U.S. patent application number 11/234645 was filed with the patent office on 2006-10-12 for monoclonal antibody therapy for pancreas cancer.
Invention is credited to Myron Arlen, Kwong Y. Tsang.
Application Number | 20060228363 11/234645 |
Document ID | / |
Family ID | 23056011 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060228363 |
Kind Code |
A1 |
Arlen; Myron ; et
al. |
October 12, 2006 |
Monoclonal antibody therapy for pancreas cancer
Abstract
The present invention relates to the use of binding equivalents
of monoclonal antibody 31.1, including chimerized and/or humanized
versions thereof, antibody fragments as well as competitively
binding and co-specific antibodies and antibody fragments, in the
treatment of pancreatic cancer.
Inventors: |
Arlen; Myron; (Great Neck,
NY) ; Tsang; Kwong Y.; (Bethesda, MD) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
44TH FLOOR
NEW YORK
NY
10112
US
|
Family ID: |
23056011 |
Appl. No.: |
11/234645 |
Filed: |
September 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10472008 |
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PCT/US02/09193 |
Mar 15, 2002 |
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11234645 |
Sep 22, 2005 |
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60276284 |
Mar 15, 2001 |
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Current U.S.
Class: |
424/155.1 ;
435/320.1; 435/338; 435/69.1; 530/388.8; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 39/39558 20130101; C07K 2317/56 20130101; A61K 49/16 20130101;
C07K 2319/00 20130101; C07K 2317/24 20130101; A61P 35/00 20180101;
G01N 33/57438 20130101; A61K 49/04 20130101; A61P 1/18 20180101;
C07K 2317/732 20130101; C07K 16/303 20130101; A61K 47/6859
20170801; G01N 2333/705 20130101; A61K 49/221 20130101; C07K
2317/14 20130101 |
Class at
Publication: |
424/155.1 ;
536/023.53; 435/069.1; 435/338; 530/388.8; 435/320.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 16/30 20060101 C07K016/30 |
Claims
1. An isolated nucleic acid molecule comprising the nucleotide
sequences of FIG. 2 (SEQ ID NOS:1 and 2) or FIG. 4 (SEQ ID NOS:4
and 5).
2. An isolated nucleic acid molecule (SEQ ID NOS:1 and 4)
comprising a nucleotide sequence that encodes the amino acid
sequence shown in FIG. 2 (SEQ ID NO:3) or FIG. 4 (SEQ ID NO:6).
3. An isolated nucleic acid molecule comprising a nucleotide
sequence that (i) binds to the nucleotide sequence of FIG. 2 (SEQ
ID NOS:1 and 2) or FIG. 4 (SEQ ID NOS:4 and 5) under the following
stringent hybridization conditions: 0.5 M NaHP0.sub.4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. and (ii) encodes for a
light or heavy chain variable region capable of binding with the
same immunospecificity as the chimeric 31.1 monoclonal
antibody.
4. An isolated polypeptide comprising the amino acid sequence of
FIG. 2 (SEQ ID NO:3).
5. An isolated polypeptide comprising the amino acid sequence of
FIG. 4 (SEQ ID NO:6).
6. An isolated polypeptide comprising the amino acid sequence
encoded by a nucleotide sequence that hybridizes to the nucleotide
sequence of claim 1 or 2 under stringent conditions and encodes a
functionally equivalent gene product.
7. A chimeric protein comprising the amino acid sequence of FIG. 2
(SEQ ID NO:3), or a fragment thereof, fused to an amino acid
sequence of a second protein, in which the second protein is not a
light chain variable region protein.
8. A chimeric protein comprising the amino acid sequence of FIG. 4
(SEQ ID NO:6), or a fragment thereof, fused to an amino acid
sequence of a second protein in which the second protein is not a
heavy chain variable region protein.
9. The chimeric protein of claim 7 or 8 wherein the second protein
comprises an immunoglobulin constant region.
10. The chimeric protein of claim 9 wherein the immunoglobulin
constant region is a human immunoglobulin constant region.
11. A recombinant vector comprising the nucleic acid of claim 1, 2
or 3.
12. A recombinant expression vector comprising the nucleic acid
molecule of claim 1, 2 or 3.
13. A recombinant cell containing the nucleic acid of claim 1, 2 or
3.
14. A recombinant cell comprising the vector of claim 11 or 12.
15. A method of producing a chimeric antibody with the same
immunoreactivity as the 31.1 monoclonal antibody, comprising
growing a recombinant cell comprising the nucleic acid of claim 1,
2 or 3 such that the encoded light and heavy chain variable regions
are expressed by the cell, and recovering the expressed
antibody.
16. An immunoassay method for detecting a pancreatic carcinoma
associated antigen capable of binding to monoclonal antibody 31.1,
or functional equivalent thereof, -in a sample, comprising: (a)
contacting said sample with monoclonal antibody 31.1 or equivalent
thereof, and (b) detecting said antigen by detecting the binding of
antibody.
17. An imaging method for detecting a pancreatic carcinoma antigen
in a subject, comprising: (a) contacting a labeled 31.1 antibody,
or functional equivalent thereof; and (b) detecting the labeled
antibody wherein detection of said labeled antibody indicates the
presence of a pancreatic carcinoma antigen.
18. A method for diagnosing pancreatic cancer in a subject
comprising: (a) removing a specimen from a patient suspected of
having a pancreatic carcinoma; (b) contacting the specimen with a
31.1 monoclonal antibody or functional equivalent thereof; (c)
staining the specimen with an immunohistochemical stain; and (d)
detecting the presence of the antigen-antibody complex wherein the
presence of an antigen-antibody complex indicates the presence of
pancreatic cancer.
19. A method of killing cells carrying a pancreatic carcinoma
associated antigen, comprising delivering to said cells an
effective amount of 31.1 monoclonal antibody, or functional
equivalent thereof.
Description
1. INTRODUCTION
[0001] The present invention relates to the use of monoclonal
antibody 31.1 and its equivalents and co-specific antibodies in the
treatment of pancreas cancer. It is based, at least in part, on the
discovery that monoclonal antibody 31.1 is reactive with malignant,
but not non-malignant, pancreatic cells. The present invention
further provides polynucleotide and amino acid sequences comprising
the light chain variable region and heavy chain variable region of
Mu-31.1 as set forth in FIG. 2 and FIG. 4, respectively. Such
polynucleotide sequences may be used to recombinantly express 31.1
equivalent antibodies for use in the methods of the invention.
2. BACKGROUND OF THE INVENTION
2.1. Pancreas Cancer
[0002] Pancreas cancer is the fifth leading cause of cancer death
in the United States, with approximately 28,000 Americans expected
to die from the disease this year Pancreas Cancer Web, The Johns
Hopkins Medical Institutions,
http://162.129.103.69:80/PANCREAS_INTRO). At present, the only
potentially curative treatment is surgical removal of the cancer,
in the context of an extensive and complex procedure which removes
the head, neck and uncinate process of the pancreas as well as the
majority of the duodenum (the "Whipple operation"). Without
treatment, the overall 5 year survival rate is only 3 percent
(Id.).
