U.S. patent application number 11/704925 was filed with the patent office on 2007-07-05 for development of human monoclonal antibodies and uses thereof.
This patent application is currently assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. Invention is credited to Ilya Trakht.
Application Number | 20070154995 11/704925 |
Document ID | / |
Family ID | 21913224 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070154995 |
Kind Code |
A1 |
Trakht; Ilya |
July 5, 2007 |
Development of human monoclonal antibodies and uses thereof
Abstract
The present invention provides a heteromyeloma cell other than
B6B11, capable of producing a trioma cell when fused with a human
lymphoid cell, wherein the trioma cell is capable of producing a
tetroma cell capable of producing a monoclonal antibody having
specific binding affinity for an antigen, when fused with a second
human lymphoid cell, the second human lymphoid cell being capable
of producing antibody having specific binding affinity for the
antigen. The invention provides a trioma cell fusion partner which
does not produce any antibody obtained by fusing a hetermomyeloma
cell which does not produce any antibody with a human lymphoid
cell. The invention provides a tetroma cell capable of producing a
monoclonal antibody having specific binding affinity for an antigen
obtained by fusing a trioma cell which does not produce any
antibody with a human lymphoid cell capable of producing antibody
having specific binding affinity for the antigen. The invention
provides a method of producing a monoclonal antibody specific for
an antigen associated with a condition. The invention provides a
method of identifying an antigen associated with a condition using
the trioma fusion partner. The invention provides a method of
diagnosing a condition using the trioma fusion partner. The
invention provides a method for preventing a condition.
Compositions and therapeutic compositions are also provided, using
monoclonal antibodies produced using the trioma fusion partner.
Inventors: |
Trakht; Ilya; (New York,
NY) |
Correspondence
Address: |
John P. White;Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
THE TRUSTEES OF COLUMBIA UNIVERSITY
IN THE CITY OF NEW YORK
|
Family ID: |
21913224 |
Appl. No.: |
11/704925 |
Filed: |
February 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09664485 |
Sep 18, 2000 |
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11704925 |
Feb 9, 2007 |
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PCT/US99/05828 |
Mar 18, 1999 |
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09664485 |
Sep 18, 2000 |
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09040833 |
Mar 18, 1998 |
6197582 |
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PCT/US99/05828 |
Mar 18, 1999 |
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Current U.S.
Class: |
435/70.21 ;
435/326; 435/366; 530/388.1 |
Current CPC
Class: |
C07K 16/2863 20130101;
C12N 2510/04 20130101; G01N 33/6893 20130101; A61P 35/00 20180101;
C12N 2510/02 20130101; C07K 16/3069 20130101; A61P 31/00 20180101;
A61P 37/06 20180101; C07K 16/1235 20130101; G01N 33/57484 20130101;
Y10S 530/809 20130101; G01N 33/56983 20130101; C07K 2317/21
20130101; C12N 5/163 20130101; C07K 16/3015 20130101; C12N 5/166
20130101; Y10S 530/808 20130101 |
Class at
Publication: |
435/070.21 ;
530/388.1; 435/326; 435/366 |
International
Class: |
C12P 21/08 20060101
C12P021/08; C12N 5/08 20060101 C12N005/08; C12N 5/06 20060101
C12N005/06; C07K 16/18 20060101 C07K016/18 |
Claims
1. (canceled)
2. A trioma cell which does not produce any antibody obtained by
fusing a heteromyeloma cell which does not produce any antibody
with a human lymphoid cell.
3. The trioma cell of claim 2, wherein the heteromyeloma cell is
the cell designated B6B11 (ATCC accession number HB-12481).
4. The trioma cell of claim 2, wherein the heteromyeloma cell is a
B6B11-like cell.
5. The trioma cell of claim 2, wherein the human lymphoid cell is a
myeloma cell.
6. The trioma cell of claim 2, wherein the human lymphoid cell is a
splenocyte or a lymph node cell.
7. The trioma cell of claim 2, wherein the trioma is the cell
designated MFP-2 (ATCC accession number HB-12482).
8-28. (canceled)
29. A method of producing a monoclonal antibody comprising: (a)
fusing a lymphoid cell capable of producing antibody with the
trioma cell of claim 2, thereby forming tetroma cells; and (b)
incubating the tetroma cells formed in step (a) under conditions
permissive to the production of antibody by the tetroma cell,
thereby producing the monoclonal antibody.
30. A method of producing a monoclonal antibody specific for an
antigen associated with a condition in a subject comprising: (a)
fusing a lymphoid cell capable of producing antibody with the
trioma cell of claim 2, thereby forming tetroma cells; (b)
incubating the tetroma cells formed in step (a) under conditions
permissive for the production of antibody by the tetroma cells; (c)
selecting a tetroma cell producing a monoclonal antibody; (d)
contacting the monoclonal antibody of step (c) with (1) a sample
from a subject with the condition or (2) a sample from a subject
without the condition under conditions permissive to the formation
of a complex between the monoclonal antibody and the sample; (e)
detecting the complex formed between the monoclonal antibody and
the sample; (f) determining the amount of complex formed in step
(e); and (g) comparing the amount of complex determined in step (f)
for the sample from the subject with the condition with the amount
determined in step (f) for the sample from the subject without the
condition, a greater amount of complex formation for the sample
from the subject with the condition indicating that a monoclonal
antibody specific for the antigen specific for the condition is
produced.
31. The method of claim 29, step (a) further comprising freezing
the lymphoid cell.
32. The method of claim 29, step (b) further comprising incubating
the selected tetroma cells under conditions permissive for cell
replication.
33. The method of claim 32, wherein tetroma replication is effected
in vitro or in vivo.
34. The method of claim 29, wherein the trioma cell is the cell
designated MFP-2 (ATCC Accession No. HB-12482).
35-78. (canceled)
Description
[0001] This application is a continuation-in-part application of
U.S. Ser. No. 09/040,833, filed Mar. 18, 1998, the content of which
is hereby incorporated by reference into this application.
[0002] Throughout this application, various publications are
referenced by author and date. Full citations for these
publications may be found listed alphabetically at the end of the
specification immediately preceding the claims. The disclosures of
these publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art.
BACKGROUND OF THE INVENTION
[0003] The seminal discovery by Kohler and Milstein (Kohler, G. and
Milstein, C., 1975) of mouse "hybridomas" capable of secreting
specific monoclonal antibodies (mabs) against predefined antigens
ushered in a new era in experimental immunology. Many problems
associated with antisera were circumvented. Clonal selection and
immortality of hybridoma cell lines assured monoclonality and
permanent availability of antibody products. At the clinical level,
however, the use of such antibodies is clearly limited by the fact
that they are foreign proteins and act as antigens in humans.
[0004] Since the report of Kohler and Milstein (Kohler, G. and
Milstein, C., 1975), the production of mouse monoclonal antibodies
has become routine. However, the application of xenogenic
monoclonal antibodies for in vivo diagnostics and therapy is often
associated with undesirable effects such as a human anti-mouse
immunoglobulin response. In addition, monoclonal antibodies have
great potential as tools for imaging. Moreover, therapeutic
treatment has motivated the search for means for the production of
human monoclonal antibodies (humAbs)(Levy, R., and Miller R A.,
1983). However, progress in this area has been hampered by the
absence of human myelomas suitable as fusion partners with
characteristics similar to those of mouse myeloma cells (Posner M
R, et al., 1983). The use of Epstein-Barr virus (EBV) has proved to
be quite efficient for human lymphocyte immortalization (Kozbor D,
and Roder J., 1981; Casual O, 1986), but has certain limitations
such as low antibody secretion rate, poor clonogenicity of
antibody-secreting lines, and chromosomal instability requiring
frequent subcloning. Undifferentiated human lymphoblastoid cell
lines appear more attractive. In contrast to differentiated myeloma
cells, these cell lines are readily adapted to culture conditions,
though the problems of low yield and unstable secretion remain
unresolved (Glassy M C, 1983; Olison L, et al., 1983). The best
potential fusion partners are syngenic myeloma cells with
well-developed protein synthesis machinery (Nilsson K. and Ponten
J., 1975). However, due to culturing difficulties few lines have
been conditioned for in vitro growth and capability to produce
viable hybrids (Goldman-Leikin R E, 1989). Existing myelomas have
low fusion yield and slow hybrid growth, although monoclonal
antibody production is relatively stable (Brodin T, 1983). Genetic
instability is a major disadvantage of interspecies hybrids. This
is the case, for example, when a mouse myeloma is used as the
immortalizing partner. Production of mouse-human cell hybrids is
not difficult, and these cells have growth characteristics In vitro
similar to those of conventional mouse-mouse hybridomas (Teng N N H
, 1983). However, spontaneous elimination of human chromosomes
considerably reduces the probability of stable mAb secretion (Weiss
M C, and Green H., 1967). In order to improve growth
characteristics and stability of human monoclonal antibody
production, heterohybrids between mouse myeloma cells and human
lymphocyte (Oestberg L, and Pursch E., 1983) as well as
heteromyelomas (Kozbor D, et. al., 1984) are used as fusion
partners.
