U.S. patent application number 11/497785 was filed with the patent office on 2006-12-07 for antibodies to treat cancer.
This patent application is currently assigned to UNIVERSITY OF MASSACHUSETTS. Invention is credited to Gary R. Fanger, Neil Fanger, David King, Marc W. Retter, Kenneth L. Rock.
Application Number | 20060275307 11/497785 |
Document ID | / |
Family ID | 27502609 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060275307 |
Kind Code |
A1 |
Fanger; Gary R. ; et
al. |
December 7, 2006 |
Antibodies to treat cancer
Abstract
Compositions and methods for the treatment of cancer,
particularly melanoma, myeloma, small cell lung cancer, thymic
lymphoma, T-cell lymphoma, B-cell lymphoma, osteosarcoma, and acute
T-cell leukemia, are disclosed. Illustrative compositions include
one or more anti-ganglioside antibodies and polynucleotides that
encode such anti-ganglioside antibodies. These antibodies may be
for example, hamster antibodies, chimeric human/hamster antibodies,
or humanized antibodies. The disclosed compositions are useful, for
example, in the treatment of cancer and can be used to induce
apoptosis in a cancer cell.
Inventors: |
Fanger; Gary R.; (Southeast
Mill Creek, WA) ; Fanger; Neil; (Seattle, WA)
; King; David; (Belmont, CA) ; Retter; Marc
W.; (Carnation, WA) ; Rock; Kenneth L.;
(Chestnut Hill, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
UNIVERSITY OF MASSACHUSETTS
Boston
MA
Corixa Corp
Seattle
WA
|
Family ID: |
27502609 |
Appl. No.: |
11/497785 |
Filed: |
August 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10317747 |
Dec 11, 2002 |
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11497785 |
Aug 1, 2006 |
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60339736 |
Dec 11, 2001 |
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60388956 |
Jun 14, 2002 |
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60399103 |
Jul 26, 2002 |
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60400958 |
Aug 1, 2002 |
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Current U.S.
Class: |
424/155.1 ;
435/320.1; 435/338; 435/69.1; 435/7.23; 530/388.8; 536/23.53;
536/53 |
Current CPC
Class: |
A61P 35/04 20180101;
A61K 2039/505 20130101; C07K 2317/56 20130101; C07K 2317/73
20130101; C07K 16/30 20130101; C07K 2317/24 20130101; C07K 2317/732
20130101 |
Class at
Publication: |
424/155.1 ;
435/007.23; 435/069.1; 435/320.1; 435/338; 530/388.8; 536/023.53;
536/053 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; C08B 37/00 20060101
C08B037/00; C12P 21/06 20060101 C12P021/06; A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30 |
Claims
1-45. (canceled)
46. A method for reducing the tumor burden in a human subject in
need thereof, comprising administering to a human subject
therapeutically effective amount of a monoclonal antibody, or
antigen-binding fragment thereof, that specifically binds to
monoisalo-GM2 but does not bind to asialo-GM2, disialo-GM2,
monosialo-GM3, disialo-GD1a, disialo-GD1b, asialo-GM1,
monosialo-GM1, lysosialo-GM1, trisialo-GT1b, and disialo-GD3, such
that tumor burden is reduced in the human subject in need
thereof.
47. The method of claim 46, wherein the monoclonal antibody, or
antigen-binding fragment thereof, specifically binds to a living
cancer cell.
48. The method of claim 47, wherein the cancer cell is selected
from a group consisting of a hematological malignancy tumor cell, a
breast cancer tumor cell, an ovarian cancer tumor cell, a uterine
cancer tumor cell, a lung cancer tumor cell, a gastrointestinal
cancer tumor cell, a pancreatic cancer tumor cell, a liver cancer
tumor cell, a biliary cancer tumor cell, a kidney cancer tumor
cell, a skin cancer tumor cell, an adrenal cancer tumor cell, an
endocrine cancer tumor cell, a brain cancer tumor cell, a neural
cancer tumor cell, a bladder cancer tumor cell, a bone cancer tumor
cell, a connective tissue cancer tumor cell, a squamous cell
carcinoma tumor cell, an adenocarcinoma tumor cell and a
mesothelioma cancer tumor cell.
49. The method of claim 47, wherein the cancer cell is selected
from a group consisting of a melanoma, a T cell leukemia, an
osteosarcoma, a T cell lymphoma, a renal cancer, a myeloma, a
prostate cancer, a small cell lung cancer, a breast cancer, a
pancreatic cancer, an ovarian cancer.
50. The method of claim 47, wherein the cancer cell is selected
from a group consisting of a myeloma, a prostate cancer, a small
cell lung cancer, a breast cancer, a pancreatic cancer, an ovarian
cancer.
51. The method of claim 47, wherein the antibody, or
antigen-binding fragment thereof, inhibits proliferation of the
cancer cell upon binding to the cell.
52. The method of claim 47, wherein the antibody, or
antigen-binding fragment thereof, induces apoptosis in the cancer
cell upon binding to the cell.
53. The method of claim 47, wherein the antibody, or
antigen-binding fragment thereof, stimulates antibody-dependent
cell-mediated cytotoxicity in the subject upon binding to the
cell.
54. The method of claim 53, wherein the antibody, or
antigen-binding fragment thereof, induces death of the cancer
cell.
55. The method of claim 51, wherein the cancer cell is selected
from the group consisting of a myeloma cancer cell, a melanoma
cancer cell, and a small cell lung cancer cell.
56. The method of claim 51, wherein the cancer cell is a T-cell
lymphoma cancer cell.
57. The method of claim 52, wherein the cancer cell is selected
from the group consisting of a small cell lung cancer cell, a
myeloma cancer cell, a melanoma cancer cell, and a leukemia cancer
cell.
58. The method of claim 52, wherein the cancer cell is a melanoma
cell.
59. The method of claim 46, wherein the monoclonal antibody, or
antigen-binding fragment thereof, binds to the same epitope of
monosialo-GM2 as does the monoclonal antibody DMF10.167.4 produced
by a hybridoma cell line deposited under ATCC No. PTA-405.
60. The method of claim 46, wherein the antibody, or
antigen-binding fragment thereof, is chimeric, humanized,
primatized, veneered, or fully human.
61. The method of claim 46, wherein the antibody, or
antigen-binding fragment thereof, comprises an antibody heavy chain
comprising the amino acid sequence of SEQ ID NO:22.
62. The method of claim 46, wherein the antibody, or
antigen-binding fragment thereof, comprises a purified antibody
heavy chain comprising a modification to an amino acid shown in SEQ
ID NO:22, the modification comprising the substitution of threonine
at linear position 78 of the sequence of the heavy chain variable
region of SEQ ID NO:22 with lysine.
63. The method of claim 46, wherein the antibody, or
antigen-binding fragment thereof, comprises a purified antibody, or
antigen-binding fragment thereof, wherein the antibody, or
antigen-binding fragment thereof, comprises at least one
complementary determinant region (CDRs) amino acid sequence
selected from the amino acid sequences shown in the group
consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, and SEQ ID NO:18.
64. The method of claim 46, wherein the antibody, or
antigen-binding fragment thereof, is conjugated to moiety selected
from the group consisting of an anti-tumor agent, a
chemotherapeutic drug, a toxin, an immunological response
modulator, a cytokine, an enzyme, a radioisotope and a detectable
label.
65. A method for inhibiting proliferation of a myeloma tumor cell
expressing a ganglioside antigen, the method comprising contacting
a myeloma tumor cell expressing a ganglioside antigen with an
effective amount of an antibody, wherein the antibody is an IgG
that specifically binds to the ganglioside, such that proliferation
of the myeloma tumor cell expressing the ganglioside is
inhibited.
66. A method for causing cell death of a myeloma tumor cell
expressing a ganglioside antigen, the method comprising contacting
a myeloma tumor cell expressing a ganglioside antigen with an
effective amount of an antibody, wherein the antibody is an IgG
that specifically binds to the ganglioside, such that the myeloma
tumor cell expressing the ganglioside dies.
67. A method for causing cell death of a melanoma tumor cell
expressing a ganglioside antigen, the method comprising contacting
a melanoma tumor cell expressing a ganglioside antigen with an
effective amount of an antibody, wherein the antibody is an IgG
that specifically binds to the ganglioside, such that the melanoma
tumor cell expressing the ganglioside dies.
68. A method for causing cell death of a small cell lung cancer
(SCLC) tumor cell expressing a ganglioside antigen, the method
comprising contacting a SCLC tumor cell expressing a ganglioside
antigen with an effective amount of an antibody, wherein the
antibody is an IgG that specifically binds to the ganglioside, such
that the SCLC tumor cell expressing the ganglioside dies.
69. A method for causing cell death of a lymphoma or leukemia tumor
cell expressing a ganglioside antigen, the method comprising
contacting a lymphoma or leukemia tumor cell expressing a
ganglioside antigen with an effective amount of an antibody,
wherein the antibody is an IgG that specifically binds to the
ganglioside, such that the lymphoma or leukemia tumor cell
expressing the ganglioside dies.
70. A method for inhibiting proliferation of a lymphoma tumor cell
expressing a ganglioside antigen, the method comprising contacting
a lymphoma tumor cell expressing a ganglioside antigen with an
effective amount of an antibody, wherein the antibody is an IgG
that specifically binds to the ganglioside, such that proliferation
of the lymphoma tumor cell expressing the ganglioside is
inhibited.
71. The method of claim 65, wherein the antigen is selected from
the group consisting of monosialo-GM2, asialo-GM2, disialo-GM2,
monosialo-GM3, disialo-GD1a, disialo-GD1b, asialo-GM1,
monosialo-GM1, lysosialo-GM1, trisialo-GT1b, and disialo-GD3.
72. The method of claim 65, wherein the antibody specifically binds
the monosialo-GM2 antigen.
73. The method of claim 65, wherein the antibody is selected from
the group consisting of a chimeric antibody, a humanized antibody,
a human antibody, a veneered antibody, and a primatized
antibody.
74. The method of claim 66, wherein the tumor cell undergoes
apoptotic cell death.
Description
REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority from the following U.S.
Provisional Patent Applications: Ser. No. 60/339,736, filed on Dec.
11, 2001, Ser. No. 60/388,956, filed Jun. 14, 2002, Ser. No.
60/399,103, filed Jul. 26, 2002, and Ser. No. 60/400,958, filed
Aug. 01, 2002. The contents of these provisional applications are
incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies that can be used
in the treatment of cancer, including myeloma, melanoma, and small
cell lung cancer. It also relates to methods for the use of such
antibodies that specifically bind to tumor cells, in which they can
inhibit cellular proliferation, and induce apotosis.
BACKGROUND OF THE INVENTION
[0003] Gangliosides are glycosphingolipids that are present in high
numbers on cells of neural crest origin as well as on a wide
variety of tumor cells of neuroectodermal origin. Portoukalian et
al;, Eur. J. Biochem.94:19-23 (1979); Yates et al., J. Lipid Res.
20:428-436 (1979). More specifically, expression of the
gangliosides GD2, GD3, and GM2 has been reported in neuroblastoma,
lung small cell carcinoma, and melanoma, each of which are highly
malignant neuroectodermal tumors. J. Exp. Med., 155:1133 (1982); J.
Biol. Chem. 257:12752 (1982); CancerRes. 47:225 (1987); Cancer Res.
47:1098 (1987); Cancer Res. 45:2642 (1985); Proc. Natl. Acad. Sci.
U.S.A. 80:5392 (1983).
[0004] The chemical structure of gangliosides includes a
hydrophilic carbohydrate portion (one or more sialic acids linked
to an oligosaccharide) attached to a hydrophobic lipid moiety
composed of a long-chain base (sphingosine) and a fatty acid
(ceramide). (G is an abbreviation for ganglioside and M, D, and T
are abbreviations for mono, di, and tri, respectively; for further
discussion of ganglioside nomenclature, see, Lehninger,
Biochemistry, pp. 294-296 (Worth Publishers, 1981) and Wiegandt, in
Glycolipids: New Comprehensive Biochemistry (Neuberger et al., ed.,
Elsevier, 1985), pp. 199-260; see, also, FIG. 1 for a schematic of
ganglioside biosynthesis).
[0005] Gangliosides are believed to be involved in cell
recognition, immunosuppression, adhesion and signal transduction.
The ceramide portion anchors the ganglioside into the cell membrane
and may, thereby, modulate intracellular signal transduction as a
second messenger. The ganglioside designated GM2 is one of a group
of sialic acid residue-containing glycolipids and is uniquely
characterized by its presence in only trace amounts in normal cells
and its upregulation in a variety of cancer cells such as, for
example, lung small cell carcinoma, melanoma, and
neuroblastoma.
[0006] Because they are immunogenic, gangliosides have received
much attention as possible vaccine targets. For example,
vaccination with a GM2 ganglioside, has been shown to stimulate
high levels of anti-GM2 antibodies in melanoma patients. GM2
vaccines comprising either bacilli Calmette-Gueriii (BCG) or, more
recently, keyhole limpet hemagglutinin (KLH) as adjuvant have been
tested in human clinical trials. Livingston et al., Proc. Natl.
Acad. Sci U.S.A. 84:2911-2915 (1987); Livingston, In "Immunity to
Cancer II." Eds MS Mitchell, Pub Alan L. Liss, Inc., NY; Irie et
al. U.S. Patent No. 4,557,931; Kirkwood et al. J. Clin. Oncol.
19(5):1430-1436 (2001); Chapman et al. Clin. Cancer Res.
6(3):874-879 (2000).
[0007] In an effort to develop a therapeutic agent against
GM2-positive cells, a number of investigators have reported the
production of anti-GM2 antibodies. For example, Yamaguchi et al.,
described the isolation of lymphocytes from a GM2-vaccinated
patient and the transformation of those lymphocytes with
Epstein-Barr virus to produce an antibody (designated 3-207)
simultaneously reactive for both GM2 and GD2. Proc. Natl. Acad.
Sci. USA 87:3333-3337 (1990). Similarly, Irie et al., disclosed a
human monoclonal anti-GM2 antibody for melanoma treatment. Lancet
1:786-787 (1989); see, also, Tai et al., Proc. Nat. Acad. Sci.
U.S.A. 80:5392-5396 (1983) (disclosing a human anti-GM2 monoclonal
antibody designated L55) and Yamasaki et al. U.S. Pat. No.
4,965,498 (disclosing a monoclonal antibody specific to a sugar
chain containing an N-glycolylneuramine acid and having the ability
to bind to at least N-glycolyl GM2 ganglioside). Furthermore,
Ritter et al., disclosed antibodies produced following immunization
with a lipopolysaccharide antigen of Campylobacter jejuni that
reportedly binds to monosialogangliosides, including both GM2 and
GM1. Int. J. Cancer 66(2):184-190 (1996).
[0008] Nakamura et al. have described two murine anti-GM2
monoclonal IgM antibodies, KM696 and KM697, as well as
corresponding chimeric antibodies, KM966 and KM967, constructed by
replacing the variable domain heavy and light chain cDNAs of a
human IgGI with the corresponding variable domain heavy and light
chain cDNAs of KM696 and KM697, respectively. Cancer Research
54:1511-1516 (1994); U.S. Pat. Nos. 5,830,470 and 5,874,255. These
investigators also reported CDR-grafted variants of the KM696/KM966
and the KM697/KM967 antibodies designated KM8966 and KM8967,
respectively. U.S. Pat. Nos. 5,939,532 and 6,042,828. Indirect
immunofluorescence staining of tumor cell lines with the KM966
chimeric antibody demonstrated that GM2 was expressed on pulmonary
tumor cells and leukemia cells as well as neuroectodermal origin
tumor cells.
SUMMARY OF THE INVENTION
[0009] The invention is based on the discovery that certain
antibodies, e.g., monoclonal antibodies, specifically bind to
monosialo-GM2 on the surface of numerous types of tumor cells, but
do not bind to other gangliosides. These monoclonal antibodies can
block proliferation and induce apoptosis of tumor cells to which
they specifically bind.
[0010] The invention features a method of inhibiting proliferation
of a myeloma tumor cell expressing a ganglioside antigen (e.g.,
monosialo-GM2, asialo-GM2, disialo-GM2, monosialo-GM3,
disialo-GD1a, disialo-GD1b, asialo-GM1, monosialo-GM1,
lysosialo-GM1, trisialo-GT1b, and disialo-GD3) in which the method
includes contacting the cell with an antibody (e.g., chimeric
antibody, a humanized antibody, a human antibody, a primatized
antibody, DMF10.167.4, DMF10.62.3, a chimeric antibody having a
variable region of DMF10.167.4, a chimeric antibody having a
variable region of DMF10.62.3, a humanized antibody having all
complementary determinant regions of DMF 10.67.4, and a chimeric
antibody with a light chain amino acid sequence of SEQ ID NO:21
(e.g., isoleucine at linear position 52 is replaced with valine),
in which and a heavy chain amino acid sequence of SEQ ID NO:22
(e.g., threonine at linear position 78 of SEQ ID NO:22 is replaced
with lysine), and the antibody specifically binds to the
ganglioside.
