one which induces
programmed cell death by any means, illustrated by but not limited
to binding of annexin V, caspase activity, fragmentation of DNA,
cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).
[0138] As used herein "antibody induced cytotoxicity" is understood
to mean the cytotoxic effect derived from the hybridoma supernatant
or antibody produced by the hybridoma deposited with the IDAC as
accession number 141205-05 which effect is not necessarily related
to the degree of binding.
[0139] Throughout the instant specification, hybridoma cell lines,
as well as the isolated monoclonal antibodies which are produced
therefrom, are alternatively referred to by their internal
designation, AR47A6.4.2 or Depository Designation, IDAC
141205-05.
[0140] As used herein "antibody-ligand" includes a moiety which
exhibits binding specificity for at least one epitope of the target
antigen, and which may be an intact antibody molecule, antibody
fragments, and any molecule having at least an antigen-binding
region or portion thereof (i.e., the variable portion of an
antibody molecule), e.g., an Fv molecule, Fab molecule, Fab'
molecule, F(ab').sub.2 molecule, a bispecific antibody, a fusion
protein, or any genetically engineered molecule which specifically
recognizes and binds at least one epitope of the antigen bound by
the isolated monoclonal antibody produced by the hybridoma cell
line designated as IDAC 141205-05 (the IDAC 141205-05 antigen).
[0141] As used herein "cancerous disease modifying antibodies"
(CDMAB) refers to monoclonal antibodies which modify the cancerous
disease process in a manner which is beneficial to the patient, for
example by reducing tumor burden or prolonging survival of tumor
bearing individuals, and antibody-ligands thereof.
[0142] A "CDMAB related binding agent", in its broadest sense, is
understood to include, but is not limited to, any form of human or
non-human antibodies, antibody fragments, antibody ligands, or the
like, which competitively bind to at least one CDMAB target
epitope.
[0143] A "competitive binder" is understood to include any form of
human or non-human antibodies, antibody fragments, antibody
ligands, or the like which has binding affinity for at least one
CDMAB target epitope.
[0144] Tumors to be treated include primary tumors and metastatic
tumors, as well as refractory tumors. Refractory tumors include
tumors that fail to respond or are resistant to treatment with
chemotherapeutic agents alone, antibodies alone, radiation alone or
combinations thereof. Refractory tumors also encompass tumors that
appear to be inhibited by treatment with such agents but recur up
to five years, sometimes up to ten years or longer after treatment
is discontinued.
[0145] Tumors that can be treated include tumors that are not
vascularized, or not yet substantially vascularized, as well as
vascularized tumors. Examples of solid tumors, which can be
accordingly treated, include breast carcinoma, lung carcinoma,
colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma.
Some examples of such tumors include epidermoid tumors, squamous
tumors, such as head and neck tumors, colorectal tumors, prostate
tumors, breast tumors, lung tumors, including small cell and
non-small cell lung tumors, pancreatic tumors, thyroid tumors,
ovarian tumors, and liver tumors. Other examples include Kaposi's
sarcoma, CNS neoplasms, neuroblastomas, capillary
hemangioblastomas, meningiomas and cerebral metastases, melanoma,
gastrointestinal and renal carcinomas and sarcomas,
rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme,
and leiomyosarcoma.
[0146] As used herein "antigen-binding region" means a portion of
the molecule which recognizes the target antigen.
[0147] As used herein "competitively inhibits" means being able to
recognize and bind a determinant site to which the monoclonal
antibody produced by the hybridoma cell line designated as IDAC
141205-05, (the IDAC 141205-05 antibody) is directed using
conventional reciprocal antibody competition assays. (Belanger L.,
Sylvestre C. and Dufour D. (1973), Enzyme linked immunoassay for
alpha fetoprotein by competitive and sandwich procedures. Clinica
Chimica Acta 48, 15).
[0148] As used herein "target antigen" is the IDAC 141205-05
antigen or portions thereof.
[0149] As used herein, an "immunoconjugate" means any molecule or
CDMAB such as an antibody chemically or biologically linked to
cytotoxins, radioactive agents, cytokines, interferons, target or
reporter moieties, enzymes, toxins, anti-tumor drugs or therapeutic
agents. The antibody or CDMAB may be linked to the cytotoxin,
radioactive agent, cytokine, interferon, target or reporter moiety,
enzyme, toxin, anti-tumor drug or therapeutic agent at any location
along the molecule so long as it is able to bind its target.
Examples of immunoconjugates include antibody toxin chemical
conjugates and antibody-toxin fusion proteins.
[0150] Radioactive agents suitable for use as anti-tumor agents are
known to those skilled in the art. For example, 131I or 211At is
used. These isotopes are attached to the antibody using
conventional techniques (e.g. Pedley et al., Br. J. Cancer 68,
69-73 (1993)). Alternatively, the anti-tumor agent which is
attached to the antibody is an enzyme which activates a prodrug. A
prodrug may be administered which will remain in its inactive form
until it reaches the tumor site where it is converted to its
cytotoxin form once the antibody complex is administered. In
practice, the antibody-enzyme conjugate is administered to the
patient and allowed to localize in the region of the tissue to be
treated. The prodrug is then administered to the patient so that
conversion to the cytotoxic drug occurs in the region of the tissue
to be treated. Alternatively, the anti-tumor agent conjugated to
the antibody is a cytokine such as interleukin-2 (IL-2),
interleukin-4 (IL-4) or tumor necrosis factor alpha (TNF-.alpha.).
The antibody targets the cytokine to the tumor so that the cytokine
mediates damage to or destruction of the tumor without affecting
other tissues. The cytokine is fused to the antibody at the DNA
level using conventional recombinant DNA techniques. Interferons
may also be used.
[0151] As used herein, a "fusion protein" means any chimeric
protein wherein an antigen binding region is connected to a
biologically active molecule, e.g., toxin, enzyme, fluorescent
proteins, luminescent marker, polypeptide tag, cytokine,
interferon, target or reporter moiety or protein drug.
[0152] The invention further contemplates CDMAB of the present
invention to which target or reporter moieties are linked. Target
moieties are first members of binding pairs. Anti-tumor agents, for
example, are conjugated to second members of such pairs and are
thereby directed to the site where the antigen-binding protein is
bound. A common example of such a binding pair is avidin and
biotin. In a preferred embodiment, biotin is conjugated to the
target antigen of the CDMAB of the present invention, and thereby
provides a target for an anti-tumor agent or other moiety which is
conjugated to avidin or streptavidin. Alternatively, biotin or
another such moiety is linked to the target antigen of the CDMAB of
the present invention and used as a reporter, for example in a
diagnostic system where a detectable signal-producing agent is
conjugated to avidin or streptavidin.
[0153] Detectable signal-producing agents are useful in vivo and in
vitro for diagnostic purposes. The signal producing agent produces
a measurable signal which is detectable by external means, usually
the measurement of electromagnetic radiation. For the most part,
the signal producing agent is an enzyme or chromophore, or emits
light by fluorescence, phosphorescence or chemiluminescence.
Chromophores include dyes which absorb light in the ultraviolet or
visible region, and can be substrates or degradation products of
enzyme catalyzed reactions.
[0154] Moreover, included within the scope of the present invention
is use of the present CDMAB in vivo and in vitro for investigative
or diagnostic methods, which are well known in the art. In order to
carry out the diagnostic methods as contemplated herein, the
instant invention may further include kits, which contain CDMAB of
the present invention. Such kits will be useful for identification
of individuals at risk for certain type of cancers by detecting
over-expression of the CDMAB's target antigen on cells of such
individuals.
Diagnostic Assay Kits
[0155] It is contemplated to utilize the CDMAB of the present
invention in the form of a diagnostic assay kit for determining the
presence of a tumor. The tumor will generally be detected in a
patient based on the presence of one or more tumor-specific
antigens, e.g. proteins and/or polynucleotides which encode such
proteins in a biological sample, such as blood, sera, urine and/or
tumor biopsies, which samples will have been obtained from the
patient.
[0156] The proteins function as markers which indicate the presence
or absence of a particular tumor, for example a colon, breast, lung
or prostate tumor. It is further contemplated that the antigen will
have utility for the detection of other cancerous tumors. Inclusion
in the diagnostic assay kits of binding agents comprised of CDMABs
of the present invention, or CDMAB related binding agents, enables
detection of the level of antigen that binds to the agent in the
biological sample. Polynucleotide primers and probes may be used to
detect the level of mRNA encoding a tumor protein, which is also
indicative of the presence or absence of a cancer. In order for the
binding assay to be diagnostic, data will have been generated which
correlates statistically significant levels of antigen, in relation
to that present in normal tissue, so as to render the recognition
of binding definitively diagnostic for the presence of a cancerous
tumor. It is contemplated that a plurality of formats will be
useful for the diagnostic assay of the present invention, as are
known to those of ordinary skill in the art, for using a binding
agent to detect polypeptide markers in a sample. For example, as
illustrated in Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. Further contemplated are any
and all combinations, permutations or modifications of the
afore-described diagnostic assay formats.
[0157] The presence or absence of a cancer in a patient will
typically be determined by (a) contacting a biological sample
obtained from a patient with a binding agent; (b) detecting in the
sample a level of polypeptide that binds to the binding agent; and
(c) comparing the level of polypeptide with a predetermined cut-off
value.
[0158] In an illustrative embodiment, it is contemplated that the
assay will involve the use of a CDMAB based binding agent
immobilized on a solid support to bind to and remove the
polypeptide from the remainder of the sample. The bound polypeptide
may then be detected using a detection reagent that contains a
reporter group and specifically binds to the binding
agent/polypeptide complex. Illustrative detection reagents may
include a CDMAB based binding agent that specifically binds to the
polypeptide or an antibody or other agent that specifically binds
to the binding agent, such as an anti-immunoglobulin, protein G,
protein A or a lectin. In an alternative embodiment, it is
contemplated that a competitive assay may be utilized, in which a
polypeptide is labeled with a reporter group and allowed to bind to
the immobilized binding agent after incubation of the binding agent
with the sample. Indicative of the reactivity of the sample with
the immobilized binding agent, is the extent to which components of
the sample inhibit the binding of the labeled polypeptide to the
binding agent. Suitable polypeptides for use within such assays
include full length tumor-specific proteins and/or portions
thereof, to which the binding agent has binding affinity.
[0159] The diagnostic kit will be provided with a solid support
which may be in the form of any material known to those of ordinary
skill in the art to which the protein may be attached. Suitable
examples may include a test well in a microtiter plate or a
nitrocellulose or other suitable membrane. Alternatively, the
support may be a bead or disc, such as glass, fiberglass, latex or
a plastic material such as polystyrene or polyvinylchloride. The
support may also be a magnetic particle or a fiber optic sensor,
such as those disclosed, for example, in U.S. Pat. No.
5,359,681.
[0160] It is contemplated that the binding agent will be
immobilized on the solid support using a variety of techniques
known to those of skill in the art, which are amply described in
the patent and scientific literature. The term "immobilization"
refers to both noncovalent association, such as adsorption, and
covalent attachment, which, in the context of the present
invention, may be a direct linkage between the agent and functional
groups on the support, or may be a linkage by way of a
cross-linking agent. In a preferred, albeit non-limiting
embodiment, immobilization by adsorption to a well in a microtiter
plate or to a membrane is preferable. Adsorption may be achieved by
contacting the binding agent, in a suitable buffer, with the solid
support for a suitable amount of time. The contact time may vary
with temperature, and will generally be within a range of between
about 1 hour and about 1 day.
[0161] Covalent attachment of binding agent to a solid support
would ordinarily be accomplished by first reacting the support with
a bifunctional reagent that will react with both the support and a
functional group, such as a hydroxyl or amino group, on the binding
agent. For example, the binding agent may be covalently attached to
supports having an appropriate polymer coating using benzoquinone
or by condensation of an aldehyde group on the support with an
amine and an active hydrogen on the binding partner (see, e.g.,
Pierce Immunotechnology Catalog and Handbook, 1991, at A12
A13).
[0162] It is further contemplated that the diagnostic assay kit
will take the form of a two-antibody sandwich assay. This assay may
be performed by first contacting an antibody, e.g. the instantly
disclosed CDMAB that has been immobilized on a solid support,
commonly the well of a microtiter plate, with the sample, such that
polypeptides within the sample are allowed to bind to the
immobilized antibody. Unbound sample is then removed from the
immobilized polypeptide-antibody complexes and a detection reagent
(preferably a second antibody capable of binding to a different
site on the polypeptide) containing a reporter group is added. The
amount of detection reagent that remains bound to the solid support
is then determined using a method appropriate for the specific
reporter group.
[0163] In a specific embodiment, it is contemplated that once the
antibody is immobilized on the support as described above, the
remaining protein binding sites on the support will be blocked, via
the use of any suitable blocking agent known to those of ordinary
skill in the art, such as bovine serum albumin or Tween 20.TM.
(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody
would then be incubated with the sample, and polypeptide would be
allowed to bind to the antibody. The sample could be diluted with a
suitable diluent, such as phosphate-buffered saline (PBS) prior to
incubation. In general, an appropriate contact time (i.e.,
incubation time) would be selected to correspond to a period of
time sufficient to detect the presence of polypeptide within a
sample obtained from an individual with the specifically selected
tumor. Preferably, the contact time is sufficient to achieve a
level of binding that is at least about 95 percent of that achieved
at equilibrium between bound and unbound polypeptide. Those of
ordinary skill in the art will recognize that the time necessary to
achieve equilibrium may be readily determined by assaying the level
of binding that occurs over a period of time.
[0164] It is further contemplated that unbound sample would then be
removed by washing the solid support with an appropriate buffer.
The second antibody, which contains a reporter group, would then be
added to the solid support. Incubation of the detection reagent
with the immobilized antibody-polypeptide complex would then be
carried out for an amount of time sufficient to detect the bound
polypeptide. Subsequently, unbound detection reagent would then be
removed and bound detection reagent would be detected using the
reporter group. The method employed for detecting the reporter
group is necessarily specific to the type of reporter group
selected, for example for radioactive groups, scintillation
counting or autoradiographic methods are generally appropriate.
Spectroscopic methods may be used to detect dyes, luminescent
groups and fluorescent groups. Biotin may be detected using avidin,
coupled to a different reporter group (commonly a radioactive or
fluorescent group or an enzyme). Enzyme reporter groups may
generally be detected by the addition of substrate (generally for a
specific period of time), followed by spectroscopic or other
analysis of the reaction products.
