U.S. patent application number 13/764724 was filed with the patent office on 2013-11-28 for cytotoxicity mediation of cells evidencing surface expression of cd44.
The applicant listed for this patent is Sheung Tat Fan, Terence Kin Wah Lee, Ronnie Tung Ping Poon, Daad Sayegh, David S.F. Young. Invention is credited to Sheung Tat Fan, Terence Kin Wah Lee, Ronnie Tung Ping Poon, Daad Sayegh, David S.F. Young.
Application Number | 20130315896 13/764724 |
Document ID | / |
Family ID | 39760460 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315896 |
Kind Code |
A1 |
Young; David S.F. ; et
al. |
November 28, 2013 |
Cytotoxicity Mediation of Cells Evidencing Surface Expression of
CD44
Abstract
This invention relates to the diagnosis and treatment of
cancerous diseases, particularly to the mediation of cytotoxicity
of primary and metastatic human tumor cells; and most particularly
to the use of an isolated monoclonal antibody or cancerous disease
modifying antibodies (CDMAB) thereof, optionally in combination
with one or more chemotherapeutic agents, as a means for initiating
the cytotoxic response in such human tumors, e.g. any primary or
metastatic tumor sites which arise from hepatocytes. The invention
further relates to binding assays which utilize the CDMAB of the
instant invention.
Inventors: |
Young; David S.F.; (Canada,
CA) ; Sayegh; Daad; (Mississauga, CA) ; Fan;
Sheung Tat; (Hong Kong, CN) ; Poon; Ronnie Tung
Ping; (Hong Kong, CN) ; Lee; Terence Kin Wah;
(Hong Kong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Young; David S.F.
Sayegh; Daad
Fan; Sheung Tat
Poon; Ronnie Tung Ping
Lee; Terence Kin Wah |
Canada
Mississauga
Hong Kong
Hong Kong
Hong Kong |
|
CA
CA
CN
CN
CN |
|
|
Family ID: |
39760460 |
Appl. No.: |
13/764724 |
Filed: |
February 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13243870 |
Sep 23, 2011 |
8388961 |
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13764724 |
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11786165 |
Apr 11, 2007 |
8048416 |
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13243870 |
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11716216 |
Mar 9, 2007 |
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11786165 |
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11364013 |
Feb 28, 2006 |
7947496 |
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11716216 |
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10810165 |
Mar 26, 2004 |
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11364013 |
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10647818 |
Aug 22, 2003 |
7189397 |
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10810165 |
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10603000 |
Jun 23, 2003 |
7252821 |
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10647818 |
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09727361 |
Nov 29, 2000 |
6657048 |
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10603000 |
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09415278 |
Oct 8, 1999 |
6180357 |
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09727361 |
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Current U.S.
Class: |
424/133.1 ;
424/155.1; 424/174.1; 435/7.23 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 37/04 20180101; C07K 16/30 20130101; G01N 33/574 20130101;
G01N 2333/70585 20130101; G01N 33/57492 20130101; A61K 49/0013
20130101; C07K 16/2884 20130101; A61P 43/00 20180101; A61P 35/02
20180101; A61P 35/04 20180101; C07K 2317/24 20130101; A61K 2039/505
20130101; A61K 47/6849 20170801; A61P 1/16 20180101 |
Class at
Publication: |
424/133.1 ;
424/174.1; 424/155.1; 435/7.23 |
International
Class: |
C07K 16/30 20060101
C07K016/30; G01N 33/574 20060101 G01N033/574 |
Claims
1.-7. (canceled)
8. A method of treating primary human tumor sites and metastatic
sites susceptible to antibody induced cellular cytotoxicity in a
mammal, wherein said primary human tumor or metastasis expresses at
least one epitope of an antigen which specifically binds to the
isolated monoclonal antibody produced by a clone deposited with the
ATCC as accession number PTA-4621 or a CDMAB thereof, which is
characterized by an ability to competitively inhibit binding of
said isolated monoclonal antibody or CDMAB thereof to its target
antigen, comprising administering to said mammal said isolated
monoclonal antibody or said CDMAB thereof in an amount effective to
result in a reduction of said mammal's tumor burden.
9. The method of claim 8 wherein said isolated monoclonal antibody
or CDMAB thereof is conjugated to a cytotoxic moiety.
10. The method of claim 8 wherein said cytotoxic moiety is a
radioactive isotope.
11. The method of claim 8 wherein said isolated monoclonal antibody
or CDMAB thereof activates complement.
12. The method of claim 8 wherein said isolated monoclonal antibody
or CDMAB thereof mediates antibody dependent cellular
cytotoxicity.
13. The method of claim 8 wherein said isolated monoclonal antibody
is a humanized antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the ATCC as accession
number PTA-4621 or a CDMAB thereof.
14. The method of claim 8 wherein said isolated monoclonal antibody
is a chimeric antibody of the isolated monoclonal antibody produced
by the hybridoma deposited with the ATCC as accession number
PTA-4621 or a CDMAB thereof.
15.-16. (canceled)
17. A binding assay to determine a presence of cancerous cells in a
tissue sample selected from a human cancerous tumor, which express
an epitope or epitopes of human CD44 antigen which is specifically
bound by the isolated monoclonal antibody produced by hybridoma
cell line H460-16-2 having ATCC Accession No. PTA-4621, comprising:
providing at least one isolated monoclonal antibody or CDMAB
thereof that recognizes the same epitope or epitopes as those
recognized by the isolated monoclonal antibody produced by
hybridoma cell line H460-16-2 having ATCC Accession No. PTA-4621;
contacting said at least one isolated monoclonal antibody or CDMAB
thereof with said human tissue sample; and determining binding of
said at least one isolated monoclonal antibody or CDMAB thereof
with said tissue sample; whereby the presence of said cancerous
cells in said tissue sample is indicated.
18. (canceled)
19. The process of claim 8, wherein said primary human tumor sites
and/or metastatic sites arise from hepatocytes.
20.-21. (canceled)
22. The process of claim 17, wherein said human cancerous tumor
arises from hepatocytes.
23. 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
ATCC as accession number PTA-4621 or a CDMAB thereof, which CDMAB
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 ATCC as accession number PTA-4621 or a
CDMAB thereof, and means for detecting whether the monoclonal
antibody, or a CDMAB thereof, is bound to a polypeptide whose
presence, at a particular cut-off level, is diagnostic of said
presence of said human cancerous tumor.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part to U.S. patent
application Ser. No. 11/716,216, filed on Mar. 9, 2007, which is a
continuation-in-part to U.S. patent application Ser. No.
11/364,013, filed on Feb. 28, 2006, which is a continuation-in-part
to U.S. patent application Ser. No. 10/810,165, filed Mar. 26,
2004, now abandoned, which is a continuation-in-part to U.S. patent
application Ser. No. 10/647,818, filed Aug. 22, 2003, which is a
continuation-in-part to U.S. patent application Ser. No.
10/603,000, filed Jun. 23, 2003, which is a continuation-in-part to
U.S. patent application Ser. No. 09/727,361 now U.S. Pat. No.
6,657,048, issued Dec. 2, 2003, which is a continuation-in-part to
U.S. patent application Ser. No. 09/415,278 now U.S. Pat. No.
6,180,357, issued Jan. 30, 2001, the contents of each 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] CD44 in Cancer: Raising monoclonal antibodies against human
white blood cells led to the discovery of the CD44 antigen; a
single chain hyaluronic acid (HA) binding glycoprotein expressed on
a wide variety of normal tissue and on all types of hematopoietic
cells. It was originally associated with lymphocyte activation and
homing. Currently, its putative physiological role also includes
activation of inflammatory genes, modulation of cell cycle,
induction of cell proliferation, induction of differentiation and
development, induction of cytoskeletal reorganization and cell
migration and cell survival/resistance to apoptosis.
[0004] In humans, the single gene copy of CD44 is located on the
short arm of chromosome 11, 11p13. The gene contains 19 exons; the
first 5 are constant, the next 9 are variant, the following 3 are
constant and the final 2 are variant. Differential splicing can
lead to over 1000 different isoforms. However, currently only
several dozen naturally occurring variants have been
identified.