SUMMARY OF THE INVENTION
[0005] The present invention provides a heteromyeloma cell which
does not produce any antibody and is capable of producing a trioma
cell which does not produce any antibody when fused with a human
lymphoid cell; wherein the trioma cell so produced is capable of
producing a tetroma cell which produces a monoclonal antibody
having specific binding affinity for an antigen when fused with a
second human lymphoid cell and such second human lymphoid cell
produces an antibody having specific binding affinity for the
antigen, with the proviso that the heteromyeloma cell is not B6B11
(ATCC accession number HB-12481).
[0006] The present invention further provides a trioma cell which
does not produce any antibody obtained by fusing a heteromyeloma
cell with a human lymphoid cell.
[0007] The present invention also provides a tetroma cell capable
of producing a monoclonal antibody having specific binding affinity
for an antigen, obtained by fusing the above-described trioma cell
which does not produce any antibody with a human lymphoid cell
capable of producing an antibody having specific binding affinity
for the antigen.
[0008] The present invention additionally provides a monoclonal
antibody produced by the above-described tetroma.
[0009] The present invention further provides a method of
generating the above-described trioma cell comprising: (a) fusing a
heteromyeloma cell which does not produce any antibody with a human
lymphoid cell thereby forming trioma cells; (b) incubating the
trioma cells formed in step (a) under conditions permissive for the
production of antibody by the trioma cells; and (c) selecting a
trioma cell that does not produce any antibody.
[0010] Still further, the present invention provides a method of
generating tetroma cells comprising: (a) fusing the described
trioma cell with a human lymphoid cell, thereby forming tetroma
cells; (b) incubating the tetroma cells formed in step (a) under
conditions permissive for the production of antibody by the tetroma
cells; and (c) selecting a tetroma cell capable of producing a
monoclonal antibody.
[0011] The present invention also provides a method of producing a
monoclonal antibody comprising (a) fusing a lymphoid cell capable
of producing antibody with the above-described trioma cell, thereby
forming tetroma cells; and (b) incubating the tetroma cell formed
in step (a) under conditions permissive for the production of
antibody by the tetroma cells; (c) selecting a tetroma cell capable
of producing the monoclonal antibody; and (d) culturing the tetroma
cell of step (c) so as to produce the monoclonal antibody.
[0012] Also, the present invention provides a method of producing a
monoclonal antibody specific for an antigen associated with a given
condition in a subject comprising: (a) fusing a lymphoid cell
capable of producing antibody with the above-described trioma cell,
thereby forming tetroma cells; (b) incubating the tetroma cell
formed in step (a) under conditions permissive for the production
of antibody by the tetroma cells; (c) selecting a tetroma cell
producing a monoclonal antibody; (d) contacting the monoclonal
antibody of step (c) with (1) a sample from a subject with the
given condition or (2) a sample from a subject without the given
condition, so as to form a complex between the monoclonal antibody
and the sample; (e) detecting any complex formed between the
monoclonal antibody and the sample; (f) determining the amount of
complex formed in step and (e); and (g) comparing the amount of
complex determined in step (f) for the sample from the subject with
the given condition with amount determined in step (f) for the
sample from the subject without the given condition, a greater
amount of complex formation for the sample from the subject with
the given condition indicating that a monoclonal antibody specific
for an antigen specific for the condition has been produced.
[0013] Additionally, the present invention provides a method of
identifying an antigen associated with a given condition in a
sample comprising: (a) contacting the monoclonal antibody produced
by the above-described method with the sample, under conditions
permissive for the formation of a complex between the monoclonal
antibody and the sample; (b) detecting any complex formed in step
(a); and (c) isolating any complex detected in step (b), so as to
thereby identify the antigen associated with the condition in the
sample.
[0014] The present invention additionally provides a method of
diagnosing a given condition in a subject comprising: (a)
contacting a sample from the subject with a monoclonal antibody
produced by the above-described method under conditions permissive
for the formation of a complex between the monoclonal antibody and
the sample; and (b) detecting the formation of any complex formed
between the monoclonal antibody and the sample, detection of
complex so formed indicating the presence of an antigen specific
for the given condition in the sample, and thus providing a
diagnosis of the given condition in the subject.
[0015] The present invention further provides a composition
comprising a monoclonal antibody described by the method described
herein and a suitable carrier.
[0016] Further, the present invention also provides a therapeutic
composition comprising a therapeutically effective amount of a
monoclonal antibody of this invention and a pharmaceutically
acceptable carrier.
[0017] Also, the present invention further provides a method of
treating a given condition in a subject comprising administering to
the subject an amount of the above-described therapeutic
composition effective to treat the condition in the subject.
[0018] Finally, the present invention provides a method of
preventing a given condition in a subject comprising administering
to the subject an amount of the above-described therapeutic
composition effective to prevent the condition in the subject.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIGS. 1A-1C Distribution of cells according to the number of
chromosomes. The X-axis indicates the amount of chromosomes. The
Y-axis indicates the percentage of cells with appropriate number of
chromosomes. The data represent the average ones based on the
analysis of more than 50 metaphase plates for each line:
P3.X63.Ag8.653 FIG. 1A, RPMI 8226 FIG. 1B, B6B11 FIG. 1C.
[0020] FIG. 2 Fragment of G-banded karyotype of B6B11 line. The
arrows indicate genetic material presumably of human origin; 3 p
portion of chromosome 3 and chromosome 19.
[0021] FIG. 3 B6B11 fusion efficiency with fresh isolated and
cultured splenocytes. SPL were isolated in LSM, immediately after a
portion of the cells were fused with B6B11 cells and the remaining
SPL were cultivated in vitro for 7-9 days in RPMI-C containing 15%
FCS in the presence of ConA, LPS, PHA, PWM or without mitogens,
then these cells were also fused with B6B11. PWM in the
concentration of 5 .mu.g/ml influenced effectively the fusion
efficiency.
[0022] FIGS. 4A-4D DNA histograms of parental cells 653 (FIG. 4A)
and 8226 (FIG. 4B), heteromyeloma B6B11 (FIG. 4C) and
B6B11-splenocyte hybrid (FIG. 4D). The amount of B6B11 DNA
constitutes about 100% of the total amount of 653 DNA plus 8226
DNA. The DNA content of B6B11-SPL hybrid is greater than that of
B6B11.
[0023] FIGS. 5A-5B Immunoglobulin production by hybridomas
(tetromas) derived from the fusion of PBLs with MFP-2. FIG. 5A
shows results of fusing fresh lymphocyte suspensions with MFP-2.
FIG. 5B shows results of fusing frozen/thawed lymphocyte
suspensions with MFP-2. The dark rectangles indicate IgM
production. The gray rectangles indicate IgG production. The Y-axis
indicates optical density at A.sub.490 for different hybridoma
samples (tetromas) generated from fusion with the MFP-2 trioma line
(X-axis). The dotted line indicates the optical density at
A.sub.490 for a 1:500 dilution of IgM antibody. The dashed line
indicates the optical density at A4.sub.90for a 1:500 dilution of
IgG antibody.