[0011] The invention also features a method of inhibiting the
proliferation of a cancer cell (e.g., a thymic lymphoma, T-cell
lymphoma, B-cell lymphoma, melanoma, osteosarcoma, acute T-cell
leukemia, small cell lung cancer, and myeloma cell) in which the
method includes contacting the cell with a chimeric antibody (e.g.,
administered in vivo to a mammal,) that includes a light chain
amino acid sequence of SEQ ID NO:21 (e.g., in which isoleucine at
linear position 52 is replace with valine) and a heavy chain amino
acid sequence of SEQ ID NO:22 (e.g., in which threonine at linear
position 78 is replaced with lysine).
[0012] The invention further features a purified chimeric antibody
(e.g., an antibody that is effective in inhibiting cell
proliferation of a tumor cell to which the antibody specifically
binds, an antibody that is effective in inducing apoptosis in a
tumor cell to which the antibody specifically binds, an antibody
that binds specifically to monosialo-GM2 on the surface of a tumor
cell and induces apoptosis in a monolayer, a single-cell suspension
of the tumor cells, or both, an antibody that includes a light
chain having the amino acid sequence of SEQ ID NO:21 and a heavy
chain having the amino acid sequence of SEQ ID NO:22, or an
antibody that binds specifically to monosialo-GM2 on the surface of
a tumor cell and induces apoptosis in a monolayer, a single-cell
suspension of tumor cells, or both), or antigen-binding fragment
thereof, in which the chimeric antibody includes an amino acid
sequence comprising SEQ ID NO:21 (e.g., in which isoleucine at
linear position 52 is replaced with valine) or SEQ ID NO:22 (e.g.,
in which threonine at linear position 78 is replaced with
lysine).
[0013] The invention includes an purified antibody light chain that
includes the sequence of SEQ ID NO:21.
[0014] Additionally, the invention includes an isolated nucleic
acid molecule that includes a nucleotide sequence (e.g., the
nucleotide sequence comprises SEQ ID NO:8) including that encodes
the antibody light chain of a purified antibody light chain (e.g.,
in which isoleucine at linear position 52 of the light chain
consisting of SEQ ID NO:21 is replaced with valine) that includes
the sequence of SEQ ID NO:21.
[0015] The invention further includes an isolated nucleic acid
molecule that includes a nucleotide sequence that encodes the
antibody light chain in which isoleucine at linear position 52 of
SEQ ID NO:21 is replaced with valine.
[0016] The invention also includes a purified antibody that
includes the light chain of claim that includes the sequence of SEQ
ID NO:21 and a heavy chain.
[0017] Also featured in the invention is a purified antibody heavy
chain having the amino acid sequence of SEQ ID NO:22 (e.g., wherein
a threonine at linear position 78 is replaced with a lysine).
[0018] The invention further features an isolated nucleic acid
molecule in which the nucleic acid molecule includes a nucleic acid
sequence that encodes the antibody heavy chain having the amino
acid sequence of SEQ ID NO:22 (e.g., wherein a threonine at linear
position 78 is replaced with a lysine).
[0019] Also featured as part of the invention is an isolated
nucleic acid molecule in which the nucleic acid molecule (e.g.,
including the nucleic acid sequence of SEQ ID NO:7) encodes the
antibody heavy chain of the amino acid sequence of SEQ ID
NO:22.
[0020] The invention also includes a purified antibody comprising
the heavy chain having he amino acid sequence of SEQ ID NO:22.
[0021] The invention additionally features a purified antibody
(e.g., an antibody effective in inhibiting cell proliferation of a
tumor cell to which the antibody specifically binds, an antibody
effective in inducing apoptosis in a tumor cell to which the
antibody specifically binds, or an antibody that binds specifically
to monosialo-GM2 on the surface of a tumor cell and induces
apoptosis in a monolayer, a single-cell suspension of the tumor
cells, or both), or antigen-binding fragment thereof, wherein the
antibody comprises a complementary determinant region (CDR) with an
amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
[0022] The invention also includes a method of inhibiting growth of
a tumor cell (e.g., in which growth of the tumor cell is inhibited
by inhibiting proliferation of the cell, in which the growth of the
tumor cell is inhibited by inducing apoptosis of the cell) in which
the method includes contacting the tumor cell with a chimeric
antibody having a light chain amino acid sequence of SEQ ID NO:21
and a heavy chain amino acid sequence of SEQ ID NO:22.
[0023] The invention further includes an isolated nucleic acid that
includes the nucleic acid sequence of SEQ ID NO:5, 6, 9, 10, 11, or
12.
[0024] Additionally, the invention includes a polypeptide that
includes the amino acid sequence of SEQ ID NO:19, 20, 23, 24, 25,
or 26.
[0025] The invention also includes a purified monoclonal antibody,
or antigen-binding fragment thereof, in which the monoclonal
antibody comprises an amino acid sequence of SEQ ID NO:19, 20, 23,
24, 25, or 26.
[0026] The invention further features an antibody, or
antigen-binding fragment thereof, in which the antibody comprises a
complementary determinant region (CDR) with an amino acid sequence
of SEQ. ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, or SEQ ID NO:18.
[0027] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0028] An "isolated nucleic acid sequence" is a nucleic acid
sequence that is substantially free of the genes that flank the
nucleic acid sequence in the genome of the organism in which it
naturally occurs. The term therefore includes a recombinant nucleic
acid sequence incorporated into a vector, into an autonomously
replicating plasmid or virus, or into the genomic nucleic acid
sequence of a prokaryote or eukaryote. It also includes a separate
molecule such as a cDNA, a genomic fragment, a fragment produced by
polymerase chain reaction (PCR), or a restriction fragment.
[0029] An antibody that "specifically binds" to monosialo-GM2 binds
to monosialo-GM2, but that does not recognize and bind to other
molecules in a sample, such as a biological sample that naturally
includes monosialo-GM2.
[0030] "Conservative" amino acid substitutions are substitutions in
which one amino acid residue is replaced with another amino acid
residue having a similar side chain. Families of amino acid
residues having similar side chains have been defined in the art.
These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Any
one of a family of amino acids can be used to replace any other
members of the family in a conservative substitution.
[0031] The terms "polypeptide, peptide, and protein" are used
interchangeably herein to refer to a chain of amino acid
residues.
[0032] An "antigen-binding fragment" of an antibody is a portion of
the antibody that is capable of binding to an epitope on an antigen
(for example, monosialo-GM2) bound by the full antibody.
[0033] An "epitope" is a particular region of an antigen (for
example, monosialo-GM2) to to which an antibody binds and which is
capable of eliciting an immune response.
[0034] An "isolated" antibody is an antibody that is substantially
free from other naturally-occurring organic molecules with which it
is naturally associated.
[0035] An antibody or other molecule that blocks cell proliferation
is an antibody or molecule that inhibits cell cycle, division, or
both.
[0036] A "reporter group" is a molecule or compound that has a
physical or chemical characteristic such as luminescence,
fluorescence, enzymatic activity, electron density, or
radioactivity that can be readily measured or detected by
appropriate detector systems or procedures.
[0037] "Contacting" a cell with an antibody includes both in vivo
and in vitro methods whereby an antibody may specifically bind to
an antigen. The antigen can be expressed on the surface of a cell.
Such methods include for example administering a solution
containing the antibody (e.g. through an injection or other methods
known in the art) to a mammal. Additionally, in vitro methods of
contacting a cell with an antibody include adding the antibody to a
solution or cell culture dish in which the test cells are
growing.
[0038] An antibody may inhibit the proliferation of a cell though
mechanisms including, but not limited to, inducing apoptosis,
mediating antibody dependent cellular cytotoxicity, mediating
complement dependent cytotoxicty, blocking angeogensis or
destroying vasculature.
[0039] The practice of the present invention will employ, unless
indicated specifically to the contrary, conventional methods of
virology, immunology, microbiology, molecular biology and
recombinant DNA techniques within the skill of the art, many of
which are described below for the purpose of illustration. Such
techniques are explained more fully in the literature. See, e.g.,
Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory
Manual (1982); DNA Cloning: A Practical Approach, vol. I & II
(D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984);
Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);
Transcription and Translation (B. Hames & S. Higgins, eds.,
1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A
Practical Guide to Molecular Cloning (1984).
[0040] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0041] The invention features antibodies that recognize
monosialo-GM2 expressed on tumor cells. The antibodies can be used
to inhibit proliferation of tumor cells and induce apoptosis of
tumor cells to which they specifically bind. The monoclonal
antibodies can be used diagnostically (for example, to determine
the presence of malignant cells), or can be used therapeutically to
treat tumor cells by themselves or through their delivery of an
attached antitumor agent.
[0042] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING
[0043] FIG. 1 is a diagrammatic representation of ganglioside
biosynthesis. It employs the following abbreviations: Cer,
ceramide; Glc, glucose; Gal, galactose; GalNAc,
N-acetylgalactosamine; Sia, sialic acid; LacCer, lactosylceramide.
This figure is adapted from Takamiya et al., Proc. Natl. Acad. Sci.
USA 93:10662 (1996).
[0044] FIG. 2 is a bar graph depicting the ganglioside binding
activity of the hamster monoclonal antibodies designated DMF
10.167.4 and DMF 10.62.3. It employs the following abbreviations:
As=asialo, Ms=monosialo, Ds=disialo, Ts=trisialo, and Ls=lysosialo
and Chl/Meth=Chloroform Methanol diluent.
[0045] SEQ ID NO: 1 is the polynucleotide sequence of an antisense
primer for the light chain of a chimeric anti-monosialo-GM2
antibody.
[0046] SEQ ID NO: 2 is the polynucleotide sequence of a sense
primer for the light chain of a chimeric anti-monosialo-GM2
antibody.
[0047] SEQ ID NO: 3 is the polynucleotide sequence of an anti-sense
primer for the heavy chain of a chimeric anti-monosialo-GM2
antibody.
[0048] SEQ ID NO: 4 is the polynucleotide sequence of a sense
primer for the heavy chain of a chimeric anti-monosial6-GM2
antibody.
[0049] SEQ ID NO: 5 is the polynucleotide sequence of the VJ of
DMF10.62.3.
[0050] SEQ ID NO: 6 is the polynucleotide sequence of the VDJ of
DMF10.62.3.
[0051] SEQ ID NO: 7 is the polynucleotide sequence the VJ-C (light
chain) of a chimeric anti-monosialo-GM2 antibody.
[0052] SEQ ID NO: 8 is the polynucleotide sequence of the VDJ-C
(heavy chain) of a chimeric anti-monosialo-GM2 antibody.
[0053] SEQ ID NO: 9 is the polynucleotide sequence of the VJ of
DMF10.167.4.
[0054] SEQ ID NO: 10 is the polynucleotide sequence of the VDJ of
DMF10.167.4.
[0055] SEQ ID NO: 11 is the polynucleotide sequence of the VJ of
DMF10.167.4 with its endogenous leader sequence.
[0056] SEQ ID NO: 12 is the polynucleotide sequence of the VDJ of
DMF10.167.4 with its endogenous leader sequence.
[0057] SEQ ID NO: 13 is the amino acid sequence of CDR3 of the
light chain of DMF10.167.4 and DMF10.62.3.
[0058] SEQ ID NO: 14 is the amino acid sequence of CDR2 of the
light chain of DMF10.167.4 and DMF10.62.3.
[0059] SEQ ID NO: 15 is the amino acid sequence of CDR1 of the
light chain of DMF10.167.4 and DMF10.62.3.
[0060] SEQ ID NO: 16 is the amino acid sequence of CDR3 of the
heavy chain of DMF10.167.4 and DMF10.62.3.
[0061] SEQ ID NO: 17 is the amino acid sequence of CDR2 of the
heavy chain of DMF10.167.4 and DMF10.62.3.
[0062] SEQ ID NO: 18 is the amino acid sequence of CDR1 of the
heavy chain of DMF10.167.4 and DMF10.62.3.
[0063] SEQ ID NO: 19 is the amino acid sequence of the VJ of
DMF10.62.3.
[0064] SEQ ID NO: 20 is the amino acid sequence of the VDJ of
DMF10.62.3.
[0065] SEQ ID NO: 21 is the amino acid sequence of the VJ-C (light
chain) of a chimeric anti-monosialo-GM2 antibody.
[0066] SEQ ID NO: 22 is the amino acid sequence ofthe VDJ-C (heavy
chain) of a chimeric anti-monosialo-GM2 antibody.
[0067] SEQ ID NO: 23 is the amino acid sequence of the VJ of
DMF10.167.4.
[0068] SEQ ID NO: 24 is the amino acid sequence the VDJ of
DMF10.167.4.
[0069] SEQ ID NO: 25 is the amino acid sequence of the VJ of
DMF10.167.4 with its endogenous leader sequence.
[0070] SEQ ID NO: 26 is the amino acid sequence of the VDJ of
DMF10.167.4 with its endogenous leader sequence.
DETAILED DESCRIPTION
[0071] The present invention features antibodies, e.g., monoclonal
antibodies that specifically bind to monosialo-GM2. Monosialo-GM2
occurs on a variety of types of tumor cells, including thymic
lymphoma, T-cell tumor, a B-cell lymphoma, melanoma, osteosarcoma,
and acute T-cell leukemia. Importantly, it was demonstrated that
monosialo-GM2 is over-expressed on certain myeloma cell lines.
Expression of monosialo-GM2 has also been observed in a variety of
different species, including humans, monkeys, and mice. Upon
binding of an antibody of the invention to a cell over-expressing
monosialo-GM2, the cell stops proliferating and undergoes
apoptosis. The antibodies of the invention (e.g., DMF10.167.4 and
ChGM2) can be used to treat myeloma. They can be used to treat
melanoma and small cell lung carcinoma. Additionally, they can be
used to treat other cancers, e.g., hematological malignancies,
breast cancers, ovarian cancers, uterine cancers, lung cancers, GI
cancers (including those affecting the oropharynx, esophagus,
stomach, small and large intestine, rectum), pancreatic cancer,
liver cancer, biliary cancers, kidney cancer, skin cancers, adrenal
cancers, endocrine cancers, brain cancers, neural cancers, bladder
cancer, bone cancer, connective tissue cancers, squamous cell
carcinoma, adenocarcinoma, and mesothelioma, provided that the
cancer expresses or over-expresses monosialo-GM2.
[0072] Three hybridoma cell lines that produce monoclonal
antibodies that specifically bind to monosialo-GM2 have been
deposited with the ATCC under Accession No. PTA-377 (DMF10.62.3),
Accession No. PTA-405 (DMF10.167.4), or Accession No. PTA-404
(DMF10.34.36).
[0073] The antibodies described herein have a variety of uses. The
antibodies can be used in in vitro diagnostic assays to determine
the presence of malignant cells in mammalian, e.g., human, tissues.
The antibodies can also be used to localize and image tumors in
vivo by administering to a subject an isolated antibody described
herein which is labeled with a reporter group. The antibodies also
have therapeutic applications, such as to treat tumors or deliver
an anti-tumor agent (e.g., as a treatment for myeloma, small cell
lung carcinoma, or melanoma).
Methods of Making Antibodies
[0074] Antibodies are immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules. Examples of fragments
of immunoglobulin molecules include fragments of an antibody, e.g.,
F(ab) and F(ab').sub.2 portions, which can specifically bind to
monosialo-GM2. Fragments can be generated by treating the antibody
with an enzyme such as pepsin. The term monoclonal antibody or
monoclonal antibody composition refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope of, e.g., a
ganglioside, polypeptide, or protein. A monoclonal antibody
composition thus typically displays a single binding affinity for
the epitope to which it specifically binds.
[0075] Immunization Polyclonal and monoclonal antibodies against
monosialo-GM2 can be raised by immunizing a suitable subject (e.g.,
a hamster, rabbit, goat, mouse, or other mammal) with an
immunogenic preparation which contains a suitable immunogen.
Immunogens include cells such as cells from immortalized cell lines
E710.2.3, RMA-S, CTLL, LB17.4, A20, WEHI-231, PBK101A2, C2.3, B16,
MC57, WOP-3027, 293T, 143Btk, Jurkat, or Cos, that have all been
shown to express monosialo-GM2. Alternatively, the immunogen can be
purified or isolated monosialo-GM2.
[0076] The antibodies raised in the subject can then be screened to
determine if the antibodies bind to fetal thymocytes while not
binding to adult thymocytes. Such antibodies can be further
screened in the assays described herein. For example, these
antibodies can be assayed to determine if they inhibit cell
proliferation of cells to which they bind; induce homotypic
aggregation of cells; and/or induce apoptosis in cells to which
they bind. Suitable methods to identify an antibody with the
desired characteristics are described herein. For example, the
ability of an antibody to induce cell death upon binding to a cell
can be assayed using commercially available kits from R&D
(Minneapolis, Minn.) or Pharmingen (San Diego, Calif.).