[0005] The CD44 standard glycoprotein consists of a N-terminal
extracellular (including a 20 a.a. leader sequence, and a membrane
proximal region (85 a.a.)) domain (270 a.a.), a transmembrane
region (21a.a.) and a cytoplasmic tail (72 a.a.). The extracellular
region also contains a link module at the N-terminus. This region
is 92 a.a. in length and shows homology to other HA binding link
proteins. There is high homology between the mouse and human forms
of CD44. The variant forms of the protein are inserted to the
carboxy terminus of exon 5 and are located extracellularly when
expressed.
[0006] A serum soluble form of CD44 also occurs naturally and can
arise from either a stop codon (within the variable region) or from
proteolytic activity. Activation of cells from a variety of stimuli
including TNF-.alpha. results in shedding of the CD44 receptor.
Shedding of the receptor has also been seen with tumor cells and
can result in an increase in the human serum concentration of CD44
by up to 10-fold. High CD44 serum concentration suggests malignancy
(ovarian cancer being the exception).
[0007] The standard form of CD44 exists with a molecular weight of
approximately 37 kD. Post-translational modifications increase the
molecular weight to 80-90 kD. These modifications include amino
terminus extracellular domain N-linked glycosylations at asparagine
residues, O-linked glycosylations at serine/threonine residues at
the carboxy terminus of the extracellular domain and
glycosaminoglycan additions. Splice variants can range in size from
80-250 kD.
[0008] HA, a polysaccharide located on the extracellular matrix
(ECM) in mammals, is thought to be the primary CD44 ligand.
However, CD44 has also been found to bind such proteins as
collagen, fibronectin, laminin etc. There appears to be a
correlation between HA binding and glycosylation. Inactive CD44
(does not bind HA) has the highest levels of glycosylation, active
CD44 (binding HA) the lowest while inducible CD44 (does not or
weakly binds HA unless activated by cytokines, monoclonal
antibodies, growth factors, etc.) has glycoslyation levels
somewhere in between the active and inactive forms.
[0009] CD44 can mediate some of its functions through signal
transduction pathways that depend on the interaction of the cell,
stimulus and the environment. Some of these pathways include the
NF.kappa.B signaling cascade (involved in the inflammatory
response), the Ras-MAPK signal transduction pathway (involved with
activating cell cycling and proliferation), the Rho family of
proteins (involved with cytoskeleton reorganization and cell
migration) and the PI3-K-related signaling pathway (related to cell
survival). All of the above-mentioned functions are closely
associated with tumor disease initiation and progression. CD44 has
also been implicated in playing a role in cancer through a variety
of additional mechanisms. These include the presentation of growth
factors, chemokines and cytokines by cell surface proteoglycans
present on the cell surface of CD44 to receptors involved in
malignancy. Also, the intracellular degradation of HA by lysosomal
hyaluronidases after internalization of the CD44-HA complex can
potentially increase the likelihood of tumor invasiveness and
induction of angiogenesis through the ECM. In addition, the
transmission of survival or apoptotic signals has been shown to
occur through either the standard or variable CD44 receptor. CD44
has also been suggested to be involved in cell differentiation and
migration. Many, if not all, of these mechanisms are environment
and cell dependent and several give rise to variable findings.
Therefore, more research is required before any conclusions can be
drawn.
[0010] In order to validate a potential functional role of CD44 in
cancer, expression studies of CD44 were undertaken to determine if
differential expression of the receptor correlates with disease
progression. However, inconsistent findings were observed in a
majority of tumor types and this is probably due to a combination
of reagents, technique, pathological scoring and cell type
differences between researchers. Renal cell carcinoma and
non-Hodgkin's lymphoma appear to be the exception in that patients
with high CD44 expressing tumors consistently had shorter survival
times than their low or non-CD44 expressing counterparts.
[0011] Due to its association with cancer, CD44 has been the target
of the development of anti-cancer therapeutics. There is still
controversy as to whether the standard or the variant forms of CD44
are required for tumor progression. There is in vivo animal data to
support both views and again it may be tumor type and even cell
type dependent. Different therapeutic approaches have included
injection of soluble CD44 proteins, hyaluronan synthase cDNA,
hyaluronidase, the use of CD44 antisense and CD44 specific
antibodies. Each approach has led to some degree of success thereby
providing support for anti-CD44 cancer therapeutics.
[0012] Both variant and standard CD44 specific monoclonal
antibodies have been generated experimentally but for the most part
these antibodies have no intrinsic biological activity, rather they
bind specifically to the type of CD44 they recognize. However,
there are some that are either active in vitro or in vivo but
generally not both. Several anti-CD44 antibodies have been shown to
mediate cellular events. For example the murine antibody A3D8,
directed against human erythrocyte Lutheran antigen CD44 standard
form, was shown to enhance CD2 (9-1 antibody) and CD3 (OKT3
antibody) mediated T cell activation; another anti-CD44 antibody
had similar effects. A3D8 also induced IL-1 release from monocytes
and IL-2 release from T lymphocytes. Interestingly, the use of A3D8
in conjunction with drugs such as daunorubicin, mitoxantrone and
etoposide inhibited apoptosis induction in HL60 and NB4 AML cells
by abrogating the generation of the second messenger ceramide. The
J 173 antibody, which does not have intrinsic activity and is
directed against a similar epitope of CD44s, did not inhibit
drug-induced apoptosis. The NIH44-1 antibody, directed against an
85-110 kD and 200 kD form of CD44, augmented T-cell proliferation
through a pathway the authors speculated as either cross-linking or
aggregation of CD44. Taken together, there is no evidence that
antibodies such as these are suitable for use as cancer
therapeutics since they either are not directed against cancer
(e.g. activate lymphocytes), induce cell proliferation, or when
used with cytotoxic agents inhibited drug-induced death of cancer
cells.
[0013] Several anti-CD44 antibodies have been described which
demonstrate anti-tumor effects in vivo. The antibody 1.1 ASML, a
mouse IgG 1 directed to the v6 variant of CD44, has been shown to
decrease the lymph node and lung metastases of the rat pancreatic
adenocarcinoma BSp73ASML. Survival of the treated animals was
concomitantly increased. The antibody was only effective if
administered before lymph node colonization, and was postulated to
interfere with cell proliferation in the lymph node. There was no
direct cytototoxicity of the antibody on the tumor cells in vitro,
and the antibody did not enhance complement-mediated cytotoxicity,
or immune effector cell function. Utility of the antibody against
human cells was not described.
[0014] Breyer et al. described the use of a commercially-available
antibody to CD44s to disrupt the progression of an
orthotopically-implanted rat glioblastoma. The rat glioblastoma
cell line C6 was implanted in the frontal lobe, and after 1 week,
the rats were given 3 treatments with antibody by intracerebral
injection. Treated rats demonstrated decreased tumor growth, and
higher body weight than buffer or isotype control treated rats. The
antibody was able to inhibit adhesion of cells in vitro to
coverslips coated with extracellular matrix components, but did not
have any direct cytotoxic effects on cells. This antibody was not
tested against human cells.
[0015] A study was carried out which compared the efficacy of an
antibody to CD44s (IM-7.8.1) to an antibody to CD44v10 (K926). The
highly metastatic murine melanoma line B16F10, which expresses both
CD44 isoforms, was implanted intravenously into mice. After 2 days,
antibodies were given every third day for the duration of the
study. Both antibodies caused a significant reduction of greater
than 50 percent in the number of lung metastases; there was no
significant difference in efficacy between the two antibodies. The
antibody did not affect proliferation in vitro, and the authors,
Zawadzki et al., speculated that the inhibition of tumor growth was
due to the antibody blocking the interaction of CD44 with its
ligand. In another study using IM-7.8.1, Zahalka et al.
demonstrated that the antibody and its F(ab').sub.2 fragment were
able to block the lymph node infiltration by the murine T-cell
lymphoma LB. This conferred a significant survival benefit to the
mice. Wallach-Dayan et al. showed that transfection of LB-TRs
murine lymphoma, which does not spontaneously form tumors, with
CD44v4-v10 conferred the ability to form tumors. IM-7.8.1
administration decreased tumor size of the implanted transfected
cells in comparison to the isotype control antibody. None of these
studies demonstrated human utility for this antibody.