[0024] FIG. 6 Anti-thyroglobulin antibody production by thyroid
cancer lymph node lymphocytes fused to fusion partner MFP-2 cells.
The Y-axis indicates optical density at A.sub.405 (OD.sub.405) for
different hybridoma samples (tetromas) generated from fusion with
the MFP-2 trioma line (X-axis). Thirty-three tetromas produced
antibody which reacted positively against thyroglobulin; eight were
particularly strongly reactive.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides a heteromyeloma cell which
does not produce any antibody and is capable of producing a trioma
cell which does not produce any antibody when fused with a human
lymphoid cell; wherein the trioma cell so produced is capable of
producing a tetroma cell which produces a monoclonal antibody
having specific binding affinity for an antigen when fused with a
second human lymphoid cell and such second human lymphoid cell of
produces an antibody having specific binding affinity for the
antigen, with the proviso that the heteromyeloma cell is not B6B11
(ATCC accession number HB-12481).
[0026] The present invention also provides a trioma cell which does
not produce any antibody obtained by fusing a heteromyeloma cell
with a human lymphoid cell. In one embodiment of this invention,
the heteromyeloma cell is the cell designated B6B11 (ATCC accession
number HB-12481). In another embodiment, the trioma is a B6B11-like
cell. For purposes of this invention a B6B11-like cell includes a
cell which is substantially identical to the B6B11 cell at the
genetic level and a functionally equivalent thereto. B6B11-like
cells thus specifically include clones or other cells derived from
B6B11 including mutants of the B6B11 and of clones thereof. In
certain embodiments of this invention, the human lymphoid cell is a
myeloma cell. In other embodiments of this invention, the human
lymphoid cell is a splenocyte or a lymph node cell (lymphocyte).
According to certain embodiments of this invention, the trioma cell
is the cell designated MFP-2 (ATCC accession number 12482).
[0027] The present invention also provides a tetroma cell capable
of producing a monoclonal antibody having specific binding affinity
for an antigen, obtained by fusing the above-described trioma cell
which does not produce any antibody with a human lymphoid cell
capable of producing antibody having specific binding affinity for
the antigen. The human lymphoid cell may be a peripheral blood
lymphocyte, a splenocyte, a lymph node cell, a B cell, a T cell, a
tonsil gland lymphocyte, a monocyte, a macrophage, an
erythroblastoid cell or a Peyer's patch cell. In one embodiment of
this invention, the trioma cell is the cell designated MFP-2 (ATCC
accession number HB-12482).
[0028] According to certain embodiments of this invention, the
antigen is a tumor-associated antigen, a cell-specific antigen, a
tissue-specific antigen, an enzyme, a nucleic acid, an
immunoglobulin, a toxin, a viral antigen, a bacterial antigen or a
eukaryotic antigen. In one embodiment, the antigen is a mammalian,
insect, fungal, E. coli or Klebsiella antigen.
[0029] The present invention provides a monoclonal antibody
produced by the above-described tetroma. The present invention also
provides an isolated nucleic acid encoding the monoclonal antibody
produced by the described tetroma. The nucleic acid may include,
but is not limited to DNA, RNA, cDNA, oligonucleotide analogs,
vectors, expression vectors or probes. Additionally, the present
invention contemplates the expression of the nucleic acid encoding
the monoclonal antibody introduced into a host cell capable of
expression the monoclonal antibody or portions thereof.
[0030] The present invention also provides isolated nucleic acids
including all or a portion of the antibody binding regions of such
monoclonal antibodies and the use of such nucleic acid to express
portions of such antibodies, for example, single chain antibodies
per se or phage-displayed single chain antibodies (sFv-a
antibody).
[0031] Moreover, nucleic acids encoding all or a portion of such
nucleic acids may be used to transfect mammalian cells such as
mouse myeloma or CHO cells to permit increased production of such
monoclonal antibody or portion thereof.
[0032] The present invention further provides a method of
generating the described trioma cell comprising: (a) fusing a
heteromyeloma cell which does not produce any antibody with a human
lymphoid cell thereby forming trioma cells; (b) incubating the
trioma cells formed in step (a) under conditions permissive for the
production of antibody by the trioma cells; and (c) selecting a
trioma cell that does not produce any antibody.
[0033] According to one embodiment of this invention, the
heteromyeloma cell of step (a) is designated B6B11 (ATCC accession
number HB-12481). According to other embodiments of this invention,
the human lymphoid cell is a lymph node lymphocyte or a splenocyte.
According to certain embodiments of the present invention, the
method further comprises selecting a trioma cell capable of growth
in serum-free media. Other embodiments comprise selecting a trioma
cell that is capable of fusing with a peripheral blood lymphocyte
or lymph node lymphocyte. The present invention further provides a
trioma cell generated by the above-described method.
[0034] Still further, the present invention provides a method of
generating a tetroma cell comprising: (a) fusing the
above-described trioma cell with a human lymphoid cell thereby
forming tetroma cells; (b) incubating the tetroma cell formed in
step (a) under conditions permissive to the production of antibody
by the tetroma cells; and (c) selecting a tetroma cell capable of
producing a monoclonal antibody. According to one embodiment of
this invention, the trioma cell of step (a) the cell is designated
MFP-2 (ATCC accession number HB-12482). According to an embodiment
of this invention, the human lymphoid cell is a peripheral blood
lymphocyte, a splenocyte, a lymph node cell, a B cell, a T cell, a
tonsil gland lymphocyte, a monocyte, a macrophage, an
erythroblastoid cell or a Peyer's patch cell. In some embodiments
of this invention, the human lymphoid cell produces antibodies
having specific binding affinity for an antigen and the tetroma
cell produces a monoclonal antibody having specific binding
affinity for such antigen. According to certain embodiments of this
invention, the antigen is a tumor-associated antigen, a
cell-specific antigen, a tissue-specific antigen, an enzyme, a
nucleic acid, an immunoglobulin, a toxin, a viral antigen, a
bacterial antigen, or a eukaryotic antigen. In some embodiments of
this invention, the antigen is a mammalian, insect, E. coli or
Klebsiella antigen. The present invention further provides a
tetroma cell generated by the above-described method.
[0035] This invention also provides human hybridoma fusion partner
cell line heteromyeloma B6B11, and human hybridoma fusion partner
cell line trioma MFP-2. These hybridoma cell lines were deposited
on Mar. 17, 1998 with the American Type Culture Collection (ATCC),
12301 Parklawn Drive, Rockville, Md. 20852, U.S.S. under the
provision of the Budapest Treaty for the International Recognition
of the Deposit of Microorganism for the Purposes of Patent
Procedure. These hybridoma have been accorded with ATCC Accession
Nos. HB-12481 and HB-12482 respectively.
[0036] The present invention also provides a method of producing a
monoclonal antibody comprising (a) fusing a lymphoid cell capable
of producing antibody with the described trioma cell, thereby
forming a tetroma cell; and (b) incubating the tetroma cell formed
in step (a) under conditions permissive for the production of
antibody by the tetroma cell so as to thereby produce the
monoclonal antibody.
[0037] Also, the present invention provides a method of producing a
monoclonal antibody specific for an antigen associated with a given
condition in a subject comprising: (a) fusing a lymphoid cell
capable of producing antibody with the above-described trioma cell,
thereby forming tetroma cells; (b) incubating the tetroma cell
formed in step (a) under conditions permissive for the production
of antibody by the tetroma cells; (c) selecting a tetroma cell
producing a monoclonal antibody; (d) contacting the monoclonal
antibody of step (c) with (1) a sample from a subject with the
given condition or (2) a sample from a subject without the given
condition under conditions permissive to the formation of a complex
between the monoclonal antibody and the sample; (e) detecting the
complex formed between the monoclonal antibody and the sample; (f)
determining the amount of complex formed in step (e); and (g)
comparing the amount of complex determined in step (f) for the
sample from the subject with the condition with amount determined
in step (f) for the sample from the subject without the condition,
a greater amount of complex formation for the sample from the
subject with the condition indicating that a monoclonal antibody
specific for the antigen specific for the condition has been
produced.