[0077] The unit dose of immunogen (e.g., purified monosialo-GM2,
tumor cell expressing monosialo-GM2) and the immunization regimen
will depend upon the subject to be immunized, its immune status,
and the body weight of the subject. To enhance an immune response
in the subject, an immunogen can be administered with an adjuvant,
such as Freund's complete or incomplete adjuvant.
[0078] Immunization of a subject with an immunogen as described
above induces a polyclonal antibody response. The antibody titer in
the immunized subject can be monitored over time by standard
techniques such as an ELISA using an immobilized antigen, e.g.,
monosialo-GM2.
[0079] Other methods of raising antibodies against monosialo-GM2
include using transgenic mice which express human immunoglobin
genes (see, e.g., Wood et al. PCT publication WO 91/00906,
Kucherlapati et al. PCT publication WO 91/10741; or Lonberg et al.
PCT publication WO 92/03918). Alternatively, human monoclonal
antibodies can be produced by introducing an antigen into immune
deficient mice that have been engrafted with human
antibody-producing cells or tissues (e.g;, human bone marrow cells,
peripheral blood lymphocytes (PBL), human fetal lymph node tissue,
or hematopoietic stem cells). Such methods include raising
antibodies in SCID-hu mice (see Duchosal et al. PCT publication WO
93/05796; U.S. Pat. No. 5,411,749; or McCune et al. (1988) Science
241:1632-1639)) or Rag-1/Rag-2 deficient mice. Human
antibody-immune deficient mice are also commercially available. For
example, Rag-2 deficient mice are available from Taconic Farms
(Germantown, N.Y.).
Hybridomas
[0080] Monoclonal antibodies can be generated by immunizing a
subject with an immunogen. At the appropriate time after
immunization, e.g., when the antibody titers are at a sufficiently
high level, antibody producing cells can be harvested from an
immunized animal and used to prepare monoclonal antibodies using
standard techniques. For example, the antibody producing cells can
be fused by standard somatic cell fusion procedures with
immortalizing cells to yield hybridoma cells. Such techniques are
well known in the art, and include, for example, the hybridoma
technique as originally developed by Kohler and Milstein, (1975)
Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar
et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma
technique to produce human monoclonal antibodies (Cole et al.,
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
pp. 77-96). The technology for producing monoclonal antibody
hybridomas is well known.
[0081] Monoclonal antibodies can also be made by harvesting
antibody producing cells, e.g., splenocytes, from transgenic mice
expressing human immunogloulin genes and which have been immunized
with monosialo-GM2. The splenocytes can be immortalized through
fusion with human myelomas or through transformation with
Epstein-Barr virus (EBV). These hybridomas can be made using human
B cell-or EBV-hybridoma techniques described in the art (see, e.g.,
Boyle et al., European Patent Publication No. 0 614 984).
[0082] Hybridoma cells producing a monoclonal antibody which
specifically binds to monosialo-GM2 are detected by screening the
hybridoma culture supernatants by, for example, screening to select
antibodies that specifically bind to the immobilized monosialo-GM2,
or by testing the antibodies as described herein to determine if
the antibodies have the desired characteristics, e.g., the ability
to inhibit cell proliferation.
[0083] Hybridoma cells that produce monoclonal antibodies that test
positive in the screening assays described herein can be cultured
in a nutrient medium under conditions and for a time sufficient to
allow the hybridoma cells to secrete the monoclonal antibodies into
the culture medium, to thereby produce whole antibodies. Tissue
culture techniques and culture media suitable for hybridoma cells
are generally described in the art (see, e.g., R. H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980). Conditioned
hybridoma culture supernatant containing the antibody can then be
collected.
Recombinant Combinatorial Antibody Libraries
[0084] Monoclonal antibodies can be engineered by constructing a
recombinant combinatorial immunoglobulin library and screening the
library with monosialo-GM2. Kits for generating and screening phage
display libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Briefly,
the antibody library is screened to identify and isolate phages
that express an antibody that specifically binds to monosialo-GM2.
In a preferred embodiment, the primary screening of the library
involves screening with an immobilized monosialo-GM2.
[0085] Following screening, the display phage is isolated and the
nucleic acid encoding the selected antibody can be recovered from
the display phage (e.g., from the phage genome) and subcloned into
other expression vectors by well known recombinant DNA techniques.
The nucleic acid can be further manipulated (e.g., linked to
nucleic acid encoding additional immunoglobulin domains, such as
additional constant regions) and/or expressed in a host cell.
Chimeric and Humanized Antibodies
[0086] Recombinant forms of antibodies, such as chimeric and
humanized antibodies, can also be prepared to minimize the response
by a human patient to the antibody. When antibodies produced in
non-human subjects or derived from expression of non-human antibody
genes are used therapeutically in humans, they are recognized to
varying degrees as foreign, and an immune response may be generated
in the patient. One approach to minimize or eliminate this immune
reaction is to produce chimeric antibody derivatives, i.e.,
antibody molecules that combine a non-human animal variable region
and a human constant region. Such antibodies retain the epitope
binding specificity of the original monoclonal antibody, but may be
less immunogenic when administered to humans, and therefore more
likely to be tolerated by the patient.
[0087] Chimeric monoclonal antibodies can be produced by
recombinant DNA techniques known in the art. For example, a gene
encoding the constant region of a non-human antibody molecule is
substituted with a gene encoding a human constant region. (see
Robinson et al., PCT Patent Publication PCT/US86/02269; Akira, et
al., European Patent Application 184,187; or Taniguchi, M.,
European Patent Application 171,496).
[0088] A chimeric antibody can be further "humanized" by replacing
portions of the variable region not involved in antigen binding
with equivalent portions from human variable regions. General
reviews of "humanized" chimeric antibodies are provided by
Morrison, S. L. (1985) Science, 229:1202-1207 and by Oi et al.
(1986) BioTechniques, 4:214. Such methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of an immunoglobulin variable region from at least one
of a heavy or light chain. The cDNA encoding the humanized chimeric
antibody, or fragment thereof, can then be cloned into an
appropriate expression vector. Suitable "humanized" antibodies can
be alternatively produced by (complementarity determining region
(CDR) substitution (see U.S. Pat. No. 5,225,539; Jones et al.
(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science
239:1534; and Beidler et al. (1988) J Immunol. 141:4053-4060).
[0089] Epitope imprinting can also be used to produce a "human"
antibody polypeptide dimer that retains the binding specificity of
the hamster antibodies specific for monosialo-GM2produced by the
hybridoma deposited as ATCC Accession No. PTA-377, Accession No.
PTA-405, or Accession No. PTA-404. Briefly, a gene encoding a
non-human variable region (VH) with specific binding to an antigen
and a human constant region (CH1), is expressed in E. coli and
infected with a phage library of human V.kappa.C.kappa. genes.
Phage displaying antibody fragments are then screened for binding
to monosialo-GM2. Selected human V.kappa. genes are recloned for
expression of V.kappa.C.kappa. chains and E. coli harboring these
chains are infected with a phage library of human VHCH1 genes and
the library is subject to rounds of screening with antigen coated
tubes. See Hoogenboom et al. PCT publication WO 93/06213.
Chimeric Anti-Monosialo-GM2 Antibodies
[0090] Among other things, the present invention provides chimeric
antibodies that are highly specific for monosialo-GM2. These
chimeric anti-monosialo-GM2 antibodies ("ChGM2 mAb") are effective
at inducing apoptosis in tumor cell monolayers and/or single-cell
suspensions and in inhibiting the in vivo proliferation of tumor
cells.
[0091] As part of the present invention, it was determined that
several hamster monoclonal antibodies specifically recognize the
ganglioside monosialo-GM2. This ganglioside is present on the
surface of many tumor cells. In general, the ChGM2 antibodies of
the present invention have the respective Fab and CDR regions from
the hamster mAbs designated DMF 10.62.3 and DMF 10.167.4. A
chimeric anti-monosialo-GM2 antibody of the present invention can
also have Fab or CDR regions of the hamster mAb designated DMF
10.34.36. Also provided are methods of using these chimeric
anti-monosialo-GM2 antibodies in the treatment of cancer.
[0092] The present invention provides chimeric anti-monosialo-GM2
antibodies that exhibit tumor cell-specific binding and that both
induce apoptosis and inhibit proliferation in the cells to which
they bind. An antibody, or antigen-binding fragment thereof, is
said to "specifically bind," "immunogically bind," and/or is
"immunologically reactive" to monosialo-GM2 if it reacts at a
detectable level (within, for example, an ELISA assay) to
monosialo-GM2, but not to asialo-GM2, disialo-GM2, monosialo-GM3,
disialo-GD1a, disialo-GD1b, asialo-GM1, monosialo-GM1,
lysosialo-GM1, trisialo-GT1b, and/or disialo-GD3.
[0093] "Immunological binding," as used herein, generally refers to
the non-covalent interactions of the type that occurs between an
antibody, or fragment thereof, and an antigen for which the
antibody is specific. Immunological binding properties of selected
antibodies can be quantified using methods well known in the art.
See, generally, Davies et al. Annual Rev. Biochem. 59:439-473
(1990).
[0094] An "antigen-binding site," or "binding portion" of an
antibody refers to the part of the immunoglobulin molecule that
participates in antigen binding. The antigen binding site is formed
by amino acid residues of the N-terminal variable ("V") regions of
the heavy ("H") and light ("L") chains. Three highly divergent
stretches within the V regions of the heavy and light chains are
referred to as "hypervariable regions" which are interposed between
more conserved flanking stretches known as "framework regions," or
"FWRs". Thus, the term "FWR" refers to amino acid sequences which
are naturally found between and adjacent to hypervariable regions
in immunoglobulins. In an antibody molecule, the three
hypervariable regions of a light chain and the three hypervariable
regions of a heavy chain are disposed relative to each other in
three-dimensional space to form an antigen-binding surface. The
antigen-binding, surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementary determinant region," or "CDRs."
[0095] A monoclonal antibody may be cleaved into various fragments
by methods known in the art. The proteolytic enzyme papain
preferentially cleaves IgG molecules to yield several fragments,
two of which (the "Fab" fragments) each comprise a covalent
heterodimer that includes an intact antigen-binding site. Another
fragment produced from papain cleavage of an antibody is the Fc
(fragment crystallizable). The Fc fragment is constant among a
given class of antibodies and mediates binding of a cell or
complement to an antibody when the antigen binding sites (Fabs) are
occupied by an antigen.
[0096] The Fc regions are particularly constant within a given
species. When a therapeutic monoclonal antibody from a first
species is administered to a second species, the immune system of
the second species may mount an immune response to the Fc region of
the mAb. Such an immune response can lead to the rapid destruction
and clearing of the mAb from the second species. Such clearing can
limit the efficacy of a therapeutic antibody.
[0097] The chimeric mAbs of the present invention have a Fab
portion that is derived from a hamster mAb that specifically binds
to monosialo-GM2 on tumor cells. The Fc region of this hamster
antibody is replaced with the Fc region of a human antibody, which
limits undesirable immunological response toward non-human
antibodies.
[0098] Antibodies and antigen-binding fragments have a heavy chain
and a light chain complementary determinant region (CDR) set, which
are respectively interposed between a heavy chain and a light chain
FWR set that provide support to the CDRs and define the spatial
relationship of the CDRs relative to each other. As used herein,
the term "CDR set" refers to the three hypervariable regions of a
heavy or light chain V region. Proceeding from the N-terminus of a
heavy or light chain, these regions are denoted as "CDR1,", "CDR2,"
and "CDR3" respectively. An antigen-binding site, therefore,
includes six CDRs, comprising the CDR set from each of a heavy and
a light chain V region. Crystalfographic analysis of a number of
antigen-antibody complexes has demonstrated that the amino acid
residues of CDRs form extensive contact with bound antigen, wherein
the most extensive antigen contact is with the heavy chain CDR3.
Thus, the molecular recognition units are primarily responsible for
the specificity of an antigen-binding site.
[0099] As used herein, the term "FWR set" refers to the four
flanking amino acid sequences that frame the CDRs of a CDR set of a
heavy or light chain V region. Some FWR residues may contact bound
antigen; however, FWRs are primarily responsible for folding the V
region into the antigen-binding site. The FWR residues directly
adjacent to the CDRs are particularly important for the folding of
the V region. Within FWRs, certain amino acid residues and certain
structural features are very highly conserved. In this regard, all
V region sequences contain an internal disulfide loop of around 90
amino acid residues. When the V regions fold into a binding-site,
the CDRs are displayed as projecting loop motifs, which form an
antigen-binding surface. It is generally recognized that there are
conserved structural regions of FWRs that influence the folded
shape of the CDR loops into certain "canonical" structures,
regardless of the precise CDR amino acid sequence. Further, certain
FWR residues are known to participate in non-covalent interdomain
contacts which stabilize the interaction of the antibody heavy and
light chains.
[0100] Both the light chain and heavy chain variable regions of the
chimeric anti-monosialo-GM2 antibodies have three complementary
determinant regions, CDRs, joined by framework regions, FWR. A
ChGM2 monoclonal antibody of the present invention may have a heavy
chain CDR1 with an amino acid sequence of THYVS (SEQ ID NO: 18), a
heavy chain CDR 2 with an amino acid sequence of WIFGGSARTNYNQKFQG
(SEQ ID NO: 17), and a heavy chain CDR3 with an amino acid sequence
of QVGWDDALDF (SEQ ID NO: 16).
[0101] Additionally, the ChGM2 mAb may have a light chain CDR1 with
an amino acid sequence of RSSQSLFSGNYNYLA (SEQ ID NO: 15), a heavy
chain CDR 2 with an amino acid sequence of YASTRHT (SEQ ID NO: 14),
and a heavy chain CDR3 with an amino acid sequence of QQHYSSPRT
(SEQ ID NO: 13).
Humanized Antibodies
[0102] In addition to the chimeric antibodies made for the present
invention, other humanized antibodies may be produced that reduce
the undesirable immunological response toward non-human antibodies
in a human patient. These humanized antibody molecules can have an
antigen-binding site derived from the hamster antibodies. For
example, the non-human CDRs described above, can be grafted into
human FWR and fused to a human constant domain. Winter et al.
Nature 349:293-299 (1991); Lobuglio et al. Proc. Nat. Acad. Sci.
USA 86:4220-4224 (1989); Shaw et al. J Immunol. 138:4534-4538
(1987); and Brown et al. Cancer Res. 47:3577-3583 (1987). The
hamster CDRs may be grafted into a human supporting FWR prior to
fusion with an appropriate human antibody constant domain.
Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et al.
Science 239:1534-1536 (1988); and Jones et al. Nature 321:522-525
(1986). Non-human CDRs can be supported by recombinantly-veneered
FWRs. European Patent Publication No. 519,596, published Dec. 23,
1992. These "humanized" molecules are designed to minimize unwanted
immunological response toward non-human antihuman antibody
molecules that limits the duration and effectiveness of therapeutic
applications of those moieties in human recipients. Similarly,
"primatized" antibodies are designed to minimize unwanted
immunological response toward non-primate anti-primate antibody
molecules the limits the duration and effectiveness of therapeutic
applications of those moieties in primate recipients (e.g., humans,
chimpanzees, gorillas, orangutans, etc.).
[0103] The terms "veneered FWRs" and "recombinantly veneered FWRs"
refer to the selective replacement of FWR residues from, e.g., a
hamster heavy or light chain V region, with human FWR residues in
order to provide a xenogeneic molecule comprising an
antigen-binding site which retains substantially all of the native
FWR folding structure. Veneering techniques are based on the
understanding that the ligand-binding characteristics of an
antigen-binding site are determined primarily by the structure and
relative disposition of the heavy and light chain CDR sets within
the antigen-binding surface (Dayies et al. Ann. Rev. Biochem.
59:439-473 (1990)). Thus, antigen binding specificity can be
preserved in a humanized antibody only wherein the CDR structures,
their interaction with each other, and their interaction with the
rest of the V region domains are carefully maintained. By using
veneering techniques, exterior (eg., solvent-accessible) FWR
residues, which are readily encountered by the immune system, are
selectively replaced with human residues to provide a hybrid
molecule that comprises either a weakly immunogenic, or
substantially non-immunogenic, veneered surface.
[0104] The process of veneering makes use of the available sequence
data for human antibody variable domains compiled by Kabat et al.,
in Sequences of Proteins of Immunological Interest, 4th ed., (U.S.
Dept. of Health and Human Services, U.S. Government Printing
Office, 1987), updates to the Kabat database, and other accessible
U.S. and foreign databases (both nucleic acid and protein). Solvent
accessibilities of V region amino acids can be deduced from the
known three-dimensional structure for human and non-human antibody
fragments.
[0105] There are two general steps in veneering a non-human
antigen-binding site. Initially, the FWRs of the variable domains
of an antibody molecule of interest are compared with corresponding
FWR sequences of human variable domains available databases. The
most homologous human V regions are then compared residue by
residue to corresponding non-human amino acids. The residues in the
non-human FWR that differ from the human counterpart are replaced
by the residues present in the human moiety using recombinant
techniques well known in the art. Residue switching is carried out
with moieties that are at least partially exposed (solvent
accessible), and care is exercised in the replacement of amino acid
residues that may have a significant effect on the tertiary
structure of V region domains, such as proline, glycine and charged
amino acids.