[0016] GKW.A3, a mouse IgG2a, is specific for human CD44 and
prevents the formation and metastases of a human melanoma xenograft
in SCID mice. The antibody was mixed with the metastastic human
cell line SMMU-2, and then injected subcutaneously. Treatments were
continued for the following 3 weeks. After 4 weeks, only 1 of 10
mice developed a tumor at the injection site, compared to 100
percent of untreated animals. F(ab').sub.2 fragments of the
antibody demonstrated the same inhibition of tumor formation,
suggesting that the mechanism of action was not dependent on
complement or antibody-dependent cellular cytotoxicity. If the
tumor cells were injected one week prior to the first antibody
injection, 80 percent of the animals developed tumors at the
primary site. However, it was noted that the survival time was
still significantly increased. Although the delayed antibody
administration had no effect on the primary tumor formation, it
completely prevented the metastases to the lung, kidney, adrenal
gland, liver and peritoneum that were present in the untreated
animals. This antibody does not have any direct cytotoxicity on the
cell line in vitro nor does it interfere with proliferation of
SMMU-2 cells, and appears to have its major effect on tumor
formation by affecting metastasis or growth. One notable feature of
this antibody was that it recognized all isoforms of CD44, which
suggests limited possibilities for therapeutic use.
[0017] Strobel et al. describe the use of an anti-CD44 antibody
(clone 515) to inhibit the peritoneal implantation of human ovarian
cancer cells in a mouse xenograft model. The human ovarian cell
line 36M2 was implanted intraperitoneally into mice in the presence
of the anti-CD44 antibody or control antibody, and then treatments
were administered over the next 20 days. After 5 weeks, there were
significantly fewer nodules in the peritoneal cavity in the
antibody treated group. The nodules from both the anti-CD44 and
control treated groups were the same size, suggesting that once the
cells had implanted, the antibody had no effect on tumor growth.
When cells were implanted subcutaneously there was also no effect
on tumor growth indicating that the antibody itself did not have an
anti-proliferative or cytotoxic effect. In addition, there was no
effect of the antibody on cell growth in vitro.
[0018] VFF-18, also designated as BIWA 1, is a high-affinity
antibody to the v6 variant of CD44 specific for the 360-370 region
of the polypeptide. This antibody has been used as a
.sup.99mTechnetium-labelled conjugate in a Phase 1 clinical trial
in 12 patients. The antibody was tested for safety and targeting
potential in patients with squamous cell carcinoma of the head and
neck. Forty hours after injection, 14 percent of the injected dose
was taken up by the tumor, with minimal accumulation in other
organs including the kidney, spleen and bone marrow. The highly
selective tumor binding suggests a role for this antibody in
radioimmunotherapy, although the exceptionally high affinity of
this antibody prevented penetration into the deeper layers of the
tumor. Further limiting the application of BIWA 1 is the
immunogenicity of the murine antibody (11 of 12 patients developed
human anti-mouse antibodies (HAMA)), heterogenous accumulation
throughout the tumor and formation of antibody-soluble CD44
complexes. WO 02/094879 discloses a humanized version of VFF-18
designed to overcome the HAMA response, designated BIWA 4. BIWA 4
was found to have a significantly lower antigen binding affinity
than the parent VFF 18 antibody. Surprisingly, the lower affinity
BIWA 4 antibody had superior tumor uptake characteristics than the
higher affinity BIWA 8 humanized VFF-18 antibody. Both
.sup.99mTechnetium-labelled and .sup.186Rhenium-labelled BIWA 4
antibodies were assessed in a 33 patient Phase 1 clinical trial to
determine safety, tolerability, tumor accumulation and maximum
tolerated dose, in the case of .sup.186Re-labelled BIWA 4. There
appeared to be tumor related uptake of .sup.99mTc-labelled BIWA 4.
There were no tumor responses seen with all doses of
.sup.186Re-labelled BIWA 4, although a number had stable disease;
the dose limiting toxicity occurred at 60 mCi/m.sup.2. There was a
50-65 percent rate of adverse events with 12 of 33 patients deemed
to have serious adverse events (thrombocytopenia, leucopenia and
fever) and of those 6, all treated with .sup.186Re-labelled BIWA 4,
died in the course of treatment or follow-up due to disease
progression. Two patients developed human anti-human antibodies
(HAHA). A Phase 1 dose escalation trial of .sup.186Re-labelled BIWA
4 was carried out in 20 patients. Oral mucositis and dose-limiting
thrombocytopenia and leucocytopenia were observed; one patient
developed a HAHA response. Stable disease was seen in 5 patients
treated at the highest dose of 60 mCi/m.sup.2. Although deemed to
be acceptable in both safety and tolerablility for the efficacy
achieved, these studies have higher rates of adverse events
compared to other non-radioisotope conjugated biological therapies
in clinical studies. U.S. Patent Application US 2003/0103985
discloses a humanized version of VFF-18 conjugated to a
maytansinoid, designated BIWI 1, for use in tumor therapy. A
humanized VFF 18 antibody, BIWA 4, when conjugated to a toxin, i.e.
BIWI 1, was found to have significant anti-tumor effects in mouse
models of human epidermoid carcinoma of the vulva, squamous cell
carcinoma of the pharynx or breast carcinoma. The unconjugated
version, BIWA 4, did not have anti-tumor effects and the conjugated
version, BIWI 1, has no evidence of safety or efficacy in
humans.
[0019] Mab U36 is a murine monoclonal IgG1 antibody generated by
UM-SCC-22B human hypopharyngeal carcinoma cell immunization and
selection for cancer and tissue specificity. Antigen
characterization through cDNA cloning and sequence analysis
identified the v6 domain of keratinocyte-specific CD44 splice
variant epican as the target of Mab U36. Immunohistochemistry
studies show the epitope to be restricted to the cell membrane.
Furthermore, Mab U36 labeled 94 percent of the head and neck
squamous cell carcinomas (HNSCC) strongly, and within these tumors
there was uniformity in cell staining. A 10 patient
.sup.99mTc-labelled Mab U36 study showed selective accumulation of
the antibody to HNSCC cancers (20.4+/-12.4 percent injected dose/kg
at 2 days); no adverse effects were reported but two patients
developed HAMA. In a study of radio-iodinated murine Mab U36 there
were 3 cases of HAMA in 18 patients and selective homogenous uptake
in HNSCC. In order to decrease the antigenicity of Mab U36 and
decrease the rate of HAMA a chimeric antibody was constructed.
Neither the chimeric nor the original murine Mab U36 has ADCC
activity. There is no evidence of native functional activity of Mab
U36. .sup.186Re-labelled chimeric Mab U36 was used to determine the
utility of Mab U36 as a therapeutic agent. In this Phase 1
escalating dose trial 13 patients received a scouting dose of
.sup.99mTc-labelled chimeric Mab U36 followed by
.sup.186Re-labelled chimeric Mab U36. There were no acute adverse
events reported but following treatment dose limiting myelotoxcity
(1.5 GBq/m.sup.2) in 2 of 3 patients, and thrombocytopenia in one
patient treated with the maximum tolerated dose (1.0 GBq/m.sup.2)
were observed. Although there were some effects on tumor size these
effects did not fulfill the criteria for objective responses to
treatment. A further study of .sup.186Re-labelled chimeric Mab U36
employed a strategy of using granulocyte colony-stimulating factor
stimulated whole blood reinfusion to double the maximum-tolerated
activity to 2.8 Gy. In this study of nine patients with various
tumors of the head and neck, 3 required transfusions for drug
related anemia. Other toxicity includes grade 3 myelotoxicity, and
grade 2 mucositis. No objective tumor responses were reported
although stable disease was achieved for 3-5 months in 5 patients.
Thus, it can be seen that although Mab U36 is a highly specific
antibody the disadvantage of requiring a radioimmunoconjugate to
achieve anti-cancer effects limits its usefulness because of the
toxicity associated with the therapy in relation to the clinical
effects achieved.
[0020] To summarize, a CD44v6 (1.1ASML) and CD44v10 (K926)
monoclonal antibody have been shown to reduce metastatic activity
in rats injected with a metastatic pancreatic adenocarcinoma or
mice injected with a malignant melanoma respectively. Another
anti-CD44v6 antibody (VFF-18 and its derivatives), only when
conjugated to a maytansinoid or a radioisotope, has been shown to
have anti-tumor effects. Anti-standard CD44 monoclonal antibodies
have also been shown to suppress intracerebral progression by rat
glioblastoma (anti-CD44s), lymph node invasion by mouse T cell
lymphoma (IM-7.8.1) as well as inhibit implantation of a human
ovarian cancer cell line in nude mice (clone 515), lung metastasis
of a mouse melanoma cell line (IM-7.8.1) and metastasis of a human
melanoma cell line in SCID mice (GKW.A3). The radioisotope
conjugated Mab U36 anti-CD44v6 antibody and its derivatives had
anti-tumor activity in clinical trials that were accompanied by
significant toxicity. These results, though they are encouraging
and support the development of anti-CD44 monoclonal antibodies as
potential cancer therapeutics, demonstrate limited effectiveness,
safety, or applicability to human cancers.