[0165] In order to utilize the diagnostic assay kit of the present
invention to determine the presence or absence of a cancer, such as
prostate cancer, the signal detected from the reporter group that
remains bound to the solid support would generally be compared to a
signal that corresponds to a predetermined cut-off value. For
example, an illustrative cut-off value for the detection of a
cancer may be the average mean signal obtained when the immobilized
antibody is incubated with samples from patients without the
cancer. In general, a sample generating a signal that is about
three standard deviations above the predetermined cut-off value
would be considered positive for the cancer. In an alternate
embodiment, the cut-off value might be determined by using a
Receiver Operator Curve, according to the method of Sackett et al.,
Clinical Epidemiology. A Basic Science for Clinical Medicine,
Little Brown and Co., 1985, p. 106-7. In such an embodiment, the
cut-off value could be determined from a plot of pairs of true
positive rates (i.e., sensitivity) and false positive rates (100
percent-specificity) that correspond to each possible cut-off value
for the diagnostic test result. The cut-off value on the plot that
is the closest to the upper left-hand corner (i.e., the value that
encloses the largest area) is the most accurate cut-off value, and
a sample generating a signal that is higher than the cut-off value
determined by this method may be considered positive.
Alternatively, the cut-off value may be shifted to the left along
the plot, to minimize the false positive rate, or to the right, to
minimize the false negative rate. In general, a sample generating a
signal that is higher than the cut-off value determined by this
method is considered positive for a cancer.
[0166] It is contemplated that the diagnostic assay enabled by the
kit will be performed in either a flow-through or strip test
format, wherein the binding agent is immobilized on a membrane,
such as nitrocellulose. In the flow-through test, polypeptides
within the sample bind to the immobilized binding agent as the
sample passes through the membrane. A second, labeled binding agent
then binds to the binding agent-polypeptide complex as a solution
containing the second binding agent flows through the membrane. The
detection of bound second binding agent may then be performed as
described above. In the strip test format, one end of the membrane
to which binding agent is bound will be immersed in a solution
containing the sample. The sample migrates along the membrane
through a region containing second binding agent and to the area of
immobilized binding agent. Concentration of the second binding
agent at the area of immobilized antibody indicates the presence of
a cancer. Generation of a pattern, such as a line, at the binding
site, which can be read visually, will be indicative of a positive
test. The absence of such a pattern indicates a negative result. In
general, the amount of binding agent immobilized on the membrane is
selected to generate a visually discernible pattern when the
biological sample contains a level of polypeptide that would be
sufficient to generate a positive signal in the two-antibody
sandwich assay, in the format discussed above. Preferred binding
agents for use in the instant diagnostic assay are the instantly
disclosed antibodies, antigen-binding fragments thereof, and any
CDMAB related binding agents as herein described. The amount of
antibody immobilized on the membrane will be any amount effective
to produce a diagnostic assay, and may range from about 25
nanograms to about 1 microgram. Typically such tests may be
performed with a very small amount of biological sample.
[0167] Additionally, the CDMAB of the present invention may be used
in the laboratory for research due to its ability to identify its
target antigen.
[0168] In order that the invention herein described may be more
fully understood, the following description is set forth.
[0169] The present invention provides CDMAB (i.e., IDAC 141205-05
CDMAB) which specifically recognize and bind the IDAC 141205-05
antigen.
[0170] The CDMAB of the isolated monoclonal antibody produced by
the hybridoma deposited with the IDAC as accession number 141205-05
may be in any form as long as it has an antigen-binding region
which competitively inhibits the immunospecific binding of the
isolated monoclonal antibody produced by hybridoma IDAC 141205-05
to its target antigen. Thus, any recombinant proteins (e.g., fusion
proteins wherein the antibody is combined with a second protein
such as a lymphokine or a tumor inhibitory growth factor) having
the same binding specificity as the IDAC 141205-05 antibody fall
within the scope of this invention.
[0171] In one embodiment of the invention, the CDMAB is the IDAC
141205-05 antibody.
[0172] In other embodiments, the CDMAB is an antigen binding
fragment which may be a Fv molecule (such as a single-chain Fv
molecule), a Fab molecule, a Fab' molecule, a F(ab')2 molecule, a
fusion protein, a bispecific antibody, a heteroantibody or any
recombinant molecule having the antigen-binding region of the IDAC
141205-05 antibody. The CDMAB of the invention is directed to the
epitope to which the IDAC 141205-05 monoclonal antibody is
directed.
[0173] The CDMAB of the invention may be modified, i.e., by amino
acid modifications within the molecule, so as to produce derivative
molecules. Chemical modification may also be possible. Modification
by direct mutation, methods of affinity maturation, phage display
or chain shuffling may also be possible.
[0174] Affinity and specificity can be modified or improved by
mutating CDR and/or phenylalanine tryptophan (FW) residues and
screening for antigen binding sites having the desired
characteristics (e.g., Yang et al., J. Mol. Biol., (1995) 254:
392-403). One way is to randomize individual residues or
combinations of residues so that in a population of otherwise
identical antigen binding sites, subsets of from two to twenty
amino acids are found at particular positions. Alternatively,
mutations can be induced over a range of residues by error prone
PCR methods (e.g., Hawkins et al., J. Mol. Biol., (1992) 226:
889-96). In another example, phage display vectors containing heavy
and light chain variable region genes can be propagated in mutator
strains of E. coli (e.g., Low et al., J. Mol. Biol., (1996) 250:
359-68). These methods of mutagenesis are illustrative of the many
methods known to one of skill in the art.
[0175] Another manner for increasing affinity of the antibodies of
the present invention is to carry out chain shuffling, where the
heavy or light chain are randomly paired with other heavy or light
chains to prepare an antibody with higher affinity. The various
CDRs of the antibodies may also be shuffled with the corresponding
CDRs in other antibodies.
[0176] Derivative molecules would retain the functional property of
the polypeptide, namely, the molecule having such substitutions
will still permit the binding of the polypeptide to the IDAC
141205-05 antigen or portions thereof.
[0177] These amino acid substitutions include, but are not
necessarily limited to, amino acid substitutions known in the art
as "conservative".
[0178] For example, it is a well-established principle of protein
chemistry that certain amino acid substitutions, entitled
"conservative amino acid substitutions," can frequently be made in
a protein without altering either the conformation or the function
of the protein.
[0179] Such changes include substituting any of isoleucine (I),
valine (V), and leucine (L) for any other of these hydrophobic
amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa; glutamine (Q) for asparagine (N) and vice versa; and serine
(S) for threonine (T) and vice versa. Other substitutions can also
be considered conservative, depending on the environment of the
particular amino acid and its role in the three-dimensional
structure of the protein. For example, glycine (G) and alanine (A)
can frequently be interchangeable, as can alanine and valine (V).
Methionine (M), which is relatively hydrophobic, can frequently be
interchanged with leucine and isoleucine, and sometimes with
valine. Lysine (K) and arginine (R) are frequently interchangeable
in locations in which the significant feature of the amino acid
residue is its charge and the differing pK's of these two amino
acid residues are not significant. Still other changes can be
considered "conservative" in particular environments.
Example 1
Hybridoma Production--Hybridoma Cell Line AR47A6.4.2
[0180] The hybridoma cell line AR47A6.4.2 was deposited, in
accordance with the Budapest Treaty, with the International
Depository Authority of Canada (IDAC), Bureau of Microbiology,
Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada,
R3E 3R2, on Dec. 14, 2005, under Accession Number 141205-05. In
accordance with 37 CFR 1.808, the depositors assure that all
restrictions imposed on the availability to the public of the
deposited materials will be irrevocably removed upon the granting
of a patent. The deposit will be replaced if the depository cannot
dispense viable samples.
[0181] To produce the hybridoma that produces the anti-cancer
antibodies AR47A6.4.2, a single cell suspension of frozen human
ovarian tumor tissue (endometroid adenocarcinoma; Genomics
Collaborative, Cambridge, Mass.) was prepared in PBS. IMMUNEASY.TM.
(Qiagen, Venlo, Netherlands) adjuvant was prepared for use by
gentle mixing. Five to seven week old BALB/c mice were immunized by
injecting subcutaneously 2 million cells in 50 microliters of the
antigen-adjuvant. Recently prepared antigen-adjuvant was used to
boost the AR47A6.4.2 immunized mice intraperitoneally, 2 and 5
weeks after the initial immunization, with approximately 2 million
cells in 50-60 microliters. A spleen was used for fusion three days
after the last immunization. The hybridomas were prepared by fusing
the isolated splenocytes with NSO-1 myeloma partners. The
supernatants from the fusions were tested from subclones of the
hybridomas.
[0182] To determine whether the antibodies secreted by the
hybridoma cells are of the IgG or IgM isotype, an ELISA assay was
employed. 100 microliters/well of goat anti-mouse IgG+IgM (H+L) at
a concentration of 2.4 micrograms/mL in coating buffer (0.1 M
carbonate/bicarbonate buffer, pH 9.2-9.6) at 4.degree. C. was added
to the ELISA plates overnight. The plates were washed thrice in
washing buffer (PBS+0.05 percent Tween-20). 100 microliters/well
blocking buffer (5 percent milk in wash buffer) was added to the
plate for 1 hour at room temperature and then washed thrice in
washing buffer. 100 microliters/well of hybridoma supernatant was
added and the plate incubated for 1 hour at room temperature. The
plates were washed thrice with washing buffer and 1/100,000
dilution of either goat anti-mouse IgG or IgM horseradish
peroxidase conjugate (diluted in PBS containing 1 percent milk),
100 microliters/well, was added. After incubating the plate for 1
hour at room temperature the plate was washed thrice with washing
buffer. 100 microliters/well of TMB solution was incubated for 1-3
minutes at room temperature. The color reaction was terminated by
adding 50 microliters/well 2M H.sub.2S0.sub.4 and the plate was
read at 450 nm with a Perkin-Elmer HTS7000 plate reader. As
indicated in FIG. 1, the AR47A6.4.2 hybridoma secreted primarily
antibodies of the IgG isotype.
[0183] To determine the subclass of antibody secreted by the
hybridoma cells, an isotyping experiment was performed using a
Mouse Monoclonal Antibody Isotyping Kit (HyCult Biotechnology,
Frontstraat, Netherlands). 500 microliters of buffer solution was
added to the test strip containing rat anti-mouse subclass specific
antibodies. 500 microliters of hybridoma supernatant was added to
the test tube, and submerged by gentle agitation. Captured mouse
immunoglobulins were detected directly by a second rat monoclonal
antibody which is coupled to colloid particles. The combination of
these two proteins creates a visual signal used to analyse the
isotype. The anti-cancer antibody AR47A6.4.2 is of the IgG2a, kappa
isotype.
[0184] After one round of limiting dilution, hybridoma supernatants
were tested for antibodies that bound to target cells in a cell
ELISA assay. Two human ovarian cancer cell lines, and 1 human
normal skin cell line were tested: OCC-1, OVCAR-3 and CCD-27sk
respectively. All cell lines, except for OCC-1, were obtained from
the American Type Tissue Collection (ATCC, Manassas, Va.). The
OCC-1 ovarian cancer cell line was obtained from the Ottawa
Regional Cancer Center (Ottawa, ON).
[0185] The plated cells were fixed prior to use. The plates were
washed thrice with PBS containing MgCl.sub.2 and CaCl.sub.2 at room
temperature. 100 microliters of 2 percent paraformaldehyde diluted
in PBS was added to each well for 10 minutes at room temperature
and then discarded. The plates were again washed with PBS
containing MgCl.sub.2 and CaCl.sub.2 three times at room
temperature. Blocking was done with 100 microliters/well of 5
percent milk in wash buffer (PBS+0.05 percent Tween-20) for 1 hour
at room temperature. The plates were washed thrice with wash buffer
and the hybridoma supernatant was added at 75 microliters/well for
1 hour at room temperature. The plates were washed 3 times with
wash buffer and 100 microliters/well of 1/25,000 dilution of goat
anti-mouse IgG antibody conjugated to horseradish peroxidase
(diluted in PBS containing 1 percent milk) was added. After 1 hour
incubation at room temperature the plates were washed 3 times with
wash buffer and 100 microliter/well of TMB substrate was incubated
for 1-3 minutes at room temperature. The reaction was terminated
with 50 microliters/well 2M H.sub.2S0.sub.4 and the plate read at
450 nm with a Perkin-Elmer HTS7000 plate reader. The results as
tabulated in FIG. 1 were expressed as the number of folds above
background compared to an in-house IgG isotype control that has
previously been shown not to bind to the cell lines tested. The
antibodies from the hybridoma AR47A6.4.2 showed binding to the
ovarian cancer cell line OVCAR-3. AR47A6.4.2 did not display a
detectable level of binding to the normal skin cell line
CCD-27sk.
[0186] In conjunction with testing for antibody binding, the
cytotoxic effect of the hybridoma supernatants was tested in the
cell lines: OCC-1, OVCAR-3 and CCD-27sk. Calcein AM was obtained
from Molecular Probes (Eugene, Oreg.). The assays were performed
according to the manufacturer's instructions with the changes
outlined below. Cells were plated before the assay at the
predetermined appropriate density. After 2 days, 75 microliters of
supernatant from the hybridoma microtiter plates were transferred
to the cell plates and incubated in a 5 percent CO.sub.2 incubator
for 5 days. The wells that served as the positive controls were
aspirated until empty and 100 microliters of sodium azide
(NaN.sub.3) or cycloheximide was added. After 5 days of treatment,
the plates were then emptied by inverting and blotting dry. Room
temperature DPBS (Dulbecco's phosphate buffered saline) containing
MgCl.sub.2 and CaCl.sub.2 was dispensed into each well from a
multichannel squeeze bottle, tapped 3 times, emptied by inversion
and then blotted dry. 50 microliters of the fluorescent calcein dye
diluted in DPBS containing MgCl.sub.2 and CaCl.sub.2 was added to
each well and incubated at 37.degree. C. in a 5 percent CO.sub.2
incubator for 30 minutes. The plates were read in a Perkin-Elmer
HTS7000 fluorescence plate reader and the data was analyzed in
Microsoft Excel. The results are tabulated in FIG. 1. Supernatant
from the AR47A6.4.2 hybridoma produced specific cytotoxicity of 20
percent on the OVCAR-3 cells. This was 29 and 31 percent of the
cytotoxicity obtained with the positive controls sodium azide and
cycloheximide, respectively. Results from FIG. 1 demonstrate that
the cytotoxic effects of AR47A6.4.2 are proportional to the binding
levels on the cancer cell types. There was a greater level of
cytotoxicity produced in the OVCAR-3 cells as compared to the OCC-1
cells, coinciding with the higher level of binding in the OVCAR-3
cells. As tabulated in FIG. 1, AR47A6.4.2 did not produce
cytotoxicity in the CCD-27sk normal cell line. The known
non-specific cytotoxic agents cycloheximide and sodium azide
generally produced cytotoxicity as expected.