[0038] In one embodiment of the present invention, step (a) further
comprises freezing the lymphoid cell. According to one embodiment
of the present invention, step (c) further comprises incubating the
selected tetroma cell under conditions permissive for cell
replication. According to certain embodiments of this invention,
the tetroma replication is effected in vitro or in vivo. According
to one embodiment of this invention, the trioma cell is the cell
designated MFP-2 (ATCC Accession No. HB-12482). The present
invention provides a monoclonal antibody specific for an antigen
associated with a condition, identified by the described method.
The present invention also provides an isolated nucleic acid
encoding the described monoclonal antibody. The nucleic acid may
include, but is not limited to DNA, RNA, cDNA, oligonucleotide
analogs, vectors, expression vectors or probes. Additionally, the
present invention contemplates the expression of the nucleic acid
encoding the monoclonal antibody introduced into a host cell
capable of expression the monoclonal antibody or portions
thereof.
[0039] The present invention also provides isolated nucleic acids
including all or a portion of the antibody binding regions of such
monoclonal antibodies and the use of such nucleic acid to express
portions of such antibodies, for example, single chain antibodies
per se or phage-displayed single chain antibodies (sFv-a
antibody).
[0040] Moreover, nucleic acids encoding all or a portion of such
nucleic acids may be used to transfect mammalian cells such as
mouse myeloma or CHO cells to permit increased production of such
monoclonal antibody or portion thereof.
[0041] According to an embodiment of this invention, the given
condition as is associated with a cancer, a tumor, a toxin, an
infectious agent, an enzyme dysfunction, a hormone dysfunction, an
autoimmune disease, an immune dysfunction, a viral antigen, a
bacterial antigen, a eukaryotic antigen, rejection of a
transplanted tissue, poisoning,. or venom intoxication.
Additionally, the condition may be any other abnormality, including
that resulting from infection, cancer, autoimmune dysfunction,
cardiovascular disease, or transplantation. In an embodiment of
this invention, the given condition is septicemia, sepsis, septic
shock, viremia, bacteremia or fungemia. In certain embodiments of
this invention, the cancer may be, but is not limited to lung
cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,
meningioma, bone cancer, thyroid cancer, ovarian cancer, bladder
cancer, pancreatic cancer, breast cancer, or prostate cancer.
According to certain embodiments of this invention, the infectious
agent may be, but is not limited to Hanta virus, HTLV I, HTLV II,
HIV, herpes virus, influenza virus, Ebola virus, human papilloma
virus, Staphlococcus, Streptococcus, Klebsiella, E. coli, anthrax,
or cryptococcus. According to certain embodiments of this
invention, the toxin is tetanus, anthrax, botulinum snake venom or
spider venom. In one embodiment of this invention, the tumor is
benign. In another embodiment, the enzyme dysfunction is
hyperactivity or overproduction of the enzyme. In still another
embodiment, the hormone dysfunction is hyperactivity or
overproduction of the hormone. In yet other embodiments of this
invention, the immune dysfunction is CD3 or CD4 mediated. In still
other embodiments of this invention, the autoimmune disease is
lupus, thyroidosis, graft versus host disease, transplantation
rejection, or rheumatoid arthritis. In still other embodiments of
the invention, the condition is any abnormality. In still other
embodiments, the condition is the normal condition.
[0042] Additionally, the present invention provides a method of
identifying an antigen associated with a given condition in a
sample comprising: (a) contacting the monoclonal antibody produced
by the above-described method with the sample under conditions
permissive for the formation of a complex between the monoclonal
antibody and the sample; (b) detecting any complex formed in step
(a); and (c) isolating the complex detected in step (b), thereby
identifying the antigen associated with the condition in the
sample.
[0043] In one embodiment of the above-described method, the
condition is a tumor.
[0044] In another embodiment of the above-identified method, the
antigen is not previously known.
[0045] This invention also provides a tumor antigen identified by
the above-described method where the antigen is not previously
known.
[0046] This invention also provides a method for diagnosing a tumor
in a sample comprising detecting the presence of the tumor antigen
identified by the above-described method wherein the condition is a
tumor, the presence of said antigen indicating the presence of
tumor in the subject.
[0047] This invention also provides the above-described method,
wherein the detecting comprises: (a) obtaining an apropriate sample
which contains the tumor antigen from the subject; (b) contacting
the sample with an antibody which is capable of specifically
binding to the tumor antigen under conditions permitting the
formation of a complex between the antibody and the antigen; and
(c) detecting the complex formed, thereby detecting the presence of
the tumor antigen.
[0048] In certain embodiments of this invention, the method further
comprises separating the monoclonal antibody from the monoclonal
antibody-antigen complex. In some embodiments the separation is by
size fractionation, e.g. the size fractionation effected by
polyacrylamide or agarose gel electrophoresis.
[0049] According to certain embodiments of this invention, the
given condition is associated with, a cancer, a tumor, a toxin, an
infectious agent, an enzyme dysfunction, a hormone dysfunction, an
autoimmune disease, an immune dysfunction, a viral antigen, a
bacterial antigen, a eukaryotic antigen, rejection of a
transplanted tissue, poisoning, or venom intoxication.
Additionally, the condition may be any other abnormality, including
one resulting from infection, cancer, autoimmune dysfunction,
cardiovascular disease, or transplantation. In an embodiment of
this invention, the condition is septicemia, sepsis, septic shock,
viremia, bacteremia or fungemia. In some embodiments of this
invention, the cancer may be but is not limited to lung cancer,
liver cancer, leukemia, lymphoma, neuroblastoma, glioma,
meningioma, bone cancer, thyroid cancer, colon cancer, ovarian
cancer, bladder cancer, pancreatic cancer, breast cancer or
prostate cancer. According to some embodiments of this invention,
the infectious agent may be but is not limited to Hanta virus, HTLV
I, HTLV II, HIV, herpes virus, influenza virus, Ebola virus, human
papilloma virus, Staphlococcus, Streptococcus, Klebsiella, E. coli,
anthrax or cryptococcus. According to some embodiments of this
invention, the toxin is tetanus, anthrax, botulinum, snake venom or
spider venom. In one embodiment of this invention, the tumor is
benign. In other embodiments, the enzyme dysfunction is
hyperactivity or overproduction of the enzyme. In still other
embodiments, the hormone dysfunction is hyperactivity or
overproduction of the hormone. In yet other embodiments of this
invention, the immune dysfunction is CD3 or CD4 mediated. In still
other embodiments of this invention, the autoimmune disease is
lupus, thyroidosis, graft versus host disease, transplantation
rejection or rheumatoid arthritis. In still other embodiments of
the invention, the condition is any abnormality. In still other
embodiments, the condition is the normal condition.
[0050] The present invention additionally provides a method of
diagnosing a condition in a subject comprising: (a) contacting a
sample from the subject with a monoclonal antibody produced by the
above-described method under conditions permissive for the
formation of a complex between the monoclonal antibody and the
sample; and (b) detecting the formation of any complex formed
between the monoclonal antibody and the sample, positive detection
of such complex indicating the presence of an antigen specific for
the condition in the sample which correlates with diagnosing the
condition in the subject.
[0051] According to an embodiment of this invention, the monoclonal
antibody is coupled to a detectable marker. In an embodiment of
this invention, the detectable marker is a radiolabel, a fluorofor,
or fluorescent molecule, an enzyme, a ligand, a colorimetric
marker, or a magnetic bead.
[0052] According to some embodiments of this invention, the given
condition is or is associated with, a cancer, a tumor, a toxin, an
infectious agent, an enzyme dysfunction, a hormone dysfunction, an
autoimmune disease, an immune dysfunction, a viral antigen, a
bacterial antigen, a eukaryotic antigen, rejection of a
transplanted tissue, poisoning, or venom intoxication. Additionally
the condition may be any other abnormality, including one resulting
from infection, cancer, autoimmune dysfunction, cardiovascular
disease, or transplantation. In certain embodiments of this
invention, the condition is septicemia, sepsis, septic shock,
viremia, bacteremia or fungemia. In some embodiments of this
invention, the cancer may be, but is not limited to lung cancer,
liver cancer, leukemia, lymphoma, neuroblastoma, glioma,
meningioma, bone cancer, thyroid cancer, ovarian cancer, bladder
cancer, pancreatic cancer, breast cancer or prostate cancer.