[0021] Thus, if an antibody composition were isolated which
mediated cancerous cell cytotoxicity, as a function of its
attraction to cell surface expression of CD44 on said cells, a
valuable diagnostic and therapeutic procedure would be
realized.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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 (1HC) 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.
[0030] 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.
[0031] 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.
[0032] 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-fluourouracil 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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:
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen
characteristic of human carcinomas and not dependent upon the
epithelial tissue of origin.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen
characteristic of human carcinomas and not dependent upon the
epithelial tissue of origin.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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 2-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.
[0056] U.S. Pat. No. 5,780,033 discloses the use of autoantibodies
for tumor therapy and prophylaxis. However, this antibody is an
anti-nuclear 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.
[0057] U.S. Pat. No. 5,916,561 discloses a specific antibody,
VFF-18, and its variants directed against the variant exon v6 of
the CD44 gene. This antibody is an improvement over the comparator
antibody in that it recognizes a human CD44 v6 variant rather than
a rat CD44 v6 variant. In addition this antibody discloses
diagnostic assays for CD44 v6 expression. There was no in vitro or
in vivo function disclosed for this antibody.
[0058] U.S. Pat. No. 5,616,468 discloses a monoclonal antibody,
Var3.1, raised against a synthetic peptide containing a sequence
encoded by the human exon 6A of the CD44 gene. Specifically this
antibody does not bind to the 90 kD form of human CD44 and is
distinguished from the Hermes-3 antibody. A method for detection of
the v6 variant of CD44 is provided, as well as a method for
screening and assaying for malignant transformation based on this
antigen. A method for screening for inflammatory disease based on
detecting the antigen in serum is also provided.
[0059] U.S. Pat. No. 5,879,898 discloses a specific antibody that
binds to a 129 by exon of a human CD44 variant 6 that produces a 43
amino acid peptide. The monoclonal antibody is produced by a number
of hybridoma cell lines: MAK<CD44>M-1.1.12,
MAK<CD44>M-2.42.3, MAK<CD44>M-4.3.16. The antibody is
generated from a fusion protein that contains at least a
hexapeptide of the novel CD44 v6 amino acid sequence. Further,
there is a disclosure of an immunoassay for the detection of exon 6
variant that can be used as a cancer diagnostic. Significantly,
there is no in vitro or in vivo function of this antibody
disclosed.
[0060] U.S. Pat. No. 5,942,417 discloses a polynucleotide that
encodes a CD44 like polypeptide, and the method of making a
recombinant protein using the polynucleotide and its variants.
Antibodies are claimed to these polypeptides however there are no
specific examples and there are no deposited clones secreting such
antibodies. Northern blots demonstrate the appearance of the
polynucleotide in several types of tissues, but there is no
accompanying evidence that there is translation and expression of
this polynucleotide. Therefore, there is no evidence that there
were antibodies to be made to the gene product of this
polynucleotide, that these antibodies would have either in vitro or
in vivo function, and whether they would be relevant to human
cancerous disease.
[0061] U.S. Pat. No. 5,885,575 discloses an antibody that reacts
with a variant epitope of CD44 and methods of identifying the
variant through the use of the antibody. The isolated
polynucleotide encoding this variant was isolated from rat cells,
and the antibody, mAb1.1ASML, directed against this variant
recognizes proteins of molecular weight 120 kD, 150 kD, 180 kD, and
200 kD. The administration of monoclonal antibody 1.1ASML delayed
the growth and metastases of rat BSp73ASML in isogenic rats.
Significantly 1.1ASML does not recognize human tumors as
demonstrated by its lack of reactivity, to LCLC97 human large-cell
lung carcinoma. A human homolog was isolated from LCLC97 but no
equivalent antibody recognizing this homolog was produced. Thus,
although an antibody specific to a variant of rat CD44 was produced
and shown to affect the growth and metastasis of rat tumors there
is no evidence for the effect the this antibody against human
tumors. More specifically the inventors point out that this
antibody does not recognize human cancers.
SUMMARY OF THE INVENTION
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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/Hematolgy
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).
[0074] Using substantially the process of U.S. Pat. No. 6,180,357,
the mouse monoclonal antibody H460-16-2 was obtained following
immunization of mice with cells from a patient's lung tumor biopsy
and from the lung cancer cell line NCI-H460 (ATCC, Virginia,
Mass.). The H460-16-2 antigen was expressed on the cell surface of
a broad range of human cell lines from different tissue origins.
The breast cancer cell line MDA-MB-231 (MB-231) and skin cancer
cell line A2058 were susceptible to the cytotoxic effects of
H460-16-2 in vitro.
[0075] The result of H460-16-2 cytotoxicity against MB-231 cells in
culture was further extended by its anti-tumor activity towards
these cancer cells when transplanted into mice (as disclosed in
Ser. No. 10/603,000). Pre-clinical xenograft tumor models are
considered valid predictors of therapeutic efficacy.
[0076] In the preventative in vivo model of human breast cancer,
H460-16-2 treatment was significantly (p<0.0001) more effective
in suppressing tumor growth during the treatment period than an
isotype control antibody. At the end of the treatment phase, mice
given H460-16-2 had tumors that grew to only 1.3 percent of the
control group. During the post treatment follow-up period, the
treatment effects of H460-16-2 were sustained and the mean tumor
volume in the treated groups continued to be significantly smaller
than controls until the end of the measurement phase. Using
survival as a measure of antibody efficacy, it was estimated that
the risk of dying in the 11460-16-2 treatment group was about 71
percent of the antibody buffer control group (p=0.028) at 70 days
post-treatment. These data demonstrated that H40-16-2 treatment
conferred a survival benefit compared to the control-treated
groups. H460-16-2 treatment appeared safe, as it did not induce any
signs of toxicity, including reduced body weight and clinical
distress. Thus, H460-16-2 treatment was efficacious as it both
delayed tumor growth and enhanced survival compared to the
control-treated groups in a well-established model of human breast
cancer.
[0077] In addition, H460-16-2 demonstrated anti-tumor activity
against MB-231 cells in an established in vivo tumor model (as
disclosed in Ser. No. 10/603,000). Treatment with H460-16-2 was
compared to the standard chemotherapeutic drug, Cisplatin, and it
was shown that the Cisplatin and H460-16-2 treatment groups had
significantly (p<0.001) smaller mean tumor volumes compared with
groups treated with either antibody dilution buffer or the isotype
control antibody. H460-16-2 treatment mediated tumor suppression
that was approximately two-thirds that of Cisplatin chemotherapy
but without the significant (19.2 percent) weight loss (p<0.003)
and clinical distress, including 2 treatment-associated deaths,
observed with Cisplatin treatment. The anti-tumor activity of
H460-16-2 and its minimal toxicity make it an attractive
anti-cancer therapeutic agent.
[0078] In the post-treatment period, H460-16-2 showed a significant
survival benefit (p<0.02) as the risk of dying in the H460-16-2
group was about half of that in the isotype control antibody group
at >70 days after treatment. The observed survival benefit
continued past 120 days post-treatment where 100 percent of the
isotype control and Cisplatin treated mice had died compared to 67
percent of the H460-16-2 treatment group. H460-16-2 maintained
tumor suppression by delaying tumor growth by 26 percent compared
to the isotype control antibody group. At 31 days post treatment,
H460-16-2 limited tumor size by reducing tumor growth by 48 percent
compared to the isotype control group, which is comparable to the
49 percent reduction observed at the end of the treatment. In the
established tumor model of breast cancer, these results indicated
the potential of H460-16-2 to maintain tumor suppression beyond the
treatment phase and demonstrated the ability of the antibody to
reduce the tumor burden and enhance survival in a mammal.