Example 2
In Vitro Binding
[0187] AR47A6.4.2 monoclonal antibodies were produced by culturing
the hybridoma in CL-1000 flasks (BD Biosciences, Oakville, ON) with
collections and reseeding occurring twice/week. Standard antibody
purification procedures with Protein G Sepharose 4 Fast Flow
(Amersham Biosciences, Baie d'Urfe, QC) were followed. It is within
the scope of this invention to utilize monoclonal antibodies that
are de-immunized, humanized, chimeric or murine.
[0188] Binding of AR47A6.4.2 to pancreatic (BxPC-3, AsPC-1 and
PL45), colon (DLD-1, Lovo, SW1116, HT-29 and Colo-205), breast
(MDA-MB-468 and MCF-7), prostate (PC-3 and DU-145), lung (NCI-H520
and A549), esophageal (T.Tn), thyroid (SW579), head and neck (FaDu)
and ovarian (OCC-1, C-13, OVCA-429, Sk-OV-3, OV2008, Hey, A2780-cp,
A2780-s and OVCAR-3) cancer cell lines, and non-cancer cell lines
from skin (CCD-27sk) and lung (Hs888.Lu) was assessed by flow
cytometry (FACS). All cell lines, except for the majority of
ovarian cancer cell lines, were obtained from the American Type
Tissue Collection (ATCC, Manassas, Va.). C-13, OV2008, Hey,
A2780-cp, A2780-s, OCC-1 and OVCA-429 ovarian cancer cell lines
were obtained from the Ottawa Regional Cancer Center (Ottawa,
ON).
[0189] Cells were prepared for FACS by initially washing the cell
monolayer with DPBS (without Ca.sup.++ and Mg.sup.++). Cell
dissociation buffer (INVITROGEN, Burlington, ON) was then used to
dislodge the cells from their cell culture plates at 37.degree. C.
After centrifugation and collection, the cells were resuspended in
DPBS containing MgCl.sub.2, CaCl.sub.2 and 2 percent fetal bovine
serum at 4.degree. C. (staining media) and counted, aliquoted to
appropriate cell density, spun down to pellet the cells and
resuspended in staining media at 4.degree. C. in the presence of
test antibody (AR47A6.4.2) or control antibodies (isotype control,
anti-EGFR) at 20 micrograms/mL on ice for 30 minutes. Prior to the
addition of Alexa Fluor 546-conjugated secondary antibody the cells
were washed once with staining media. The Alexa Fluor
546-conjugated antibody in staining media was then added for 30
minutes at 4.degree. C. The cells were then washed for the final
time and resuspended in fixing media (staining media containing 1.5
percent paraformaldehyde). Flow cytometric acquisition of the cells
was assessed by running samples on a FACSarray.TM. using the
FACSarray.TM. System Software (BD Biosciences, Oakville, ON). The
forward (FSC) and side scatter (SSC) of the cells were set by
adjusting the voltage and amplitude gains on the FSC and SSC
detectors. The detectors for the fluorescence (Alexa-546) channel
was adjusted by running unstained cells such that cells had a
uniform peak with a median fluorescent intensity of approximately
1-5 units. For each sample, approximately 10,000 gated events
(stained fixed cells) were acquired for analysis and the results
are presented in FIG. 3.
[0190] FIG. 2 presents the mean fluorescence intensity fold
increase above isotype control. Representative histograms of
AR47A6.4.2 antibodies were compiled for FIG. 3. AR47A6.4.2 showed
strong binding to the pancreatic cancer cell lines BxPC-3 and PL45
(31.6-fold and 26.4-fold respectively), colon cancer cell lines
DLD-1, HT-29 and Colo-205 (91.5-fold, 22.1-fold and 44.9-fold
respectively), breast cancer cell line MDA-MB-468 (35.9-fold), head
and neck cancer cell line FaDu (77.1-fold), esophageal cancer cell
line T.Tn (26.9-fold) and ovarian cancer cell lines OV2008, OVCAR-3
and C-13 (78.4-fold, 28.4-fold and 43.6-fold respectively). Binding
was also observed on the breast cancer cell line MCF-7 (5.4-fold),
prostate cancer cell lines PC-3 and DU-145 (3.3-fold and 5.1-fold
respectively), lung cancer cell line NCI-H520 (10.7-fold), colon
cancer cell line SW1116 (1.8-fold) and ovarian cancer cell lines
Hey, Sk-OV-3, OCC-1 and OVCAR-429 (6.6-fold, 1.9-fold, 10-fold and
4.2-fold respectively). Binding to the non-cancer cell lines from
skin (CCD-27sk) and lung (Hs888.Lu) was not detectable under these
conditions. These data demonstrate that AR47A6.4.2 exhibited
functional specificity in that although there was clear binding to
a variety of cancer cell lines there was only associated
cytotoxicity with some of the lines tested.
Example 3
In Vivo Prophylactic Tumor Experiments with BxPC-3 Cells
[0191] Examples 1 and 2 demonstrated that AR47A6.4.2 had
anti-cancer properties against a human cancer cell line with
detectable binding across several different cancer indications.
With reference to FIGS. 4 and 5, 6 to 8 week old female SCID mice
were implanted with 5 million human pancreatic cancer cells
(BxPC-3) in 100 microliters saline injected subcutaneously in the
scruff of the neck. The mice were randomly divided into 3 treatment
groups of 5. On the day after implantation, 20 mg/kg of AR47A6.4.2
test antibody or buffer control was administered intraperitoneally
to each cohort in a volume of 300 microliters after dilution from
the stock concentration with a diluent that contained 2.7 mM KCl, 1
mM KH.sub.2PO.sub.4, 137 mM NaCl and 20 mM Na.sub.2HPO.sub.4. The
antibody and control samples were then administered once per week
for a period of 7 weeks in the same fashion. Tumor growth was
measured about every seventh day with calipers for 8 weeks or until
individual animals reached Canadian Council for Animal Care (CCAC)
endpoints. Body weights of the animals were recorded once per week
for the duration of the study. At the end of the study all animals
were euthanised according to CCAC guidelines.
[0192] AR47A6.4.2 prevented tumor growth and reduced tumor burden
in an in vivo prophylactic model of human pancreatic cancer. On day
49 post-implantation, the last day of treatment, the mean tumor
volume in the AR47A6.4.2 treated group was 53 percent less than
that of the buffer control-treated group (p<0.05; FIG. 4).
[0193] There were no clinical signs of toxicity throughout the
study. Body weight, shown in FIG. 5, was used as a surrogate for
well-being and failure to thrive. Within groups, there was a
nonsignificant 10 percent increase in body weight in the control
group over the duration of the study. As well, there was a
nonsignificant increase in the body weight of the AR47A6.4.2
treated group; a 14 percent increase from a mean of 20 g to 22.8 g.
There was no significant difference in body weight between the
groups at the end of the treatment period.
[0194] In summary, AR47A6.4.2 was well-tolerated and decreased the
tumor burden in this human pancreatic cancer xenograft model.
Example 4
In Vivo Established Tumor Experiments with BxPC-3 Cells
[0195] To further determine the efficacy of AR47A6.4.2 on the
BxPC-3 model of human pancreatic cancer, the antibody was tested on
an established BxPC-3 xenograft model. With reference to FIGS. 6
and 7, 6 to 8 week old female SCID mice were implanted with 5
million human pancreatic cancer cells (BxPC-3) in 100 microliters
saline injected subcutaneously in the scruff of the neck. Tumor
growth was measured with calipers every week. When the majority of
the cohort reached an average tumor volume of 85 mm.sup.3 (range
56-111) at 33 days post-implantation 9 mice were randomly assigned
into each of 2 treatment groups. AR47A6.4.2 test antibody or buffer
control was administered intraperitoneally to each cohort, with
dosing at 20 mg/kg of antibody in a volume of 300 microliters after
dilution from the stock concentration with a diluent that contained
2.7 mM KCl, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 20 mM
Na.sub.2HPO.sub.4. The antibodies were then administered 3 times
per week for a total of 10 doses in the same fashion until day 53
post-implantation. Tumor growth was measured about every seventh
day with calipers until day 63 post-implantation or until
individual animals reached the CCAC end-points. Body weights of the
animals were recorded once per week for the duration of the study.
At the end of the study all animals were euthanised according to
CCAC guidelines.
[0196] AR47A6.4.2 significantly reduced tumor burden in an
established model of human pancreatic cancer. On day 54, one day
after the last dose of antibody was administered,
AR47A6.4.2-treated animals had a mean tumor volume that was 40
percent of the mean tumor volume in control-treated animals
(p<0.0001; FIG. 6). These results correspond to a mean T/C of 30
percent for AR47A6.4.2.
[0197] Body weight measured at weekly intervals was used as a
surrogate for well-being and failure to thrive. As seen in FIG. 7,
there was no significant difference in mean body weight between the
antibody-treated group and the control at the end of the study. In
addition, body weight in all groups did not vary significantly over
the course of the study.
[0198] In summary, AR47A6.4.2 was well-tolerated and decreased the
tumor burden in this established human pancreatic cancer xenograft
model. AR47A6.4.2 has demonstrated efficacy in both a preventative
and established model of human pancreatic cancer.
Example 5
In Vivo Prophylactic Tumor Experiments with PL45 Cells
[0199] Examples 3 and 4 demonstrated that AR47A6.4.2 had
anti-cancer properties against a human pancreatic cancer cell line.
To determine the efficacy of AR47A6.4.2 against another human
pancreatic cell line, the antibody was tested on a xenograft model
of PL45 human pancreatic cancer. With reference to FIGS. 8, 9 and
10, 8 to 10 week old female SCID mice were implanted with 5 million
human pancreatic cancer cells (PL45) in 100 microliters PBS
solution injected subcutaneously in the scruff of the neck. The
mice were randomly divided into 2 treatment groups of 10. On the
day after implantation, 20 mg/kg of AR47A6.4.2 test antibody or
buffer control was administered intraperitoneally to each cohort in
a volume of 300 microliters after dilution from the stock
concentration with a diluent that contained 2.7 mM KCl, 1 mM
KH.sub.2PO.sub.4, 137 mM NaCl and 20 mM Na.sub.2HPO.sub.4. The
antibody and control samples were then administered once per week
for the duration of the study. Tumor growth was measured about
every 7 day with calipers. The treatment was completed after 9
doses of antibody. Body weights of the animals were recorded once
per week for the duration of the study. At the end of the study all
animals were euthanized according to CCAC guidelines.
[0200] AR47A6.4.2 completely inhibited tumor growth in the PL45 in
vivo prophylactic model of human pancreatic cancer. Treatment with
ARIUS antibody AR47A6.4.2 reduced the growth of PL45 tumors by
nearly 100 percent (p=0.0005, t-test), compared to the
buffer-treated group, as determined on day 77, 20 days after the
last dose of antibody (FIG. 8) when almost all mice in control and
antibody-treated group were living. The study was still ongoing at
day 102, 45 days after last dose, at which point all mice in the
control group had been removed from the study due to tumor volume.
However AR47A6.4.2 still demonstrated almost complete inhibition of
tumor growth and 4 mice in that group were still alive (FIG. 9). It
should be noted that between days 42 and 48, one mouse in the
AR47A6.4.2-treated group died from causes not related to antibody
treatment. In addition, between days 48 and 55, 5 mice in the
antibody-treated group died due to a water bottle leakage in the
cage. All 6 mice that died in the AR47A6.4.2-treated group had yet
to develop measurable subcutaneous PL45 tumors. Consequently, the 4
mice that were still alive at day 102 is an under representation of
the survival benefit of antibody treatment.
[0201] There were no obvious clinical signs of toxicity throughout
the study. Body weight measured at weekly intervals was a surrogate
for well-being and failure to thrive. The mean body weight
increased in all groups over the duration of the study (FIG. 10).
The mean weight gain between day 13 and day 77 was 3.39 g (17.4
percent) in the control group and 1.7 g (8.6 percent) in the
AR47A6.4.2-treated group. There were no significant differences
between the groups during the treatment period and at day 77, 20
days after the last dose.
[0202] In summary, AR47A6.4.2 was well-tolerated and almost
completely inhibited the tumor growth in this human pancreatic
cancer xenograft model. AR47A6.4.2 treatment also demonstrated
increased survival in comparison to buffer treatment. AR47A6.4.2
therefore has demonstrated efficacy in two different models of
human pancreatic cancer.
Example 6
In Vivo Prophylactic Tumor Experiments with PC-3 Cells
[0203] Examples 3, 4 and 5 demonstrated that AR47A6.4.2 had
anti-cancer properties against two different human pancreatic
cancer xenograft models. To determine the efficacy of AR47A6.4.2
against a different human cancer xenograft model, the antibody was
tested on a PC-3 prostate cancer xenograft model. With reference to
FIGS. 11, 12, and 13, 8 to 10 week old female SCID mice were
implanted with 5 million human prostate cancer cells (PC-3) in 100
microliters PBS solution injected subcutaneously in the scruff of
the neck. The mice were randomly divided into 2 treatment groups of
10. On the day after implantation, 20 mg/kg of AR47A6.4.2 test
antibody or buffer control was administered intraperitoneally to
each cohort in a volume of 300 microliters after dilution from the
stock concentration with a diluent that contained 2.7 mM KCl, 1 mM
KH.sub.2PO.sub.4, 137 mM NaCl and 20 mM Na.sub.2HPO.sub.4. The
antibody and control samples were then administered once per week
for the duration of the study. Tumor growth was measured about
every 7 day with calipers. The study was completed after 8 doses of
antibody. Body weights of the animals were recorded once per week
for the duration of the study. At the end of the study all animals
were euthanized according to CCAC guidelines when reaching
endpoint.
[0204] AR47A6.4.2 inhibited tumor growth in the PC-3 in vivo
prophylactic model of human prostate adenocarcinoma cells.
Treatment with ARIUS antibody AR47A6.4.2 reduced the growth of PC-3
tumors by 60.9 percent (p=0.00037, t-test), compared to the buffer
treated group, as determined on day 32 after 5 doses of treatment
with antibody (FIG. 11) when almost all mice in control and
antibody-treated group were still alive. All mice in the control
group had been removed from the study by day 47, 3 days before the
last dose of antibody, due to tumor volume/lesions. However, the
study was still ongoing at day 77; 27 days after last dose of
antibody where 40 percent of the mice in the AR47A6.4.2-treated
group still were still alive (FIG. 12).
[0205] There were no obvious clinical signs of toxicity throughout
the study. Body weight measured at weekly intervals was a surrogate
for well-being and failure to thrive. The mean body weight remained
relatively constant in all groups over the duration of the study
(FIG. 13). There were no significant differences between the groups
during the treatment period or at day 32, after 5 doses of
antibody.