According to other embodiments of this invention, the infectious
agent may be, but os not limited to Hanta virus, HTLV I, HTLV II,
HIV, herpes virus, influenza virus, Ebola virus, human papilloma
virus, Staphlococcus, Streptococcus, Klebsiella, E. coli, anthrax
or cryptococcus. According to some embodiments of this invention,
the toxin is tetanus, anthrax, botulinum, snake venom or spider
venom. In one embodiment of this invention, the tumor is benign. In
other embodiments, the enzyme dysfunction is hyperactivity or
overproduction of the enzyme. In still other embodiments, the
hormone dysfunction is hyperactivity or overproduction of the
hormone. In yet other embodiments of this invention, the immune
dysfunction is CD3 or CD4 mediated. In still other embodiments of
this invention, the autoimmune disease is lupus, thyroidosis, graft
versus host disease, transplantation rejection or rheumatoid
arthritis. In still other embodiments of the invention, the
condition is any abnormality. In still other embodiments, the
condition is the normal condition.
[0053] The present invention further provides a composition
comprising a monoclonal antibody produced by the method described
herein and a suitable carrier.
[0054] Further, the present invention also provides a therapeutic
composition comprising a therapeutically effective amount of a
monoclonal antibody of this invention and a pharmaceutically
acceptable carrier.
[0055] According to certain embodiments of this invention, the
condition is cancer and the amount of monoclonal antibody is
sufficient to inhibit the growth of or eliminate the cancer.
According to certain embodiments, the condition is an infection and
the amount of monoclonal antibody is sufficient to inhibit the
growth of or kill the infectious agent. According to certain
embodiments of this invention, the condition is associate with a
toxin and the amount of monoclonal antibody is sufficient to reduce
the amount of or destroy the toxin. In still other embodiments, the
condition is an autoimmune disease and the amount of monoclonal
antibody is sufficient to reduce the amount of or destroy the
offending antibody or subunit(s) thereof. In still other
embodiments, the condition is a cardiovascular disease and the
amount of monoclonal antibody is sufficient to reduce the
condition. In yet other embodiments, the condition is a
transplantation rejection, and the amount of monoclonal antibody is
sufficient to reduce the condition.
[0056] According to certain embodiments of this invention, the
monoclonal antibody is coupled to an effector compound. In certain
embodiments of this invention, the effector compound is a cytotoxic
agent, drug, enzyme, dye, or radioisotope. In certain embodiments
of this invention, the monoclonal antibody is coupled to a carrier.
According to other embodiments of this invention, the carrier is a
liposome.
[0057] Also, the present invention further provides a method of
treating a given condition in a subject comprising administering to
the subject an amount of the above-described therapeutic
composition effective to treat the condition in the subject.
According to one embodiment of this invention, the therapeutic
composition is administered to a second subject.
[0058] According to an embodiment of this invention, the given
condition is or is associated with a cancer, a tumor, a toxin, an
infectious agent, an enzyme dysfunction, a hormone dysfunction, an
autoimmune disease, an immune dysfunction, a viral antigen, a
bacterial antigen, a eukaryotic antigen, rejection of a
transplanted tissue, poisoning, or venom intoxication.
Additionally, the condition may be any other abnormality, including
that resulting from infection, cancer, autoimmune dysfunction,
cardiovascular disease, or transplantation. In an embodiment of
this invention, the given condition is septicemia, sepsis, septic
shock, viremia, bacteremia or fungemia. In certain embodiments of
this invention, the cancer may be but is not limited to lung
cancer, liver cancer, leukemia, lymphoma, neuroblastoma, glioma,
meningioma, bone cancer, thyroid cancer, colon cancer, ovarian
cancer, bladder cancer, pancreatic cancer, breast cancer or
prostate cancer. According to an embodiment of this invention, the
infectious agent may be, but is not limited to Hanta virus, HTLV I,
HTLV II, HIV, herpes virus, influenza virus, Ebola virus, human
papilloma virus, Staphlococcus, Streptococcus, Klebsiella, E. coli,
anthrax or cryptococcus. According to certain embodiments of this
invention, the toxin is tetanus, anthrax, botulinum, snake venom or
spider venom. In one embodiment of this invention, the tumor is
benign. In another embodiment, the enzyme dysfunction is
hyperactivity or overproduction of the enzyme. In still another
embodiment, the hormone dysfunction is hyperactivity or
overproduction of the hormone. In yet other embodiments of this
invention, the immune dysfunction is CD3 or CD4 mediated. In still
other embodiments of this invention, the autoimmune disease is
lupus, thyroidosis, graft versus host disease, transplantation
rejection or rheumatoid arthritis. In still other embodiments of
the invention, the condition is any abnormality. In still other
embodiments, the condition is the normal condition.
[0059] Finally, the present invention provides a method of
preventing a given condition in a subject comprising administering
to the subject an amount of the above-described therapeutic
composition effective to prevent the condition in the subject. In
one embodiment of this invention, the subject previously exhibited
the condition. According to one embodiment of this invention, the
therapeutic composition is administered to a second subject.
[0060] According to certain embodiments of this invention, the
condition is or is associated with a cancer, a tumor, a toxin, an
infectious agent, an enzyme dysfunction, a hormone dysfunction, an
autoimmune disease, an immune dysfunction, a viral antigen, a
bacterial antigen, a eukaryotic antigen, rejection of a
transplanted tissue, poisoning, or venom intoxication.
Additionally, the condition may be any other abnormality, including
one resulting from infection, cancer, autoimmune dysfunction,
cardiovascular disease, or transplantation. In certain embodiments
of this invention, the condition is septicemia, sepsis, septic
shock, viremia, bacteremia or fungemia. In some embodiments of this
invention, the cancer may be but is not limited to lung cancer,
liver cancer, leukemia, lymphoma, neuroblastoma, glioma,
meningioma, bone cancer, thyroid cancer, colon cancer, ovarian
cancer, bladder cancer, pancreatic cancer, breast cancer or
prostate cancer. According to an embodiment of this invention, the
infectious agent may be but is not limited to Hanta virus, HTLV I,
HTLV II, HIV, herpes virus, influenza virus, Ebola virus, human
papilloma virus, Staphlococcus, Streptococcus, Klebsiella, E. coli,
anthrax or cryptococcus. According to some embodiments of this
invention, the toxin is tetanus, anthrax, botulinum, snake venom or
spider venom. In one embodiment of this invention, the tumor is
benign. In other embodiments, the enzyme dysfunction is
hyperactivity or overproduction of the enzyme. In still other
embodiments, the hormone dysfunction is hyperactivity or
overproduction of the hormone. In yet other embodiments of this
invention, the immune dysfunction is CD3 or CD4 mediated. In still
other embodiments of this invention, the autoimmune disease is
lupus, thyroidosis, graft versus host disease, transplantation
rejection or rheumatoid arthritis. In still other embodiments of
the invention, the condition is any abnormality. In still other
embodiments, the condition is the normal condition.
[0061] The present invention also provides the production of
antibodies for antigens which are not associated with a given
condition, but more properly constitute a component of the entire
repetoire of antibodies in a human immune system.
[0062] In addition, the present invention provides identification
of novel antigens relevant to a given condition in a subject and
the use thereof for diagnosis and treatment of the given condition
in the subject. The invention also provides identification of the
repetoire of naturally occurring antibodies in normal subjects and
subjects having a pathological condition. In one embodiment, the
condition may be venom detoxicification (neutralization). For
example, the condition may result from scorpion, spider, rattle
snake or poison toad bites or venom exposure. The present invention
provides antibodies to act as antidote for such conditions.
[0063] The trioma cell of the present invention may also be fused
with macrophages, monocytes, T-lymphocytes, and erythroblastoid
cells. Hybridoma cells resulting from such fusions may produce
growth factors, cytokines, enzymes, hemoglobin.