[0079] In addition to the beneficial effects in the established in
vivo tumor model of breast cancer, H460-16-2 treatment in
combination with a chemotherapeutic drug (Cisplatin) had anti-tumor
activity against PC-3 cells in an established in vivo prostate
cancer model (as disclosed in Ser. No. 10/810,165). Using a paired
t-test, H460-16-2 plus Cisplatin treatment was significantly more
effective in suppressing tumor growth shortly after the treatment
period than buffer control (p<0.0001), Cisplatin treatment alone
(p=0.004) or H460-16-2 treatment alone (p<0.0001). At the end of
the treatment phase, mice given H460-16-2 plus Cisplatin had tumors
that grew to only 28.5 percent of the buffer control group. For
PC-3 SCID xenograft models, body weight can be used as a surrogate
indicator of disease progression. Mice in all the groups
experienced severe weight loss. In this study, mice in all groups
showed a weight loss of approximately 23 to 35 percent by the end
of the treatment period. The group treated with H460-16-2 showed
the smallest degree of weight loss (21.7 percent). After treatment,
day 48, there was no significant increase in weight loss associated
with the treatment of H460-16-2 and Cisplatin in comparison to
buffer control (p=0.5042). Thus, H460-16-2 plus Cisplatin treatment
was efficacious as it delayed tumor growth compared to the isotype
control treated group in a well-established model of human prostate
cancer.
[0080] In order to validate the H460-16-2 epitope as a drug target,
the expression of H460-16-2 antigen in normal human tissues was
previously determined (Ser. No. 10/603,000). This work was extended
by comparison with the anti-CD44 antibodies; clone L178 (disclosed
in Ser. No. 10/647,818) and clone BU75 (disclosed in Ser. No.
10/810,165). By IHC staining with H460-16-2, the majority of the
tissues failed to express the H460-16-2 antigen, including the
cells of the vital organs, such as the liver, kidney (except for
marginal staining of tubular epithelial cells), heart, and lung.
Results from tissue staining indicated that H460-16-2 showed
restricted binding to various cell types but had binding to
infiltrating macrophages, lymphocytes, and fibroblasts. The BU75
antibody showed a similar staining pattern. However, there was at
least one difference of note; staining of lymphocytes was more
intense with BU75 in comparison to H460-16-2.
[0081] Localization of the H460-16-2 antigen and determining its
prevalence within the population, such as among breast cancer
patients, is important in assessing the therapeutic use of
H460-16-2 and designing effective clinical trials. To address
H460-16-2 antigen expression in breast tumors from cancer patients,
tumor tissue samples from 50 individual breast cancer patients were
previously screened for expression of the H460-16-2 antigen (Ser.
No. 10/603,000) and was compared to L178 (Ser. No. 10/647,818),
BU75 (Ser. No. 10/810,165) and the anti-Her2 antibody c-erbB-2
(Ser. No. 10/810,165). The results of these studies were similar
and showed that 62 percent of tissue samples stained positive for
the H460-16-2 antigen while 73 percent of breast tumor tissues were
positive for the BU75 epitope. Expression of H460-16-2 within
patient samples appeared specific for cancer cells as staining was
restricted to malignant cells. H460-16-2 stained 4 of 10 samples of
normal tissue from breast cancer patients while BU75 stained 8.
Breast tumor expression of both the H460-16-2 and BU75 antigen
appeared to be mainly localized to the cell membrane of malignant
cells, making CD44 an attractive target for therapy. H460-16-2
expression was further evaluated based on breast tumor expression
of the receptors for the hormones estrogen and progesterone, which
play an important role in the development, treatment, and prognosis
of breast tumors. No correlation was apparent between expression of
the H460-16-2 antigen and expression of the receptors for either
estrogen or progesterone. When tumors were analyzed based on their
stage, or degree to which the cancer advanced, again there was no
clear correlation between H460-16-2 antigen expression and tumor
stage. Similar results were obtained with BU75. In comparison to
c-erbB-2, H460-16-2 showed a completely different staining profile
where 52 percent of the breast tumor tissue samples that were
positive for the H460-16-2 antigen were negative for Her2
expression indicating a yet unmet targeted therapeutic need for
breast cancer patients. There were also differences in the
intensity of staining between the breast tumor tissue sections that
were positive for both H460-16-2 and Her2. The c-erbB-2 antibody
also positively stained one of the normal breast tissue
sections.
[0082] To further extend the potential therapeutic benefit of
H460-16-2, the frequency and localization of the antigen within
various human cancer tissues was also previously determined (Ser.
No. 10/603,000) and was compared to clone L178 (Ser. No.
10/647,818). The majority of these tumor types were also positive
for the L178 antigen. As with human breast tumor tissue, H460-16-2
and L178 localization occurred on the membrane of tumor cells.
However, there was substantially more membrane localization with
the L178 compared to the H460-16-2 antibody. Also, of the tumor
types that were stained by both H460-16-2 and L178, 43 percent of
the tissues showed higher intensity staining with the L178
antibody.
[0083] In addition, the frequency and localization of the antigen
within prostate and liver normal and cancer tissues was previously
determined (Ser. No. 11/364,013). From the prostate cancer array,
19/53 (36 percent) of the tested tumors were positive for
H460-16-2. H460-16-2 was specific for tumor cells and stroma
fibroblasts. Cellular localization was mostly membranous and
cytoplasmic membranous with or without luminal localization. The
percentage of positive cells ranged from <10 percent->50
percent indicating heterogenous binding of the antibody to tumor
cells. The relation of the antibody binding to tumors' stages could
not be assessed properly due to a discrepancy in the number of
tumors among different tumor stages, being 1/1 (100 percent), 4/12
(33 percent), 0/2 (0 percent) and 11/33 (33 percent) to stage I,
II, III and IV, respectively. There was higher binding to Gleason
score G3-G4 (36 percent) than to G1-G2 (25 percent). The Gleason
score is a system of grading prostate cancer. The Gleason grading
system assigns a grade to each of the two largest areas of cancer
in the tissue samples. Grades range from 1 to 5 with 1 being the
least aggressive and 5 the most aggressive. Grade 3 tumors, for
example, seldom have metastases, but metastases are common with
grade 4 or grade 5. The two grades are then added together to
produce a Gleason score. A score of 2 to 4 is considered low grade;
5 through 7, intermediate grade; and 8 through 10, high grade. A
tumor with a low Gleason score typically grows slowly enough that
it may not pose a significant threat to the patient in his
lifetime. All 3 normal prostate tissue sections were positive for
the antibody. However, the tissue specificity was for myoepithelium
and stromal fibroblasts and spared the glandular epithelium. FIG.
12 demonstrates the heterogeneity of the binding of H460-16-2 to
tested prostate tumors: 10/53, 6/53, 3/53 positive tumors were in
the categories of <10-10 percent, <50-50 percent and >50
percent, respectively. As a result of its binding to prostate
cancer cells, the therapeutic benefit of H460-16-2 can potentially
be extended to the treatment of prostate cancer.
[0084] From the liver cancer array, H460-16-2 antibody showed
binding to 21/49 (43 percent) of tested liver cancers, including
11/37 (30 percent) of primary, 7/8 (88 percent) of metastatic
hepatocellular carcinoma, 1/2 (50 percent) of primary and 2/2 (100
percent) of metastatic cholangiocarcinomas. The antibody showed
significant higher binding to advanced tumors' stages III and IV in
comparison with early stages I and II (p=0.03) [stage I, 0/2 (0
percent); stage II, 2/17 (12 percent); stage III, 8/16 (50 percent)
and stage IV, 6/8 (75 percent)]. H460-16-2 was specific for tumor
cells and infiltrating inflammatory cells. Cellular localization
was mainly membranous. Some tumors also displayed a diffuse
cytoplasmic staining pattern. The antibody bound to 9/9 of
non-neoplastic liver tissues. However, the binding was restricted
to the sinusoidal cells and infiltrating lymphocytes. The H460-16-2
antigen appears to be specifically expressed on advanced liver
tumor tissue. H460-16-2 therefore has potential as a therapeutic
drug in the treatment of liver cancer.
[0085] There appears to be no form of CD44 that exactly matches the
IHC data presented herein based on comparisons with the IHC data
from the literature. The standard form of CD44 is normally
expressed in the human brain; the H460-16-2 antigen is not.