[0003] Chemotherapy (often using gemcitabine (Gemzar.RTM.)) and
radiation therapy are the main treatments offered to patients with
unresectable tumors (Id.). An experimental immunotherapy is
currently being studied in which a patient's own cells are
genetically modified to express the immune stimulatory protein,
granulocyte-macrophage colony stimulating factor, irradiated to
prevent tumor growth, and then reintroduced into the patient, where
they will hopefully stimulate an immune response (1997, Cancer Res.
57:1537-1546; Pancreas Cancer Web, The Johns Hopkins Medical
Institutions, http://162.129.103.69:80/PANCREAS_MEDICAL_TX)).
2.2. Monoclonal Antibody 31.1
[0004] Antibody 31.1 represents a protein-directed monoclonal
antibody derived by immunizing BALB(c) mice with a preparation of
membrane obtained from pooled (human) allogeneic colon carcinoma
specimens. The cells used to prepare the antigen were fragmented
using a nitrogen (Parr) bomb and then subjected to
ultracentrifugation. Membrane material was initially tested by
electron microscopy to guarantee consistency from batch to batch,
ruling out cytoplasmic and nuclear components. It was then
sonicated and fractionated with sephadex G200. Discontinuous
polyacrylamide gel electrophoresis was used for the initial partial
purification (approximately 80%) and 30 .mu.gm tested for delayed
cutaneous hypersensitivity (DHR), (3). BALB mice were immunized by
intraperitoneal injection of 50 micrograms of colon carcinoma
associated antigen. A second injection was given 10 days later and
the mice then sacrificed to obtain spleen cells for fusion. Fusion
was performed by incubating 5.times.10.sup.7 mouse spleen cells
with 10.sup.7 sp2/0-AG 14 myeloma cells in 40% PEG. The antigen
defined by the monoclonal antibody 31.1 has been shown to have M.W.
of 72,000. Studies using immunoperoxidase have suggested that the
antigen recognized by 31.1 is seen with greater frequency in the
higher grade colon tumors. Specificity for the antibody is high, so
that in a study of shed colonocytes at the Mayo Clinic, sensitivity
and specificity were superior when compared with anti-CEA,
anti-MUC1 and B72.3.
[0005] Several candidate antibodies were isolated and tested from
the 1st generation TAA. All proved to be protein derived and
relatively specific for colon carcinoma. Antibody 31.1 corresponded
to one of the two antigens that have been shown to migrate closely
on gel-electrophoresis and related to the immunogenic glycoprotein
inducing the DHR. The murine version is of IgG2a isotype which
converts to an IgG1 isotype on chimerization. 31.1 was found to
have strong localization indices. As such, this antibody was the
first to be chimerized.
[0006] For chimerization of monoclonal antibody 31.1, the protein
coding exon of 31.1 heavy chain variable region gene was spliced to
the protein coding exons of human gamma 1 chain constant region.
PCR was employed. The 31.1 VH cDNA was amplified by the PCR using
the degenerate backward primers synthesized based on the consensus
first framework (FR1) region DNA sequences and a forward primer
synthesized according to the consensus J-C junction region DNA
sequences. The amplified 31.1 V.sub.H DNA was cloned into the
pBluescript vector and sequenced. Chimeric 31.1 was produced by
transfecting SP2/0 AG14 cells with the vector.
[0007] Monoclonal antibody 31.1 and a chimeric (humanized) version
of that antibody are described in U.S. Pat. No. 5,688,657 issued
Nov. 18, 1997, now the subject of a reissue application, the
contents of which are hereby incorporated by reference in its
entirety herein. Furthermore, monoclonal antibody 31.1 has been
deposited with the American Type Culture Collection ("ATCC"),
having an address at 10801 University Blvd., Manassas, Va.,
20110-2209 and assigned accession number ATCC PTA-2497 and the
chimerized version has been deposited with the ATCC and assigned
accession number 12316.
3. SUMMARY OF THE INVENTION
[0008] The present invention relates to the use of binding
equivalents of monoclonal antibody 31.1, including chimerized
and/or humanized versions thereof, antibody fragments and
competitively binding antibodies and antibody fragments, as well as
co-specific antibodies, derivatives and fragments in the treatment
of pancreas cancer.
[0009] It is based, at least in part, on in vitro studies using
both murine and chimeric versions of 31.1 which compared the ADCC
activites of the 31.1 antibodies with D6-12 and 17.1a (Panorex).
While the murine version of 31.1 can induce a 35% ADCC response,
the chimeric version has been shown to result in 80% of tumor cells
being destroyed every three hours, using a chromium release assay.
This compares with a 30% rate of destruction associated with D6-12
and a 15% rate for Panorex. Using xenograft models with human colon
cancer cell lines LC-174T and Colo205, chimeric 31.1 was found to
cause regression of established tumor lines (well defined
molecules) after inoculating two million tumor cells into the hind
legs of nude mice and administering intra-peritoneal antibody at 10
days along with human effector cells. At 30 days the volume of
tumor in the treated animals when compared to controls was reduced
by more than 95%. Similar results may be expected when the antibody
is directed toward pancreatic cancer cells, as the 31.1 antibody
has been shown to bind to antigen present in pancreatic cancer
cells, but not non-malignant pancreatic tissues.
[0010] The present invention provides polynucleotide and amino acid
sequences comprising the light chain variable region and heavy
chain variable region of Mu-31.1 which may be used to express
chimerized 31.1 antibodies. The nucleotide sequences of the
invention include: (a) the nucleotides sequences shown in FIG. 2 or
FIG. 4; (b) a nucleotide sequence that encodes the amino acid
sequence shown in FIG. 2 or FIG. 4; and (c) any nucleotide sequence
that (i) binds to the nucleotide sequence of FIG. 2 or FIG. 4 under
stringent hybridization conditions, e.g., hybridization to filter
boun DNA in 0.5 M NaBPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1
mM EDTA at 65.degree., and washing in 0.1.times.SSC/0.1% SDS at
68.degree. and (ii) encodes for a light and heavy chain variable
region capable of binding with the same immunospecificity as the
chimeric 31.1 monoclonal antibody.
[0011] The invention further provides for a new expression
construct of chimerized 31.1 antibody, termed pRc/CMV 31.1 which
has been depostied with the ATCC and assigned accession no. ATCC [
]. This plasmid carries a dihydrofolate reductase ("dhfr")
expression unit driven by an enhancer-deficient SV40 early promoter
that allows expresion at greater than 200 mg/liter in dihyfrofolate
reductase deficient Chinese hamster ovary cells.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A-F. Series of plasmids used to construct pRc/CMV 31.1
vector by inserting the Chi31.1-1 light chain and heavy chain genes
into plasmid pDCM-dhfr (FIG. 1F).