[0064] As used herein, a human-murine hybridoma (the "immortalizing
hybridoma") is an immortal cell line which results from the fusion
of a murine myeloma or other murine tumor cell with a human
lymphoid cell derived from a normal subject. As described herein
below, by careful selection and mutation, an immortalizing
hybridoma which provides improved chromosomal stability, has human
characteristics, and which does not secrete immunoglobulin may be
obtained. The antibody secreting capability of such a resulting
trioma may be provided by the third cell fusion which is typically
derived either from B cells of an immunized human individual, or
with B cells which have been immortalized.
[0065] As used herein, a "B6B11" cell is a hybrid cell produced by
the fusion of mouse myeloma 653 and human myeloma RPMI 8226.
[0066] As used herein, a "B6B11-like" cell is a a hybrid cell
produced by the fusion of mouse myeloma 653-related cell and human
myeloma RPMI 8226-related cell.
[0067] As used herein, a "MFP" cell is a hybrid cell produced by
the fusion of a B6B11 cell and a human lymphocyte. B6B11-like cells
share function properties and characteristics with B6B11
heteromyeloma cells.
[0068] As used herein, a "MFP-like" cell is a hybrid cell produced
by the fusion of a B6B11-like cell and a human lymphocyte. MFP-like
cells share function properties and characteristics with MFP trioma
cells.
[0069] As used herein, "non-secreting" or "non-producing" hybridoma
refers to a hybridoma which is capable of continuous reproduction
and, therefore, is immortal, and which does not produce
immunoglobulin.
[0070] As used herein, a hybridoma "having human characteristics"
refers to a hybridoma which retains detectable human-derived
chromosomes such as those producing human HLA antigen which may be
expressed on the cell surface.
[0071] As used herein, lymphoid cells "immunized against a
predefined determinant" refers to lymphoid cells derived from an
subject who has been exposed to an antigen having the determinant.
For example, a subject can be induced to produce (from its lymphoid
B cells) antibodies against the antigenic determinants of various
blood types, by exposure, through transfusions or previous
pregnancy, or against the antigenic determinants of specific
viruses or of bacteria by virus of exposure through past infections
or vaccinations.
[0072] As used herein, "cell line" refers to various embodiments
including but not limited to individual cells, harvested cells and
cultures containing cells so long as these are derived from cells
of the cell line referred to may not be precisely identical to the
ancestral cells or cultures and any cell line referred to include
such variants.
[0073] As used herein, "trioma" refers to a cell line which
contains generic components originating in three originally
separate cell linages. These triomas are stable, immortalized cells
which result from the fusion of a human-murine hybridoma with a
human lymphoid cell.
[0074] As used herein, "tetroma" refers to a a cell line which
contains generic components originating in four originally separate
cell lineages. These tetromas are stable, immortalized antibody
producing cells which result from the fusion of a trioma with a
human lymphoid cell which is capable of producing antibody.
[0075] As used herein, "autologously" refers to a situation where
the same subject is both the source of cell immunoglobulin and the
target for cells, or immunoglobulin or therapeutic composition.
[0076] As used herein, "heterologously" refers to a situation where
one subject is the source of cells or immunoglobulin and another
subject is the target for the cell, immunoglobulin or therapeutic
composition.
[0077] In the practice of any of the methods of the invention or
preparation of any of the pharmaceutical compositions a
"therapeutically effective amount" is an amount which is capable of
binding. to an antigen associated with the condition. Accordingly,
the effective amount will vary with the subject being treated, as
well as the condition to be treated. For the purposes of this
invention, the methods of administration are to include, but are
not limited to, administration cutaneously, subcutaneously,
intravenously, parenterally, orally, topically, or by aerosol.
[0078] As used herein, the term "suitable pharmaceutically
acceptable carrier" encompasses any of the standard
pharmaceutically accepted carriers, such as phosphate buffered
saline solution, water, emulsions such as an oil/water emulsion or
a triglyceride emulsion, various types of wetting agents,
liposomes, tablets, coated tablets, capsules and RBC shadows. An
example of an acceptable triglyceride emulsion useful in
intravenous and intraperitoneal administration of the compounds is
the triglyceride emulsion commercially known as
Intralipid.RTM..
[0079] Typically such carriers contain excipients such as starch,
milk, sugar, certain types of clay, gelatin, stearic acid, talc,
vegetable fats or oils, gums, glycols, or other known excipients.
Such carriers may also include flavor and color additives or other
ingredients.
[0080] This invention also provides for pharmaceutical compositions
capable of binding to an antigen associated with the condition
together with suitable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such compositions are
liquids or lyophilized or otherwise dried formulations and include
diluents of various buffer content (e.g., Tris-HCl., acetate,
phosphate), pH and ionic strength, additives such as albumin or
gelatin to prevent absorption to surfaces, detergents (e.g., Tween
20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents
(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.,
ascorbic acid, sodium metabisulfite), preservatives (e.g.,
Thimerosal, benzyl alcohol, parabens), bulking substances or
tonicity modifiers (e.g., lactose, mannitol), covalent attachment
of polymers such as polyethylene glycol to the compound,
complexation with metal ions, or incorporation of the compound into
or onto particulate preparations of polymeric compounds such as
polylactic acid, polglycolic acid, hydrogels, etc, or onto
liposomes, micro emulsions, micelles, unilamellar or multi lamellar
vesicles, erythrocyte ghosts, or spheroplasts. Such compositions
will influence the physical state, solubility, stability, rate of
in vivo release, and rate of in vivo clearance of the compound or
composition.
[0081] Controlled or sustained release compositions include
formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
Also comprehended by the invention are particulate compositions
coated with polymers (e.g., poloxamers or poloxamines) and the
compound coupled to antibodies directed against tissue-specific
receptors, ligands or antigens or coupled to ligands of
tissue-specific receptors. Other embodiments of the compositions of
the invention incorporate particulate forms protective coatings,
protease inhibitors or permeation enhancers for various routes of
administration, including parenteral, pulmonary, nasal and
oral.
[0082] When administered, compounds are often cleared rapidly from
the circulation and may therefore elicit relatively short-lived
pharmacological activity. Consequently, frequent injections of
relatively large doses of bioactive compounds may by required to
sustain therapeutic efficacy. Compounds modified by the covalent
attachment of water-soluble polymers such as polyethylene glycol,
copolymers of polyethylene glycol and polypropylene glycol,
carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinylpyrrolidone or polyproline are known to exhibit
substantially longer half-lives in blood following intravenous
injection than do the corresponding unmodified compounds
(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al.,
1987). Such modifications may also increase the compound's
solubility in aqueous solution, eliminate aggregation, enhance the
physical and chemical stability of the compound, and greatly reduce
the immunogenicity and reactivity of the compound. As a result, the
desired in vivo biological activity may be achieved by the
administration of such polymer-compound adducts less frequently or
in lower doses than with the unmodified compound.
[0083] Attachment of polyethylene glycol (PEG) to compounds is
particularly useful because PEG has very low toxicity in mammals
(Carpenter et al., 1971). For example, a PEG adduct of adenosine
deaminase was approved in the United States for use in humans for
the treatment of severe combined immunodeficiency syndrome. A
second advantage afforded by the conjugation of PEG is that of
effectively reducing the immunogenicity and antigenicity of
heterologous compounds. For example, a PEG adduct of a human
protein might be useful for the treatment of disease in other
mammalian species without the risk of triggering a severe immune
response. The carrier includes a microencapsulation device so as to
reduce or prevent an host immune response against the compound or
against cells which may produce the compound. The compound of the
present invention may also be delivered microencapsulated in a
membrane, such as a liposome.
[0084] Polymers such as PEG may be conveniently attached to one or
more reactive amino acid residues in a protein such as the
alpha-amino group of the amino terminal amino acid, the epsilon
amino groups of lysine side chains, the sulfhydryl groups of
cysteine side chains, the carboxyl groups of aspartyl and glutamyl
side chains, the alpha-carboxyl group of the carboxy-terminal amino
acid, tyrosine side chains, or to activated derivatives of glycosyl
chains attached to certain asparagine, serine or threonine
residues.