[0106] In this manner, the resultant "veneered" non-human
antigen-binding sites are thus designed to retain the non-human CDR
residues, the residues substantially adjacent to the CDRs, the
residues identified as buried or mostly buried (solvent
inaccessible), the residues believed to participate in non-covalent
(e.g., electrostatic and hydrophobic) contacts between heavy and
light chain domains, and the residues from conserved structural
regions of the FWRs which are believed to influence the "canonical"
tertiary structures of the CDR loops. These design criteria are
then used to prepare recombinant nucleotide sequences that combine
the CDRs of both the heavy and light-chain of a non-human
antigen-binding site into human-appearing FWRs that can be used to
transfect mammalian cells for the expression of recombinant human
antibodies that exhibit the antigen specificity of the non-human
antibody molecule.
Antibody Fragments
[0107] The present invention encompasses new antitumor antibodies
and any fragments thereof containing the active binding region of
the antibody, such as Fab, F(ab')2, and Fv fragments. Such
fragments can be produced from the antibody using techniques well
established in the art (see, e.g., Rousseaux et al., in Methods
Enzymol., 121:663-69 Academic Press, (1986)). For example, the
F(ab').sub.2 fragments can be produced by pepsin digestion of the
antibody molecule, and the Fab fragments can be generated by
reducing the disulphide bridges of the F(ab').sub.2 fragments.
Utility of Antibodies
[0108] The antibodies described herein have a variety of uses. The
antibodies can be used in vitro for diagnostic purposes to
determine the presence of malignant cells in human tissues. The
method involves examining a tissue sample for the presence of
monosialo-GM2. For example, the tissue sample can be contacted with
the monoclonal antibody produced by the hybridoma cell line ATCC
Accession NO PTA-377, Accession No. PTA-405, or Accession No.
PTA-404, and the ability of the antibody to specifically bind to
the cells in the tissue sample is determined. Binding indicates the
presence of a tumor cell. Alternatively, the antibody can also be
used to screen blood samples for released antigen.
[0109] The antibodies can also be used to localize a tumor in vivo
by administering to a subject an isolated antibody of the present
invention that is labeled with a reporter group which gives a
detectable signal. The bound antibodies are then detected using
external scintigraphy, emission tomography, or radionuclear
scanning. The method can be used to stage a cancer in a patient
with respect to the extent of the disease and to monitor changes in
response to therapy.
[0110] The antibodies also have therapeutic applications. The new
antibodies can be used to treat tumors, because specific binding of
the antibody to the tumor cell causes the cell to stop
proliferating and to die. The antibodies of the invention can be
used to treat myeloma, small cell lung carcinoma, or melanoma. They
can be used to treat other cancers, e.g., hematological
malignancies, breast cancers, ovarian cancers, uterine cancers,
lung cancers, GI cancers (including those affecting the oropharynx,
esophagus, stomach, small and large intestine, rectum), pancreatic
cancer, liver cancer, biliary cancers, kidney cancer, skin cancers,
adrenal cancers, endocrine cancers, brain cancers, neural cancers,
bladder cancer, bone cancer, connective tissue cancers, squamous
cell carcinoma, adenocarcinoma, and mesothelioma, provided that the
cancer expresses or over-expresses monosialo-GM2.
[0111] The antibodies can also be used therapeutically, e.g., as
targeting agents, to deliver antitumor agents to the tumors. Such
anti-tumor agents include chemotherapeutic drugs, toxins,
immunological response modulators, enzymes, and radioisotopes.
Detectable Labels
[0112] The antibodies that react with monosialo-GM2 can be used
diagnostically, e.g., to detect the presence of a tumor in a sample
in vitro, or to locate and/or image a tumor in a subject. Detection
can be facilitated by coupling the antibody to a detectable label.
Examples of detectable labels include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials,
bioluminescent materials, electron dense labels, labels for MRI,
and radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
Antibodies as Targeting Agents
[0113] The antibodies and antibody fragments described herein can
be conjugated to a moiety and the antibody can be used to direct
the moiety to the site of a tumor cell which expresses
monosialo-GM2. Examples of moieties include toxins, radionuclides,
or chemotherapeutic agents which can be used to kill tumor cells,
or imaging agents which can be used to locate and size tumors
expressing monosialo-GM2. The antibodies used to direct the moiety
to the tumor in humans are preferably monoclonal antibodies, e.g.,
a humanized monoclonal antibodies.
[0114] The antibody can be fused to the moiety, e.g., the toxin,
either by virtue of the moiety and the antibody being encoded by a
fused gene which encodes a hybrid protein molecule, or by means of
conjugation, e.g., a non-peptide covalent bond, e.g., a non-amide
bond, which is used to join separately produced antibody and the
moiety.
[0115] The antibody described herein can also be fused to another
antibody that is specific for immune cells and stimulates the
immune cells to kill the tumor.
Toxins
[0116] Useful toxin molecules that can be linked to the new
antibodies include peptide toxins, which are significantly
cytotoxic when present intracellularly. "Linked" mean attached or
bound covalently or non-covalently with a bond that cannot by
disrupted under normal physiological conditions for at least 24
hours. Examples of other toxins include cytotoxins, metabolic
disrupters (inhibitors and activators) that disrupt enzymatic
activity and thereby kill tumor cells, and radioactive molecules
that kill all cells within a defined radius of the effector
portion. A metabolic disrupter is a molecule, e.g., an enzyme or a
cytokine, that changes the metabolism of a cell such that its
normal function is altered. Broadly, the term toxin includes any
effector that causes death to a tumor cell.
[0117] Many peptide toxins have a generalized eukaryotic receptor
binding domain; in these instances the toxin must be modified to
prevent killing cells not bearing the targeted protein (e.g., to
prevent killing of cells not bearing monosialo-GM2 but having a
receptor for the unmodified toxin). Such modifications must be made
in a manner that preserves the cytotoxic function of the molecule.
Potentially useful toxins include, but are not limited to:
diphtheria toxin, cholera toxin, ricin, .alpha.-Shiga-like toxin
(SLT-I, SLT-II, SLT-II.sub.v), LT toxin, C3 toxin, Shiga toxin,
pertussis toxin, tetanus toxin, Pseudomonas exotoxin, alorin,
saponin, modeccin, and gelanin. Other toxins include tumor necrosis
factor alpha (TNF-.alpha.) and lymphotoxin (LT). Another toxin
which has antitumor activity is calicheamicin gamma 1, a diyne-ene
containing antitumor antibiotic with considerable potency against
tumors (Zein, N., et al., Science, 240:1198-201 (1988)).
[0118] As an example, diphtheria toxin can be conjugated to the
antibodies described herein. Diphtheria toxin, whose sequence is
known, is described in detail in Murphy, U.S. Pat. No. 4,675,382,
which is incorporated herein by reference. The natural diphtheria
toxin molecule secreted by Corynebacterium diphtheriae consists of
several functional domains that can be characterized, starting at
the amino terminal end of the molecule, as enzymatically-active
Fragment A (amino acids Gly.sub.1-Arg.sub.193) and Fragment B
(amino acids Ser.sub.194-Ser.sub.535), which includes a
translocation domain and a generalized cell binding domain (amino
acid residues 475 through 535).
Linkage of Toxins to Antibodies
[0119] The antibody and the toxin moiety can be linked in any of
several ways. If the compound is produced by expression of a fused
gene, a peptide bond serves as the link between the cytotoxin and
the antibody. Alternatively, the toxin and the antibody can be
produced separately and later coupled by means of a non-peptide
covalent bond. For example, the covalent linkage may take the form
of a disulfide bond. In this case, the DNA encoding this antibody
can be engineered, by conventional methods, to contain an extra
cysteine codon.
[0120] For a disulfide bond linkage, the toxin molecule is also
derivatized with a sulfhydryl group reactive with the cysteine of
the modified antibody. In the case of a peptide toxin this linkage
can be accomplished by inserting a cysteine codon into the DNA
sequence encoding the toxin. Alternatively, a sulfhydryl group,
either by itself or as part of a cysteine residue, can be
introduced using solid phase polypeptide techniques. For example,
the introduction of sulfhydryl groups into peptides is described by
Hiskey, Peptides, 3:137 (1981).
[0121] Derivatization can also be carried out according to the
method described for the derivatization of a peptide hormone in
Bacha et al., U.S. Pat. No. 4,468,382. The introduction of
sulfhydryl groups into proteins is described in Maasen et al., Eur.
J. Biochem., 134:32 (1983). Once the required sulfhydryl groups are
present, the cytotoxin and the antibody are purified, both sulfur
groups are reduced, cytotoxin and antibody are mixed (in a ratio of
about 1:5 to 1:20), and disulfide bond formation is allowed to
proceed to completion (generally 20 to 30 minutes) at room
temperature. The mixture is then dialyzed against phosphate
buffered saline to remove unreacted antibody and toxin molecules.
Sephadex.sup.R chromatography or the like is used to separate the
desired toxin-antibody conjugate compounds from toxin-toxin and
antibody-antibody conjugates on the basis of size.
Immune Response Modulators
[0122] The antitumor moiety can also be a modulator of the immune
system that either activates or inhibits the body's immune system
at the local level. For example, cytokines, e.g., lymphokines such
as IL-2, delivered to a tumor can cause the proliferation of
cytotoxic T-lymphocytes or natural killer cells in the vicinity of
the tumor.
Radioactive Molecules
[0123] The moiety or reporter group can also be a radioactive
molecule, e.g., a radionucleotide, or a so-called sensitizer, e.g.,
a precursor molecule, that becomes radioactive under specific
conditions, e.g., boron when exposed to a beam of low-energy
neutrons, in the so-called "boron neutron capture therapy" (BNCT).
Barth et al., Scientific American, October 1990:100-107 (1990).
Compounds with such radioactive effector portions can be used both
to inhibit tumor cell proliferation and to label the tumor cells
for imaging purposes.
[0124] Radionuclides are single atom radioactive molecules that can
emit either .alpha., .beta., or .gamma. particles. Alpha particle
emitters are preferred to .beta. or U particle emitters, because
they release far higher energy emissions over a shorter distance,
and are therefore efficient without significantly penetrating, and
harming, normal tissues. Suitable particle emitting radionuclides
include .sup.211At, .sup.212Pb, and .sup.212Bi.
[0125] The radioactive molecule must be tightly linked to the
antibody, e.g., either directly or by a bifunctional chelate. This
chelate must not allow elution and thus premature release ofthe
radioactive molecule in vivo. (see, e.g.,Waldmann, Science,
252:1657-62-(1991)).
[0126] For example, to adapt BNCT to the present invention, a
stable isotope of boron, e.g., boron 10, is selected as the
antitumor moiety or effector portion of the compound. The boron is
delivered to and concentrates in or on the tumor cells by the
specific binding of the antibody to the tumor cell. After a time
that allows a sufficient amount of the boron to accumulate, the
tumor is imaged and irradiated with a beam of low-energy neutrons,
having an energy of about 0.025 eV. While this neutron irradiation,
by itself, causes little damage to either the healthy tissue
surrounding the tumor, or the tumor itself, boron 10 (e.g., on the
surface of a tumor cell) captures the neutrons, thereby forming an
unstable isotope, boron 11. Boron 11 instantly fissions yielding
lithium 7 nuclei and energetic particles, about 2.79 million Ev.
These heavy particles are a highly lethal, but very localized, form
of radiation, because particles have a path length of only about
one cell diameter (10 microns).
[0127] Calculations have shown that to destroy a tumor cell, about
one billion boron atoms are required along with a flow of thermal
neutrons of from 10.sup.12 to 10.sup.13 neutrons per square
centimeter, so that the radiation generated by the particles
exceeds the background radiation generated by neutron capture
reactions with nitrogen and hydrogen.
Imaging Moieties
[0128] The antibodies described herein specifically bind to
monosialo-GM2 and are thus also useful to detect and/or image human
tumors. One such approach involves the detection of tumors in vivo
by tumor imaging techniques using the antibody labeled with an
appropriate moiety or reporter group, e.g., an imaging reagent that
produces a detectable signal. Imaging reagents and procedures for
labeling antibodies with such reagents are well known (see, e.g.,
Wensel and Meares, Radio Immunoimaging and Radioimmunotherapy,
Elsevier, N.Y. (1983); Colcher et al., Meth. Enzymol., 121:802-16
(1986)). The labeled antibody can be detected by a technique such
as radionuclear scanning (see, e.g., Bradwell et al. in Monoclonal
Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.),
pp. 65-85, Academic Press (1985)), or magnetic resonance
imaging.
Administration
[0129] The antibodies described herein can be administered to a
subject, e.g., an animal or a human, to image or treat tumors. The
antibodies can be administered alone, or in a mixture, e.g., in the
presence of a pharmaceutically acceptable excipient or carrier
(e.g., physiological saline). The excipient or carrier is selected
on the basis of the mode and route of administration. Suitable
pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences (E. W. Martin), a well known reference text in this field,
and in the USP/NF (United States Pharmacopeia and the National
Formularly).
Pharmaceutical Compositions
[0130] In some embodiments, the present invention concerns
formulation of one or more of the anti-monosialo-GM2 antibodies
disclosed herein in pharmaceutically-acceptable carriers for
administration to a cell or an animal, either alone, or in
combination with one or more other modalities of therapy.
[0131] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes, or multiple dose vials made of glass or plastic.
Carriers and Buffers
[0132] It will be understood that, if desired, a composition as
disclosed herein may be administered in combination with other
agents as well, such as, e.g., other proteins or various
pharmaceutically-active agents. The compositions may thus be
delivered along with various other agents as required in the
particular instance. Such compositions may be purified from host
cells or other biological sources, or alternatively may be
chemically synthesized.
[0133] Therefore, in another aspect of the present invention,
pharmaceutical compositions are provided comprising one or more of
the anti-monsialo-GM2 antibodies described herein in combination
with a physiologically acceptable carrier. In certain preferred
embodiments, the pharmaceutical compositions of the invention
include a ChGM2 mAb for use in prophylactic and/or therapeutic
applications.
[0134] It will be apparent that any of the pharmaceutical
compositions described herein can contain pharmaceutically
acceptable salts of the anti-monosialo-GM2 antibodies. Such salts
can be prepared, for example, from pharmaceutically acceptable
non-toxic bases, including organic bases (e.g., salts of primary,
secondary and tertiary amines and basic amino acids) and inorganic
bases (e.g., sodium, potassium, lithium, ammonium, calcium and
magnesium salts).
[0135] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will typically vary depending
on the mode of administration. Compositions of the present
invention may be formulated for any appropriate manner of
administration, including for example, topical, oral, nasal,
mucosal, intravenous, intracranial, intraperitoneal, subcutaneous
and intramuscular administration.
[0136] Carriers for use within such pharmaceutical compositions are
biocompatible, and may also be biodegradable. In certain
embodiments, the formulation may provide a relatively constant
level of active component release. In other embodiments, however, a
more rapid rate of release immediately upon administration may be
desired. The formulation of such compositions is well within the
level of ordinary skill in the art using known techniques.
Illustrative carriers useful in this regard include microparticles
of poly(lactide-co-glycolide), polyacrylate, latex, starch,
cellulose, dextran and the like. Other illustrative delayed-release
carriers include supramolecular biovectors, which comprise a
non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or
oligosaccharide) and, optionally, an external layer comprising an
amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat.
No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO
96/06638). The amount of active compound contained within a
sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0137] The pharmaceutical compositions of the invention will often
further comprise one or more buffers (e.g., neutral buffered saline
or phosphate buffered saline), carbohydrates (e.g., glucose,
mannose, sucrose or dextrans), mannitol, proteins, gangliosides or
amino acids such as glycine, antioxidants, bacteriostats, chelating
agents such as EDTA or glutathione, adjuvants (e.g., aluminum
hydroxide), solutes that render the formulation isotonic, hypotonic
or weakly hypertonic with the blood of a recipient, suspending
agents, thickening agents and/or preservatives. Alternatively,
compositions of the present invention may be formulated as a
lyophilizate.
Polynucleotide-based Compositions
[0138] In another embodiment, illustrative compositions of the
present invention comprise DNA encoding one or more of the
anti-ganglioside antibodies as described above, such that the
antibody is generated in vivo. The polynucleotide may be
administered within any of a variety of delivery systems known to
those of ordinary skill in the art. Indeed, numerous gene delivery
techniques are Well known in the art, such as those described by
Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998,
and references cited therein. Appropriate polynucleotide expression
systems will, of course, contain the necessary DNA regulatory
sequences for expression in a patient (such as a suitable promoter
and terminating signal).
Packaging
[0139] The pharmaceutical compositions described herein may be
presented in unit-dose or multi-dose containers, such as sealed
ampoules or vials, along with instructions for use, e.g., to treat
a specific cancer. Such containers are typically sealed in such a
way to preserve the sterility and stability of the formulation
until use. In general formulations may be stored as suspensions,
solutions or emulsions in oily or aqueous vehicles. Alternatively,
a pharmaceutical composition may be stored in a freeze-dried
condition requiring only the addition of a sterile liquid carrier
immediately prior to use.