[0013] FIG. 2. Nucleic acid sequence (double stranded) and possible
amino acid sequences (depending on reading frame) of the 31.1 light
chain variable region, showing restriction enzyme cleavage
sites.
[0014] FIG. 3. List of non-cutting enzymes of the light chain
variable region nucleic acid sequence shown in FIG. 2.
[0015] FIG. 4. Nucleic acid sequence (double stranded) and possible
amino acid sequences (depending on reading frame) of the 31.1 heavy
chain variable region, showing restriction enzyme cleavage
sites.
[0016] FIG. 5. List of non-cutting enzymes of the heavy chain
variable region nucleic acid sequence shown in FIG. 4.
[0017] FIG. 6A-B. An antibody-dependent cellular cytotoxicity
(ADCC) assay was conducted to test the effector funtion of the CHO
chi 31.1 antibody against target cells SW1643 and PANC-1. As cell
lysis occurs in the presence of 31.1 antibody (FIG. 6A) but not in
the presence of control antibody (FIG. 6B).
5. DETAILED DESCRIPTION OF THE INVENTION
[0018] For purposes of clarity of presentation and not by way of
limitation, the detailed description is divided into the following
two subsections:
[0019] i) monoclonal antibody 31.1 and its equivalents; and
[0020] ii) treatment protocol.
5.1. Monoclonal Antibody 31.1 and its Equivalents
[0021] Monoclonal antibody 31.1 is a murine monoclonal antibody
(hereinafter referred to as Mu-3L), originally generated by
immunization with purified material from colon carcinoma cell
membranes. Hybridoma cells secreting this antibody have been
deposited with the American Type Culture Collection ("ATCC") and
assigned accession no. ATCC PTA 2497.
[0022] The present invention provides nucleic acid molecules and
polypeptides comprising the light chain variable region and heavy
chain variable region of Mu-31.1. The nucleotide sequences of the
invention include: (a) the DNA sequences shown in FIG. 2 or FIG. 4;
(b) a nucleotide sequence that encodes the amino acid sequence
shown in FIG. 2 or FIG. 4; (c) any nucleotide sequence that (i)
hybridizes to the nucleotide sequence set forth in (a) or (b) under
stringent conditions, e.g., hybridization to filter-bound DNA in
0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. (Ausubel F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol., Green Publishing Associates, Inc., and
John Wiley & sons, Inc., New York, at p. 2.10.3) and (ii)
encodes a functionally equivalent gene product. Functional
equivalent gene products include those polypeptides which compete
with 31.1 for binding to its target antigen. The invention also
encompasses nucleotide sequences that encode peptide fragments of
the heavy and light chain variable regions, and fusion proteins
thereof.
[0023] The nucleotides of the invention may be isolated using a
variety of different methods known to those skilled in the art. For
example, a cDNA library constructed using RNA from cells or tissue
known to express the 31.1 monoclonal antibody or its equivalent,
can be screened using a labeled nucleic acid probe derived from the
sequences depicted in FIG. 2 or FIG. 4. Further, nucleic acid
sequences encoding the heavy and light chain variable regions may
be derived by performing PCR using two oligonucleotide primers
designed on the basis of the nucleotide sequences disclosed herein.
The template for the reaction may be cDNA obtained by reverse
transcription of mRNA prepared from cell lines or tissue known to
express the 31.1. monoclonal antibody.
[0024] The invention also encompasses (a) DNA vectors that contain
any of the foregoing heavy and light chain variable region
sequences and/or their complements (i.e., antisense); (b) DNA
expression vectors that contain any of the foregoing heavy and
light chain variable region sequences operatively associated with a
regulatory element that directs the expression of the heavy and
light chain variable region coding sequences; and (c) genetically
engineered host cells that contain any of the foregoing heavy and
light chain variable region sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences in the host cell. As used herein, regulatory elements
include but are not limited to inducible and non-inducible
promoters, enhancers, operators and other elements known to those
skilled in the art that drive and regulate expression.
[0025] FIG. 2 shows the deduced amino acid sequence of the 31.1
light chain variable region and FIG. 4 shows the deduced amino acid
sequence of the 31.1 heavy chain variable region. Thus, the amino
acid sequences of the invention include the amino acid sequence
shown in FIG. 2 and FIG. 4.
[0026] The invention also encompasses proteins that are
functionally equivalent to proteins encoded by the nucleotide
sequences described above, as judged by any of a number of
criteria, including but not limited to the ability to bind to the
epitope recognized by the 31.1 monoclonal antibody.
[0027] Peptides corresponding to one or more domains of the heavy
and light chain variable regions, as well as fusion proteins in
which the full length or a portion of the heavy and light chain
variable region is fused to an unrelated protein are also within
the scope of the invention and can be designed on the basis of the
nucleotide and amino acid sequences disclosed herein (see, FIG. 2
and FIG. 4).
[0028] While the heavy and light chain variable regions can be
chemically synthesized (e.g., see Creighton, 1983, Proteins:
Structures and Molecular Principles, W.H. Freeman & Co., N.Y.),
the regions may be advantageously produced by recombinant DNA
technology using techniques well known in the art for expressing a
nucleic acid containing heavy and light chain variable region gene
sequences and/or coding sequences. Such methods can be used to
construct expression vectors containing the nucleotide sequences
described above and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. (See, for example, the techniques described
in Sambrook et al., 1989, supra, and Ausubel et al., 1989,
supra).
[0029] A variety of host-expression vector systems may be utilized
to express the nucleotide sequences of the invention. Where the
heavy and light chain variable regions are expressed as a soluble
derivative and are not secreted, the peptide or polypeptide can be
recovered from the host cell. Alternatively, where the heavy and
light chain variable regions are secreted the peptide or
polypeptides may be recovered from the culture media.
[0030] The expression systems that may be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid or
cosmid DNA expression vectors containing nucleotide sequences
encoding the 31.1 heavy and light chain variable regions; yeast
transformed with recombinant yeast expression vectors containing
nucleotide sequences encoding for the 31.1 heavy and light chain
variable regions or mammalian cell systems harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells or from mammalian viruses.
[0031] Appropriate expression systems can be chosen to ensure that
the correct modification, processing and sub-cellular localization
of the heavy and light chain variable region protein occurs. To
this end, host cells which possess the ability to properly modify
and process antibodies for secretion are preferred. For long-term,
high yield production of recombinant proteins, such as that desired
for development of cell lines for production of chimeric
antibodies, stable expression is preferred. Rather than using
expression vectors which contain origins of replication, host cells
can be transformed with DNA controlled by appropriate expression
control elements and a selectable marker gene, i.e., tk; hgprt,
dhfr, neo, and hygro gene, to name a few. Following the
introduction of the foreign DNA, engineered cells may be allowed to
grow for 1-2 days in enriched media, and then switched to a
selective media.