Antibodies directed against pan-CD44 isoforms do not stain the
liver (including Kuppfer cells) and positively stain the
endometrial glands in all phases of the reproductive cycle. The
H460-16-2 antigen is clearly present on Kuppfer cells and is only
present on the secretory endometrial glands of the reproductive
cycle. H460-16-2 antigen is clearly present on tissue macrophages
and only the variant forms V4/5 and V8/9 show occasional macrophage
staining. The similar yet distinct binding pattern seen with
H460-16-2 in comparison to anti-CD44 L178 and now BU75 indicates
that the H460-16-2 antigen is an unique epitope of CD44.
[0086] As disclosed previously (Ser. No. 10/647,818), additional
biochemical data also indicated that the antigen recognized by
H460-16-2 is one of the forms of CD44. This was supported by
studies that showed a monoclonal antibody (L178) reactive against
CD44 identifies proteins that were bound to H460-16-2 by
immunoprecipitation. Western blotting studies also suggested that
the epitope of CD44 recognized by H460-16-2 was not present on v6
or v10. The H460-16-2 epitope was also distinguished by being
carbohydrate and conformation dependent, whereas many anti-CD44
antibodies are directed against peptide portions of CD44. These IHC
and biochemical results demonstrated that H460-16-2 binds to a
variant of the CD44 antigen. Thus, the preponderance of evidence
showed that H460-16-2 mediates anti-cancer effects through ligation
of an unique carbohydrate dependent conformational epitope present
on a variant of CD44. For the purpose of this invention, said
epitope is defined as a "CD44 antigenic moiety" characterized by
its ability to bind with a monoclonal antibody encoded by the
hybridoma cell line H460-16-2, antigenic binding fragments thereof
or antibody conjugates thereof.
[0087] In order to further elucidate the mechanism behind
H460-16-2's anti-cancer effects, hyaluronic acid (HA) binding
assays were performed (as disclosed in Ser. No. 10/810,165). It was
determined that an average concentration of 1.87 (+/-1.01)
micrograms/mL of H460-16-2 was required to inhibit adhesion of
MDA-MB-231 cells to HA by 50 percent. These results indicated that
H460-16-2 interacts with, at least in part, the region(s) on CD44
that are responsible for binding to HA and consequently could be
mediating its anti-cancer effects through down regulation of
angiogenesis or tumor invasiveness through the ECM.
[0088] In addition to the HA binding assays, a cell cycling
experiment was performed in order to determine if the H460-16-2 in
vitro and in vivo anti-cancer effects were due to regulation of the
cell cycle (as disclosed in Ser. No. 10/810,165). After 24 hours
and with 20 micrograms/mL of H460-16-2, there was an increase in
the number of MDA-MB-231 apoptotic cells in comparison to the
isotype control. This effect also appeared to be dose dependent.
Therefore, the efficacy of H460-16-2 might be also due, in whole or
in part, to its apoptotic inducing capabilities.
[0089] To further elucidate the mechanism of action for H460-16-2,
the effect of H460-16-2 treatment upon apoptosis in MDA-MB-231
tumors grown in vivo in a xenograft model of breast cancer was
investigated (as disclosed in Ser. No. 11/364,013). Apoptotic cells
were counted using morphological criteria such as deletion of
single cells, cell shrinkage and compaction of chromatin into a
dense mass. The buffer control treatment group yielded an average
total score of 17 cells (.+-.5.29) while the H460-16-2 treated
group yielded an average total score of 22.5 cells (.+-.4.20).
Therefore, there is a trend towards increased apoptosis with
H460-16-2 treatment as determined using cellular morphology.
[0090] Two chimeric versions of H460-16-2 were generated as
disclosed in Ser. No. 11/364,013. One version is of isotype IgG1,
kappa ((ch)ARH460-16-2-IgG1) and the other is of isotype IgG2,
kappa ((ch)ARH460-16-2-IgG2). Supernatants from chimeric IgG1 and
chimeric IgG2-secreting clones were able to detect CD44 in a
Western blot, with a signal that was similar to that obtained with
the murine H460-16-2 (as disclosed in Ser. No. 11/364,013).
[0091] Both of the chimeric antibodies were compared to the murine
version of H460-16-2 in an established model of breast cancer (as
disclosed in Ser. No. 11/364,013). Both murine H460-16-2 and
(ch)ARH460-16-2-IgG1 reduced tumor growth in an established
MDA-MB-231 in vivo model of human breast cancer. At day 62, 5 days
after the last dose was administered, treatment with H460-16-2
resulted in a tumor growth inhibition of 39 percent (Mean T/C=57
percent). This reduction in tumor growth was significantly
different from the control (p=0.0037). The chimeric antibody
(ch)ARH460-16-2-IgG1 resulted in an enhanced tumor growth
inhibition (TGI) of 64 percent (Mean T/C=26.9 percent;
p<0.0001). By contrast, the IgG2 version of the chimeric
antibody, (ch)ARH460-16-2-IgG2 showed no inhibition in tumor growth
when compared with the buffer control (TGI=0 percent; Mean T/C=122
percent; p=0.7264).
[0092] Annexin-V staining was performed to determine whether the
chimeric versions of H460-16-2 were able to induce apoptosis in the
same manner as the murine counterpart on the MDA-MB-231 human
breast cancer cell line (as disclosed in Ser. No. 11/364,013).
Spontaneous apoptotic effects of cells treated with isotype control
were found to be similar to cells treated with vehicle only. The
murine and human chimeric IgG1 and IgG2 H460-16-2 antibodies were
all found to induce apoptosis in the breast cancer cell line in a
dose dependent manner in each experiment, with greater apoptotic
effect seen with both the (ch)ARH460-16-2 IgG 1 and IgG2
antibodies. Results indicate that in vitro the (ch)ARH460-16-2-IgG2
antibody has the greatest apoptotic effect when compared to the
chimeric IgG1 antibody. All 3 antibodies showed an increase in the
percentage necrotic and necrotic/apoptotic populations over their
prospective isotype controls. The largest increase in the
percentage necrotic and necrotic/apoptotic populations was seen
with (ch)ARH460-16-2-IgG2, then (ch)ARH460-16-2-IgG1 and then
H460-16-2.
[0093] In toto, this data demonstrates that the murine and chimeric
H460-16-2 antigen is a cancer associated antigen and is expressed
in humans, and is a pathologically relevant cancer target. Further,
this data also demonstrates the binding of the H460-16-2 antibody
to human cancer tissues, and can be used appropriately for assays
that can be diagnostic, predictive of therapy, or prognostic. In
addition, the cell membrane localization of this antigen is
indicative of the cancer status of the cell due to the lack of
expression of the antigen in most non-malignant cells, and this
observation permits the use of this antigen, its gene or
derivatives, its protein or its variants to be used for assays that
can be diagnostic, predictive of therapy, or prognostic.
[0094] Other studies, involving the use of anti-CD44 antibodies,
have limitations of therapeutic potential that are not exhibited by
H460-16-2. H460-16-2 demonstrates both in vitro and in vivo
anti-tumor activity. Previously described antibodies such as
MAK<CD44>M-1.1.12, MAK<CD44>M-2.42.3 and
MAK<CD44>M-4.3.16 have no in vitro or in vivo cytotoxicity
ascribed to them and VFF-18 and Mab U36 show no intrinsic tumor
cytotoxicity. In addition other anti-CD44 antibodies that have
shown in vivo tumor effects also have certain limitations that are
not evident with 1-1460-16-2. For example, ASML1.1, K926,
anti-CD44s and IM-78.1 show in vivo anti-tumor activity against
rat, murine, rat and murine tumors grown in xenograft models
respectively. H460-16-2 demonstrates anti-tumor activity in a model
of human cancer. H460-16-2 is also directed against human CD44
while antibodies such as ASML1.1 recognize only rat CD44. The clone
515 anti-CD44 antibody does inhibit peritoneal tumor implantation
of a human ovarian cell line but does not prevent or inhibit tumor
growth. H460-16-2 is capable of inhibiting human breast tumor
growth in a SCID mouse xenograft model. GKW.A3 is an anti-human
CD44 monoclonal antibody capable of inhibiting tumor growth of a
human metastasizing melanoma grown in mice in a preventative but
not an established model. H460-16-2 has demonstrated significant
anti-tumor activity in both preventative and established murine
xenograft models of human breast cancer. Consequently, it is quite
apparent that H460-16-2 has superior anti-tumor properties in
comparison to previously described anti-CD44 antibodies. It has
demonstrated both in vitro and in vivo anti-tumor activity on a
human breast tumor in SCID mice and is directed against human CD44.