Dosing, Delivery, and Treatment Regimens:
[0140] The development of suitable dosing and treatment regimens
for using the particular compositions described herein in a variety
of treatment regimens, including e.g., oral, parenteral,
intravenous, intranasal, and intramuscular administration and
formulation, is well known in the art, some of which are briefly
discussed below for general purposes of illustration.
[0141] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or intraperitoneally. Such
approaches are well known to the skilled artisan, some of whicn are
further described, for example, in U.S. Pat. No. 5,543,158; U.S.
Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain
embodiments, solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations generally will
contain a preservative to prevent the growth of microorganisms.
[0142] Illustrative pharmaceutical forms suitable for injectable
use include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions (for example, see U.S. Pat. No.
5,466,468). In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and/or vegetable oils. Proper
fluidity may be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and/or by the use of surfactants. The
prevention of the action of microorganisms can be facilitated by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0143] In some embodiments, for parenteral administration in an
aqueous solution, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, a sterile aqueous medium that can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. Moreover, for human administration, preparations
will of course preferably meet sterility, pyrogenicity, and the
general safety and purity standards as required by FDA Office of
Biologics standards.
[0144] In another embodiment of the invention, the compositions
disclosed herein may be formulated in a neutral or salt form.
Illustrative pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective.
[0145] The carriers can further comprise any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human.
[0146] The anti-monosialo-GM2 antibody compositions described
herein may be used in therapeutic methods for the treatment of
cancer. For example, a pharmaceutical composition containing the
ChGM2 antibodies of the invention, may be administered to a human
patient. Such pharmaceutical compositions may be administered
either prior to or following surgical removal of primary tumors
and/or treatment such as administration of radiotherapy or
conventional chemotherapeutic drugs. Administration of the ChGM2
antibody compositions may be by any suitable method, including
administration by intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal, intradermal, anal, vaginal, topical and
oral routes.
[0147] Anti-monosialo-GM2 specific antibodies of the present
invention may be employed in passive immunotherapeutic methods.
These treatment methods involve the delivery of ChGM2 antibodies
that can directly or indirectly mediate antitumor effects, such as
inducing apoptosis and/or inhibiting proliferation of the tumor
cell to which the antibody binds. Such immunotherapeutic methods do
not necessarily depend on an intact host immune system because of
the anti-monosialo-GM2 antibody's ability to induce apoptosis in a
tumor cell to which it binds.
[0148] Routes and frequency of administration of the therapeutic
compositions of the present invention, as well as dosage, will vary
from individual to individual, and may be readily established using
standard techniques. In general, the pharmaceutical compositions
may be administered by injection (e.g., intracutaneous,
intramuscular, intravenous or subcutaneous), intranasally (e.g., by
aspiration) or orally.
[0149] An appropriate dosage and treatment regimen provides the
active compound(s) in an amount sufficient to provide therapeutic
and/or prophylactic benefit. Such a response can be monitored by
establishing an improved clinical outcome (e.g., more frequent
remissions, complete or partial, or longer disease-free survival)
in treated patients as compared to non-treated patients. Typically,
a suitable dose is an amount of a compound that, when administered
as described above, is capable of promoting an anti-tumor response,
and is at least 10-50% above the basal (i.e., untreated) level.
[0150] The most effective mode of administration and dosage regimen
for the compositions of this invention depend upon the severity and
course of the disease, the patient's health and response to
treatment, and the judgment of the treating physician. Accordingly,
the dosages of the compositions should be titrated to the
individual patient. An effective dose of the antibody composition
of this invention is in the range of from about 1 ug to about 5000
mg, preferably about 1 to about 500 mg, or preferably about 100-200
mg.
Diagnostic Kits
[0151] The invention also encompasses diagnostic kits for carrying
out the methods disclosed above. The diagnostic kit includes (a) a
monoclonal antibody described herein, and (b) a conjugate of a
specific binding partner for the antibody and a label for detecting
bound antibody. The kit may also include ancillary agents such as
buffering agents and protein stabilizing agents, e.g.,
polysaccharides and the like. The diagnostic kit may further
include, where necessary, other components of a signal-producing
system including agents for reducing background interference,
control reagents, and an apparatus for conducting a test. In
another embodiment, the diagnostic kit includes a conjugate of a
rnonoclonal antibody of the invention and a label capable of
producing a detectable signal. Ancillary agents as mentioned above
may also be present. Instructions on how to use the diagnostic kit
are generally also included.
EXAMPLES
[0152] The following examples are given to illustrate various
embodiments that have been made of the present invention. It is to
be understood that the following examples are not comprehensive or
exhaustive of the many types of embodiments that can be prepared in
accordance with the present invention.
Example 1
Biological Characterization of Hamster Monoclonal Antibody DMF
10.167.4
[0153] The hamster monoclonal antibody (mAb) DMF 10.167.4 and the
clonally related antibody DMF10.62.3 bind to the cell surface of
murine thymic lymphoma cells. Antibodies DMF 10.167.4 and
DMF10.62.3 were derived from the same fusion. The DMF 10.167.4 mAb
induces apoptosis and inhibits cellular proliferation of cells to
which the antibody binds. Three hamster monoclonal antibodies (DMF
10.62.3, DMF 10.167.4, and DMF 10.34.36), were raised against
E710.2.3 mouse thymic lymphoma cells, that bound to a cell surface
ligand on a large number of cell lines including a few normal
primary lines as well as tumor cell lines.
[0154] The DMF 10.167.4 mAb antibody was tested for ganglioside
specificity. To determine ligand specificity, individual wells of
ELISA plates were coated overnight, with monosialo-GM2, asialo-GM2,
disialo-GM2, monosialo-GM3, disialo-GD1a, disialo-GD1b, asialo-GM1,
monosialo-GM1, lysosialo-GM1, trisialo-GT1b, and disialo-GD3
gangliosides (all from Sigma; St. Louis, Mo.). Wells were blocked
for 2 hours with blocking buffer (PBS+1% BSA) and incubated with
primary antibodies, either DMF 10.167.4, DMF 10.62.3, or hamster
immunoglobulin (Ig) as a control, diluted 1 .mu.g/ml in blocking
buffer for 1 hour. Following 5 washes in PBS, plates were incubated
with goat anti-hamster Ig-HRP (Pharmingen; San Diego, Calif.) for 1
hour in PBS+1% BSA. Plates were washed in PBS and developed in TMB
substrate for 10 minutes before quenching with 1M NaOH. Absorbance
was measured at 450 nm-570 nm by an ELISA plate reader. (FIG. 2).
These data show that the DMF 10.167.4 mAb bound specifically only
to monosialo-GM2. The structurally related ganglioside
monosialo-GM3, lacking 1 terminal galactose residue compared to
monosialo-GM2, and monosialo-GM 1, possessing one additional
galactose residue, were not recognized, nor was the sialic acid
deficient ganglioside asialo-GM2. These results suggest that the
epitope recognized by DMF10.167.4 mAb consists of a combination of
the terminal galactose sugar and the sialic acid residue. A
molecular description of these gangliosides is shown in FIG. 1.
Treatment of monosialo-GM2 with neuraminidase A, which cleaves off
the sialic acid residue, led to a loss of DMF10.167.4 binding as
measured by ELISA (data not shown), further validating the epitope
specificity of this mAb.
[0155] Cell surface binding of hamster monoclonal antibody DMF
10.167.4 was determined by flow cytometric methods (i.e. FACS
analysis). Single suspensions of murine thymic lymphoma cells were
generated, cells were washed 3 times with ice cold staining buffer
(PBS+1%BSA+Azide), and were incubated for 30 minutes on ice with 10
.mu.g/ml of Protein A/G purified hamster mAb DMF 10.167.4. The
cells were washed three times with staining buffer and then
incubated with a 1:100 dilution of an anti-hamster IgG-FITC reagent
(Pharmingen; San Diego: Calif.) for 30 minutes on ice. Following
three washes, the cells were re-suspended in staining buffer
containing Propidium Iodide (PI), a vital stain that allows for
identification of permeable cells, and analyzed by FACS.
[0156] Flow cytometric analysis using the DMF 10.167.4 mAb was
performed to determine the extent of coverage of monosialo-GM2
surface expression on several types of human tumor cell lines. As
summarized in Table 1, 90% of the SCLC lines that were tested were
positive for monosialo-GM2 expression. Three lines, NCI-H69, HTB173
and HTB 180 demonstrated weak binding, as approximately 15% of
cells were positive for DMF10.167.4 binding, whereas six lines,
including HTB 175, HTB 171, DMS79, NCI-H128, NCI-H187, and SHP-77
demonstrated strong binding with greater than 40% of cells staining
positive. The SCLC line HTB 172 was negative for monosialo-GM2
expression. For melanoma cell lines, 75% of lines tested expressed
monosialo-GM2, with CHL-1, Me1 S, and Me1 D showing strong positive
staining, whereas MTL450-5 cells were negative. In terms of
monosialo-GM2 expression on the cell surface of other types of
tumor cell lines, two kidney lines, HEK293 and HICK 10-4, were
strongly positive, as were the Jurkat T cell and K562 B cell
leukemia lines, while the HL-60and THP-1 leukemia lines and the 721
B cell line showed no monosialo-GM2 expression. Representative
lines derived from pancreas, breast, prostate, ovarian and myeloma
tumors were also analyzed and demonstrated lower extents of
coverage (33% to 20%) of monosialo-GM2 expression.
[0157] Neither the DMF10.167.4 mAb nor the DMF10.62.3 mAb was able
to recognize monosialo-GM2 in formalin-fixed or frozen tissues by
IHC analysis, suggesting that the epitope recognized by these mAbs
is destroyed during the tissue preparation process. Because of this
absence of epitope recognition, flow cytometry was used to analyze
DMF10.167.4 mAb reactivity on normal cells obtained from Clonetics
Inc. that had been dissociated from normal tissues and propagated
in vitro. This analysis revealed that lung and prostate epithelium,
as well as prostate stroma, human aortic and umbilical endothelium,
and human PBMCs demonstrated no specific binding with this mAb.
Human fibroblasts were weakly positive for monosialo-GM2 (less than
10% positive-staining cells), but were found not to apoptose when
treated with the mAb in vitro (data not shown). These data confirm
the over-expression of monosialo-GM2 on tumor cell lines and the
relative lack of monosialo-GM2 expression on normal tissues, making
this antigen an appropriate target of therapeutic mAbs.
[0158] For analysis of apoptosis induction, 2.times.10.sup.5 murine
thyrnic lymphoma cells were incubated over night with 10 .mu.g/ml
of the DMF 10.167.4 mAb or control mAbs, then assayed for annexin
positivity and active caspase content by incubating the cells with
an annexin V-Alexa488 conjugate (Molecular Probes; Eugene, Oreg.)
or with the CaspaTag.TM. reagent (Intergen; Norcross, Ga.). Cells
were subjected to flow cytometric analysis to determine the amount
of annexin positivity or caspa-tag positivity, respectively, as
measures of induced apoptotic activity.
[0159] To determine the effective of the mAb on cellular
proliferation, 3000 cells/well were plated into a 96 well plate in
triplicate or quadruplicate in a final volume of 200 .mu.l media.
CHL melanoma or K562 leukemia cells were allowed to adhere for at
least four hrs, then 10-20 .mu.g/ml of DMF 10.167.4 mAb or control
hamster mAb was added. Cells were grown for three days, after which
time 25 .mu.Ci of .sup.3H-thymidine was added per well. Following a
four hour incubation, cells were lysed then subjected to counting
on a beta-counter to determine relative levels of .sup.3H
incorporation.
[0160] Surface binding, apoptosis, caspase activation, and
proliferation data are shown in Table 1. These data revealed that
the hamster monoclonal antibody, designated DMF 10.167.4, was
capable of binding to a variety of tumor cell lines from breast,
pancreas, kidney, ovary, melanoma, leukemia, prostate, and
osteosarcoma. Furthermore, this antibody was effective in inducing
apoptosis and caspase activity as well as in decreasing
proliferation in a subset of these tumor cell lines. TABLE-US-00001
TABLE 1 Ex vivo Biological Characterization of Hamster Antibody DMF
10.167.4 Surface Apoptosis Caspase Decreased Tumor Type by FACS by
Annexin Activation Proliferation Source BT474 Breast - -/na ATCC;
Manassas, VA HTB 175 SCLC +++ ++ HTB 171 SCLC +++ DMS 79 SCLC +++
NCI-H128 SCLC +++ NCI-H1H7 SCLC +++ SHP-77 SCLC +++ NCI-H69 SCLC +
HTB 173 SCLC + HTB 180 SCLC + HTB-172 SCLC - MB415 Breast + -/na
ATCC; Manassas, VA SKBR3 Breast - ATCC; Manassas, VA CRL-1687
Pancreas - ATCC; Manassas, VA CRL-1837 Pancreas - - ATCC; Manassas,
VA PCT391-34 Pancreas + - ATCC; Manassas, VA HICK 10-4 Renal +
ATCC; Manassas, VA HEK293 Renal + -/na ATCC; Manassas, VA OTL298-95
Ovarian - ATCC; Manassas, VA ES-2 Ovarian - ATCC; Manassas, VA
OV1063 Ovarian + ATCC; Manassas, VA SKOV3 Ovarian - ATCC; Manassas,
VA CHL-1 Melanoma ++++ ++/m3 ++ 40%(2) ATCC; Manassas, VA MTL450-5
Melanoma - ATCC; Manassas, VA HL-60 Leukemia - ATCC; Manassas, VA
K562 Leukemia +++ ++ + 17%, 25% ATCC; Manassas, VA THP-1 Leukemia -
ATCC; Manassas, VA Du145 Prostate ++ -/na - ATCC; Manassas, VA
LnCap Prostate - ATCC; Manassas, VA PC-3 Prostate - - - ATCC;
Manassas, VA Mel D Melanoma + - - ATCC; Manassas, VA Mel S Melanoma
+ - - ATCC; Manassas, VA Jurkat T Cell + - - ATCC; Leukemia
Manassas, VA 721 B Cell - ATCC; Leukemia Manassas, VA Hela Cervical
- ATCC; Manassas, VA 143BTK Osteosarcoma + ATCC; Manassas, VA RMAS
Mouse +++ ++ ATCC; Manassas, VA N. Normal - - Clonetics;
PrEpithelium Prostate Verviers, Belgium N.Pr. Normal - - Clonetics;
Stromal Prostate Verviers, Belgium N.Lu. Normal Lung - - Clonetics;
Epithelium Verviers, Belgium N. Fibroblast Mouse + - Clonetics;
Verviers, Belgium Adult Spleen Mouse - Adult Mouse - Thymus Adult
BM Mouse - Fetal Mouse + Thymus Activated T Mouse - Cells Activated
B Mouse - Cells Blank cells indicate that cell line was not
tested.
Example 2
Sequence Analysis of cDNA Encoding the Hamster DMF 10.167.4 mAb
[0161] To determine the heavy and light chain variable region cDNA
sequences of the DMF-10.167.4 mAb total RNA was isolated from
.about.2 million cells by extracting the cells with Trizol reagent
(Invitrogen Corp.; Carlsbad, Calif.). First strand cDNA was
generated using the Advantage RT for PCR kit (BD Biosciences
Clontech; Franklin Lakes, N.J.), tailed with dGTP using TdT
(Invitrogen Corp.; Carlsbad, Calif.), and then used as a template
for PCR amplification. The PCR was carried out using a 5' sense
poly-dCTP oligo with a 3' anti-sense kappa or gamma chain constant
region specific oligonucleotiodes for amplification of light and
heavy chains, respectively. The products were subcloned into the
pCR-Blunt vector (Invitrogen Corp.; Carlsbad, Calif.) and subjected
to sequence analysis. Clones 10.167.4H and 10.167.4L were
determined to encode the leader sequence and the heavy chain
variable region (VDJ) and light chain variable region (VJ),
respectively, of the DMF10.167.4 mAb. The sequences are disclosed
herein as SEQ ID NOs: 12 and 11, respectively. The corresponding
variable region sequences minus the endogenous leader sequences are
disclosed herein as SEQ ID NOs: 10 and 9, respectively, and the
corresponding amino acid sequences encoded by the above DNA
sequences are disclosed herein as SEQ ID NOs: 26, 25, 24 and 23,
respectively.
Example 3
Anti-Ganglioside Antibodies-DMF 10.62.3 and DMF 10.167.4 are
Clonally Related
[0162] The DMF10.62.3 antibody disclosed and characterized in U.S.
patent application Ser. No. 09/618,421 is clonally related (i.e.
derived from the same parental B cell clone) to the DMF10.167.4
antibody disclosed therein and further characterized herein and, as
a consequence thereof, both of these antibodies share functional
properties of antigen binding specificity and affinity.