[0032] A chimeric version of murine 31.1, referred to hereinafter
as Chi-31.1-1, comprising variable region from Mu-31.1 together
with human constant region immunoglobulin sequences, is produced by
hybridoma cells deposited with the ATCC and assigned accession no.
ATCC CRL-12316.
[0033] In a specific embodiment of the invention, a second chimeric
version of murine 31.1, hereinafter referred to as Chi-31.1-2,
having variable regions from Mu-31.1 and human constant
immunoglobulin regions and derived from the Chi31.1-1 heavy and
light chain genes, may be produced by expression of vector pRc/CMV
31.1, described herein, and as shown in FIG. 1. This vector has the
advantage of producing high yields of chimeric antibody. A
description of the preparation of this vector is provided in the
example section, below.
[0034] In specific non-limiting embodiments of the invention,
further chimeric versions may be produced comprising the variable
regions of Mu-31.1. For example, the heavy chain variable region
and light chain variable region may be generated using PCR primers
designed based on the variable region sequences set forth in FIG. 2
(light chain variable region) and FIG. 4 (heavy chain variable
region) or variants thereof to alter the termini to facilitate
splicing in a vector of choice and using, as a source of template
DNA, DNA collected from a hybridoma that produces a 31.1-Ab
equivalent, such as one of the hybridomas set forth above which
have been deposited with the ATCC. The variable region encoding
sequences may then be combined with human constant-region encoding
sequences to produce "humanized" antibody.
[0035] Alternatively, nucleic acid encoding Chi31.1-1 heavy and
light chains (including human constant regions) may be inserted
into various expression vectors to facilitate expression. Specific
non-limiting examples of such PCR primers are:
[0036] a) for insertion of Chi31.1-1 light chain encoding sequences
at a BamH1/XbaI insertion site: TABLE-US-00001 i)
Chi31.1-LcBamHI(S): 5'-ATA GGA TCC ATG AAG TCA CAG ACC CAG GTC TTC
G-3' ii) Chi31.1-LcXBaI(A): 5'-TTT CTA GAC TAA CAC TCT CCC CTG TTG
AAG C-3'
[0037] b) for insertion of Chi31.1-1 heavy chain encoding sequences
at a EcoRI/NotI insertion site: TABLE-US-00002 i)
Chi31.1-HcEcoRI(S): 5'-ATA GAA TTC ATG GCT TGG GTG TGG ACC TTG
CT-3' ii) Chi31.1-HcNotI(A): 5'-TTG CGG CCG CTC ATT TAC CCG
GAG-3'.
Such primers may be used in polymerase chain reactions using, as
template, DNA prepared from hybridoma cells deposited with the ATCC
and assigned accession no. ATCC CRL-12316.
[0038] "Equivalents" of Mu-31.1 are defined herein as
immunoglobulin molecules or fragments or derivatives thereof which
compete with Mu-31.1 for binding to its target antigen, as
evaluated using standard techniques. Such equivalents may include
complete antibody molecules (i.e., having two heavy chains and two
light chains), single chain antibody molecules (see, for example,
Lee et al., 1999, Molec. Immunol. 36:61-71, incorporated by
reference herein), fragments such as F(ab) and F(ab).sub.2
fragments of Mu-31.1, Chi-31.1-1, Chi-31.1-2, or equivalent
complete antibody molecules, and derivative molecules including,
but not limited to, one or more of the foregoing immunoglobulin
molecules or fragments conjugated to a bioactive agent, or modified
to more closely resemble a human immunoglobulin molecule (see, for
example; Ryu et al., 1996, Human Antibod. Hybridomas 7:113-122).
Such equivalents, which include Mu-31.1, Chi-31.1-1, Chi-31.1-2,
are collectively referred to as "31.1-Ab equivalents".
[0039] The use of co-specific antibodies and their equivalents
(with equivalents having the same scope as that applied to the 31.1
antibody) is also envisioned according to the invention. A
co-specific antibody to Mu-31.1 (referred to as "31.1 co-specific
antibodies") may or alternatively may not compete with binding of
Mu-31.1, but recognizes (i.e., binds to) the same target antigen,
referred to herein as "31.1-Ag"). The co-specific antibodies to
31.1 and their equivalents are referred to herein as "31.1
co-specific antibody equivalents".
[0040] Any 31.1 antibody equivalent or 31.1 co-specific antibody
equivalent to be used in humans preferably has a structure which
itself does not provoke a deleterious immune reaction in humans.
For example, said 31.1 antibody equivalent or 31.1. co-specific
antibody equivalent may inherently lack such immunogenic structures
or may be the product of a "humanization" process by standard
techniques to minimize or eliminate structures which would be
recognized as non-self by a subject (e.g. chimerization and/or site
by site engineering). 31.1-Ag appears to be localized to the
membrane of colon and pancreas cancers. Its presence has not been
detected on normal human tissue obtained fresh and immediately
frozen (TABLE A). TABLE-US-00003 TABLE A Cross-reactivity to normal
fresh frozen human tissues. Tissue (number) Staining paraffin
Staining frozen samples Colon (3) Negative (3) Negative (2) Trace
Positive (1) Small bowel (3) Negative (3) Negative (3) Esophagus
(3) Negative (3) Negative (3) Oral mucosa (2) Negative (2) Negative
(2) Jejunum (1) Negative (1) Negative (1) Stomach (1) Negative (1)
Negative (1) Liver (3) Negative (3) Negative (3) Pancreas (3)
Negative (3) Negative (3) Thymus (3) Negative (3) Negative (3)
Heart (2) Negative (2) Negative (2) Prostate (2) Negative (2)
Negative (2) Breast (3) Negative (3) Negative (3) Testis (1)
Negative (1) Negative (1) Ovary (2) Negative (2) Negative (2)
Salivary gland (3) Negative (3) Negative (3) Spleen (2) Negative
(2) Negative (2) Brain (3) Negative (3) Negative (3) Lymph node (2)
Negative (2) Negative (2) Adrenal (1) Negative (1) Negative (1)
Vagina (1) Negative (1) Negative (1) WBC (1) Negative (1) Negative
(1)
[0041] 31.1-Ag is, however, found on the surface of colon and
pancreas cancers obtained fresh at the moment of surgery and frozen
(TABLE B). TABLE-US-00004 TABLE B Localization of 31.1 antigen on
colon and pancreas cancers Staining frozen Cancer (number) Staining
paraffin samples Adenocarcinoma of colon (3) Positive (3) Positive
(3) Adenocarcinoma of pancreas (3) Positive (3) Positive (3)
[0042] It should be noted that this result differs from that
presented in Table 2 of U.S. Pat. No. 5,688,657 (at column 24,
lines 1-26), which indicates that antibody Mu-31.1 did not bind to
either of two pancreas tumor samples tested. Table 1 of U.S. Pat.