[0206] In summary, AR47A6.4.2 was well-tolerated and significantly
inhibited the tumor growth in this human prostate cancer xenograft
model. Treatment with antibody also demonstrated a survival benefit
in comparison to the control group. AR47A6.4.2 has demonstrated
efficacy against two different human cancer indications; pancreatic
and prostate.
Example 7
In Vivo Prophylactic Tumor Experiments with MCF-7 Cells
[0207] Examples 3, 4, 5 and 6 demonstrated that AR47A6.4.2 had
anti-cancer properties against two different human pancreatic and a
prostate cancer xenograft model. To determine the efficacy of
AR47A6.4.2 against another human cancer xenograft model, the
antibody was tested on a MCF-7 cancer xenograft model. With
reference to FIGS. 14, 15 and 16, Balb/C nude mice were irradiated
for 24 hours (2.5 Gy, Co.sup.60) and 20 million human breast cancer
cells (MCF-7) in 200 microliters RPMI 1640 were injected
subcutaneously in the right flank of the mice. The mice were
randomly divided into 2 treatment groups of 12. On the day after
implantation, 20 mg/kg of AR47A6.4.2 test antibody or buffer
control was administered intraperitoneally to each cohort in a
volume of 200 microliters per mouse of 20 g. The antibody and
control samples were then administered once per week for the
duration of the study in the same fashion. Tumor growth was
measured about twice a week with calipers. The study was completed
after 8 injections of antibody. Body weights of the animals were
recorded twice per week for the duration of the study. Mice were
sacrificed when the tumor volume reached 2000 mm.sup.3 and all
remaining mice were sacrificed on day 91 of the study.
[0208] AR47A6.4.2 reduced tumor growth in the MCF-7 in vivo
prophylactic model of human breast cancer. Treatment with ARIUS
antibody AR47A6.4.2 resulted in a marked tumor growth delay.
AR47A6.4.2 induced T/C percent values that were lower than 42
percent from day 18 to day 35 of treatment and close to 42 percent
up to day 49 (optimal T/C percent value of 10.9 percent at day 18)
(FIG. 14). At day 53, after treatment was terminated, efficacy with
treatment of AR47A6.4.2 was still observed with a T/C of 57
percent. At the end of the study (day 91), 2 mice from the
AR47A6.4.2 treatment group remained tumor-free.
[0209] There were no clinical signs of toxicity throughout the
study. Body weight was measured twice a week and was a surrogate
for well-being and failure to thrive. A reduced body weight gain
was only observed during the first week of treatment in the
AR47A6.4.2 treatment group. After that, no significant body weight
changes were detected between the AR47A6.4.2 treated and buffer
control group (FIG. 15). There were no significant differences
between groups at the end of the treatment period.
[0210] A post-treatment survival benefit (FIG. 16) was associated
with AR47A6.4.2 administration. The buffer control group reached
100 percent mortality by day 85 post-treatment while 33.3 percent
of the AR47A6.4.2 mice were still alive at day 91
post-treatment.
[0211] In summary, AR47A6.4.2 was well-tolerated, reduced tumor
growth and provided a survival benefit in this human breast cancer
xenograft model. AR47A6.4.2 has demonstrated efficacy against three
different human cancer indications; pancreatic, prostate and
breast.
Example 8
In Vivo Prophylactic Tumor Experiments with Colo 205 Cells
[0212] Examples 3, 4, 5, 6 and 7 demonstrated that AR47A6.4.2 had
anti-cancer properties against two different human pancreatic, a
prostate and a breast cancer xenograft model. To determine the
efficacy of AR47A6.4.2 against another human cancer xenograft
model, the antibody was tested on a Colo 205 colon cancer xenograft
model. With reference to FIGS. 17 and 18, 8 to 10 week old female
SCID mice were implanted with 5 million human colon cancer cells
(Colo 205) in 100 microliters PBS solution injected subcutaneously
in the right flank of each mouse. The mice were randomly divided
into 2 treatment groups of 10. One day after implantation, 20 mg/kg
of AR47A6.4.2 test antibody or buffer control was administered
intraperitoneally to each cohort in a volume of 300 microliters
after dilution from the stock concentration with a diluent that
contained 2.7 mM KCl, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 20 mM
Na.sub.2HPO.sub.4. The antibody and control samples were then
administered once per week for the first two weeks and twice per
week for another 3 weeks. Tumor growth was measured about every 3-4
days with calipers. The treatment was completed after 8 doses of
antibody. Body weights of the animals were recorded when tumors
were measured for the duration of the study. At the end of the
study all animals were euthanized according to CCAC guidelines when
reaching endpoint.
[0213] AR47A6.4.2 inhibited tumor growth in the Colo 205 in vivo
prophylactic model of human colorectal adenocarcinoma cells.
Treatment with ARIUS antibody AR47A6.4.2 reduced the growth of Colo
205 tumors by 60.2 percent (p=0.0003851, t-test), compared to the
buffer treated group, as determined on day 27, 4 days before the
last dose of antibody (FIG. 17).
[0214] There were no obvious clinical signs of toxicity throughout
the study. Body weight measured at weekly intervals was a surrogate
for well-being and failure to thrive. There were no significant
differences in mean body weight between the groups during the
treatment period (FIG. 18).
[0215] In summary, AR47A6.4.2 was well-tolerated and significantly
inhibited the tumor growth in this human colon cancer xenograft
model. AR47A6.4.2 has demonstrated efficacy against four different
human cancer indications; pancreatic, prostate, breast and colon.
Treatment benefits were observed in several well-recognized models
of human cancer disease suggesting pharmacologic and pharmaceutical
benefits of this antibody for therapy in other mammals, including
man.
Example 9
Identification of Binding Proteins by Western Immunoblotting
[0216] To identify the antigen(s) recognized by the antibody
AR47A6.4.2, cell membrane preparations were subjected to sodium
dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), and
transferred to membranes. The latter were probed with the antibody
AR47A6.4.2 to visualize the proteins detected by this antibody.
1. Total Membrane Fraction Preparation
[0217] Total cell membranes were prepared from confluent cultures
of MDA-MB-231 (MB-231) breast cancer cells. Media was removed from
cell stacks and the cells were washed with phosphate buffered
saline (PBS). Cells were dissociated with dissociation buffer
(Gibco-BRL; Grand Island, N.Y.) for 20 minutes at 37.degree. C. on
a platform shaker. Cells were collected and centrifuged at 900 g
for 10 minutes at 4.degree. C. After centrifugation, cell pellets
were washed by resuspending in PBS and centrifuging again at 900 g
for 10 minutes at 4.degree. C. Pellets were then stored at
-80.degree. C. until required. To prepare membranes, cell pellets
were thawed and resuspended in homogenization buffer containing 1
tablet per 50 mL of complete protease inhibitor cocktail (Roche;
Laval QC) at a ratio of 3 mL buffer per gram of cells. The cell
suspension was subjected to homogenization using a polytron
homogenizer on ice in order to lyse the cells. The cell homogenate
was centrifuged at 15,000 g for 10 minutes at 4.degree. C. to
remove the nuclear particulate. Supernatant was harvested, divided
into tubes and then centrifuged at 75,600 g for 90 minutes at
4.degree. C. Supernatant was carefully removed and each membrane
pellet was resuspended in approximately 5 mL of homogenization
buffer. The membrane pellets from all tubes were combined, divided
one more time, and centrifuged at 75,600 g for 90 minutes at
4.degree. C. Supernatant was carefully removed and the pellets were
weighed. Solubilization buffer containing 1 percent Triton X-100
was added to the pellets at a ratio of 3 mL buffer per gram of
membrane pellet. Membranes were solubilized by shaking on a
platform shaker at 300 rpm, for 1 hour on ice. The membrane
suspension was centrifuged at 75,600 g to pellet insoluble
material. The supernatant, containing the solubilized membrane
proteins, was carefully removed from the tubes, assayed for protein
concentration, and stored at -80.degree. C.
2. Immunoprecipitation, 1-Dimensional SDS-PAGE and Western
Immunoblotting
[0218] Immunoprecipitation of AR47A6.4.2 antigen was carried out as
follows: total membrane fraction was diluted to a 1 mg/mL final
protein concentration, with 1.times. RIPA buffer containing
protease inhibitors. Protein G Sepharose beads chemically
conjugated to 8A3B.6 isotype control antibody (conjugated at a
ratio of 2 micrograms of antibody per 1 microliters of drained
beads), were added to the total membrane fraction and incubated at
4.degree. C. for 3 hour, with rotation. After, the sample was
centrifuged at 20000.times.g for 8 sec. The supernatant (unbound
fraction) was removed and the beads were stored on ice. An
identical volume of Protein G Sepharose beads conjugated to
AR47A6.4.2 (conjugated at a ratio of 2 micrograms of antibody per 1
microliters of drained beads) was added to the TM protein mixture
supernatant from the previous step. The sample was incubated for 3
hours at 4.degree. C., with rotation. After incubation, the sample
was centrifuged as described above and the beads were saved. The
isotype control and AR47A6.4.2 beads were then washed 3.times.1 mL
with RIPA buffer and rinsed with 1.times.PBS. These two samples,
and an identical volume of AR47A6.4.2 and 8A3B.6-Protein G
Sepharose-conjugated beads (`mock IP` samples) beads were prepared
for SDS-PAGE by boiling in non-reducing sample buffer. Proteins
from the total membrane fraction of MB-231 cells were separated by
1-dimensional SDS-PAGE (1D SDS-PAGE), on a 5 and 10 percent
stacking and separating gel, respectively. Proteins were
transferred overnight, at 4.degree. C., by electroblotting onto
PVDF membranes (Millipore; Billerica, Mass.). Complete transfer was
determined by assessing the transfer of prestained molecular weight
markers onto the membrane. After transfer, the membranes were
blocked with 5 percent (w/v) skim milk in TBST, for 1 hour at room
temperature (RT), and two replicate blots were then probed as
follows: one blot was probed with the antibody AR47A6.4.2 (5 mg/mL,
in 5 percent skim milk in TBST) and the replicate blot was probed
with an IgG2a isotype control (5 mg/mL, in 5 percent skim milk in
TBST). Blots were washed 3 times for 10 minutes in TBST and then
incubated with horseradish HRP-conjugated goat anti-mouse IgG (Fc)
(Bio-Rad Laboratories; Hercules, Calif.), for 1 hour at RT. After
washing 3 times for 10 minutes each with TBST, the blots were
developed with the ECL Plus.TM. kit (GE Healthcare, Life Sciences
formerly Amersham Biosciences; Piscataway, N.J.) following the
manufacturers' instructions. The blots were rinsed with water and
images were acquired with a gel documentation system (Bio-Rad;
Hercules, Calif.). Blots were imaged under the same conditions of
camera focus, aperture and image acquisition time.
[0219] In FIG. 19, when used as a probe on a Western blot, the
antibody AR47A6.4.2 clearly bound to a protein with an apparent
molecular weight of approximately 50 kDa in the total membrane
fraction from MB-231 cells, but not to the whole cell lysate of
either PC-3 or CCD-27sk cells. In FIG. 20, the antibody AR47A6.4.2
specifically recognized a protein of apparent molecular weight of
around 50 kDa that was immunoprecipitated, by AR47A6.4.2-conjugated
Protein G Sepharose beads, from MB-231 total membrane fraction. It
can be observed that the band recognized by the probe AR47A6.4.2 is
very distinct from those observed by cross reactivity of the
secondary antibody alone, which represent IgG and IgG heavy chain
that leaked off the Protein G beads.
[0220] It was then determined if the disperse nature of the
antigen, as detected by Western immunoblotting, was due to
heterogeneous glycosylation. Immunocomplexes obtained by
immunoprecipitation with AR47A6.4.2, or with isotype control
antibody, from MB-231 total membrane fraction were subjected to
treatment, under non-reducing conditions, with either Enzyme
Deglycosylation Kit (Prozyme, San Leandro, Calif.), which contained
a mixture of glycopeptidase F, O-glycanase, sialidase,
.beta.(1-4)galactosidase and .beta.-N-Acetylglucosaminidase which
removed specific carbohydrate groups, or with deglycosylation
buffer only. After 24 hours incubation at 37.degree. C., the
samples were subjected to 1D SDS-PAGE and Western blotting. It was
expected that if some of the enzymes removed a portion of
carbohydrate that accounted for a significant amount of the mass of
the antigen(s) recognized by the antibody AR47A6.4.2, that it would
be possible to detect that difference by SDS-PAGE. FIG. 21 shows
that glycosidase treatment of immunocomplexes obtained from MB-231
TM fraction resulted in a significant decrease in the mass of the
recognized antigen(s). This indicated that the antigen recognized
by the AR47A6.4.2 antibody was comprised of at least one
glycoprotein.
Example 10
Identification of Antigen Bound by AR47A6.4.2
[0221] 1. Large Scale Immunoprecipitation of Antigens from MB-231
Total Membrane Fraction
[0222] Total membrane fraction extract from MB-231 cells (9.4 mg)
was prepared by dilution, 1 mg/mL final concentration, with
1.times. RIPA buffer containing a protease inhibitors cocktail.
Total membrane fraction extract was pre-cleared by incubation with
protein G Sepharose beads (5 mL drained beads) for 2 hour, at
4.degree. C. with rotation. After centrifugation the beads were
removed and stock bovine serum albumin (BSA) (10 mg/mL) was added
to a 0.5 mg/mL final BSA concentration. While extract was being
pre-cleared, AR47A6.4.2 and 8A3B.6 isotype control
antibody-conjugated protein G-Sepharose beads (120 micrograms of
antibody chemically cross-linked to 60 microliters of protein G
Sepharose) were blocked with 1 mL of 0.5 mg/mL BSA, by incubation
at 4.degree. C., also for 2 hours. After blocking, the
antibody-conjugated beads were washed twice for 5 minutes with
1.times. RIPA buffer. The total membrane extract was then incubated
with the isotype control (8A3B.6)-conjugated protein G Sepharose
beads (60 microliters of drained beads, 120 micrograms of IgG) at
4.degree. C. for 2 hours, with rotation, on an end-over-end
rotator. After centrifugation at 20,000 g, for 10 seconds, at
4.degree. C., the supernatant (unbound fraction) was removed and
saved, and the beads were washed 3 times for 5 minutes, with 1 mL
of RIPA buffer in each wash step. The beads were then rinsed once
with 1.5 mL of PBS and then were stored on ice. The saved
supernatant (unbound fraction) was then incubated with the
AR47A6.4.2-conjugated protein G Sepharose beads (60 microliters of
drained beads, 120 micrograms of IgG) at 4.degree. C., for 2 hours,
with rotation. After centrifugation at 20,000 g, for 10 seconds, at
4.degree. C., the supernatant (unbound fraction) was removed and
saved, and the beads were washed 3 times for 5 minutes, with 1 mL
of RIPA buffer in each wash step. The beads were then rinsed once
with 1.5 mL of PBS and TM fraction extract was saved at -80.degree.