[0163] The nucleotide sequences of the immunoglobulin heavy and
light chains of the anti-monosialo-GM2 hamster mAb DMF10.62.3 were
determined and compared to the corresponding sequences of hamster
mAb DMF10.167.4. Approximately two million DMF10.62.3 hybrid cells
were used to isolate MRNA using Tri-reagent (Gibco; San Diego,
Calif.). First strand cDNA synthesis was carried out using the
Advantage RT for PCR kit (BD Biosciences Clontech; Franklin Lakes,
N.J.). PCR amplification was performed using specific constant
region and degenerate consensus leader oligonucleotide primers
provided in the Ig-prime kit (Novagen, Inc.; Madison, Wis.). PCR
products were suboloned into the pCR-Blunt vector (Invitrogen
Corp.; Carlsbad, Calif.) and subjected to sequencing analysis.
Clones 10.62.3L and 10.62.3H were determined to encode the light
variable region (VJ) and heavy variable region (VDJ), respectively,
of the DMF10.62.3 mAb. Thes sequence of the light chain and heavy
chain variable regions of the DMF10.167.4 mAb were compared to
another hamster mAb, DMF10.62.3, which also was determined to bind
to monosialo-GM2.
[0164] The amino acid sequence of the VJ and VDJ of DMF10.62.3 are
disclosed as SEQ ID NOS: 19 and 20 respectively. The nucleotide
sequence of the VJ and the VDJ of DMF10.62.3 are disclosed as SEQ
ID NOS: 5 and 6 respectively. The sequence of DMF10.62.3 and
DMF10.167.4 were substantially similar with a single residue change
in the light chain variable region and a single residue change in
the heavy chain variable region. The isoleucine at linear position
52 of the of the light chain variable region of DMF10.167.4 as
defined in SEQ ID NO: 23 is replaced with valine in SEQ ID NO: 19,
and the threonine at linear position 78 of the heavy chain variable
region of DMF10.167.4 as defined in SEQ ID NO: 24 is replaced with
lysine in SEQ ID NO: 20. These modifications are not within the
CDRs of the hamster antibodies.
[0165] The near identity of the variable regions and identity of
CDR3 confirm that the DMF10.62.3and DMF10.167.4 mAbs are clonally
related and, consequently, share functional properties such as
antigen binding specificity and affinity.
Example 4
Comparision of DMF10.167.4 mAb and L6-20-4 mAb Ligand
Specificity
[0166] The ligand specificity of the anti-monosialo-GM2 specific
hamster antibody, DMF10.167.4, is distinct from that of the
anti-asialo-GM2 specific mouse antibody, L6-20-4 (ATCC No. HB-8677)
described in U.S. Pat. Nos. 4,935,495 and 5,091,177 to Hellstrom,
et al. The specificity of the DMF10.167.4 mAb was further
characterized by flow cytometry and ELISA and shown to be different
than the specificity of the anti-asialo-GM2 L6-20-4 mAb.
[0167] For flow cytometric analysis single cell suspensions of HEK
and CHL-1 cells were generated. Cells were then washed 3 times with
ice cold staining buffer (PBS+1%BSA+azide). Next, the cells were
incubated for 30 minutes on ice with DMF10.167.4 supernatant or
16-20-4 supernatant. The cells were washed 3 times with staining
buffer and then incubated with a 1:100 dilution of an anti-hamster
IgG-FITC reagent (Pharmingen; San Diego, Calif.) or anti-mouse
IgG-FITC (Southern Biotech; Birmingham, Ala.) for 30 minutes on
ice. Following 3 washes, the cells were resuspended in staining
buffer containing Propidium Iodide (PI), a vital stain that allows
for identification of permeable cells, and analyzed by flow
cytometry. The DMF10.167.4 mAb recognized both HEK and CHL-1 cells
by FACS as demonstrated by the increase in the mean fluorescent
intensity (MFI) of 30 for DMF 10.167.4 mAb vs. Hamster Ig
MFI.about.3 for both HEK and CHL cells whereas L6-20-4 mAb bound
CHL-1 cells (MFI.about.40 vs. irrelevant mouse Ig MFI.about.3) but
not HEK cells (MFI.about.3 vs irrelevant mouse Ig MFI.about.3)
demonstrating that the antibody has binding activity.
[0168] For ELISA analysis monosialo-GM3, monosial-GM2, and
asialo-GM2 were coated onto wells of 96 well plates at 0.5
.mu.g/well in chloroform/methanol. Wells were blocked for 2 hours
with PBS+1% BSA and incubated with primary antibody supernatants,
either DMF 10.167.4 or the L6-20-4 mAb, for 1 hour. Following 5
washes in PBS, plates were incubated with goat anti-hamster or goat
anti-mouse Ig-HRP (Pharningen; San Diego, Calif.) for 1 hour.
Plates were washed in PBS and developed in TMB substrate for 10
minutes before quenching with 1M,NaOH. Absorbance was measured at
450 nm-570 nm by an ELISA plate reader. These results demonstrate
that DMF10.167.4 specifically bound monosialo-GM2 (OD.sub.450 2.64
vs OD.sub.450=0.099 for monosialo-GM3 and 0.139 for asialo-GM2 and
0.159 for methanol) whereas L6-20-4 showed no specific binding to
monosialo-GM2 (OD.sub.450=0.032for methanol, 0.0245 for
monosialo-GM3, 0.026 for monosialo-GM2, and 0.03 for asialo-GM2).
These data collectively confirmed that the DMF10.167.4 mAb bound
specifically to monosialo-GM2, and is of a different specificity
than that of the L6-20-4 mAb.
Example 5
The In Vivo Effect of the DMF10.62.3 Antibody
[0169] AKR mice were injected IV or IP with 5.times.10.sup.6
syngeneic E710.2.3 tumor cells and received saline or an injection
IP of 0.5 mg of control hamster antibody or DMF10.62.3 antibody on
the initial day and again 10 days later. The survival of animals
was followed for 50 days (Table 2). These data show that the
DMF10.62.3 mAb can block tumor formation and prolong viability in
vivo. TABLE-US-00002 TABLE 2 Treatment Survival Average time to
death 1. saline 0% 35 days 2. control antibody 0% 33 days 3.
DMF62.3 100% (No deaths)
Example 6
Human Myeloma Cell-Surface Binding By DMF10.167.4 mAb
[0170] To determine if monosialo-GM2 is expressed on the surface of
myeloma cells and recognized by the DMF10.167.4 mAb,
1.times.10.sup.6 cells were incubated with 10 .mu.g/ml irrelevant
hamster IgG or DMF10.167.4 mAb on ice, then washed 3 times with
staining buffer (PBS+1% BSA+Azide). Cells were then incubated with
FITC-conjugated anti-hamster IgG on ice, and then washed 3 times
with staining buffer. Cells were resuspended in staining buffer
containing propidium iodide, a vital stain that distinguishes
permeable cells from viable cells, then analyzed by flow cytometry.
As demonstrated by the increase in the mean fluorescent intensity
(MFI) values of the DMF10.167.4 mAb compared to irrelevant hamster
IgG, which is a measurement of the relative binding ability, the
DMF10.167.4 mAb was shown to recognize and bind to monosialo-GM2 on
the surface of several human myeloma cell lines, including DP-6
(Irrelevant: 3.03 vs. DMF10.167.4: 93.81), OPM-2 (2.82 vs. 488.1),
U266 (1.86 vs. 232.7), RPMI-8226 (6.14 vs. 150.8) and NCI-H929 (4;2
vs. 62.7). These data indicate that the DMF10.167.4 mab recognizes
monosialo-GM2 on a number of myeloma cell lines and suggest that
the mAb could be used for the therapeutic treatment of multiple
myeloma.
Example 7
Apoptosis of Human Myeloma Cells by DMF10.167.4 mAb Treatment
[0171] To demonstrate the apoptotic potential of the DMF10.167.4
mAb on myeloma cells, 2.times.10.sup.5 U-266 cells were plated and
incubated O/N with 20 .mu.g/ml of the anti-DMF10.167.4 mAb or
irrelevant hamster IgG, then assayed for annexin positivity and
active caspase content by incubating the cells with an annexin
V-Alexa488 conjugate (Molecular Probes). Cells were subjected to
flow cytometric analysis to determine the amount of annexin
positivity as a measure of DMF10.167.4 mAb-induced apoptotic
activity. The irrelevant hamster IgG revealed a background of 9.6%
apoptosis, whereas the DMF10.167.4 mAb was responsible for inducing
31.3% of the cells to undergo apoptosis, a greater than 3-fold
increase. These data indicate that the hamster anti-monosialo-GM2
mAb could be used as a therapeutic antibpdy by targeting
monosialo-GM2 on myeloma tumors.
Example 8
Human Melanoma Cell Surface Binding BY DMF10.167.4 mAb
[0172] To determine if monsialo-GM2 is expressed on the surface of
melanoma cells and recognized by the DMF10.167.4 mAb,
1.times.10.sup.6 CHL-1 cells were incubated with 10 .mu.g/ml
irrelevant hamster IgG or DMF10.167.4 mAb on ice, then washed 3
times with staining buffer (PBS+1% BSA+Azide). Cells were then
incubated with FITC-conjugated anti-hamster IgG on ice, and then
washed 3 times with staining buffer. Cells were resuspended in
staining buffer containing propidium iodide, a vital stain that
distinguishes permeable cells from viable cells, then analyzed by
flow cytometry. As demonstrated by the increase in the mean
fluorescent intensity (MFI) values of the DMF10.167.4 mAb compared
to irrelevant hamster IgG, which is a measurement of the relative
binding ability, the DMF10.167.4 mAb was shown to recognize and
bind to monosialo-GM2 on the surface of the CHL-1 melanoma cell
line (Irrelevant: 3.2 vs. DMF10.167.4: .about.100). These data
indicate that the DMF10.167.4 mAb recognizes monosialo-GM2 on
melanoma cells and suggest that the mAb could be used for the
therapeutic treatment of melanoma.
Example 9
Apoptosis Of Human Melanoma Cells By DMF10.167.4 mAb Treatment
[0173] To demonstrate the apoptotic potential of the DMF10.167.4
mAb on melanoma cells, 2.times.10.sup.5 CHL-1 cells were plated and
incubated O/N with 20 .mu.g/ml of the DMF10.167.4 mAb or irrelevant
hamster IgG, then assayed for annexin positivity and active caspase
content by incubating the cells with an annexin V-Alexa488
conjugate (Molecular Probes). Cells were subjected to flow
cytometric analysis to determine the amount of annexin positivity
as a measure of DMF10.167.4 mAb induced apoptotic activity. The
irrelevant hamster IgG revealed a background of 4.6% apoptosis,
whereas the DMF10.167.4 mAb was responsible for inducing 42.1% of
the cells to undergo apoptosis, a greater than 9-fold increase.
These data indicate that the hamster anti-monosialo-GM2 mAb could
be used as a therapeutic antibody by targeting monosialo-GM2 on
melanoma tumors.
Example 10
Human Small Cell Lung Cancer (SCLC) Surface Binding By DMF10.167.4
mAb
[0174] To determine if monosialo-GM2 is expressed on the surface of
SCLC cells and recognized by the DMF10.167.4 mAb, 1.times.10.sup.6
cells from numerous SCLC cell lines were incubated with 10 .mu.g/ml
irrelevant hamster IgG or DMF10.167.4 mAb on ice, then washed 3
times with staining buffer (PBS+1% BSA+Azide). Cells were then
incubated with FITC-conjugated anti-hamster IgG on ice, and then
washed 3 times with staining buffer. Cells were resuspended in
staining buffer containing propidium iodide, a vital stain that
distinguishes permeable cells from viable cells, then analyzed by
flow cytometry. As demonstrated by the increase in the mean
fluorescent intensity (MFI) values of the DMF10.167.4 mAb compared
to irrelevant hamster IgG, which is a measurement of the relative
binding ability, the DMF10.167.4 mAb was shown to recognize and
bind to monosialo-GM2 on the surface of the SCLC cell lines,
including NCI-H69 (Irrelevant: 3.6 vs. DMF10.167.4: 22.3), NCI-H128
(4.79 vs 115.49), HTB 171(10.44 vs. 673.85), HTB 173 (4.04 vs
20.91), HTB 175 (6.18 vs 730.46), DMS79 (8.38 vs 31.65), HTB 180
(6.21 vs 39.98), NCI-H187 (7.37 vs 374.5) and SHP-77 (5.69 vs
140.1). These data indicate that the DMF10.167.4 mAb recognizes
monosialo-GM2 on SCLC cells and suggest that the mAb could be used
for the therapeutic treatment of small cell lung cancer.
Example 11
DMF10.167.4 mAb Suppresses Human Melanoma Tumor Formation In
Vivo
[0175] To determine if the anti-monosialo-GM2 mAb would suppress
tumor formation (prophylactic model) in mice, we performed tumor
model studies in vivo using the CHL-1 melanoma cell line. Fifteen
female SCID mice were injected sub-cutaneously with
5.times.10.sup.6 CHL-1 cells, and then separated into 3 groups of 5
animals. One group of five mice was untreated, one group received
100 .mu.g of the hamster DMF10.167.4 anti-monosialo-GM2 mAb
intravenously, and one group received 100 .mu.g of an irrelevant
hamster IgG i.v. at day 0 (time of tumor cell injection). On day 4,
the mAb groups received an additional intravenous injection of the
indicated antibodies. Tumor size was measured by caliper for 45
days, using the formula (length.times.width) to measure the tumor
area. All five of the animals that received no treatment or
received irrelevant hamster IgG developed easily detectable tumors
by day 8 (mean tumor area=60 mm.sup.2) which continued to expand
throughout the course of the study, leading to their sacrifice at
day 21 (mean tumor area=130 mm.sup.2). Mice that had received
hamster DMF10.167.4 mAb showed greater than 3-fold repression in
tumor size (mean tumor area=40 mm2) at day 21, and continued to
show repressed growth, for at 45 days, the mean tumor area was 100
mm.sup.2, which was still smaller than controls at day 21. These
data demonstrate the potent anti-tumor formation activity in vivo
of the DMF10.167.4 hamster anti-monosialo-GM2 mAb.
Example 12
Reactivity of Human Myeloma Cells with DMF 10.167.4 Antibody
[0176] The reactivity of human myeloma cells with the antibody DMF
10.167.4 was evaluated by indirect immunofluorescence and flow
cytometry. Human myeloma cell lines were incubated with
DMF.10.167.4 antibody or control hamster Ig on ice for 30-45
minutes. The cells were then washed and incubated with FITC-labeled
anti-hamster Ig on ice for 30-45 minutes. The cells were
subsequently washed and fixed with paraformaldehyde (1%.times.10').
The binding of fluorescent antibody was quantified with a flow
cytometer.
[0177] In total, 6 myeloma cells lines were analyzed. As
demonstrated by the increase in mean fluorescent intensity (MFI)
values of the DMF10.167.4 mAb compared to irrelevant hamster IgG,
which is a measurement of the relative binding ability of the mAb,
the DMF10.167.4 mAb was shown to recognize and bind to
monosialo-GM2 on the surface of myeloma cells lines, including
MM-1S (irrelevant hamster IgG MFI.about.4 vs. DMF10.167.4
MFI.about.40) RPMI-8226 (.about.4 vs. .about.25) and LP1 (.about.4
vs. .about.80). Two additional lines, OCI-My5 and EJM, both
demonstrated weak binding (.about.3 vs. .about.7) and one line,
MM-1R was shown not to express monosialo-GM2 as it demonstrated no
detectable binding.
[0178] The ability of the DMF10.167.4 mAb to inhibit the
proliferation of myeloma cells in vitro was also tested. MM-1S
cells were plated into a 96 well plate in triplicate in a final
volume of 200 ul media. The cells were allowed to adhere for 4
hours, then 2.5-10 ug/ml of DMF10.167.4 mAb or control hamster IgG
was added to the wells. Cells were grown for three days, after
which time 25uCi of .sup.3H-thymidine was added per well. Following
a four hour incubation, cells were lysed and then subjected to
counting on a beta-counter to determine relative levels of .sup.3H
incorporation. The DMF10.167.4 mAb was shown to inhibit the
proliferation of the MM-1S in a dose-dependent manner, as no
treatment of cells yielded a CPM of 23,758, hamster IgG treatment
of 2.5 ug/ml, 5 ug/ml, 10 ug/ml, and 20 ug/ml yielded 22,407,
21334, 16893, and 10818 CPM, respectively, whereas DMF10.167.4
treatment of 2.5 ug/ml, 5 ug/ml, 10 ug/ml, and 20 ug/ml yielded
7414, 2038, 984, and 544 CPM, respectively. Based on the sarne
methods, similar results were observed for RPMI cells: no treatment
of cells yielded a CPM of 2543, hamster IgG treatment of 2.5 ug/ml,
5 ug/ml, 10 ug/ml, and 20 ug/ml yielded 9343, 8036, 9748, and 5296
CPM, respectively, whereas DMF10.167.4 treatment of 2.5 ug/ml, 5
ug/ml, 10 ug/ml, and 20 ug/ml yielded 2626, 1269, 424, and 154 CPM,
respectively. Collectively, these data demonstrate that DMF10.167.4
binds to GM2 expressed on myeloma cells arid can block the
proliferation of said myeloma cells, indicating that these tumors
are a suitable target for DMF10.167.4 mAb immunotherapy.