No. 5,688,657 (at column 23 lines 1-38) shows that Mu-31.1 reacted
with two out of three pancreatic cancer-derived cell lines. Based
on the information contained in U.S. Pat. No. 5,688,657, one may
have concluded that 31.1 Ag only appeared after passage of the
cells in culture, and was not present on fresh pancreatic cancer
tissue. It is therefore unexpected, based on the disclosure of U.S.
Pat. No. 5,688,657, that 31.1-Ag would be present on 3/3 pancreatic
tumor samples, as set forth in TABLE B herein.
[0043] Mu-31.1 is secreted from a hybridoma cell line developed by
fusion with the murine SP2 cell line cell-line. Mu-31.1,
Chi-31.1-1, and Chi-31.1-2, 31.1-Ab equivalents, and 31.1
co-specific antibodies may be manufactured, for example and not by
way of limitation, for clinical use by standard in vitro cell
culture and down-stream purification processes. For example,
hybridoma cells may be grown in Geneticin (0.2 mg/ml) since the
presence of the antibiotic has been observed to allow the hybridoma
cells to grow better.
[0044] Preferably, compositions comprising the forgoing 31.1-Ab
equivalents and 31.1 co-specific antibodies may be made without the
addition of human additives. For example, the preparations may be
filtered through a bacterial Millipore 0.2 micron filter to
eliminate contaminants and verified as sterile for bacteria and
fungi by streaking blood agar plates and culture media with
positive controls for 14 days. The preparation may be determined to
be free of Mycoplasma by, for example, PCR Mycoplasma assays and by
Mycoplasma Agar plates (Life Technology cat #18042-010) and Myco
Test Kit (Life Technology Cat # 15672-017) using 3T6 control
cells.
[0045] Media containing one or more of the foregoing 31.1-Ab
equivalents or 31.1 co-specific antibodies may be filtered through
a Pall endotoxin filter and the glassware heat sterilized to
eliminate endotoxin. Desirably, but not by way of limitation, an
appropriate endotoxin level may be 0.125 units/ml or less, as
measured by the BioWhittaker Pyrogent 03,250 test kit.
[0046] In preferred, non-limiting embodiments of the invention, one
of the foregoing preparations may be treated so as to inactivate
virus. For example, retrovirus may be inactivated by acetic acid
treatment at pH3 for one hour during column chromatography and
filtration through a Pall Ultipor Grade DV50 Virus Removal Filter
of 10-40 nm.
[0047] In a specific, non-limiting embodiment of the invention, 50
mg of Chi-31.1-1 is contained in a vial at a concentration of 2
mg/ml in phosphate buffered saline ("PBS").
5.2. Treatment Protocols
[0048] The present invention provides for the use of 31.1-Ab
equivalents and/or 31.1 co-specific antibody equivalents, used
singly or in combination, in the treatment of pancreas cancer in a
subject in need of such treatment. The method involves
administering, to the subject, a therapeutically effective dose of
one or more 31.1-Ab equivalent and/or 31.1 co-specific antibody
equivalent. A therapeutically effective dose is defined, herein, as
a dose which achieves one or more of the following in the subject:
produces detectable pancreatic carcinoma cell lysis in the subject;
causes a decrease in the growth, or invasiveness, or size of a
pancreas tumor; causes an improvement in clinical symptoms; and/or
causes an increase in survival time. Preferably, but not by way of
limitation, a single dose of 31.1-Ab equivalent and/or 31.1
co-specific antibody equivalent may range from about 25 mg to about
1000 mg, and preferably from about 100 mg to 250 mg. The magnitude
of the dose may be adjusted on a patient-by-patient basis to avoid
undesirable side effects and/or toxicity. It is preferred that the
31.1-Ab equivalent and/or 31.1 co-specific antibody equivalent is
administered as a series (plurality) of single doses, administered
at intervals of between about 1 and 4 weeks, preferably every two
weeks, until side effects rise to an undesirable level or disease
progresses to an undesirable level. The 31.1-Ab equivalent and/or
31.1 co-specific antibody equivalent may be administered via any
standard route; preferably, to test whether a patient tolerates the
formulation (i.e., the patient does not manifest an undesirable
allergic and/or other toxic reaction), it may first be administered
subcutaneously, and once adequate tolerance is shown, it may be
administered intravenously.
[0049] In one specific, non-limiting example, a protocol according
to the invention may be as follows.
[0050] Using aseptic procedures, a "humanized" 31.1-Ab equivalent
and/or 31.1 co-specific antibody equivalent, produced using
standard biotechnology techniques, may be filtered through a 0.22
micron low protein filter into a glass infusion bottle or
non-DEHP-containing infusion bag containing 0.9% sodium chloride to
a final concentration of 0.4 mg/ml. The infusate may be mixed
gently. If the infusion is observed to be cloudy, it should not be
administered.
[0051] To determine whether a patient tolerates treatment with the
"humanized" 31.1-Ab equivalent or 31.1 co-specific antibody
equivalent, the patient may be pre-medicated with diphenhydramine
25 mg i.v. and paracetamol 650 mg p.o., and then 30 micrograms of
31.1-Ab equivalent or 31.1 co-specific antibody equivalent may be
injected subcutaneously. If no allergic toxicity or a grade 1
allergic toxicity occurs, intravenous treatment will proceed. If a
grade 1 allergic toxicity occurs, resolution of the toxicity will
be necessary prior to proceeding with the intravenous
injection.
[0052] If the patient tolerates the subcutaneous test dose
described in the preceding paragraph, the patient may be treated
with a first infusion of 25 mg of the 31.1-Ab equivalent or 31.1
co-specific antibody equivalent over 2 hours. Pre-medication in the
form of diphenhydramine 25 mg i.v. and paracetamol 650 mg p.o. may
be given. The patient may then be observed for any potential side
effects for 6 hours after the injection. The patient may be
monitored with vital signs prior to the injection, and every 15
minutes during the first hour of treatment, every 30 minutes for
two hours thereafter, and every hour thereafter until 6 hours after
completion of the infusion.
[0053] If the first infusion has been found to be tolerated, after
2 weeks, the patient may then receive an infusion of 50 mg of the
31.1-Ab equivalent or 31.1 co-specific antibody equivalent, in a
volume of 100 cc PBS or other suitable diluent, over 4 hours using
the same clinical protocol as set forth in the preceding paragraph.