[0085] Numerous activated forms of PEG suitable for direct reaction
with proteins have been described. Useful PEG reagents for reaction
with protein amino groups include active esters of carboxylic acid
or carbonate derivatives, particularly those in which the leaving
groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or
1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing
maleimido or haloacetyl groups are useful reagents for the
modification of protein free sulfhydryl groups. Likewise, PEG
reagents containing amino hydrazine or hydrazide groups are useful
for reaction with aldehydes generated by periodate oxidation of
carbohydrate groups in proteins.
[0086] The present invention describes the production of human
monoclonal antibodies directed to tumor-associated antigens, tumor
cells, infectious agents, infection-specific antigens, and self
antigens using a modified cell fusion partner, trioma cell line and
human lymphocytes derived from lymph nodes, spleen, Peyer's
patches, or any other lymph tissue or peripheral blood of the human
subjects.
[0087] Antibodies are selected using cultured cells, purified
antigens, primary human cells and tissues and combinatorial
libraries relevant to the antibody screening including cells and
tissues obtained from autologous donor of lymphoid cells.
[0088] This invention is illustrated by examples set forth in the
Experimental Details section which follows. This section is
provided to aid in an understanding of the invention but is not
intended to, and should not be construed to, limit in any way the
invention as set forth in the claims which follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Construction of Mouse-Human Heteromyeloma for the Production of
Human Monoclonal Antibodies
INTRODUCTION
[0089] B6B11 or B6B11-like cells may be produced by the fusion of
mouse myeloma cells with human myeloma cells selected for
non-secretion of antibody. The specific generation and application
of heteromyeloma B6B11, is described herein below. B6B11 was
obtained by fusing the mouse HAT-sensitive and G-418 resistant
myeloma X63.Ag8.653 with the subclone of human myeloma RPMI 8226
selected for non secretion of lambda light chains. Fusion of human
splenocytes and B6B11 cells resulted in a fusion frequency of 30-50
hybrids per 10.sup.7 cells. This is similar to the frequency of
murine hybridoma formation. The hybrids are readily cloned by
limiting dilution, produce antibodies for at least 10 month and
grow in serum-free media. Two clones were obtained which secreted
human IgM reactive against lipopolysaccharide (LPS) of
Gram-negative bacteria. These clones were obtained by fusing in
vitro immunized human splenocytes with the B6B11 cells. Anti-lipid
A murine mAb is known to prevent development of septic shock
(Shnyra AA, et al., 1990). Human mAbs have important clinical
applications.
Results
Heteromyeloma B6B11.
[0090] Heteromyeloma, B6B11, was generated by PEG-fusion of mouse
myeloma 653 (HAT-sensitive, G-418) with human RPMI 8226, which was
selected for non-secretion of lambda chains. Hybrids were selected
in the presence of HAT and G-418. Selection for 8-Ag resistance was
done by gradually increasing the 8-Ag concentration from 2 ug/ml to
20 ug/ml for 2.5-3 weeks. The HAT-sensitive hybrid population
653.times.8226 was twice cloned. Clones were tested for the ability
to produce hybrids with human lymphocytes. One clone, designated as
B6B11, was selected. B6B11 cells died in medium containing
aminopterine, during a period of 5-6 days; no revertants were
detected for more than 18 months. In RPMI 1640 supplemented with
10% fetal calf serum (FCS), the line had the doubling time of about
25-30 hours, the maximal density in 75 cm.sup.2 flasks was
approximately 1.5.times.10.sup.6 cells/ml (in a volume of 30 ml).
B6B11 culture medium was tested for the presence of human
immunoglobulin by enzyme linked immunoassay (ELISA) using rabbit
anti-human immunoglobulin. B6B11 exhibited secretion of IgG, IgM or
IgA. Staining the cell preparations with MAH-L,H by PAP-technique
detected no traces of cytoplasmic light and heavy chain human
immunoglobulin.
Karyotyping.
[0091] FIG. 1 illustrates the distribution of parental and B6B11
cells by chromosomal content. Chromosomal analysis of the
heteromyeloma cells indicated that chromosomal number varies from
60 to 82.
[0092] FIG. 2 shows a fragment of the G-banded karyotype of B6B11
cells. Normal mouse chromosomes constitute about 84% of the
karyotype. There are several rearranged chromosomes. There are some
markers for mouse myeloma chromosomes as well as rearranged
heteromyeloma (human-mouse chimeric) chromosomes. One large
telocentric chromosome was represented in all B6B11 metaphase
plates examined. This suggested that the proximal portion of this
chromosome contains mouse and the distal portion contains human
genetic material of chromosome 3 (3p21.1-3p ter). Localization of
human material was performed as described (33). In some of analyzed
B6B11, cells human chromosome 19 and human chromosome 7 was
deleted.
Fusion of B6B11 Cells With Human Lymphocytes.
[0093] Fusion of B6B11 cells with freshly isolated peripheral blood
lymphocytes (PBL) and splenic lymphocytes (SPL) was performed as
described herein below in the Experimental Procedures Section.
Fusion of peripheral blood lymphocytes (PBL) and pokeweed mitogen
(PWM) treated peripheral blood lymphocytes (PBL) resulted in low
hybridoma yield (1-5 hybrids per 10.sup.7 lymphocytes), while
fusion with splenic lymphocytes (SPL) and pokeweed mitogen (PWM)
treated splenic lymphocytes (SPL) yielded 30-60 hybrids per
10.sup.7 cells (see Table 1) After the fusion, cells were seeded at
a density of 1.5.times.10.sup.5 cells per well. Variations in the
cell ratios of 1:1 to 1:2 (heteromyeloma:lymphocyte) had no effect
on the fusion efficiency for PBL or SPL. However, fusion efficiency
was dramatically reduced at B6B11:lymphocyte ratios of 1:4 to 1:8.
TABLE-US-00001 TABLE 1 Fusion of human lymphocytes with B6B11
cells. LYMPHOCYTES PBL PBL-PWM SPL SPL-PWM Number of fusion 4 6 10
8 Number of wells 1536 2304 4800 3072 Growth.sup.2, % 4 6.9 55 72
Hybrid populations.sup.3 per 1-3 3-5 30-50 40-60 10.sup.7
lymphocytes Wells with Ig 95 92 84 82 secretion.sup.4, % .sup.1
Fresh isolated peripheral blood lymphocytes (PBL) and splenocytes
(SPL) were activated with PWM (5 ug/ml) for 7-9 days in complete
RPMI 1640 supplemented with 15% FCS. .sup.2Wells with hybrids (% of
the total well number) .sup.3After fusion cells were seeded at a
density of 15 .times. 10.sup.4 cells/well .sup.4Total Ig production
was determined by ELISA with mouse monoclonal antibodies to H- and
L-chains of human Ig
[0094] The effects of splenocyte stimulation with various mitogens
on the fusion efficiency are illustrated in FIG. 3. PWM treatment
significantly increased the efficiency of SPL hybridization
compared with ConA-treatment, PHA-treatment, LPS-treatment or
untreated SPL. Fusion efficiency was dependent on the timing of the
HAT addition. When HAT was added immediately following fusion, the
yield decreased to 10-15 hybrids per 10.sup.7 lymphocytes (for
SPL).
[0095] Cloning of hybrids with SPL and PBL (stimulated and
non-stimulated) indicated that PBL could not be used for hybridoma
formation. Cloning was performed 4-6 weeks after fusion in 50%
epithelial conditioned media (ECM) (pre-incubated for 24 hours at
37.degree. C. in 96-well plates) and 50% RPMI 1640 containing 15%
FCS. Results were determined at in 2-2.5 weeks. Cloning efficiency
(1.5-2 cells per well) was 50-80% for SPL and 10-30% for PBL. ELISA
using rabbit anti-human immunoglobulin and MAH-L,H indicated that
the total immunoglobulin production was present in 90-95% of
growing hybrids with PBL and 80-85% with SPL hybridomas. Based on
SPL was selected for PWM stimulation and in vitro immunization.