C. and the beads were stored on ice. The isotype control beads and
two aliquots containing AR47A6.4.2-protein G Sepharose conjugated
beads (one being that used in the immunoprecipitation step and a
second aliquot containing identical volume of beads, but not used
in any IP (designated as `mock` IP)). The beads were then stored
overnight at -85.degree. C. To prepare the samples for SDS-PAGE,
each sample containing antibody-conjugated Protein G Sepharose
beads (samples AR47A6.4.2 IP, AR47A6.4.2 `mock` IP and 8A3B.6
isotype control IP) were divided in two 30 microliter aliquots. To
one of the aliquots from each sample was added 60 microliters of
1.times. non-reducing SDS-PAGE sample buffer. After boiling for 4
minutes the sample buffer was removed and transferred into the tube
containing the second aliquot from the same sample. This pooled
sample was then boiled for 4 minutes. After cooling down on ice,
each sample was loaded onto two separate gels ( 1/10th of the
sample in one gel, for detection by Western blotting, the remaining
9/10th on the other gel, for detection by staining with Colloidal
Blue). The gel designated for Western blotting was transferred onto
a PVDF membrane for 2 hours at 320 mA, rinsed with deionized water,
blocked for 1 hour at RT with 5 percent milk in TBST and then
incubated for 2 hours in 5 percent milk in TBST, also at RT. Blots
were washed 3 times for 10 minutes in TBST and incubated with an
HRP-conjugated Fc-specific goat anti-mouse IgG (1:50000) in 5
percent milk in TBST, for 1 hour at room temperature. Blots were
then washed 3 times for 10 minutes and were developed by using an
enhanced chemiluminescence detection system, following the
manufacturer's recommendations. The gel designated for protein
staining was incubated overnight with the Coomassie Colloidal Blue
stain and destained with ultrapure water, for 48 hour.
2. Peptide Mapping, and Antigen Identification by Mass
Spectrometry
[0223] From the experiment above, the image of the Western blot and
of the Coomassie Colloidal Blue stained gel were lined up using the
bands from the molecular weight markers lanes as reference (FIG.
22). A specific band from the lane, on the Coomasie Colloidal
Blue-stained gel, containing the AR47A6.4.2 immunoprecipitate was
cored using a glass pasteur pipette. The equivalent regions of all
the control lanes (AR47A6.4.2 `mock IP` and 8A3B.6 isotype control
IP) and from a region of the gel that did not contain any sample
were also cored as shown in FIG. 23, where left and right panels
are the images of the gel before and after coring, respectively.
Gel plugs were divided in two aliquots containing similar amounts
of gel plugs. One of the sets of aliquots was stored at 4.degree.
C. while the replicate aliquots were subjected to in-gel tryptic
digestion using a commercially available kit (Pierce, Rockford,
Ill.).
[0224] Aliquots from each digest were subjected to mass
spectrometry analysis on a SELDI-TOF Ciphergen PBSIIc reader
(Ciphergen Biosystems Inc., Fremont, Calif.). Briefly, an aliquot
from each digest was manually spotted onto an H4 chip (Ciphergen
Biosystems Inc., Fremont, Calif.). After drying, an aliquot of CHCA
matrix (a-cyano 4-hydroxy cinnaminic acid; Ciphergen Biosystems
Inc., Fremont, Calif.) was added onto the same spot on the chip and
allowed to dry. The samples were then analyzed on the PBSIIc
reader. Similar sized bands from parallel regions on isotype
control lanes and blank gel region were processed side-by-side with
the gel plug from the AR47A6.4.2 IP, so as to enable determination
of unique peptide fragments generated by the digestion of the
antigen immunoprecipitated by AR47A6.4.2 (FIG. 24). The masses of
the unique peptide fragments were searched using PROFOUND, a
publicly accessible online tool for searching protein sequence
databases using information from mass spectra. The unique peptides
in the sample from the AR47A6.4.2 IP digest were then subjected to
MS/MS analysis on a QSTAR (Applied Biosystems, Foster City, Calif.)
equipped with an interface that enabled analysis of the same sample
spots that were previously analyzed on the PBSIIc reader. The MS/MS
data was then analyzed with MASCOT, a publicly accessible online
tool for searching protein databases using information from MS/MS
spectra. The only protein that was suggested as a putative
candidate, with a significant degree of confidence was TROP-2. FIG.
25 is a summary from the MASCOT search. SEQ ID NO:9 is shown. The
only protein that was identified with a high degree of probability
was TROP-2, supporting the previous identification by MS peptide
mass fingerprinting.
3. AR47A6.4.2 Antigen ID Confirmation
[0225] Confirmation of the ID of the putative antigen for
AR47A6.4.2 was carried out through determination of whether a known
anti-human TROP-2 monoclonal antibody (clone 77220.11, R&D
Systems, Minneapolis, Minn.) would react with the protein(s)
immunoprecipitated by AR47A6.4.2. Further confirmation was also
carried out by Western immunoblotting of recombinant human TROP-2
purified from transfected eukaryotic cells. Immunoprecipitates from
an MB-231 total membrane extract in 1.times. RIPA buffer, prepared
with the monoclonal antibodies AR47A6.4.2 and 8A3B.6 IgG2a isotype
control, and the AR47A6.4.2 `mock IP` negative control (described
above) were analyzed by 1D SDS-PAGE followed by Western
immunoblotting. Equal volume fractions from each immunocomplex
sample were analyzed on replicate gels. After electroblotting onto
PVDF membranes, the blots from the replicate gels were probed in
parallel with the monoclonal antibodies AR47A6.4.2, anti-human
TROP-2 and with the IgG2a isotype control. FIG. 26 demonstrates the
result from the cross-IP experiments, in which the material
immunoprecipitated by the test monoclonal antibodies AR47A6.4.2 was
analyzed by Western immunoblotting. Each of the monoclonal
antibodies AR47A6.4.2 and anti-human TROP-2 clone 7220.11
specifically cross-reacted with similar antigen(s)
immunoprecipitated by AR47A6.4.2. However the isotype control
antibody 8A3B.6 did not cross-react with any specific band. In
addition, the antibodies used to probe the Western blots
cross-reacted with no bands on the negative control
immunocomplexes. This data indicated that the epitope recognized by
the AR47A6.4.2 antibody was contained within the TROP-2
antigen.
[0226] To further confirm that AR47A6.4.2 was directly binding to
the human TROP-2 antigen, its reactivity was assessed, by Western
immunoblotting against recombinant fusion polypeptides containing
the extracellular domain of human TROP-2 and the Fc region of human
IgG1, and expressed by the mouse myeloma cell line NSO(R&D
Systems, Minneapolis, Minn.).
[0227] The results illustrated by FIG. 27 revealed that AR47A6.4.2
specifically recognized the recombinant form of human TROP-2 (lane
1 of the blot probed by AR47A6.4.2) and did not recognize a
recombinant GST-fusion construct of the extracellular domain 2
(GST-EC2) of human CD63. The specificity of the antibody against
the recombinant human TROP-2 was further confirmed by the
observation that a commercially available anti-human TROP-2
antibody (clone 77220.11) also recognized similar sized bands and
did not recognize the GST-EC2 domain of human CD63. In addition, an
anti-human CD63 antibody (clone 1A245.6) specifically recognized
the GST-EC2 fusion construct of human CD63 but failed to recognize
the recombinant human TROP-2 protein. The above results demonstrate
that AR47A6.4.2 recognized and directly bound to human TROP-2, and
specifically to its extracellular domain encompassing amino acids
27-274.
Example 11
Deglycosylation Studies
[0228] In order to determine the effects of glycosylation on the
binding of AR47A6.4.2, deglycosylation reactions were set up as per
manufacturer's (Enzymatic Deglycosylation Kit, Prozyme, San
Leandro, Calif.) instructions under both denaturing and
non-denaturing conditions. For denaturing reactions, 0.4 micrograms
recombinant TROP-2 (rhTROP-2; R&D Systems, Minneapolis, Minn.)
or 100 micrograms of MDA-MB-231 membrane proteins (isolated as
described above) were diluted to 30 microliters with water. 10
microliters of incubation buffer (5 .times., 0.25 M
NaH.sub.2PO.sub.4, pH 7.0) and 2.5 microliters of denaturation
solution (2 percent SDS, 1 M beta-mercaptoethanol) were added, and
reactions were boiled for 5 minutes. Once reactions cooled to room
temperature, 2.5 microliters of detergent solution (15 percent
NP-40) and 1 microliter of each of the following enzymes were
added: N-Glycanase.RTM. PNGase F (.gtoreq.5 U/mL), Sialidase A.TM.
(.gtoreq.5 U/mL), O-Glycanase.RTM. (.gtoreq.1.25 U/mL), beta (1-4)
Galactosidase (3 U/mL) and beta-N-Acetylglucosaminidase (40 U/mL).
Control reactions were included which contained 5 microliters of
water instead of deglycosylation enzymes. For non-denaturing
reactions, 0.4 micrograms rhTROP-2 or 100 micrograms of MDA-MB-231
membrane proteins were diluted to 35 microliters with water. 10
microliters of incubation buffer was added, along with 1 microliter
of each enzyme listed above. Control reactions were included which
contained 5 microliters of water instead of deglycosylation
enzymes. All reactions were incubated at 37.degree. C. for 24
hours.
[0229] Following deglycosylation, reactions were prepared for
SDS-PAGE. 16.7 microliters of reducing or non-reducing sample
loading buffer (4.times.) was added to the denatured and
non-denatured reactions, respectively. Samples were boiled for 5
minutes, then cooled to room temperature. 16.7 microliters of each
reaction was loaded onto quadruplicate 12 percent SDS-PAGE gels.
Gels were run at 150 V until the dye front ran off. Proteins were
transferred to PVDF membranes overnight at 40 V. Membranes were
blocked with 5 percent milk prepared with TBST (Tris-buffered
saline with 0.05 percent Tween-20) for 1 hour, followed by
incubation with primary antibodies for 2 hours. Each primary
antibody was diluted to 5 micrograms/mL in 5 percent milk, except
anti-human TROP-2, which was diluted to 2 micrograms/mL. Blots were
incubated with one of AR47A6.4.2, anti-human TROP-2 (R&D
Systems, Minneapolis, Minn.) or IgG isotype control. Following
primary antibody incubation, blots were washed 3 times, 10 minutes
each, with TBST. Blots were incubated with goat anti-mouse IgG Fc
HRP secondary antibody diluted to 1:50,000 in 5 percent milk for 1
hour, then washed 3 times, 10 minutes each, with TBST. Blots were
developed with ECL Plus Western Blotting Detection Reagents (GE
Healthcare, Life Sciences formerly Amersham Biosciences;
Piscataway, N.J.) and an X-ray developer.
[0230] FIGS. 28-30 show the results of the 3 blots probed with
AR47A6.4.2, anti-human TROP-2 and IgG isotype control respectively.
FIG. 28 (blot probed with AR47A6.4.2) shows a weak band in lane 1
(non-denatured and non-deglycosylated MB-231 total membrane
fraction) at .about.52 kDa. This band is not detectable in FIG. 29
(blot probed with anti-human TROP-2). The reactive band in the
sample from lane 2 (non-denatured and deglycosylated MB-231 total
membrane fraction) shows a shift to around 37 kDa and was
recognized by AR47A6.4.2 (FIG. 28) and by the commercial
anti-TROP-2 antibody (FIG. 29) blot, but not by the isotype control
(FIG. 30). No bands were detected in lanes 5 or 6 (denatured and
non-deglycosylated and deglycosylated MB-231 total membrane
fraction) in any of the blots, indicating that the antibodies did
not detectably bind to the target protein in the total membrane
fraction under reducing conditions.
[0231] Recombinant human TROP-2 appears as a very intense, very
high molecular weight band (apparent molecular weight larger than
220 kDa) in lanes 3 and 4 (non-denatured non-deglycosylated and
deglycosylated rhTROP-2, respectively) in FIGS. 28 and 29
(AR47A6.4.2 and anti-human TROP-2, respectively) correspond to
disulfide-bond linked multimers of rhTROP-2. Less intense bands
appear at .about.70 kDa in lane 3 (non-denatured non-deglycosylated
rhTROP-2) and .about.55 kDa in lane 4 (non-denatured and
deglycosylated rhTROP-2) in FIG. 28 (blots probed with AR47A6.4.2)
and correspond to the monomeric forms of rhTROP-2. The shift in
apparent molecular weight of both the multimer and monomer bands
from larger than 220 kDa and 70 kDa to lower than 220 kDa and 55
kDa, respectively (lanes 3 and 4) result form the loss of
carbohydrate groups due to deglycosylation. Under reducing
conditions (lanes 7 and 8), rhTROP-2 is detected only as the
smaller monomeric polypeptide, with a decrease of approximately 20
kDa in apparent molecular weight upon treatment with the
glycosidase mixture (FIG. 29). FIG. 30 (blot probed with the
isotype control antibody) does not display reactivity in any of the
lanes.
[0232] The two anti-human TROP-2 antibodies used, AR47A6.4.2 and
the commercial anti-human TROP-2 antibody, recognized the human
TROP-2 antigen in a total membrane preparation from MDA-MB-231, and
the purified recombinant human TROP-2, prior to, and after,
treatment with a mixture of glycosidases. This result suggests that
the antibodies may recognize a non-carbohydrate epitope, possibly a
polypeptide epitope, although it is not possible to rule out that
binding may be occurring to a carbohydrate group that was not
removed by the particular mixture of glycosidases used in this
experiment.
Example 12
Competition Experiments
[0233] In order to further characterize the binding properties of
AR47A6.4.2 and AR52A301.5 (another antibody generated in-house
which also binds to Trop-2) antibody competition experiments were
carried out by Western blot to determine if AR47A6.4.2 and
AR52A301.5 recognize similar or distinct epitopes of TROP-2. Two
micrograms of recombinant fusion polypeptides containing the
extracellular domain of human TROP-2 and the Fc region of human
IgG1, and expressed by the mouse myeloma cell line NS0 (R&D
Systems, Minneapolis, Minn.) were subjected to SDS-PAGE under
non-reducing conditions using preparative well combs that spanned
the entire length of each of two 10 percent polyacrylamide gels.