It also exhibits activity in a preventative and established (more
clinically relevant) model of human breast cancer and it exhibits
activity with Cisplatin in an established model of human prostate
cancer.
[0095] The present invention describes the development and use of
H460-16-2 developed by the process described in patent U.S. Pat.
No. 6,180,357 and identified by, its effect, in a cytotoxic assay,
in tumor growth in animal models and in prolonging survival time in
those suffering from cancerous disease.
[0096] This invention represents an advance in the field of cancer
treatment in that it describes reagents that bind specifically to
an epitope or epitopes present on the target molecule, CD44, and
that directly mediate inhibition of tumor growth and metastasis in
in vivo models of human liver cancer. The preponderance of
evidence, disclosed herein, demonstrates that chimeric H460-16-2
mediates anti-cancer effects through ligation of epitopes present
on CD44, which is expressed on liver cancer, which will broadly be
understood to encompass any primary or metastatic tumor sites which
arise from hepatocytes. This application demonstrates that
expression of CD44 is observed with metastatic versus primary human
liver cancer cell lines. This invention also discloses that
chimeric H460-16-2 reduces the tumor burden and probability of
metastasis of human liver cancer in vivo.
[0097] This is an advance in relation to any other previously
described anti-CD44 antibody, since none have been shown to have
similar properties. It also provides an advance in the field since
it clearly demonstrates the direct involvement of CD44 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.
[0098] In all, this invention teaches the use of the H460-16-2
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 (thus delaying disease progression) and the likelihood of
metastasis 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 a CDMAB (H460-16-2), and its
derivatives, ligands and antigen binding fragments thereof, to
target its antigen to reduce the tumor burden of a cancer
expressing the antigen in a mammal, and to prolong the survival of
a mammal bearing tumors that express this antigen. Furthermore,
this invention also teaches the use of detecting the H460-16-2
antigen in cancerous cells that can be useful for the diagnosis,
prediction of therapy, and prognosis of mammals bearing tumors that
express this antigen.
[0099] 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.
[0100] It is an additional objective of the invention to teach
cancerous disease modifying antibodies, ligands and antigen binding
fragments thereof.
[0101] It is a further objective of the instant invention to
produce cancerous disease modifying antibodies whose cytotoxicity
is mediated through antibody dependent cellular toxicity.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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
[0106] 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.
[0107] FIG. 1 is a summary of H460-16-2 binding on a human liver
tumor and normal tissue microarray.
[0108] FIG. 2. Representative micrographs showing the binding
pattern on liver tumor tissue obtained with H460-16-2 (A) or the
isotype control antibody (B) and on non-neoplastic liver tissue
obtained with H460-16-2 (C) or the isotype control antibody (D)
from a human tissue microarray. H460-16-2 displayed strong positive
staining for the tumor cells and was limited to staining on
sinusoidal cells (black arrows) and infiltrating lymphocytes (green
arrows) on the non-neoplastic liver tissue. Magnification is
200.times..
[0109] FIG. 3 demonstrates the correlation between CD44
over-expression and the metastatic potential of various HCC cell
lines.
[0110] FIG. 4 demonstrates the effect of (ch)ARH460-16-2-IgG1 on
HCC tumor growth and metastasis in an established orthotopic HCC
tumor model. Data points represent the mean+/-SEM.
[0111] FIG. 5 demonstrates the quantitative effect of
(ch)ARH460-16-2-IgG1 on tumor growth in an established orthotopic
HCC tumor model. The bar graph summarizes the average basal signal
of tumors from four groups of animals in
photons/s/cm.sup.2/steridian. Each column represents the average
basal level signals on day 45 following tumor inoculation.
[0112] FIG. 6 visually demonstrates the effect of
(ch)ARH460-16-2-IgG1 on primary tumor growth in an established
orthotopic HCC tumor model.
[0113] FIG. 7 is the tabulation of the number of different
metastasis that developed with and without antibody treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0114] In general, the following words or phrases have the
indicated definition when used in the summary, description,
examples, and claims.
[0115] 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).
[0116] 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.
[0117] "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).
[0118] 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.
[0119] 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., .beta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0120] 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.
[0121] "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).
[0122] "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.
[0123] 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)).
[0124] "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.
[0125] 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).
[0126] 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.
[0127] "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.
[0128] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(x) and lambda (.lamda.), based on the amino acid sequences of
their constant domains.
[0129] "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).
[0130] 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).
[0131] 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.
[0132] 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.
[0133] An antibody "which binds" an antigen of interest, e.g. CD44
antigenic moiety, 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 CD44 antigenic
moiety it will usually preferentially bind CD44 antigenic moiety 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.
[0134] 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.
[0135] "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.
[0136] 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.
[0137] 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, chlornaphazine, 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.
[0138] "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.
[0139] "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.
[0140] 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.
[0141] "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.
[0142] 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).
[0143] 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 ATCC as
accession number PTA-4621, a humanized antibody of the isolated
monoclonal antibody produced by the hybridoma deposited with the
ATCC as accession number PTA-4621, a chimeric antibody of the
isolated monoclonal antibody produced by the hybridoma deposited
with the ATCC as accession number PTA-4621, antigen binding
fragments, or antibody ligands thereof, which effect is not
necessarily related to the degree of binding.
[0144] 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, H460-16-2 (murine), (h)ARH460-16-2-IgG1,
(ch)ARH460-16-2-IgG1, or Depository Designation, ATCC PTA-4621.
[0145] 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 ATCC PTA-4621 (the ATCC PTA-4621 antigen), a
humanized antibody of the isolated monoclonal antibody produced by
the hybridoma deposited with the ATCC as accession number PTA-4621,
a chimeric antibody of the isolated monoclonal antibody produced by
the hybridoma deposited with the ATCC as accession number PTA-4621,
and antigen binding fragments thereof.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] As used herein "antigen-binding region" means a portion of
the molecule which recognizes the target antigen.
[0152] 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 ATCC
PTA-4621(the ATCC PTA-4621 antibody), a humanized antibody of the
isolated monoclonal antibody produced by the hybridoma deposited
with the ATCC as accession number PTA-4621, a chimeric antibody of
the isolated monoclonal antibody produced by the hybridoma
deposited with the ATCC as accession number PTA-4621, antigen
binding fragments, or antibody ligands thereof, 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).
[0153] As used herein "target antigen" is the ATCC PTA-4621 antigen
or portions thereof.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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
[0160] It is contemplated to utilize any suitable CDMAB in
accordance with 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.
[0161] 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, Chapters 9-14, 1988. Further
contemplated are any and all combinations, permutations or
modifications of the afore-described diagnostic assay formats.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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,
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.
[0171] 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.
[0172] Additionally, CDMABs of the present invention may be used in
the laboratory for research due to its ability to identify its
target antigen.
[0173] In order that the invention herein described may be more
fully understood, the following description is set forth.
[0174] The present invention provides CDMABs (i.e., ATCC PTA-4621
CDMAB, a humanized antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the ATCC as accession
number PTA-4621, a chimeric antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the ATCC as
accession number PTA-4621, antigen binding fragments, or antibody
ligands thereof) which specifically recognize and bind the ATCC
PTA-4621 antigen.
[0175] The CDMABs of the isolated monoclonal antibody produced by
the hybridoma deposited with the ATCC as accession number PTA-4621
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 ATCC PTA-4621 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 ATCC PTA-4621 antibody fall
within the scope of this invention.
[0176] In one embodiment of the invention, the CDMAB is the ATCC
PTA-4621 antibody.
[0177] 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').sub.2
molecule, a fusion protein, a bispecific antibody, a heteroantibody
or any recombinant molecule having the antigen-binding region of
the ATCC PTA-4621 antibody. The CDMAB of the invention is directed
to the epitope to which the ATCC PTA-4621 monoclonal antibody is
directed.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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
051206-01 antigen or portions thereof.
[0182] These amino acid substitutions include, but are not
necessarily limited to, amino acid substitutions known in the art
as "conservative".
[0183] 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.
[0184] 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
[0185] Human Liver Tumor Tissue Staining
[0186] IHC studies were conducted to further evaluate the binding
of H460-16-2 to human liver tumor tissue. IHC optimization studies
were performed previously in order to determine the conditions for
further experiments. H460-16-2 monoclonal antibodies were produced
and purified as previously disclosed in Ser. No. 10/603,000.