Example 13
Generation of an Anti-monosialo-GM2 Hamster-Human Chimeric
Monoclonal Antibody
[0179] An anti-GM2 chimeric Monoclonal antibody (ChGM2 mab) was
constructed with the variable regions of a hamster mAb and the
constant regions of a human mAb. The light chain variable region of
the ChGM2 mAb has the amino acid sequence of the light chain
variable region of DMF10.167.4 SEQ ID NO: 23; and the heavy chain
variable region of the ChGM2 mAb has the amino acid sequence of the
heavy chain variable region of DMF10.167.4 of SEQ ID NO: 24. The
constant region of the ChGM2 mAb is from a human IgG 1 isotype
determined to have significant homology to the constant region of
the anti-monosialo-GM2 hamster antibodies. The ChGM2 mAb has the
light chain nucleotide sequence of SEQ ID NO: 7 and a heavy chain
nucleotide sequence of SEQ ID NO: 8. Additionally, a chimeric
antibody of the present invention may have the variable regions of
the DMF10.62.3 monoclonal antibody.
[0180] The chimeric anti-monosialo-GM2 monoclonal antibody (mAb)
was generated by fusing the hamster anti-monosialo-GM2 DMF10.167.4
mAb variable region domains to human IgG1 and kappa constant region
domains. Multiple allotype (and isotype) constant regions could be
used including f, a, z and combinations thereof for the heavy chain
and 1, 2, 3, and combinations thereof for the light chain. Litwin,
S. D. Immunol Sel. (1989) 43:203-236.
[0181] For chimeric generation, the DMF10.167.4 heavy (H) chain
cDNA template) was PCR amplified using a 5' sense oligo
(GTCGGCCGGAAGGGCCTTGGCCCAGGTCCAGCTGCAGCAGTCTG) SEQ ID NO: 4 and a
3' anti-sense oligo
(ATGCTGGGCCCTTGGTGGAGGCTGAGGAGACAGTGACTTGGGTCCCTTGACC) SEQ ID NO:
3, restriction endonuclease digested with SfiI and Apal and
subcloned into an expression vector containing the human IgGI
constant region domains. Likewise, the DMF10.167.4 light (L) chain
cDNA template was PCR amplified using a 5' sense oligo
(ACTGGCCGGAAGGGCCTTGGCCGATATCGTGATGACACAGTCTCCA) SEQ ID NO: 2 and a
3' anti-sense oligo
(AGACAGATGGCGCCGCCACGGTCCGTTTGATTTTCAGCTTGGTGCC) SEQ ID NO: 1,
restriction endonuclease digested with SfiI and KasI and subcloned
into an expression vector containing the human kappa constant
region domain. These H and L chain expression constructs whose ORFs
are defined by SEQ ID NOS: 7, 8, 21, 22 were transfected into
CHO-K1 (ATCC No. CCL-61) cells to produce a chimeric
anti-monosialo-GM2 mab that was subsequently purified by protein A
column chromatography.
[0182] The data presented herein demonstrate that the chimeric
anti-monosialo-GM2 mAb with a hamster variable regions of the
DMF10.167.4 mAb and a human .sub..gamma.1/.kappa. constant regions
retains biological properties ofthe DMF10.167.4 mAb. More
specifically, the chimeric mAb retains the capacity to (1) bind to
tumor cells displaying monosialo-GM2 on their cell surfaces and (2)
to induce apoptosis in the tumor cells to which the antibody
specifically binds.
Example 14
Chimeric Anti-monosialo-GM2 mAb Induces Apoptosis of Several Human
Tumor Cell Lines In Vitro
[0183] In vitro cell culture experiments revealed that the hamster
DMF10.167.4 mAb induced apoptosis in several human tumor lines as
measured by annexin binding, including the Jurkat T cell leukemia
line, the CHL-1 melanoma line and the HTB 175 SCLC line. Because
annexin specifically binds to phosphatidylserine, which flips from
the internal surface of the plasma membrane to the external surface
upon initiation of apoptosis, one can use flow cytometric detection
of annexin binding to evaluate the induction of apoptosis.
[0184] To validate that the ChGM2 mAb retained the
apoptotic-inducing activity in vitro, these same experiments were
repeated using the ChGM2 mAb. Jurkat cells were maintained
overnight in normal growth media with no treatment, or incubated
with either irrelevant human IgG or the ChGM2 mab. While nearly 93%
of cells receiving no treatment or irrelevant human IgG were
viable, incubation with the ChGM2 mAb reduced the population of
live cells to 69% and increased the percentage of apoptotic cells
to 27% compared to 3% for the no treatment or irrelevant IgG
groups. All treatments yielded the same percentage of late
apoptotic or necrotic cells.
[0185] Additional in vitro annexin binding assays were performed
using the CHL-1 melanoma and HTB 175 small cell lung cancer cell
lines. As with the Jurkat cell line, the ChGM2 mAb was able to
induce apoptosis in these lines. In the CHL-1 line, the percentage
of apoptotic cells increased from 10% and 18% for no treatment and
irrelevant human IgG, respectively, to 44% for ChGM2 treated cells.
Moreover, despite the relatively high levels of basal apoptosis in
the HTB175 cell line, treatment with the ChGM2 mAb led to an
increase in the percentage of apoptotic cells compared to those
receiving irrelevant IgG or no treatment (45% vs.32%).
Collectively, these data demonstrate that the chimerization process
has not destroyed the apoptotic activity inherent to the hamster
DMF10.167.4 mAb.
Example 15
Chimeric Anti-monosialo-GM2 Monoclonal Antibody Suppresses Human
Melanoma Tumor Formation In Vivo
[0186] To determine if the anti-monosialo-GM2 mAb would suppress
tumor formation (prophylactic model) in mice, we performed tumor
model studies in vivo using the CHL-1 melanoma cell line. CHL-1
cells, grown in DMEM and 10% DMEM and 10% FCS, were lifted from
tissue culture flasks by cell dissociation solution, washed in
1.times.PBS, then resuspended in 1.times.PBS. Fifteen female SCID
mice were injected sub-cutaneously with 5.times.10.sup.6 CHL-1
cells, and then separated into 3 groups of 5 animals. One group of
five mice was untreated, one group received 100 .mu.g of the ChGM2
anti-monosialo-GM2 mAb intravenously, and one group received 100
.mu.g of an irrelevant human IgG i.v. at day 0 (time of tumor cell
injection). On days 4 and 8, the mAb groups received an additional
intravenous injection of the indicated antibodies. Tumor size was
measured by caliper for 45 days, using the formula
(length.times.width) to measure the tumor area. All five of the
animals that received no treatment or received irrelevant human IgG
developed easily detectable tumors by day 8 (mean tumor area=55-60
mm.sup.2) which continued to expand throughout the course of the
study, leading to their sacrifice at day 21 (mean tumor area=120
mm.sup.2). Mice that had received ChGM2 mAb showed greater than
6-fold repression in tumor size (mean tumor area=20 mm.sup.2) at
day 21, and continued to show repressed growth, for at 45 days, the
mean tumor area was .about.30 mm.sup.2, which was still smaller
than controls at day 21. These data demonstrate the potent
anti-tumor formation activity in vivo of the ChGm2
anti-monosialo-GM2 mAb and its effectiveness in suppressing tumor
cell growth in a prophylactic mouse tumor model system.
Example 16
Therapeutic Human Melanoma Tumor Suppression In Vivo by the
DMF10.Hamster-Human Chimeric Anti-monosialo-GM2 mAb.
[0187] The chimeric anti-monosialo-GM2 antibodies (ChGM2 mAb) of
the present invention are effective in suppressing tumor cell
growth in a therapeutic murine tumor model system employing the
human melanoma cell-line CHL-1.
[0188] A xenograft tumor model using CHL-1 human melanoma cells
implanted in SCID mice was established to test the efficacy of the
anti-monosialo-GM2 monoclonal antibody DMF10.167.4. Approximately
5.times.10.sup.6 CHL-1 tumor cells were implanted, subcutaneously,
in SCID mice. Seven days later, when tumors of approximately 20
square millimeters were established, mice with tumors were randomly
segregated into two groups of six animals. The control group
received 100 .mu.g per day of human IgG antibody on days 7, 11, 14,
18 and 22. The second group received 100 .mu.g per day of chimeric
anti-monosialo-GM2 antibody also on days 7, 11, 14, 18 and 22.
Tumor size was measured for 27 days, using the formula
length.times.width to measure tumor area. All of the six animals
that received irrelevant human IgG had tumors of mean area=100
mm.sup.2 at 26 days, while the 6 mice that received ChGM2 mAb
showed at least a five times reduction in tumor size (mean tumor
area=20 mm.sup.2) at 26 days. These results demonstrate the
suppressive effect of the ChGM2 mAb in melanoma tumors.
Example 17
In Vivo Repression of Human SCLC Tumor Growth with Chimeric
Anti-monosialo-GM2 Antibody
[0189] To extend the functional analysis of the ChGM2 mAb in vivo,
additional SCID tumor model studies were performed using HTB 175
SCLC cells to determine if this mAb could suppress the progression
of established SCLC tumors. Twenty female SCID mice were injected
subcutaneously with 4.times.10.sup.6 HTB 175 SCLC cells and tumors
were allowed to establish for 15 days. The mean tumor area for the
HTB 175 cells was 38 mm.sup.2, approximately twice as large as the
CHL-1 tumors in the previous study. At this time, the mice were
randomized into two groups of 9 mice based on tumor size, and one
group received 100 .mu.g of the ChGM2 mAb and one group received
100 .mu.g of irrelevant human IgG intravenously. On days 19 and 22,
each group received only 80 .mu.g of their respective dose of ChGM2
mAb, and it was delivered i.p. due to difficulties with i.v
delivery during this experiment. Tumor size (area) was measured for
26 days. HTB 175 tumor growth continued in all nine of the animals
that received irrelevant human IgG treatment, whereas the mice that
had received ChGM2 mAb demonstrated a reduction in the progression
of tumor growth. Collectively, these in vivo tumor model studies
demonstrate the potential therapeutic efficacy of this ChGM2 mAb
for the treatment of melanoma and SCLC tumors.
Example 18
In Vivo Apoptosis with Chimeric Anti-monosialo-GM2 Antibody
[0190] To determine if ADCC (antibody-dependent cellular
cytotoxicity) plays a role in the ChGM2 mAbs ability to inhibit
CHL-1 tumor formation in vivo, a tumor model study was carried out
comparing the activity of the chimeric anti-monosialo-GM2
antibodies in SCID mice to SCID/beige mice. Due to the beige
mutation, these mice have been reported to be deficient in
macrophages and possess selectively impaired natural killer cells,
leading to their inability to carry out ADCC. Two groups of five
SCID and two groups of five SCID/beige mice were injected with
5.times.10.sup.6 CHL-1 cells subcutaneously on day 0. The mice were
then injected i.v. with 100 .mu.g/injection of human irrelevant IgG
or ChGM2 mAb on days 0, 3, 7, and 13. Tumor size (area) was
measured for 35 days. Tumor formation was completely suppressed in
4 out of 5 mice in both the SCID and SCID/beige groups after ChGM2
treatment (tumor area=0 mm.sup.2), whereas all 5 SCID mice (tumor
area>120 mm.sup.2 each) and 4 of 5 SCID/beige (tumor area>100
mm.sup.2) formed tumors when injected with irrelevant human IgG.
These results demonstrate that the ChGM2 is able to block tumor
formation without requiring cell-mediated immune effector cells,
suggesting that the apoptotic ability of this antibody alone is
responsible for the lack of tumor formation, and possibly tumor
progression, in vivo, again-supporting the use of the mAb as a
naked, non-conjugated form for the therapeutic treatment of
monosialo-GM2-expressing myeloma tumors.
Example 19
Human Myeloma Cell-Surface Binding by ChGM2mAb
[0191] To determine if monosialo-GM2 is expressed on the surface of
myeloma cells and recognized by the ChGM2 mAb, 1.times.10.sup.6
cells were incubated with 10 .mu.g/ml irrelevant human IgG or ChGM2
mAb on ice, then washed 3 times with staining buffer (PBS+1%
BSA+Azide). Cells were then incubated with FITC-conjugated
anti-human IgG on ice, and then washed 3 times with staining
buffer. Cells were resuspended in staining buffer containing
propidium iodide, a vital stain that distinguishes permeable cells
from viable cells, then analyzed by flow cytometry. As demonstrated
by the increase in the mean fluorescent intensity (MFI) values of
the ChGM2 mAb compared to irrelevant human IgG, which is a
measurement of the relative binding ability, the ChGM2 mAb was
shown to recognize and bind to monosialo-GM2 on the surface of
several myeloma cell lines, including DP-6 (Irrelevant: 6.26 vs.
ChGM2: 24.73), OPM-2 (2.98 vs. 86.27), U266 (1.96 vs. 165.19), and
RPMI-8226 (4.94 vs. 12.38). The ChGM2 bound weakly to the NCI-H929
myeloma line (20 .mu.g/ml; 8.34 vs. 14.34). These data indicate
that the ChGM2 mAb recognizes monosialo-GM2 on a number of myeloma
cell lines and could be used for the therapeutic treatment of
multiple myeloma.
Example 20
Apoptosis of Human Myeloma Cells by ChGM2mAb mAb Treatment
[0192] To demonstrate the apoptotic potential of the ChGM2 mAb on
myeloma cells, 2.times.10.sup.5 U-266 cells were plated and
incubated overnight with 20 .mu.g/ml of the anti-ChGM2 mAb or
irrelevant human IgG, then assayed for annexin positivity by
incubating the cells with an annexin V-Alexa488 conjugate
(Molecular Probes). Cells were subjected to flow cytometric
analysis to determine the amount of annexin positivity as a measure
of ChGM2 mAb-induced apoptotic activity. The irrelevant human IgG
revealed a background of 20.5% apoptosis, whereas the ChGM2 mAb was
responsible for inducing 44% of the cells to undergo apoptosis, a
greater than 2-fold increase. These data indicate that the chimeric
anti-monosialo-GM2 mAb could be used in a naked, non-conjugated
form as a therapeutic treatment of monosialo-GM2-expressing myeloma
tumors due to the apoptotic-inducing and subsequent tumor killing
ability of the mAb.
Example 21
Antibody-Dependent Cellular Cytotoxicity of ChGM2 Antibody
[0193] The ability of an antibody to mediate antibody-dependent
cellular cytotoxicity (ADCC) is a property that can be of
importance in therapeutic methods. An experiment was conducted to
test the ADCC mediating potential in vitro of the chimeric
anti-monosialo-GM2 antibodies of the present invention. Peripheral
blood mononuclear cells (PBMCs), antibodies, and CHL-1 cells were
incubated together in various combinations and then assayed for the
ability of the PBMCs to kill CHL-1 cells in the presence of the
anti-monosialo-GM2 antibody The chimeric anti-monsialo-GM2 antibody
was determined to induce cell-mediated killing in vitro.
[0194] PBMCs were isolated from heparinized whole blood using
Histopaque-1077 (Sigma). The isolated PBMCs were washed three times
in PBS and resuspended at 5.0.times.10.sup.6 cells/100 .mu.l in
phenol red free RPMI (Invitrogen) containing 1% BSA. CHL-1 cells
were lifted off the dish in Cell Dissociation Solution (Sigma),
washed in PBS, and resuspended to 1.0.times.10.sup.4 cells/50 .mu.l
in phenol red free RPMI containing 1% BSA. Human Chimeric
anti-monosialo-GM2 antibody and Human IgG control antibody were
distributed into three 4.times. stocks each of 4 .mu.g/ml, 0.4
.mu.g/ml, and 0.04 .mu.g/ml in phenol red free RPMI containing 1%
BSA.
[0195] Samples for the experiment were prepared in quadruplicate
with the following: 100 .mu.l PBMCs, 50 .mu.l CHL-1, and 50 .mu.l
of antibody (sample subsets included the 3 different concentrations
of antibody using both antibody types, an additional set of
controls following the conditions outlined above but without PBMCs
was done to control for apoptosis induced by the anti-monosialo-GM2
antibody alone). Additionally, a sample set containing just PBMCs
and CHL-1 cells was included as a background control (BC), and
sonicated CHL-1 cells with viable PBMC's was included as the
"maximum release" control, or high control (HC). In samples where
one of the three components was missing, RPMI+1% BSA was
substituted to maintain equal volumes in all samples of 200 .mu.l.
The samples were then incubated at 37.degree. C. for 4 hours in a
7% CO.sub.2 atmosphere.