The proteins from the gels were transferred to PVDF membranes at
40V for approximately 17 hours at 4.degree. C. The membranes were
blocked with 5 percent skim milk in TBST for one hour at room
temperature on a rotating platform. The membranes were washed twice
with approximately 20 mL of TBST and were placed in a Western
multiscreen apparatus creating twenty separate channels in which
different probing solutions were applied. Previously, biotinylated
AR47A6.4.2 and AR52A301.5 had been prepared using EZ-Link NHS-PEO
Solid Phase Biotinylation Kit (Pierce, Rockford, Ill.). Primary
antibody solutions were prepared by mixing biotinylated AR47A6.4.2
or biotinylated AR52A301.5 with varying concentrations of
non-biotinylated antibodies. Specifically, solutions were prepared
containing 0.05 micrograms/mL of biotinylated AR52A301.5 in 3
percent skim milk in TBST plus 0.5 micrograms/mL, 5 micrograms/mL,
50 micrograms/mL, 500 micrograms/mL or 1000 micrograms/mL of
non-biotinylated antibody. The non-biotinylated antibodies that
were used were AR52A301.5, AR47A6.4.2 and control antibody 8A3B.6
(anti-bluetongue virus; IgG2a, kappa, purified in-house). Solutions
containing 0.05 micrograms/mL of biotinylated AR47A6.4.2 were
prepared with the same concentrations listed above of the
non-biotinylated antibodies AR52A301.5, AR47A6.4.2 and control
antibody 1B7.11 (anti-TNP; IgG1, kappa, 20 micrograms/mL, purified
in-house). A negative control solution consisting of three percent
skim milk in TBST was added to two channels on each membrane.
[0234] The primary antibody solutions were incubated in separate
channels on the membranes for 2 hours at room temperature on a
rocking platform. Each channel was washed 3 times with TBST for ten
minutes on a rocking platform. Secondary solution consisting of
0.01 micrograms/mL peroxidase conjugated streptavidin (Jackson
Immunoresearch, West Grove Pa.) in 3 percent skim milk in TBST was
applied to each channel on the membrane, except for one channel on
each membrane to which 3 percent milk in TBST alone was applied as
a negative control. The membranes were incubated in secondary
solution for 1 hour at room temperature on a rocking platform. Each
channel was washed 3 times with TBST for ten minutes on a rocking
platform. The membranes were removed from the multiscreen apparatus
and incubated with an enhanced chemiluminescence detection solution
(GE Healthcare, Life Sciences formerly Amersham Biosciences;
Piscataway, N.J.) according to manufacturer's directions. The
membranes were then exposed to film and developed.
[0235] FIGS. 31 and 32 show the results of the antibody competition
experiments. Binding of the biotinylated AR52A301.5 was completely
inhibited at a concentration of 50 micrograms/mL and greater of
non-biotinylated AR52A301.5 (1000.times. excess; FIG. 31 lanes 3-7)
while the binding of AR47A6.4.2 was completely inhibited at a
concentration of 500 micrograms/mL and greater of non-biotinylated
AR47A6.4.2 (10000.times. excess; FIG. 32 lanes 9-13). The binding
of biotinylated AR52A301.5 was not inhibited in any of the samples
containing IgG2a isotype control antibody (FIG. 31 lanes 15-19) and
the binding of biotinylated AR47A6.4.2 was not inhibited in any of
the samples containing IgG1 isotype control antibody (FIG. 32 lanes
15-19). This indicates that the inhibition of binding observed with
the biotinylated antibodies mixed with the same non-biotinylated
antibody was due to the occupation of antigen binding sites by the
non-biotinylated antibody, not by non-specific interactions of
excess antibody alone. The binding of biotinylated AR52A301.5 was
not completely inhibited in any of the samples containing
AR47A6.4.2, and the binding of biotinylated AR47A6.4.2 was not
completely inhibited in any of the samples containing AR52A301.5.
In both Western blots however, the reactivity of each biotinylated
TROP-2 antibody at 5 micrograms/mL, 50 micrograms/mL, 500
micrograms/mL, and 1000 micrograms/mL of the other non-biotinylated
TROP-2 antibody was less intense than in the corresponding lanes of
excess isotype control antibody. These results indicate that the
binding of AR52A301.5 does not prevent the binding of AR47A6.4.2 to
TROP-2 and vice versa. Overall, the results of the competition
Western blots suggest that the epitopes of the TROP-2 molecule that
are recognized by AR47A6.4.2 and AR52A301.5 are distinct from one
and other, although the binding of one antibody does affect the
binding of the other.
Example 13
Human Normal Tissue Staining
[0236] IHC studies were conducted to characterize the AR47A6.4.2
antigen distribution in frozen human normal tissues sections
(previous experiments showed no reactivity of this antibody with
formalin fixed tissues). Slides were postfixed for 10 minutes in
cold (-20.degree. C.) acetone and then allowed to come to room
temperature. Slides were rinsed in 4.degree. C. cold phosphate
buffered saline (PBS) 3 times for 2 minutes each followed by
blocking endogenous peroxidase activity with washing in 3 percent
hydrogen peroxide for 10 minutes. Slides were then rinsed in PBS 3
times for 5 minutes followed by incubation in Universal blocking
solution (Dako, Toronto, Ontario) for 5 minutes at room
temperature. AR47A6.4.2, anti-human muscle actin (Clone HHF35,
Dako, Toronto, Ontario), anti-TROP-2 clone 77220.11 (R&D System
Inc., MN, USA) or isotype control antibody (directed towards
Aspergillus niger glucose oxidase, an enzyme which is neither
present nor inducible in mammalian tissues; Dako, Toronto, Ontario)
were diluted in antibody dilution buffer (Dako, Toronto, Ontario)
to its working concentration (5 micrograms/mL for each antibody
except for anti-actin which was 0.5 micrograms/mL and commercial
anti-TROP-2 was 1 microgram/mL) and incubated overnight for 1 hour
at room temperature. The slides were washed with PBS 3 times for 2
minutes each. Immunoreactivity of the primary antibodies was
detected/visualized with HRP conjugated secondary antibodies as
supplied (Dako Envision System, Toronto, Ontario) for 30 minutes at
room temperature. Following this step the slides were washed with
PBS 3 times for 5 minutes each and a color reaction developed by
adding DAB (3,3'-diaminobenzidine tetrahydrachloride, Dako,
Toronto, Ontario) chromogen substrate solution for immunoperoxidase
staining for 10 minutes at room temperature. Washing the slides in
tap water terminated the chromogenic reaction. Following
counterstaining with Meyer's Hematoxylin (Sigma Diagnostics,
Oakville, ON), the slides were dehydrated with graded ethanols
(75-100 percent) and cleared with xylene. Using mounting media
(Dako Faramount, Toronto, Ontario) the slides were coverslipped.
Slides were microscopically examined using an Axiovert 200 (Ziess
Canada, Toronto, ON) and digital images acquired and stored using
Northern Eclipse Imaging Software (Mississauga, ON). Results were
read, scored and interpreted by a histopathologist.
[0237] Binding of antibodies to 12 human normal organs, ovary,
pancreas, thyroid, brain (cerebrum, cerebellum), lung, spleen,
uterus, cervix, heart, skin, and skeletal muscle was performed
using a human normal tissue screening array (Biochain, CA, USA).
The array contained 20 normal human organs; however, only 12 of the
organs were interpretable after staining. FIG. 33 presents a
summary of the results of AR47A6.4.2 staining of an array of human
normal tissues. The AR47A6.4.2 antibody showed binding
predominantly to epithelial tissues (endothelium of blood vessels,
follicular epithelium of thyroid, acinar and ductal epithelium of
pancreas, alveolar epithelium of lung, and epidermal keratinocytes
of skin). The antibody also showed equivocal binding to lymphoid
tissue of the spleen and binding to neural tissue of the brain
(FIG. 34). Cellular localization was cytoplasmic and membranous
with diffuse staining pattern. AR47A6.4.2 showed a similar binding
pattern when compared to the commercial anti-TROP-2 (clone
77220.11).
Example 14
Human Multi-Tumor Tissue Staining
[0238] IHC studies were conducted to characterize the AR47A6.4.2
antigen prevalence in frozen human cancer sections. Slides were
transferred from -80 to -20.degree. C. After one hour the slides
were postfixed for 10 minutes in cold (-20.degree. C.) acetone and
then allowed to come to room temperature. Slides were rinsed in
4.degree. C. cold phosphate buffered saline (PBS) 3 times for 2
minutes each followed by blocking endogenous peroxidase activity
with washing in 3 percent hydrogen peroxide for 10 minutes. Slides
were then rinsed in PBS 3 times for 5 minutes followed by
incubation in Universal blocking solution (Dako, Toronto, Ontario)
for 5 minutes at room temperature. AR47A6.4.2, anti-human muscle
actin (Clone HHF35, Dako, Toronto, Ontario), anti-cytokeratin 7
clone OV-TL 12/30 (Dako, Toronto, Ontario) or isotype control
antibody (directed towards Aspergillus niger glucose oxidase, an
enzyme which is neither present nor inducible in mammalian tissues;
Dako, Toronto, Ontario) were diluted in antibody dilution buffer
(Dako, Toronto, Ontario) to its working concentration of 5
micrograms/mL for each antibody except for anti-actin which was 0.5
micrograms/mL and anti-cytokeratin 7 which was ready to use.
Primary antibody and slides were incubated together for 1 hour at
room temperature. The slides were washed with PBS 3 times for 2
minutes each. Immunoreactivity of the primary antibodies was
detected/visualized with HRP conjugated secondary antibodies as
supplied (Dako Envision System, Toronto, Ontario) for 30 minutes at
room temperature. Following this step the slides were washed with
PBS 3 times for 5 minutes each and a color reaction developed by
adding DAB (3,3'-diaminobenzidine tetrahydrachloride, Dako,
Toronto, Ontario) chromogen substrate solution for immunoperoxidase
staining for 10 minutes at room temperature. Washing the slides in
tap water terminated the chromogenic reaction. Following
counterstaining with Meyer's Hematoxylin (Sigma Diagnostics,
Oakville, ON), the slides were dehydrated with graded ethanol
(75-100 percent) and cleared with xylene. Using mounting media
(Dako Faramount, Toronto, Ontario) the slides were coverslipped.
For the pancreatic array (Tri Star, Rockville, Md.) the same
protocol was followed except for the following modifications. The
tissue sections were initially air dried at room temperature for 2
hours and air dried again for 30 minutes after fixation with
acetone. The endogenous hydrogen peroxide was blocked using 3
percent hydrogen peroxide in methanol for 15 minutes; this step was
done after the primary antibody incubation.
[0239] Slides were microscopically examined using an Axiovert 200
(Ziess Canada, Toronto, ON) and digital images acquired and stored
using Northern Eclipse Imaging Software (Mississauga, ON). Results
were read, scored and interpreted by a histopathologist.
[0240] FIG. 35 presents a summary of the results of AR47A6.4.2
staining of various human tumors and their corresponding normal
tissue sections (10 colon cancers and 1 normal colon, 7 ovarian
cancers and 1 normal ovary, 11 breast cancers and 3 normal breast,
14 lung cancers and 3 normal lung, 13 prostate cancers and 3 normal
prostate, and 13 pancreatic cancers and 4 normal pancreas). The
tissues were distributed on three different tissue microarrays (Tri
Star, Rockville, Md.). The antibody showed moderate to strong
binding to 5/10 (50 percent), 6/7 (86 percent), 10/11 (91 percent),
11/14 (79 percent), 13/13 (100 percent) and 2/13 (15 percent) of
colon, ovarian, breast, lung, prostate and pancreatic cancers,
respectively. In addition, equivocal to weak binding was observed
in 2/10 (20 percent), 1/11 (9 percent), 3/14 (21 percent), and 2/13
(15 percent) colon, breast, lung and pancreatic cancer sections,
respectively (FIG. 36). In all of the tested tumors, the binding
was specific for the tumor cells. For the corresponding normal
tissues the antibody showed binding to 0/1, 0/1, 3/3, 3/3, 3/3 and
4/4 of normal colon, ovary, breast, lung, prostate, and pancreatic
tissues. However, the binding was predominantly to the epithelial
tissues of the normal organs. The positive control antibodies
anti-cytokeratin-7 or anti-actin showed expected positive binding
to epithelial and muscular tissues, respectively. The negative IgG
isotype control showed no detectable binding to any of the tested
tissues.
Example 15
Multi-Species Tissue Staining
[0241] IHC studies were conducted to characterize the AR47A6.4.2
antigen cross reactivity in frozen normal tissues of various
species in order to select a preclinical toxicology model. Sections
of SCID mouse normal tissues (harvested in house), a rat normal
tissue array (Biochain, CA, USA), a multi-species brain array
(Biochain, CA, USA) and a multi-species liver array (Biochain, CA,
USA) were transferred from -80 to -20.degree. C. After one hour the
slides were post fixed for 10 minutes in cold (-20.degree. C.)
acetone and then allowed to come to room temperature. Slides were
rinsed in 4.degree. C. cold phosphate buffered saline (PBS) 3 times
for 2 minutes each followed by blocking endogenous peroxidase
activity with washing in 3 percent hydrogen peroxide for 10
minutes. Slides were then rinsed in PBS 3 times for 5 minutes
followed by incubation in Universal blocking solution (Dako,
Toronto, Ontario) for 5 minutes at room temperature. AR47A6.4.2,
anti-Grp94 (Stressgen, Victoria, BC, Canada), anti-human muscle
actin (Clone HHF35, Dako, Toronto, Ontario) or isotype control
antibody (directed towards Aspergillus niger glucose oxidase, an
enzyme which is neither present nor inducible in mammalian tissues;
Dako, Toronto, Ontario) was diluted in antibody dilution buffer
(Dako, Toronto, Ontario) to its working concentration (5
micrograms/mL for each antibody except for anti-actin which was 0.5
micrograms/mL) and incubated for 1 hour at room temperature. The
slides were washed with PBS 3 times for 2 minutes each.
Immunoreactivity of the primary antibodies was detected/visualized
with HRP conjugated secondary antibodies as supplied (Dako Envision
System, Toronto, Ontario) for 30 minutes at room temperature.
Following this step the slides were washed with PBS 3 times for 5
minutes each and a color reaction developed by adding DAB
(3,3'-diaminobenzidine tetrahydrachloride, Dako, Toronto, Ontario)
chromogen substrate solution for immunoperoxidase staining for 10
minutes at room temperature. Washing the slides in tap water
terminated the chromogenic reaction. Following counterstaining with
Meyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides
were dehydrated with graded ethanols (75-100 percent) and cleared
with xylene. Using mounting media (Dako Faramount, Toronto,
Ontario) the slides were coverslipped. For the human, cynomolgus,
rabbit, hamster and rhesus individual sections (Biochain, CA, USA)
the same protocol was followed with the following modifications.
For the first step, the tissue sections were air dried at room
temperature for 30 minutes and then washed with cold PBS without
acetone fixation (the sections were acetone fixed from the
manufacturer). The endogenous hydrogen peroxide was blocked using 3
percent hydrogen peroxide in methanol for 20 minutes; this step was
done after the primary antibody incubation.