If this second infusion has also been found to be tolerated, the
patient may then receive infusions of 100 mg of the 31.1-Ab
equivalent or 31.1 co-specific antibody equivalent in 100 cc
diluent over 4 hours every two weeks, using the above-described
protocol. The patient may then continue such treatment until
intolerance develops or progression of disease occurs, and
preferably for a maximum of 4 months. If any grade 3 or higher
toxicity occurs due to the treatment, the patient may discontinue
treatment permanently. If it is deemed that the toxicity is not
treatment related, the patient may be able to resume treatment upon
recovery of the toxicity. If any grade 2 toxicity occurs during or
after treatment, the infusion may desirably be stopped. If recovery
to grade 0 occurs, the infusion may then be restarted. If recovery
has not occurred by the time of the next planned treatment,
treatment may be delayed until recovery to grade 0 has occurred. If
recovery to grade 0 does not occurred within 4 weeks, treatment may
be discontinued permanently. If any allergic reaction of grade 2 or
higher occurs, the treatment may be stopped and preferably no
further infusion may be given.
[0054] In specific non-limiting embodiments of the invention, the
following may serve as criteria for patients suitable for
treatment:
[0055] a) the patient may suffer from a histologically confirmed
recurrent or metastatic adenocarcinoma of the pancreas, where the
tumor reacts with the 31.1-Ab equivalent or 31.1 co-specific
antibody intended to be used;
[0056] b) treatment of the patient by a standard regimen for
metastatic pancreas cancer may have failed;
[0057] c) disease in the patient may be measurable by one or more
of the following: [0058] i) physical examination; [0059] ii)
computerized tomography or other radiological study, [0060] iii)
CEA levels; and/or [0061] iv) Ca 19-9 levels;
[0062] d) the patient may be 18 years of age or older;
[0063] e) the patient may exhibit a WHO performance status of 0, 1,
or 2;
[0064] f) the prognosis of the patient may indicate a life
expectancy of at least 12 weeks;
[0065] g) hematological testing of the patient may indicate the
following values: [0066] i) WBC>3,000; [0067] ii) HGB>10; and
[0068] iii) platelets>100,000;
[0069] h) clinical chemistry values may be as follows:
[0070] Creatinine, bilirubin, aspartate transaminase, alanine
transaminase, alkaline phosphatase, and bilirubin are all less than
or equal to 2 times upper limit of normal; and/or
[0071] i) the patient has adequate peripheral venous access for
repeated blood sampling.
[0072] In specific non-limiting embodiments of the invention, the
following may serve as criteria for excluding patients who may be
unsuitable for treatment:
[0073] a) less than 4 weeks may have elapsed since prior
chemotherapy (or 6 weeks for nitrosoureas or mitomycin-C), since
treatment with biological response modifiers or since radiation
therapy;
[0074] b) the patient is currently receiving steroid therapy
[0075] c) the patient is pregnant (men and women on the study, if
fertile, are counseled to practice effective contraception);
[0076] d) the patient is a lactating female;
[0077] e) the patient suffers from a debilitating non-malignant
co-morbid condition, such as active infection or an acute
intercurrent complication of malignancy;
[0078] f) there is central nervous system involvement;
[0079] g) the patient has previously received a bone marrow or
other organ transplant;
[0080] h) the patient has a history of another malignancy, except
for adequately treated non-melanoma cancer of the skin or in situ
cancer of the cervix;
[0081] i) the patient has previously been exposed to murine
monoclonal or polygonal antibodies; and/or
[0082] j) the patient is known to be HIV positive.
[0083] During the course of the study, non-limiting examples of
adverse reactions include shortness of breath, hypotension,
cyanosis, rash, bronchospasm, chills, rigors, back pain, fever,
cyanosis, nausea, vomiting, palpitations or any other adverse
reaction.
[0084] In non-limiting embodiments of the invention, the following
laboratory tests may desirably be performed to evaluate patients
being treated by the protocol. With regard to hematology tests, a
complete blood count, differential, and platelet count may be
obtained prior to each infusion and weekly during treatment until
four weeks after the last injection. With regard to clinical
chemistry tests, a complete chemistry panel measuring glucose,
sodium, potassium, bicarbonate, chloride, blood urea nitrogen,
creatinine, uric acid, calcium, inorganic phosphate, total protein,
albumin, lactate dehydrogenase, aspartate transaminase, alanine
transaminase and alkaline phosphatase may be obtained weekly during
treatment and until four weeks after the last injection. With
regard to special laboratory tests, serum samples obtained from 10
cc of blood may be collected before and within two minutes of each
injection, at times 15 min, 30 min, 60 min, 2, 4, 24 and 72 hours
after completion of the first injection and every two weeks
thereafter prior to each injection and until four weeks after the
last treatment and processed for the detection of administered
31-1-Ab equivalent and/or 31.1 co-specific antibody equivalent.
These serum samples may then be used to determine ADCC, antibody
concentration, and the presence of human antibodies directed toward
the administered antibody equivalent. Urinalysis may be performed
at enrollment and before each of the injection as well as four
weeks after the last injection, with microscopic examination
performed on any abnormal specimens.
[0085] In various embodiments of the invention, the following
safety assessments may desirably be made. For each of infusion,
vital signs including the temperature, pulse and blood pressure of
the patient may be obtained prior to and after each infusion The
pulse and blood pressure may be recorded every fifteen minutes
during the first hour of infusion and then every half hour for two
hours, followed by hourly until 6 hours after the completion of the
infusion. Patients may be observed and vital signs monitored until
six hours after the completion of the infusion or until return to
baseline of the vital signs.
[0086] An initial evaluation and subsequent evaluations of the
patient's response to treatment may be performed as follows. Tumor
measurement may be performed by physical examination and or
standard or special radiological studies such as chest X-ray,
computerized tomography, magnetic resonance imaging, or ultrasound.
If more than one measurable lesion exists, representative lesions
should be measured. The longest perpendicular measurements of the
representative lesions may be recorded prior to treatment and every
eight weeks. Levels of Ca 19.9 may be monitored regularly, for
example monthly.
[0087] Preferably written informed consent is obtained for each
patient to be treated. Each patient should be given a verbal
description of the treatment, its potential risks and benefits as
well as alternative treatments available, prior to signing the
written consent.
[0088] During the course of treatment, blood products, antibiotics,
anti-emetics, analgesics or other medications for stable coexisting
medical conditions may be administered as appropriate.
[0089] The treatment may be discontinued in a patient if there is
evidence of progressive disease, if a serious or unexpected adverse
reaction occurs, or for other medically appropriate reasons.
[0090] In addition to the therapeutic uses described herein, the
31.1 antibodies and functional equivalents thereof may be used to
diagnose pancreatic carcinoma in a subject. The diagnostic methods
of the invention are based on the discovery that the 31.1 antibody
selectively binds to an antigen expressed in pancreatic carcinoma
cells but not normal cells.