[0187] Binding of antibodies to 49 human liver tumor and 9 normal
liver tissues was performed using a human, liver normal and tumor
tissue microarray (Imgenex, San Diego, Calif.). The following
information was provided for each patient: age, sex, organ and
diagnosis. Tissue sections were deparaffinized by drying in an oven
at 58.degree. C. for 1 hour and dewaxed by immersing in xylene 5
times for 4 minutes each in Coplin jars. Following treatment
through a series of graded ethanol washes (100 percent-75 percent)
the sections were re-hydrated in water. The slides were immersed in
10 mM citrate buffer at pH 6 (Dako, Toronto, Ontario) then
microwaved at high, medium, and low power settings for 5 minutes
each and finally immersed in cold PBS. Slides were then immersed in
3 percent hydrogen peroxide solution for 6 minutes, washed with PBS
three times for 5 minutes each, dried, incubated with Universal
blocking solution (Dako, Toronto, Ontario) for 5 minutes at room
temperature. H460-16-2, anti-AFP (alpha 1 fetoprotein; clone
AFP-11, Abcam, Cambridge, Mass.) 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-PSMA which was diluted to 10
micrograms/mL) and incubated for 1 hour at room temperature. The
slides were washed with PBS 3 times for 5 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.
[0188] As disclosed in FIG. 1, the H460-16-2 antibody showed
binding to 21/49 (43 percent) of tested liver cancers, including
11/37 (30 percent) of primary, 7/8 (88 percent) of metastatic
hepatocellular carcinoma, 1/2 (50 percent) of primary and 2/2 (100
percent) of metastatic cholangiocarcinomas. The antibody showed
significant higher binding to advanced tumors' stages III and IV in
comparison with early stages I and II (p=0.03) [stage I, 0/2 (0
percent); stage II, 2/17 (12 percent); stage III, 8/16 (50 percent)
and stage IV, 6/8 (75 percent)]. H460-16-2 was specific for tumor
cells and infiltrating inflammatory cells. Cellular localization
was mainly membranous. Some tumors also displayed a diffuse
cytoplasmic staining pattern. The antibody bound to 9/9 of
non-neoplastic liver tissues (FIG. 2). However, the binding was
restricted to the sinusoidal cells and infiltrating lymphocytes.
The H460-16-2 antigen appears to be specifically expressed on
advanced liver tumor tissue. 11460-16-2 therefore has potential as
a therapeutic drug in the treatment of liver cancer.
[0189] Therefore, the H460-16-2 antigen appears to be expressed on
liver tumor tissue with binding preference for metastatic and
advanced liver tumor tissue. H460-16-2 therefore has utility as a
diagnostic reagent for hepatocellular carcinoma, and as a
therapeutic drug in the treatment of liver cancer.
Example 2
[0190] Correlation of CD44 with Metastatic Potential of Various HCC
Cell Lines
[0191] To further evaluate this correlation in vitro, six
heptaocellular carcinoma (HCC) cell lines with various metastatic
potential (Hep3B (American Type Culture Collection, Manassas, Va.),
Huh-7 (a gift from Dr. H. Nakabayashi, Hokkaido University School
of Medicine, Sapporo, Japan), PLC (Japanese Cancer Research Bank,
Tokyo, Japan), MHCC-97L, MHCC-97H and HCCLM3 (Liver Cancer
Institute, Fudan University, Shanghai, China)) were evaluated for
CD44 expression by flow cytometry using (ch)ARH460-16-2-IgG1
antibody. To detect expression of CD44 in various HCC cell lines,
cells were stained for 1 hour with (ch)ARH460-16-2-IgG1 or isotype
control antibody (10 micrograms/mL) and 30 minutes with the
appropriate secondary antibody and analyzed by FACS.
[0192] By flow cytometry, CD44 expression levels were found to be
higher in the metastatic HCC cell lines (MHCC-97L, HCCLM3 and
MHCC-97H) when compared with the primary non-metastatic cell lines
(Hep3B, Huh-7 and PLC) (FIG. 3).
Example 3
[0193] Orthotopic HCC Tumor Model with HCCLM3 Cells
Luciferase Labeling of Cells
[0194] To further evaluate this correlation in vivo,
(ch)ARH460-16-2-IgG1 was tested in an orthotopic HCC tumor model.
For the luciferase labelling of HCCLM3 (a metastatic HCC cell line
Yang et al., Cancer Genet. Cytogenet. 158(2):180-183 2005) cells,
lentiviral vector harboring the luciferase gene was constructed and
transfected into the cells using the method described previously
(Cheung et al., Cancer Res. 66(8):4357-4367 2006). Stable
transfectants were generated from a pool of greater than 20
positive clones (which were selected with blasticidin at a
concentration of 2 micrograms/mL).
Animal CCD Experiments
[0195] One million HCCLM3 luciferase labeled cells were
sub-cutaneously injected into the right flank of nude mice, which
were then observed daily for signs of tumor development. Once the
tumors reached 1 to 1.5 cm in diameter, it was removed and cut into
about 1- to 2-mm cubes, which were then subsequently implanted into
the left liver lobe of 5 week old male nude mice. Ten days later,
the nude mice were randomized into a group of four and were treated
either with isotype control or 2, 10 or 20 mg/kg
(ch)ARH460-16-2-IgG1. (ch)ARH460-16-2-IgG1 test antibody was
administered intraperitoneally to each cohort, in a volume of 200
microlitres 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 2 times per week for a total of 12 doses in the same
fashion until day 45 post-implantation.
[0196] The mice were imaged on day 7 and day 45 after tumor
inoculation. Mice were anesthetized with a ketamine-xylazine mix in
a 4:1 ratio according to the Committee on the Use of Live Animals
in Teaching and Research of the University of Hong Kong.
[0197] Imaging was done using a Xenogen IVIS 100 cooled CCD camera
(Xenogen, New Jersey, USA). The mice were injected with 200
microliters of 15 mg/mL D-luciferin intraperitoneally for 15
minutes before imaging after which they were placed in a
light-tight chamber. A gray-scale reference image was obtained
followed by the acquisition of a bioluminescent image. The
acquisition time ranged from 3 seconds to 1 minute. The images
shown are pseudoimages of the emitted light in
photons/s/cm2/steradian, superimposed over the gray-scale
photographs of the animal.
[0198] (ch)ARH460-16-2-IgG1 significantly reduced tumor burden in
an established model of human HCC (FIG. 4). On day 45 after tumor
implantation, (ch)ARH460-16-2-IgG1 decreased primary liver tumor
signal from 37.6E+7.+-.0.17 to 9E+7.+-.0.72, 2.3 E+7.+-.0.52 and
0.1E+7.+-.0.5 at the doses of 2, 10 and 20 mg/kg, respectively
(FIG. 5). Representative primary tumors from the different groups
were also photographed (FIG. 6). There was no significant
difference in mean body weight between the two groups over the
course of the study.
[0199] (ch)ARH460-16-2-IgG1 significantly suppressed intrahepatic
and lung metastases in an established orthotopic model of human
HCC. The number of mice with lung and intrahepatic metastases in
the treatment group and control group are shown in FIG. 7.
(ch)ARH460-16-2-IgG1 significantly suppressed intrahepatic
metastasis from (5/5) 100 percent to ( ) 40 percent at a dose of 2
mg/kg. (ch)ARH460-16-2-IgG1 also significantly suppressed lung
metastasis from (5/5) 100 percent to (0/5) 0 percent at a dose of 2
mg/kg (FIG. 7). Besides the liver and lung metastasis, control
groups also showed intestine (4/5) and urinary (3/5)
metastasis.
[0200] In summary, (ch)ARH460-16-2-IgG1 was well-tolerated,
decreased the tumor burden and intrahepatic and lung metastases in
this established human orthotopic HCC tumor model.
[0201] The preponderance of evidence shows that murine and chimeric
H460-16-2 mediates anti-cancer effects through ligation of epitopes
present on CD44, which is expressed on liver cancer. It has been
shown that higher expression of CD44 is observed with metastatic
versus primary human liver cancer cell lines. It has also been
shown that chimeric H460-16-2 reduces the tumor burden and
probability of metastasis of human liver cancer in vivo. Therefore,
chimeric 11460-16-2 has therapeutic potential for the diagnosis and
treatment of liver cancer, broadly understood to include any
primary or metastatic tumor sites which arise from hepatocytes.
[0202] 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.
[0203] 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. 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.
* * * * *