[0242] Immunoreactivity of the primary antibodies was
detected/visualized with anti-mouse HRP conjugated secondary
antibodies as supplied (Dako Envision System, Toronto, Ontario) for
30 minutes at room temperature. Slides were microscopically
examined using an Axiovert 200 (Ziess Canada, Toronto, ON) and
digital images acquired and stored using Northern Eclipse Imaging
Software (Mississauga, ON). Results were read, scored and
interpreted by a histopathologist.
[0243] Binding of the antibodies to a panel of SCID mouse normal
tissues (harvested in house), brain tissues of rat, guinea pig,
goat, sheep, chicken, cow, horse, dog and pig (Biochain, CA, USA),
liver tissues from rat, goat, chicken and cow (Biochain, CA, USA)
and human, cynomolgus, rhesus, rabbit, hamster and guinea pig
individual tissue sections (Biochain, CA, USA) was determined. The
positive control antibody anti-actin (Clone HHF35, Dako, Toronto,
Ontario) showed the expected specific binding to muscular tissues.
The positive control antibody anti-Grp94 (Stressgen, Victoria, BC)
showed the expected positive binding to predominantly the
epithelial tissues. The isotype negative control antibody (Dako,
Toronto, Ontario) generally showed no detectable binding to the
tested tissues. Tissue sections that showed obvious background
staining in the negative control were excluded from
interpretation.
[0244] FIG. 37 shows the tabulated results of AR47A6.4.2 staining
of the human and various species normal tissues. AR47A6.4.2 showed
no detectable binding to the tested mouse, rat, guinea pig, goat,
sheep, hamster, chicken, cow, horse or pig normal tissues. For the
normal rabbit and dog tissues, there was dissimilar binding to that
observed in the corresponding human tissues. For the cynomolgus
normal tissues, AR47A6.4.2 showed similar tissue specificity as
observed in the corresponding human normal tissues (FIG. 38) for
all of the tested organs except for the ovary and testis in which
no detectable binding was observed for the cynomolgus sections. For
the rhesus normal tissues, AR47A6.4.2 showed similar tissue
specificity as observed in the corresponding human normal tissues
(FIG. 38). It should be noted that rhesus normal tissue panel was
smaller than what was tested for the cynomolgus. Based on the
staining profiles, both the cynomolgus and rhesus monkey are
considered to be suitable toxicology models for AR47A6.4.2.
Example 16
AR47A6.4.2 Murine Sequence
[0245] 1.0 Cloning Variable Region Genes into Sequencing
Vectors
[0246] To facilitate production of antibody chimera, the genes
encoding the variable regions of both heavy and light chains were
separately cloned into the commercial sequencing vector pGEM-T
easy: (Promega Corp. Madison Wis.).
1.1 Isolation of mRNA
[0247] Messenger ribonucleic acid (mRNA) was isolated from a
culture of confluent Master Cell Bank (AR47A6.4.2) hybridoma cells
using Poly A Tract System 1000 mRNA extraction kit (Promega Corp.,
Madison, Wis.). mRNA was stored at -80.degree. C. until required
for further use.
1.2 RT-PCR Amplification of Variable Region Genes
[0248] Separate reactions were carried out to amplify the light and
heavy chain variable regions. Reverse transcriptase polymerase
chain reaction (RT-PCR) synthesized complimentary deoxynucleic acid
(cDNA) from the mRNA template and then specifically amplified the
targeted gene.
[0249] For the kappa light chain, 5.0 microliters of mRNA was mixed
with 1.0 microliter of 20 pmol/microliter MuIgG.kappa.V.sub.L-3'
primer OL040 and 5.5 microliters nuclease free water (Promega
Corp., Madison, Wis.). For the lambda light chain, 5.0 microliters
of mRNA was mixed with 1.0 microliter of 20 pmol/microliter
MuIgG.lamda.V.sub.L-3' primer OL042 and 5.5 microliters nuclease
free water (Promega Corp., Madison, Wis.). For the gamma heavy
chain, 5 microliters of mRNA was mixed with 1.0 microliter of 20
pmol/microliter MuIgGV.sub.H-3' primer OL023 and 5.5 microliters
nuclease free water (Promega Corp., Madison, Wis.). All three
reaction mixes were placed in the pre-heated block of the thermal
cycler set at 70.degree. C. for 5 minutes. The reaction mixes were
then chilled on ice for 5 minutes before adding to each 4.0
microliters ImPromII 5.times. reaction buffer (Promega Corp.,
Madison, Wis.), 0.5 microliters RNasin ribonuclease inhibitor
(Promega Corp., Madison, Wis.), 2.0 microliters 25 mM MgCl.sub.2
(Promega Corp., Madison, Wis.), 1.0 microliter 10 mM dNTP mix
(Invitrogen, Paisley, UK) and 1.0 microliter Improm II reverse
transcriptase (Promega Corp., Madison, Wis.). These reaction mixes
were incubated at room temperature for 5 minutes before being
transferred to a pre-heated PCR block set at 42.degree. C. for 1
hour. After this time the reverse transcriptase was heat
inactivated by incubating at 70.degree. C. in a PCR block for
fifteen minutes.
[0250] Heavy and light chain sequences were then specifically
amplified using pools of primers (See FIG. 39 for primer sequences;
SEQ ID NOS:10-47). The primer working solutions were made up as
follows: [0251] 1. 5' single primer (MuIgV.sub.H5'-A and B;
MuIg.kappa.V.sub.Lh5'-A, B and C; MuIg.lamda.V.sub.L5'-A) contained
each primer at a concentration of 20 micromolar; [0252] 2. 5'
primer pools (MuIgV.sub.H5'-C to F; MuIg.kappa.V.sub.Lh5'-D to G)
contained each constituent primer at a concentration of 5
micromolar.
[0253] Heavy and light chain sequences were amplified from cDNA. A
PCR master mix was prepared by adding 37.5 microliters 10.times.
Hi-Fi Expand PCR buffer (Roche, Mannheim, Germany), 7.5 microliters
10 mM dNTP mix (Invitrogen, Paisley, UK) and 3.75 microliters Hi-Fi
Expand DNA polymerase (Roche, Mannheim, Germany) to 273.75
microliters nuclease free water. This master mix was dispensed in
21.5 microliter aliquots into 15 thin walled PCR reaction tubes, on
ice. Into six of these tubes was added 2.5 microliters of
MuIgV.sub.H-3' reverse transcription reaction mix and 1.0
microliter of heavy chain 5' primer mix A to F. To another seven
tubes was added 2.5 microliters of MuIg.kappa.V.sub.L-3' reverse
transcription reaction and 1.0 microliter of light chain 5' primer
mixes A to G. Into the final tube was added 2.5 microliters of
MuIg.lamda.V.sub.L-3' reverse transcription reaction and 1.0
microliter of lambda light chain primer MuIg.lamda.V.sub.L5'-A.
Reactions were placed in the block of the thermal cycler and heated
to 95.degree. C. for 2 minutes. The polymerase chain reaction (PCR)
reaction was performed for 40 cycles of 94.degree. C. for 30
seconds, 55.degree. C. for 1 minute and 72.degree. C. for 30
seconds. Finally the PCR products were heated at 72.degree. C. for
5 minutes, and then stored at 4.degree. C. PCR product was purified
using QIAquick PCR Purification Kit (QIAGEN, Crawley, UK).
[0254] FIG. 40 shows the result of the RT-PCR reactions. The heavy
chain reactions (lanes 2-7) demonstrate a strong band at 500 bp
amplified using MuIgV.sub.H5'-C (lane 4) and MuIgV.sub.H5'-E (lane
6). Light chain reactions (lanes 8-15) demonstrate a strong 450 bp
product band amplified using primers MuIg.kappa.V.sub.L 5'-A (lane
8) and MuIg.kappa.V.sub.L5'-G (lane 14) forward primer. PCR
products from these reactions were purified using QIAquick PCR
Purification Kit (QIAGEN, Crawley, UK).
1.3 Cloning into Sequencing Vectors
[0255] Light chain A and G and heavy chain C and E purified PCR
products were separately cloned into pGEM-T easy vector using the
pGEM-T easy Vector System I (Promega Corp., Madison, Wis.). Both
the light and heavy chain reactions were prepared by adding 3.0
microliters of purified PCR product to 5.0 microliters of 2.times.
ligation buffer, 1.0 microliter pGEM-T easy vector and 1.0
microliter T4 DNA ligase. Plasmids were transformed into
sub-cloning grade XL1-blue competent E. coli (Stratgene, La Jolla,
Calif.) as per manufacturer's instructions. For both the light and
heavy chain transformations, 2.0 microliters of the ligation
reaction was used.
[0256] 100 microliters of transformed cells from each reaction was
plated onto Luria broth (LB) agar (Q-Biogene, Cambridge, UK) plates
containing 50 micrograms/mL ampicillin (Sigma, Poole, UK). The
plates were inverted and incubated at 37.degree. C. overnight.
[0257] Eight clones from each of the four plates were selected and
used to inoculate 20 microliters sterile water. A PCR master mix
was prepared by mixing 513.6 microliters sterile water, 34.0
microliters Dimethyl Sulphoximide (Sigma, Poole, UK), 68.0
microliters 10.times. Taq buffer (Invitrogen, Paisley, UK), 13.6
microliters 10 mM dNTP mix (Invitrogen, Paisley, UK), 6.8
microliters of 50 pmol/microliter primer OL001, 6.8 microliters of
50 pmol/microliter primer OL002 and 3.4 microliters Taq DNA
polymerase (Invitrogen, Paisley, UK). This master mix was dispensed
into 32 PCR reaction tubes in 19 microliter aliquots. Into to each
of these was added 1.0 microliter of the inoculated colony
suspensions. PCR reactions were placed in the block of the thermal
cycler and heated to 95.degree. C. for 5 minutes. The polymerase
chain reaction (PCR) reaction was performed for 25 cycles of
94.degree. C. for 1 minute, 55.degree. C. for 1 minute and
72.degree. C. for 1 minute. Finally the PCR products were heated at
72.degree. C. for 10 minutes. 5 microliters from each reaction was
then run into a 1 percent agarose gel.
[0258] FIG. 41 shows the PCR screening reactions from eight
colonies of AR47A6.4.2 V.sub.H-C and eight colonies of AR47A6.4.2
V.sub.H-E. Three of the eight, V.sub.HE-2 (lane 11), V.sub.HE-4
(lane 14), and V.sub.HE-7 (lane 17) were positive for a 650 bp
product band indicating the successful cloning of a 500 bp product
into pGEM-T easy vector. The remaining five V.sub.HE and all eight
of the V.sub.H-C reactions produced bands of a lower molecular
weight indicating a negative result for a 500 bp insert.
[0259] FIG. 42 shows the PCR screening reactions from eight
colonies of AR47A6.4.2 V.sub.L-A and eight colonies of AR47A6.4.2
V.sub.L-G. All eight of the V.sub.LA colonies (lanes 2-9) and 3 of
the eight V.sub.LG colonies, V.sub.LG-1 (lane 10), V.sub.LG-3 (lane
12) and V.sub.LG-5 (lane 14) were positive for a 600 bp product
band indicating the successful cloning of a 450 bp product into
pGEM-T easy vector. The remaining five V.sub.L-G reactions produced
bands of a different molecular weight indicating a negative result
for a 450 bp insert.
[0260] A maximum of 4 positive colonies from each ligation were
chosen to inoculate 5 mL 2YT (Sigma, Poole, UK) broth containing 50
mg/L ampicillin (Sigma, Poole, UK). Cultures were incubated at
37.degree. C. with shaking overnight. Plasmid DNA was extracted
from each culture using Qiagen, QIAprep Spin Miniprep Kit (Qiagen,
Crawley, UK).
1.4 DNA Sequencing
[0261] Plasmid DNA from nine AR47A6.4.2 V.sub.L and V.sub.H clones
(V.sub.L A-1, V.sub.L A-2, V.sub.L A-5, V.sub.L A-6, V.sub.L G-1,
V.sub.L G-3, V.sub.H E-2, V.sub.H E-4, and V.sub.H E-7) were
sequenced at Geneservice Ltd. DNA sequencing facility (Cambridge,
UK). Sequences are given in FIGS. 43 and 44 with the
complimentarity determining regions (CDRs) underlined. FIG. 43
shows SEQ ID NO:8 with the underlined CDRs designated SEQ ID
NOS:4-6. FIG. 44 shows SEQ ID NO:7 with the underlined CDRs
designated SEQ ID NOS:1-3. CDR definitions and amino acid sequence
numbering is done according to Kabat et al. (1991). The Kabat
numbering is listed above the amino acid sequence in FIGS. 43 and
44.
[0262] The correct AR47A6.4.2 V.sub.L sequence was found in all 4
of the clones amplified with 5' primer MuIg.kappa.V.sub.L-A. The
two V.sub.L clones amplified using 5' primer MuIg.kappa.V.sub.L-G
contained an aberrant immunoglobulin gene. The correct AR47A6.4.2
V.sub.H sequence was found in all 3 clones amplified with 5' primer
MuIgV.sub.H-E.
Example 17
Isolation of Competitive Binders
[0263] Given an antibody, an individual ordinarily skilled in the
art can generate a competitively inhibiting CDMAB, for example a
competing antibody, which is one that recognizes the same epitope
(Belanger L et al. Clinica Chimica Acta 48:15-18 (1973)). One
method entails immunizing with an immunogen that expresses the
antigen recognized by the antibody. The sample may include but is
not limited to tissues, isolated protein(s) or cell line(s).
Resulting hybridomas could be screened using a competition assay,
which is one that identifies antibodies that inhibit the binding of
the test antibody, such as ELISA, FACS or Western blotting. Another
method could make use of phage display antibody libraries and
panning for antibodies that recognize at least one epitope of said
antigen (Rubinstein J L et al. Anal Biochem 314:294-300 (2003)). In
either case, antibodies are selected based on their ability to
displace the binding of the original labeled antibody to at least
one epitope of its target antigen. Such antibodies would therefore
possess the characteristic of recognizing at least one epitope of
the antigen as the original antibody.
Example 18
Cloning of the Variable Regions of the AR47A6.4.2 Monoclonal
Antibody
[0264] The sequences of the variable regions from the heavy
(V.sub.H) and light (V.sub.L) chains of monoclonal antibody
produced by the AR47A6.4.2 hybridoma cell line were determined
(Example 16). To generate chimeric and humanized IgG, the variable
light and variable heavy domains can be subcloned into an
appropriate vector for expression.