[0091] In accordance with the invention, measurement of levels of
monoclonal antibody 31.1 reactivity in samples derived from a
subject can be used for the diagnosis of diseases such as
pancreatic carcinoma. The detection of monoclonal 31.1 antibody
reactivity in a sample from a subject can be accomplished by any of
a number of methods. Preferred diagnostic methods can involve, for
example, immunoassays wherein 31.1 reactive antigen is detected by
their interaction with an 31.1 monoclonal antibody. Immunoassays
useful in the practice of the invention include but are not limited
to assay systems using techniques such as Western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, to name but a few.
[0092] A biological sample, such as pancreatic tissue or other
biological tissue, is obtained from a subject suspected of having a
particular cancer or risk for cancer. Aliquots of whole tissues, or
cells, are solubilized using any one of a variety of solubilization
cocktails known to those skilled in the art. For example, tissue
can be solubilized by addition of lysis buffer comprising (per
liter) 8 M urea, 20 ml of Nonidet P-40 surfactant, 20 ml of
ampholytes (pH 3.5-10), 20 ml of 2-mecaptoethanol, and 0.2 mM of
phenylmethylsulfonyl fluoride (PMSF) in distilled deionized
water.
[0093] Immunoassays for detecting expression of the 31.1 reactive
antigen typically comprise contacting the biological sample, such
as a tissue sample derived from a subject, with the 31.1 monoclonal
antibody under conditions such that an immunospecific
antigen-antibody binding reaction can occur, and detecting or
measuring the amount of any immunospecific binding by the antibody.
In a specific aspect, such binding of antibody, for example, can be
used to detect the presence and increased production of 31.1
reactive antigen wherein the detection of the antigen is an
indication of a diseased condition.
6. EXAMPLE
Preparation of pRc/CMV Vector
[0094] The pRc/CMV vector was prepared using a series of plasmids,
as depicted in FIG. 1A-F. The heavy and light chains of Chi 31.1-1
were cloned into the pCR vector (FIG. 1A) by TOPO (Topoisomerase 1)
cloning. Sequences used for inserting the light and heavy chain
sequences into the pCR vector by PCR are as follows:
[0095] a) for insertion of the Chi31.1-1 light chain encoding
region at a BamH1/XbaI insertion site: TABLE-US-00005 i)
Chi31.1-LcBamH1(S): 5'-ATA GGA TCC ATG AAG TCA CAG ACC CAG GTC TTC
G-3' ii) Chi31.1-LcXBaI(A): 5'-TTT CTA GAC TAA CAC TCT CCC CTG TTG
AAG C-3'
[0096] b) for insertion of the Chi31.1-1 heavy chain encoding
region at a EcoRI/NotI insertion site: TABLE-US-00006 i)
Chi31.1-HcEcoRI(S): 5'-ATA GAA TTC ATG GCT TGG GTG TGG ACC TTG
CT-3' ii) Chi31.1 -HcNotI(A): 5'-TTG CGG CCG CTC ATT TAC CCG
GAG-3'
[0097] These were then cloned from the pCR vector into the
pDCM-dhfr vector, such that the light chain encoding region was
inserted at the BamH1/XbaI site (under the control of the
cytomegalovirus ("CMV") promoter, and the heavy chain encoding
region was inserted into the EcoRI/NotI site, under the control of
a second CMV promoter element (FIG. 1F).
[0098] The pDCM-dhfr vector was prepared using the series of steps
set forth in FIGS. 1B-E. A series of vector constructions using
some related components are described in Ryu et al., 1996, Hum.
Antibod. Hybridomas 7:113-122 (based on the pRc/CMV vector
(Invitrogen); see, for example, page 115 and FIG. 4 of Ryu et al.);
Jin et al., 1995, Virus Res. 38:269; and Lee et al., 1999, Molec.
Immunol. 36:61-71 (see, for example, FIG. 2 of that
publication).
[0099] Basically, the pcDNA3 vector (Invitrogen)(FIG. 1B) was used
as the basis for the pDCM vector (FIG. 1C), in that digestion with
pairs of restriction enzymes followed by re-ligation was used, in
parallel preparations, to destroy certain cleavage sites and
maintain others in vector downstream of the CMV promoter sequences.
Specifically, as shown in FIG. 1C, digestion of pcDNA3 with first
HindIII and BamHI, followed by religation and then digestion with
XhoI and ApaI, followed by religation, resulted in the preservation
of BstXI, EcoRI, EcoRV, BstXI, and NotI sites downstream of the
promoter; subsequent cleavage with BsmI linearized the molecule
between the ampicillin and neomycin resistance genes (component 1).
In parallel, digestion of pcDNA3 with BstxI and NotI, followed by
removal of the small fragment and re-ligation, removed the BstXI,
EcoRI, EcoRV, BstXI, and NotI sites and left the HindIII, KpnI,
BamHI, XhoI, XbaI and ApaI sites intact; cleavage with PvuII and
NruI gave rise to a fragment containing the CMV promoter, the
preserved sites, and BGHpA (component 2). Component 2 was inserted
between the ends of component 1, resulting in pDCM, having two
different insertion sites for genes downstream of two respective
CMV promoter elements. As shown in FIG. 1E, a dihydrofolate
reductase gene ("dhfr") from KC-dhfr may then be inserted into pDCM
(see Lee et al., 1999, Molec. Immunol. 36:61-71) to produce
pDCM-dhfr. Alternatively, as shown in FIG. 1D, the dhfr gene from
KC-dbfr may be incorporated into pcDNA3, to produce pCdhfr, which
may then be engineered by methods analogous to those shown in FIG.
1C to produce the two CMV promoter/insertion site cassette.
[0100] The Chi31.1-1 heavy and light chain encoding sequences were
then cloned from the pCR vector into pDCM-dhfr, to form pRc/CMV,
which may be transfected into CHO dhfr- cells, after which
expressed chimeric immunoglobulin molecules may be collected
according to standard techniques.
7. EXAMPLE
Human Immune Response to Chi31.1-1
[0101] To determine whether the 31.1 chimeric antibodies are
capable of inducing an immune response, plasma was collected from a
human subject who had been administered Chi31.1-1 chimeric
monoclonal antibody. The presence of an immune reaction in the
patient toward the chimeric antibody was tested using the following
assay.
[0102] 96 well microtiter plates were coated with Chi31.1-1
antibody, using a solution which was 10 micrograms per milliliter,
with 100 microliters per well. A preparation of Chi31.1-1 was
biotinylated. Then, either control plasma or patient plasma (50
microliters) was introduced into wells, and 50 microliters of the
biotinylated Chi31.1-1 was added. The plates were then incubated
for ninety minutes at 37 degrees centigrade and then the wells were
washed and streptavidin-horseradish peroxidase conjugate was added.
The wells were then washed three times. Then TMB substrate
(3,3',5,5' tetramethyl benzidine) was added, and the plates were
incubated for 20 minutes. Stop solution was added, and the amount
of reacted substrate was determined.