[0196] Killing of CHL-1 cells by PBMCs was assayed by screening for
the presence of lactate deyhydrogenase (LDH) in the supematant of
each sample (as a cell dies it's membrane becomes permeable, thus
allowing LDH to leak into the medium) using the LDH Cytotoxicity
Detection Kit (Takara Shuzo Co., LTD.). Briefly, 250 .mu.l of
solution A was mixed with 11.25 ml solution B immediately before
use and 100 .mu.l of this working stock (A+B) was added to 100
.mu.l of each sample supernatant (note: samples were spun for 2
minutes at 1800 RPM and 100 .mu.l of supematant was transferred to
a new 96 well plate prior to the addition of LDH substrate).
Samples were developed in the dark for 15 minutes and then read on
a 96 well plate spectrophotometer at 490 nm. Sample absorbance (A)
measurements were converted to percent killing by the following
equation: percent killing=[(A-BC)/(HC-BC)] *100.
[0197] The percent of the CHL-1 cells killed in the presence of the
chimeric anti-monosialo-GM2 was high and dose dependent. In the
samples with 1 .mu.g/ml of the chimeric anti-monosialo-GM2 antibody
and in the presence of PBMCs, about 83% killing was observed. In
the samples with 0.1 .mu.g/ml of the chimeric anti-monosialo-GM2
antibody and in the presence of PBMCs, about 66% killing was
observed. In the samples with 0.01 .mu.g/ml of the chimeric
anti-monosialo-GM2 antibody and in the presence of PBMCs, about 21%
killing was observed. In contrast the human IgG control antibodies
demonstrated low levels of PBMC killing possibly due to nonspecific
binding of the control antibodies to the CHL-1 cells. About 15%,
10%, and 8% killing was observed with the control antibodies in the
presence of PBMCs. Additionally, nonsignificant killing was
observed in the samples without PBMCs and with either the chimeric
anti-monsialo-GM2 antibodies or the control antibodies. It should
be noted that the chimeric anti-monosialo-GM2 has shown apoptotic
properties independent of PBMCs, but due to the short duration of
these assays this apoptotic ability would not be evident. These
data demonstrate that the chimeric anti-monsiaolo-GM2 antibodies of
the present invention are able to mediate antibody-dependent
cellular cytotoxicity.
Deposit Statement
[0198] The hybridoma cell line producing the monoclonal antibody
DMF10.62.3, was received by the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va., on Jul. 20,
1999, and the hybridoma cell lines producing the monoclonal
antibodies DMF10.167.4 and DMF10.34.36 were received by the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va., on Jul. 22, 1999. The hybridomas have
been deposited under conditions that assure that access to the
hybridomas will be available during the pendency of the patent
application disclosing them to one determined by the Commissioner
of Patents and Trademarks to be entitled thereto under 37 CFR 1.14
and 35 USC 122. The deposits are available as required by foreign
patent laws in countries wherein counterparts of the subject
application, or its progeny, are filed. However, it should be
understood that the availability of a deposit does not constitute a
license to practice the subject invention in derogation of patent
rights granted by governmental action. Further, the subject culture
deposits will be stored and made available to the public in accord
with the provisions of the Budapest Treaty for the Deposit of
Microorganism, i.e., they will be stored with all the care
necessary to keep them viable and uncontaminated for a period of at
least five years after the most recent request for the furnishing
of a sample of the deposits, and in any case, for a period of at
least 30 (thirty) years after the date of deposit or for the
enforceable life of any patent which may issue disclosing the
cultures plus five years after the last request for a sample from
the deposit. The depositor acknowledges the duty to replace the
deposit should the depository be unable to furnish a sample when
requested, due to the condition of the deposits. All restrictions
on the availability to the public of the subject culture deposit
will be irrevocably removed upon the granting of a patent
disclosing them.
Other Embodiments
[0199] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
26 1 46 DNA Artificial Synthetic Oligonucleotide 1 agacagatgg
cgccgccacg gtccgtttga ttttcagctt ggtgcc 46 2 46 DNA Artificial
Synthetic Oligonucleotide 2 actggccgga agggccttgg ccgatatcgt
gatgacacag tctcca 46 3 52 DNA Artificial Synthetic Oligonucleotide
3 atgctgggcc cttggtggag gctgaggaga cagtgacttg ggtcccttga cc 52 4 44
DNA Artificial Synthetic Oligonucleotide 4 gtcggccgga agggccttgg
cccaggtcca gctgcagcag tctg 44 5 336 DNA Cricetulus migratorius 5
gacatcgtga tgacacagtc tccatcttcc ttggctgtgt cagcaggaga cacggtcacc
60 atcaactgca ggtccagtca gagtcttttc tctggaaatt ataactattt
ggcttggtac 120 cagcagaaaa cagggcagac tcctaaatta ctggtctctt
acgcatccac tcggcacact 180 ggtgtccctg atcgcttcgt gggcagtgga
tctgggacag atttcattct aaccatctac 240 aatttccaga ctgaagatct
gggagattac tattgccagc agcattacag ttctcctcgg 300 acgtttggac
ctggcaccaa gctgaaaatc aaacgg 336 6 357 DNA Cricetulus migratorius 6
caggtccagc tgcagcagtc tggggctgag ctggtgaaac ccggagcctc agtgaggctg
60 tcctgcaaga cttcaggcta cacgtttacc actcactatg tgagctgggt
gaaacagaag 120 cctggacagg gactggagtg gattggatgg atttttggtg
gaagtgctag aactaattat 180 aatcagaaat tccagggcaa ggccacactg
actgtagaca catcctccag caaggcctac 240 atggatctca gaagcctgac
atctgatgac tctgcagtct atttctgtgt aagacaagta 300 gggtgggacg
atgctctgga tttctggggt caagggaccc aagtcactgt ctcctca 357 7 657 DNA
Artificial Chimeric Cricetulus migratorius and Human 7 gatatcgtga
tgacacagtc tccatcttcc ttggctgtgt cagcaggaga cacggtcacc 60
atcaactgca ggtccagtca gagtcttttc tctggaaact ataactattt ggcttggtac
120 cagcagaaaa cagggcagac tcctaaatta ctgatctctt acgcatccac
tcggcacact 180 ggtgtccctg atcgcttcgt gggcagtgga tctgggacag
atttcattct aaccatctac 240 aatttccaga ctgaagatct gggagattac
tattgccagc agcattacag ttctcctcgg 300 acgtttggac ctggcaccaa
gctgaaaatc aaacggaccg tggcggcgcc atctgtcttc 360 atcttcccgc
catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420
aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg
480 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta
cagcctcagc 540 agcaccctga cgctgagcaa agcagactac gagaaacaca
aagtctacgc ctgcgaagtc 600 acccatcagg gcctgtctag ccccgtcaca
aagagcttca accgcggaga gtgttaa 657 8 1353 DNA Artificial Chimeric
Cricetulus migratorius and Human 8 caggtccagc tgcagcagtc tggggctgag
ctggtgaaac ccggagcctc agtgaggctg 60 tcctgcaaga cttcaggcta
cacgtttacc actcactatg tgagctgggt gaagcagaag 120 cctggacagg
gactggagtg gattggatgg atttttggtg gaagtgctag aactaattat 180
aatcagaaat tccagggcaa ggccacactg actgtagaca catcctccag cacggcctac
240 atggatctca gaagcctgac atctgatgac tctgcagtct atttctgtgt
aagacaagta 300 gggtgggacg atgctctgga tttctggggt caagggaccc
aagtcactgt ctcctcagcc 360 tccaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540
ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac
600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt
tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
960 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 1020 gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg
gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1260
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320 aagagcctct ccctgtctcc gggtaaatga tga 1353 9 336 DNA Cricetulus
migratorius 9 gatatcgtga tgacacagtc tccatcttcc ttggctgtgt
cagcaggaga cacggtcacc 60 atcaactgca ggtccagtca gagtcttttc
tctggaaact ataactattt ggcttggtac 120 cagcagaaaa cagggcagac
tcctaaatta ctgatctctt acgcatccac tcggcacact 180 ggtgtccctg
atcgcttcgt gggcagtgga tctgggacag atttcattct aaccatctac 240
aatttccaga ctgaagatct gggagattac tattgccagc agcattacag ttctcctcgg
300 acgtttggac ctggcaccaa gctgaaaatc aaacgg 336 10 357 DNA
Cricetulus migratorius 10 caggtccagc tgcagcagtc tggggctgag
ctggtgaaac ccggagcctc agtgaggctg 60 tcctgcaaga cttcaggcta
cacgtttacc actcactatg tgagctgggt gaagcagaag 120 cctggacagg
gactggagtg gattggatgg atttttggtg gaagtgctag aactaattat 180
aatcagaaat tccagggcaa ggccacactg actgtagaca catcctccag cacggcctac
240 atggatctca gaagcctgac atctgatgac tctgcagtct atttctgtgt
aagacaagta 300 gggtgggacg atgctctgga tttctggggt caagggaccc
aagtcactgt ctcctca 357 11 396 DNA Cricetulus migratorius 11
atggagtcac acaatgaggt ccttgtgacc ctgctgctct gggtgtctgg tgcctgtgca
60 gatatcgtga tgacacagtc tccatcttcc ttggctgtgt cagcaggaga
cacggtcacc 120 atcaactgca ggtccagtca gagtcttttc tctggaaact
ataactattt ggcttggtac 180 cagcagaaaa cagggcagac tcctaaatta
ctgatctctt acgcatccac tcggcacact 240 ggtgtccctg atcgcttcgt
gggcagtgga tctgggacag atttcattct aaccatctac 300 aatttccaga
ctgaagatct gggagattac tattgccagc agcattacag ttctcctcgg 360
acgtttggac ctggcaccaa gctgaaaatc aaacgg 396 12 414 DNA Cricetulus
migratorius 12 atgggatgga gctggatcat cctcttcctg gtgacagcag
ctacaggtgt ccactcccag 60 gtccagctgc agcagtctgg ggctgagctg
gtgaaacccg gagcctcagt gaggctgtcc 120 tgcaagactt caggctacac
gtttaccact cactatgtga gctgggtgaa gcagaagcct 180 ggacagggac
tggagtggat tggatggatt tttggtggaa gtgctagaac taattataat 240
cagaaattcc agggcaaggc cacactgact gtagacacat cctccagcac ggcctacatg
300 gatctcagaa gcctgacatc tgatgactct gcagtctatt tctgtgtaag
acaagtaggg 360 tgggacgatg ctctggattt ctggggtcaa gggacccaag
tcactgtctc ctca 414 13 9 PRT Cricetulus migratorius 13 Gln Gln His
Tyr Ser Ser Pro Arg Thr 1 5 14 7 PRT Cricetulus migratorius 14 Tyr
Ala Ser Thr Arg His Thr 1 5 15 15 PRT Cricetulus migratorius 15 Arg
Ser Ser Gln Ser Leu Phe Ser Gly Asn Tyr Asn Tyr Leu Ala 1 5 10 15
16 10 PRT Cricetulus migratorius 16 Gln Val Gly Trp Asp Asp Ala Leu
Asp Phe 1 5 10 17 17 PRT Cricetulus migratorius 17 Trp Ile Phe Gly
Gly Ser Ala Arg Thr Asn Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly 18 5
PRT Cricetulus migratorius 18 Thr His Tyr Val Ser 1 5 19 112 PRT
Cricetulus migratorius 19 Asp Ile Val Met Thr Gln Ser Pro Ser Ser
Leu Ala Val Ser Ala Gly 1 5 10 15 Asp Thr Val Thr Ile Asn Cys Arg
Ser Ser Gln Ser Leu Phe Ser Gly 20 25 30 Asn Tyr Asn Tyr Leu Ala
Trp Tyr Gln Gln Lys Thr Gly Gln Thr Pro 35 40 45 Lys Leu Leu Val
Ser Tyr Ala Ser Thr Arg His Thr Gly Val Pro Asp 50 55 60 Arg Phe
Val Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Tyr 65 70 75 80
Asn Phe Gln Thr Glu Asp Leu Gly Asp Tyr Tyr Cys Gln Gln His Tyr 85
90 95 Ser Ser Pro Arg Thr Phe Gly Pro Gly Thr Lys Leu Lys Ile Lys
Arg 100 105 110 20 119 PRT Cricetulus migratorius 20 Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser
Val Arg Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Thr His 20 25
30 Tyr Val Ser Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Trp Ile Phe Gly Gly Ser Ala Arg Thr Asn Tyr Asn Gln
Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser
Ser Lys Ala Tyr 65 70 75 80 Met Asp Leu Arg Ser Leu Thr Ser Asp Asp
Ser Ala Val Tyr Phe Cys 85 90 95 Val Arg Gln Val Gly Trp Asp Asp
Ala Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser
Ser 115 21 218 PRT Artificial Chimeric Cricetulus migratorius and
Human 21 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser
Ala Gly 1 5 10 15 Asp Thr Val Thr Ile Asn Cys Arg Ser Ser Gln Ser
Leu Phe Ser Gly 20 25 30 Asn Tyr Asn Tyr Leu Ala Trp Tyr Gln Gln
Lys Thr Gly Gln Thr Pro 35 40 45 Lys Leu Leu Ile Ser Tyr Ala Ser
Thr Arg His Thr Gly Val Pro Asp 50 55 60 Arg Phe Val Gly Ser Gly
Ser Gly Thr Asp Phe Ile Leu Thr Ile Tyr 65 70 75 80 Asn Phe Gln Thr
Glu Asp Leu Gly Asp Tyr Tyr Cys Gln Gln His Tyr 85 90 95 Ser Ser
Pro Arg Thr Phe Gly Pro Gly Thr Lys Leu Lys Ile Lys Arg 100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115
120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 210 215 22 449 PRT Artificial Chimeric
Cricetulus migratorius and Human 22 Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys
Lys Thr Ser Gly Tyr Thr Phe Thr Thr His 20 25 30 Tyr Val Ser Trp
Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp
Ile Phe Gly Gly Ser Ala Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60
Gln Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Asp Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr
Phe Cys 85 90 95 Val Arg Gln Val Gly Trp Asp Asp Ala Leu Asp Phe
Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 Lys 23 112 PRT Cricetulus migratorius 23 Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Asp Thr
Val Thr Ile Asn Cys Arg Ser Ser Gln Ser Leu Phe Ser Gly 20 25 30
Asn Tyr Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Thr Gly Gln Thr Pro 35
40 45 Lys Leu Leu Ile Ser Tyr Ala Ser Thr Arg His Thr Gly Val Pro
Asp 50 55 60 Arg Phe Val Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu
Thr Ile Tyr 65 70 75 80 Asn Phe Gln Thr Glu Asp Leu Gly Asp Tyr Tyr
Cys Gln Gln His Tyr 85 90 95 Ser Ser Pro Arg Thr Phe Gly Pro Gly
Thr Lys Leu Lys Ile Lys Arg 100 105 110 24 119 PRT Cricetulus
migratorius 24 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Lys Thr Ser Gly Tyr
Thr Phe Thr Thr His 20 25 30 Tyr Val Ser Trp Val Lys Gln Lys Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Phe Gly Gly Ser
Ala Arg Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr
Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu
Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Val
Arg Gln Val Gly Trp Asp Asp Ala Leu Asp Phe Trp Gly Gln Gly 100 105
110 Thr Gln Val Thr Val Ser Ser 115 25 132 PRT Cricetulus
migratorius 25 Met Glu Ser His Asn Glu Val Leu Val Thr Leu Leu Leu
Trp Val Ser 1 5 10 15 Gly Ala Cys Ala Asp Ile Val Met Thr Gln Ser
Pro Ser Ser Leu Ala 20 25 30 Val Ser Ala Gly Asp Thr Val Thr Ile
Asn Cys Arg Ser Ser Gln Ser 35 40 45 Leu Phe Ser Gly Asn Tyr Asn
Tyr Leu Ala Trp Tyr Gln Gln Lys Thr 50 55 60 Gly Gln Thr Pro Lys
Leu Leu Ile Ser Tyr Ala Ser Thr Arg His Thr 65 70 75 80 Gly Val Pro
Asp Arg Phe Val Gly Ser Gly Ser Gly Thr Asp Phe Ile 85 90 95 Leu
Thr Ile Tyr Asn Phe Gln Thr Glu Asp Leu Gly Asp Tyr Tyr Cys 100 105
110 Gln Gln His Tyr Ser Ser Pro Arg Thr Phe Gly Pro Gly Thr Lys Leu
115 120 125 Lys Ile Lys Arg 130 26 138 PRT Cricetulus migratorius
26 Met Gly Trp Ser Trp Ile Ile Leu Phe Leu Val Thr Ala Ala Thr Gly
1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Lys 20 25 30 Pro Gly Ala Ser Val Arg Leu Ser Cys Lys Thr Ser
Gly Tyr Thr Phe 35 40 45 Thr Thr His Tyr Val Ser Trp Val Lys Gln
Lys Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Phe Gly
Gly Ser Ala Arg Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Gln Gly Lys
Ala Thr Leu Thr Val Asp Thr Ser Ser Ser 85 90 95 Thr Ala Tyr Met
Asp Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Phe
Cys Val Arg Gln Val Gly Trp Asp Asp Ala Leu Asp Phe Trp 115 120 125
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 130 135
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