[0265] In another embodiment, AR47A6.4.2 or its de-immunized,
chimeric or humanized version is produced by expressing a nucleic
acid encoding the antibody in a transgenic animal, such that the
antibody is expressed and can be recovered. For example, the
antibody can be expressed in a tissue specific manner that
facilitates recovery and purification. In one such embodiment, an
antibody of the invention is expressed in the mammary gland for
secretion during lactation. Transgenic animals include but are not
limited to mice, goat and rabbit.
(i) Monoclonal Antibody
[0266] DNA encoding the monoclonal antibody (as outlined in Example
1) is readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
monoclonal antibodies). The hybridoma cell serves as a preferred
source of such DNA. Once isolated, the DNA may be placed into
expression vectors, which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be
modified, for example, by substituting the coding sequence for
human heavy and light chain constant domains in place of the
homologous murine sequences. Chimeric or hybrid antibodies also may
be prepared in vitro using known methods in synthetic protein
chemistry, including those involving crosslinking agents. For
example, immunotoxins may be constructed using a disulfide exchange
reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate.
(ii) Humanized Antibody
[0267] A humanized antibody has one or more amino acid residues
introduced into it from a non-human source. These non-human amino
acid residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization can
be performed the method of Winter and co-workers by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody (Jones et al., Nature 321:522-525 (1986); Riechmann
et al., Nature 332:323-327 (1988); Verhoeyen et al., Science
239:1534-1536 (1988); reviewed in Clark, Immunol. Today 21:397-402
(2000)).
[0268] A humanized antibody can be prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e. the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the consensus and import sequence so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
(iii) Antibody Fragments
[0269] Various techniques have been developed for the production of
antibody fragments. These fragments can be produced by recombinant
host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557
(1999); Little et al., Immunol. Today 21:364-370 (2000)). For
example, Fab'-SH fragments can be directly recovered from E. coli
and chemically coupled to form F(ab').sub.2 fragments (Carter et
al., Biotechnology 10:163-167 (1992)). In another embodiment, the
F(ab').sub.2 is formed using the leucine zipper GCN4 to promote
assembly of the F(ab').sub.2 molecule. According to another
approach, Fv, Fab or F(ab') 2 fragments can be isolated directly
from recombinant host cell culture.
Example 19
A Composition Comprising the Antibody of the Present Invention
[0270] The antibody of the present invention can be used as a
composition for preventing/treating cancer. The composition for
preventing/treating cancer, which comprises the antibody of the
present invention, are low-toxic and can be administered as they
are in the form of liquid preparations, or as pharmaceutical
compositions of suitable preparations to human or mammals (e.g.,
rats, rabbits, sheep, swine, bovine, feline, canine, simian, etc.)
orally or parenterally (e.g., intravascularly, intraperitoneally,
subcutaneously, etc.). The antibody of the present invention may be
administered in itself, or may be administered as an appropriate
composition. The composition used for the administration may
contain a pharmacologically acceptable carrier with the antibody of
the present invention or its salt, a diluent or excipient. Such a
composition is provided in the form of pharmaceutical preparations
suitable for oral or parenteral administration.
[0271] Examples of the composition for parenteral administration
are injectable preparations, suppositories, etc. The injectable
preparations may include dosage forms such as intravenous,
subcutaneous, intracutaneous and intramuscular injections, drip
infusions, intraarticular injections, etc. These injectable
preparations may be prepared by methods publicly known. For
example, the injectable preparations may be prepared by dissolving,
suspending or emulsifying the antibody of the present invention or
its salt in a sterile aqueous medium or an oily medium
conventionally used for injections. As the aqueous medium for
injections, there are, for example, physiological saline, an
isotonic solution containing glucose and other auxiliary agents,
etc., which may be used in combination with an appropriate
solubilizing agent such as an alcohol (e.g., ethanol), a
polyalcohol (e.g., propylene glycol, polyethylene glycol), a
nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene
(50 mols) adduct of hydrogenated castor oil)), etc. As the oily
medium, there are employed, e.g., sesame oil, soybean oil, etc.,
which may be used in combination with a solubilizing agent such as
benzyl benzoate, benzyl alcohol, etc. The injection thus prepared
is usually filled in an appropriate ampoule. The suppository used
for rectal administration may be prepared by blending the antibody
of the present invention or its salt with conventional bases for
suppositories. The composition for oral administration includes
solid or liquid preparations, specifically, tablets (including
dragees and film-coated tablets), pills, granules, powdery
preparations, capsules (including soft capsules), syrup, emulsions,
suspensions, etc. Such a composition is manufactured by publicly
known methods and may contain a vehicle, a diluent or excipient
conventionally used in the field of pharmaceutical preparations.
Examples of the vehicle or excipient for tablets are lactose,
starch, sucrose, magnesium stearate, etc.
[0272] Advantageously, the compositions for oral or parenteral use
described above are prepared into pharmaceutical preparations with
a unit dose suited to fit a dose of the active ingredients. Such
unit dose preparations include, for example, tablets, pills,
capsules, injections (ampoules), suppositories, etc. The amount of
the aforesaid compound contained is generally 5 to 500 mg per
dosage unit form; it is preferred that the antibody described above
is contained in about 5 to about 100 mg especially in the form of
injection, and in 10 to 250 mg for the other forms.
[0273] The dose of the aforesaid prophylactic/therapeutic agent or
regulator comprising the antibody of the present invention may vary
depending upon subject to be administered, target disease,
conditions, route of administration, etc. For example, when used
for the purpose of treating/preventing, e.g., breast cancer in an
adult, it is advantageous to administer the antibody of the present
invention intravenously in a dose of about 0.01 to about 20 mg/kg
body weight, preferably about 0.1 to about 10 mg/kg body weight and
more preferably about 0.1 to about 5 mg/kg body weight, about 1 to
5 times/day, preferably about 1 to 3 times/day. In other parenteral
and oral administration, the agent can be administered in a dose
corresponding to the dose given above. When the condition is
especially severe, the dose may be increased according to the
condition.
[0274] The antibody of the present invention may be administered as
it stands or in the form of an appropriate composition. The
composition used for the administration may contain a
pharmacologically acceptable carrier with the aforesaid antibody or
its salts, a diluent or excipient. Such a composition is provided
in the form of pharmaceutical preparations suitable for oral or
parenteral administration (e.g., intravascular injection,
subcutaneous injection, etc.). Each composition described above may
further contain other active ingredients. Furthermore, the antibody
of the present invention may be used in combination with other
drugs, for example, alkylating agents (e.g., cyclophosphamide,
ifosfamide, etc.), metabolic antagonists (e.g., methotrexate,
5-fluorouracil, etc.), anti-tumor antibiotics (e.g., mitomycin,
adriamycin, etc.), plant-derived anti-tumor agents (e.g.,
vincristine, vindesine, Taxol.RTM., etc.), cisplatin, carboplatin,
etoposide, irinotecan, etc. The antibody of the present invention
and the drugs described above may be administered simultaneously or
at staggered times to the patient.
[0275] The method of treatment described herein, particularly for
cancers, may also be carried out with administration of other
antibodies or chemotherapeutic agents. For example, an antibody
against EGFR, such as ERBITUX.RTM. (cetuximab), may also be
administered, particularly when treating colon cancer. ERBITUX.RTM.
has also been shown to be effective for treatment of psoriasis.
Other antibodies for combination use include Herceptin.RTM.
(trastuzumab) particularly when treating breast cancer,
AVASTIN.RTM. particularly when treating colon cancer and SGN-15
particularly when treating non-small cell lung cancer. The
administration of the antibody of the present invention with other
antibodies/chemotherapeutic agents may occur simultaneously, or
separately, via the same or different route.
[0276] The chemotherapeutic agent/other antibody regimens utilized
include any regimen believed to be optimally suitable for the
treatment of the patient's condition. Different malignancies can
require use of specific anti-tumor antibodies and specific
chemotherapeutic agents, which will be determined on a patient to
patient basis. In a preferred embodiment of the invention,
chemotherapy is administered concurrently with or, more preferably,
subsequent to antibody therapy. It should be emphasized, however,
that the present invention is not limited to any particular method
or route of administration.
[0277] The preponderance of evidence shows that AR47A6.4.2 mediates
anti-cancer effects and prolongs survival through ligation of
epitopes present on TROP-2. It has been shown, in Examples 9, 10
and 11, AR47A6.4.2 antibodies can be used to immunoprecipitate the
cognate antigen from expressing cells such as MDA-MB-231 cells.
Further it has been shown, in Examples 1, 2 and 13-15, that the
AR47A6.4.2 antibody can be used in detection of cells and/or
tissues which express a TROP-2 antigenic moiety which specifically
binds thereto, utilizing techniques illustrated by, but not limited
to FACS, cell ELISA or IHC.
[0278] Thus, it could be shown that the immunoprecipitated
AR47A6.4.2 antigen can inhibit the binding of AR47A6.4.2 to such
cells or tissues using FACS, cell ELISA or IHC assays. Further, as
with the AR47A6.4.2 antibody, other anti-TROP-2 antibodies could be
used to immunoprecipitate and isolate other forms of the TROP-2
antigen, and the antigen can also be used to inhibit the binding of
those antibodies to the cells or tissues that express the antigen
using the same types of assays.
[0279] All patents and publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0280] It is to be understood that while a certain form of the
invention is illustrated, it is not to be limited to the specific
form or arrangement of parts herein described and shown. It will be
apparent to those skilled in the art that various changes may be
made without departing from the scope of the invention and the
invention is not to be considered limited to what is shown and
described in the specification.
[0281] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. Any oligonucleotides, peptides, polypeptides, biologically
related compounds, methods, procedures and techniques described
herein are presently representative of the preferred embodiments,
are intended to be exemplary and are not intended as limitations on
the scope. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention and are defined by the scope of the appended claims.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention which are obvious to those skilled
in the art are intended to be within the scope of the following
claims.
Sequence CWU 1
1
4715PRTHomo sapiens 1Asn Tyr Gly Met Asn1 5217PRTHomo sapiens 2Trp
Ile Asn Thr Lys Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys1 5 10
15Gly312PRTHomo sapiens 3Gly Gly Tyr Gly Ser Ser Tyr Trp Tyr Phe
Asp Val1 5 10411PRTHomo sapiens 4Lys Ala Ser Gln Asp Val Ser Ile
Ala Val Ala1 5 1057PRTHomo sapiens 5Ser Ala Ser Tyr Arg Tyr Thr1
569PRTHomo sapiens 6Gln Gln His Tyr Ile Thr Pro Leu Thr1
57121PRTHomo sapiens 7Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu
Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro
Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly
Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser
Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn
Leu Lys Lys Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Gly Arg Gly Gly
Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110Ala Gly
Thr Thr Val Thr Val Ser Ser 115 1208107PRTHomo sapiens 8Asp Ile Val
Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile 35 40
45Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Arg Val Gln
Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile
Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105929PRTHomo sapiens 9Arg Thr Leu Val Arg Pro Ser Glu His Ala Leu
Val Asp Asn Asp Gly1 5 10 15Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly
Arg Phe Cys 20 251024DNAArtificialprimer sequence used for PCR
10cgccagggtt ttcccagtca cgac 241124DNAArtificialprimer sequence
used for PCR 11agcggataac aatttcacac agga 241225DNAArtificialprimer
sequence used for PCR 12atgrasttsk ggytmarctk grttt
251326DNAArtificialprimer sequence used for PCR 13atgraatgsa
sctgggtywt yctctt 261429DNAArtificialprimer sequence used for PCR
14atggactcca ggctcaattt agttttcct 291526DNAArtificialprimer
sequence used for PCR 15atggctgtcy trgbgctgyt cytctg
261629DNAArtificialprimer sequence used for PCR 16atggvttggs
tgtggamctt gcyattcct 291726DNAArtificialprimer sequence used for
PCR 17atgaaatgca gctggrtyat sttctt 261826DNAArtificialprimer
sequence used for PCR 18atggrcagrc ttacwtyytc attcct
261926DNAArtificialprimer sequence used for PCR 19atgatggtgt
taagtcttct gtacct 262026DNAArtificialprimer sequence used for PCR
20atgggatgga gctrtatcat sytctt 262123DNAArtificialprimer sequence
used for PCR 21atgaagwtgt ggbtraactg grt 232225DNAArtificialprimer
sequence used for PCR 22atggratgga sckknrtctt tmtct
252325DNAArtificialprimer sequence used for PCR 23atgaacttyg
ggytsagmtt grttt 252425DNAArtificialprimer sequence used for PCR
24atgtacttgg gactgagctg tgtat 252523DNAArtificialprimer sequence
used for PCR 25atgagagtgc tgattctttt gtg 232628DNAArtificialprimer
sequence used for PCR 26atggattttg ggctgatttt ttttattg
282723DNAArtificialprimer sequence used for PCR 27acgaggggga
agacatttgg gaa 232826DNAArtificialprimer sequence used for PCR
28ccagggrcca rkggatarac ngrtgg 262924DNAArtificialprimer sequence
used for PCR 29atgragwcac akwcycaggt cttt 243025DNAArtificialprimer
sequence used for PCR 30atggagacag acacactcct gctat
253129DNAArtificialprimer sequence used for PCR 31atggagwcag
acacactsct gytatgggt 293232DNAArtificialprimer sequence used for
PCR 32atgaggrccc ctgctcagwt tyttggnwtc tt 323331DNAArtificialprimer
sequence used for PCR 33atgggcwtca agatgragtc acakwyycwg g
313429DNAArtificialprimer sequence used for PCR 34atgagtgtgc
ycactcaggt cctggsgtt 293531DNAArtificialprimer sequence used for
PCR 35atgtggggay cgktttyamm cttttcaatt g 313628DNAArtificialprimer
sequence used for PCR 36atggaagccc cagctcagct tctcttcc
283726DNAArtificialprimer sequence used for PCR 37atgagnmmkt
cnmttcantt cytggg 263826DNAArtificialprimer sequence used for PCR
38atgakgthcy cngctcagyt yctnrg 263925DNAArtificialprimer sequence
used for PCR 39atggtrtccw casctcagtt ccttg
254027DNAArtificialprimer sequence used for PCR 40atgtatatat
gtttgttgtc tatttct 274129DNAArtificialprimer sequence used for PCR
41atgaagttgc ctgttaggct gttggtgct 294229DNAArtificialprimer
sequence used for PCR 42atggatttwc argtgcagat twtcagctt
294327DNAArtificialprimer sequence used for PCR 43atggtyctya
tvtccttgct gttctgg 274427DNAArtificialprimer sequence used for PCR
44atggtyctya tvttrctgct gctatgg 274521DNAArtificialprimer sequence
used for PCR 45actggatggt gggaagatgg a 214625DNAArtificialprimer
sequence used for PCR 46atggcctgga ytycwctywt mytct
254723DNAArtificialprimer sequence used for PCR 47agctcytcwg
wgganggygg raa 23
* * * * *