[0103] The results are presented in TABLE C, and are expressed in
nanograms of Chi31.1-1 bound per milliliter of plasma. Results
greater than 2-fold above the pre-treatment baseline are considered
to be positive. Non-specific baseline binding values from 3 healthy
normal samples were found to be 4 plus or minus 2 nanograms per
milliliter. The standard was determined by using goat anti-human
IgG1 coated wells with various concentraions of biotinylated
Chi31.1-1 monoclonal antibody. TABLE-US-00007 TABLE C Human Immune
Response to Chi31.1-1 Monoclonal Antibody (HAMA) Time ng/ml bound 0
hour (pretreatment) 2 1 hour 3 2 hours 2 3 hours 3 4 hours 3 5
hours 2 6 hours 3 Next day 4 1 week 3 2 weeks 5
8. EXAMPLE
ADCC Activity of CHO CHI 31.1 Antibody
[0104] The following section describes experiments demonstrating
that the CHO chi 31.1 monoclonal antibody has biological activity
associated with destruction of tumors. Specifically, the antibody
was shown to have antibody-dependent cellular cytotoxicity
(ADCC).
[0105] A four hour .sup.111In release assay was used to measure
ADCC activity. Target cells were the colon tumor cell line SW1643
and pancreatic cancer cell line PANC-1. UPC-10 was used as a
control antibody. Target cells were labeled with 50 .mu.Ci of
.sup.111In-oxyquinoline for 15 minutes at room temperature. Target
cells (1.times.10.sup.4) in 50 .mu.l were added to 96-well plate.
Ratios of effector to target cells of 100:1, 50:1 and 25:1 were
assayed in the presence of CHO 31.1 (1 mg/well). The plates were
incubated for four hours at 37.degree. in a humidified atmosphere
containing 5% CO.sub.2. Supernatant was harvested for gamma
counting with the use of Skatron Harvester frames. Experiments were
carried out in triplicate. Specific lysis was calculated with the
use of the following formula: % .times. .times. lysis = 100
observed .times. .times. release .function. ( cpm ) - spontaneous
.times. .times. .times. release .function. ( cpm ) total .times.
.times. .times. release .function. ( cpm ) - spontaneous .times.
.times. release .function. ( cpm ) . ##EQU1##
[0106] As presented in FIGS. 6A and 6B, the CHO 31.1 antibody, but
not the control UPC-10 antibody, was capable of mediating
antibody-dependent cellular cytotoxicity against the target
cells.
[0107] Various publications are cited herein, the contents of which
are hereby incorporated by reference in their entireties herein.
Sequence CWU 1
1
10 1 384 DNA Homo sapien 1 atgaagtcac agacccaggt cttcgtattt
ctactgctct gtgtgtctgg tgctcatggg 60 agtattgtga tgacccagac
tcccaaattc ctgcttgtat cagcaggaga cagggttacc 120 ataacctgca
aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaaacca 180
gggcagtctc ctaaactgct gatatactat gcatccaatc gctacactgg agtccctgat
240 cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac
tgtgcaggct 300 gaagacctgg cagtttattt ctgtcagcag gattatagct
ctccgctcac gttcggtgct 360 gggaccaagc tggagctgaa acgt 384 2 384 DNA
Homo sapien misc_feature (0)...(0) Antisense strand 2 acgtttcagc
tccagcttgg tcccagcacc gaacgtgagc ggagagctat aatcctgctg 60
acagaaataa actgccaggt cttcagcctg cacagtgctg atggtgaaag tgaaatccgt
120 cccatatcca ctgccagtga agcgatcagg gactccagtg tagcgattgg
atgcatagta 180 tatcagcagt ttaggagact gccctggttt ctgttggtac
caagctacat cattactcac 240 actctgactg gccttgcagg ttatggtaac
cctgtctcct gctgatacaa gcaggaattt 300 gggagtctgg gtcatcacaa
tactcccatg agcaccagac acacagagca gtagaaatac 360 gaagacctgg
gtctgtgact tcat 384 3 128 PRT Homo sapien 3 Met Lys Ser Gln Thr Gln
Val Phe Val Phe Leu Leu Leu Cys Val Ser 1 5 10 15 Gly Ala His Gly
Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu 20 25 30 Val Ser
Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser 35 40 45
Val Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 50
55 60 Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro
Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe
Thr Ile Ser 85 90 95 Thr Val Gln Ala Glu Asp Leu Ala Val Tyr Phe
Cys Gln Gln Asp Tyr 100 105 110 Ser Ser Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Arg 115 120 125 4 411 DNA Homo sapien 4
atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtgc ccaagcacag
60 atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt
caagatctcc 120 tgcaaggctt ctgggtatac cttcacaaac tatggaatga
actgggtgaa gcaggctcca 180 ggaaagggtt taaagtggat gggctggata
aacacctaca ctggagagcc aacatatgct 240 gatgacttca agggacggtt
tgccttctct ttggaaacct ctgccagcac tgcctatttg 300 cagatcaaca
acctcaaaaa tgaggacacg gctacatatt tctgtgcaag agcctactat 360
ggtaaatact ttgactactg gggccaaggc accactctca cagtctcctc a 411 5 411
DNA Homo sapien misc_feature (0)...(0) antisense strand 5
tgaggagact gtgagagtgg tgccttggcc ccagtagtca aagtatttac catagtaggc
60 tcttgcacag aaatatgtag ccgtgtcctc atttttgagg ttgttgatct
gcaaataggc 120 agtgctggca gaggtttcca aagagaaggc aaaccgtccc
ttgaagtcat cagcatatgt 180 tggctctcca gtgtaggtgt ttatccagcc
catccacttt aaaccctttc ctggagcctg 240 cttcacccag ttcattccat
agtttgtgaa ggtataccca gaagccttgc aggagatctt 300 gactgtctct
ccaggcttct tcagctcagg tccagactgc accaactgga tctgtgcttg 360
ggcactttgg gcagctgcca tcaggaatag caaggtccac acccaagcca t 411 6 137
PRT Homo sapien 6 Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala
Ala Ala Gln Ser 1 5 10 15 Ala Gln Ala Gln Ile Gln Leu Val Gln Ser
Gly Pro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Gly Met Asn
Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Asp Asp
Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95
Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100
105 110 Tyr Phe Cys Ala Arg Ala Tyr Tyr Gly Lys Tyr Phe Asp Tyr Trp
Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser 130 135 7 34
DNA Artificial Sequence Oligonucleotide primer 7 ataggatcca
tgaagtcaca gacccaggtc ttcg 34 8 31 DNA Artificial Sequence
Oligonucleotide primer 8 tttctagact aacactctcc cctgttgaag c 31 9 32
DNA Artificial Sequence Oligonucleotide primer 9 atagaattca
tggcttgggt gtggaccttg ct 32 10 24 DNA Artificial Sequence
Oligonucleotide primer 10 ttgcggccgc tcatttaccc ggag 24
* * * * *
References