[0096] In order to increase the efficiency of hybridization,
splenocytes were treated with 2.5 mM Leu-Ome and fused with B6B11
cells at ratio of 1:1 or 1:2 (B6B11: SPL) (see Table 2). The effect
on this treatment was apparent after 18-24 hours of cultivation
with PWM; SPL without Leu-Ome treatment exhibited blasts only after
three days. The efficiency of hybridization of Leu-Ome-treated SPL
was somewhat higher (80%) compared with non-treated SPL (72%). This
treatment considerably increased (93%) the number of Ig-secreting
hybrids. TABLE-US-00002 TABLE 2 Effect of Leu-Ome treatment of
splenocytes on the efficiency of their hybridization with B6B11
cells (data from 3 spleens) Number Wells with hybrid Wells2 with Ig
Lymphocytes of wells populations, (%) secretion, (%) SPL 1440 1034
(72) 825 (80) SPL-Leu-Ome 864 691 (80) 642 (93) .sup.1 Splenocytes
were isolated in LSM. One portion was treated with Leu-Ome (2.5 mM,
40 minutes in serum-free RPMI 1640), the other served as a control.
Prior to fusion both portions were cultured for 7 days in complete
RPMI 1640 supplemented with 15% FCS in the presence of 5 .mu.g/ml
PWM. .sup.2 Ig production was determined by ELISA with mouse
monoclonal antibodies to H- and L-chains of human Ig.
[0097] The heteromyeloma cells were fused with Leu-Ome-treated
splenocytes immunized with Salmonella minnesota ReS95 (Re595) in
the presence of PWM and mouse thymocyte conditioned media (TCM)
(Table 3). The hybridoma culture supernatants were tested for
anti-bacterial antibodies at different stages of hybrid growth: (1)
after transferring responding populations to 24-well plates and (2)
after cloning and subsequent clonal expansion. Two independent
clones producing anti-bacterial antibodies were selected. ELISA
using immobilized lipoplysaccharide (LPS) or immobilized Re595 and
LPS in solution determined that the antibodies produced by both
clones reacted with LPS.
[0098] ELISA using immobilized ReS95 monoclonal mouse anti-human
isotypes and goat anti-mouse peroxidase conjugate absorbed with
human immunoglobulin, determined that the antibody isotype was
IgM-kappa. Both clones were adapted to serum free media (SFM) by
gradual replacing of the growth medium containing 10% FCS. The
maximal density upon culturing in SFM was approximately
1.2.times.10.sup.6 cells/ml. SFM-adapted cells were cloned as
described above. The efficiency and cloning time were similar to
those of the cells cultured in serum-supplemented RPMI 1640 medium.
TABLE-US-00003 TABLE 3 Fusion of in vitro immunized
splenocytes.sup.1 with B6B11 cells. Number of fusion 1 2 3 Number
of wells 288 864 576 Wells with hybrid 193 734 472 populations, (%)
(69) (85) (82) Wells with ig secretion, 173 675 420 (%) (90) (92)
(89) Primary response.sup.2 to Re595, 9 -- 17 number of wells (4.5)
(3.6) Secondary response.sup.3, 2 -- 16 number of wells Number of
responding -- -- 2 populations after cloning .sup.1Splenocytes
after treatment with Leu-Ome (2.5 mM, 40 min) were in vitro
immunized with S. minnesota Re595 (10.sup.7-10.sup.10 cells/ml) in
the presence of PWM (5 ug/ml) and TCM for 7-9 days. Fusions with
B6B11 cells were done at ratios 1:1 and 1:2 .sup.2ELISA of
hybridoma culture supenatants from 96-well plates (rabbit
anti-human Ig). .sup.3ELISA of hybridoma culture supernatants after
transferring in 24-well plates (rabbit anti-human Ig).
DNA Analysis.
[0099] FIG. 4 illustrates the distribution of the DNA content by
parental lines, B6B11 heteromyeloma and B6B11-splenocyte hybrid.
The DNA of heteromyeloma cells consists of 78.7% of the total
parental DNA. The DNA content of B6B11-splenocyte hybrid cells is
3% greater than that of B6B11 cells.
Discussion
[0100] A partner cell line for production of human monoclonal
antibodies was generated by somatic hybridization of mouse
X63.Ag8.653 and human RPMI 1640 myeloma cells. Adaptation to medium
with 8-Ag, subsequent cloning and selection by hybridization
efficiency led to a heterohybrid clone which was designated B6B11.
Fusion between heterohybrid lines and lymphocytes gives essentially
stable productive hybrids (Raison R L, et al., 1982). The
mechanisms underlying this phenomenon are unknown. It is suggested
that human chromosomes or their fragments retained in the partner
line after the first fusion modify the intracellular environment in
such a way that the human lymphocyte chromosomes or fragments after
the second fusion are stabilized (Oestberg L, and Pursch E., 1983).
The large number of chromosomes, the presence of hybrid marker
chromosomes and increased DNA content observed in the experiments
described herein, confirmed the hybrid nature of B6B11 cells. The
DNA content of B6B11-SPL hybrid cells was also increased.
Immunocytochemical testing for intracellular heavy and light chains
and ELISA testing for immunoglobulin secretion demonstrated that
B6B11 cells produce neither immunoglobulins nor heavy and light
chains. Fusion of B6B11 with SPL resulted in more hybrids than
fusion with PBL (30-50 per 10.sup.7 SPL compound to 1-5 per id PBL)
Cloning efficiency with SPL was 50-80% as compared to 10-30% with
PBL. Thus SPL were the more preferable partner for fusion. The
culture media was conditioned by endothelial cells; which was
deemed crucial for viability and clonogeneity of the hybrids. In
the case of B6B11-PBL hybrids, immunoglobulin secretion was
detected in up to 95% of the hybrids. To increase the yield of
immunoglobulin-secreting hybrids after fusion with SPL (up to 93%)
Leu-Ome was used. Almost all hybrids secreted antibodies of unknown
specificity. The antibody production by B6B11 hybrids was stable
for at least 10 months. The hybrids were readily adapted to
serum-free media, thereby facilitating a ex-vivo antibody
production.
[0101] Two antibody-producing clones (with probably similar
specificity to LPS of S. minnesota Re595) were obtained after
fusion of immunized SPL with B6B11 cells. As demonstrated herein,
human-mouse heteromyeloma, B6B11, is useful for producing human
monoclonal antibodies to various antigens. Proper in vitro
sensitization of lymphocytes is also of critical importance for
generating human antibodies.
Experimental Procedures
Cell Culture.
[0102] 8-Azaguanine (8-Ag) resistant mouse myeloma X63.Ag8.653
(653) cells were transfected with plasmid pBgl-neoR (Dr. A.
Ibragimov) as described below. The myeloma cells were maintained in
DMEM medium supplemented with 10% fetal calf serum (FCS), 4 mM
L-glutamine, 1 mM Sodium pyruvate, non-essential amino acids and
vitamins (Flow Laboratories). Prior to fusion the cells were
passaged 3 times in the presence of 20 .mu.g/ml 8-Ag (Sigma) and
500 .mu.g/ml G-418 (Gibco).
[0103] Human myeloma cell line RPMI 8226 (8226) was cultured in
RPMI 1640 medium with above-mentioned supplements (regular RPMI
1640). The hybrid heteromyeloma B6B11 was cultured either in
regular RPMI 1640 with 10% FCS or in serum-free media which
represented 1:1 mixture of Iscove's modification of Dulbecco medium
(IMDM) and HAM F-12 (Flow Laboratories) supplemented with bovine
serum albumin fraction #5, 2 mg/ml, (BSA) (Sigma), bovine insulin,
5 .mu.g/ml (Serva), human transferrin, 5 .mu.g/ml (Sigma),
progesterone, 6 ng/ml (Gibco), hydrocortisone, 60 ng/ml (Gibco).
Hybridomas were adapted to this serum free medium (SFM) by gradual
replacement of the growth medium containing 10% FCS. All cells were
cultured in a humidified atmosphere of 5.5% CO.sub.2/94.5% air at
37.degree. C.
[0104] Human peripheral blood lymphocytes (PBL) were isolated using
lymphocytes separation medium (LSM) (Flow Laboratories) as per
manufacturer instructions. Spleens collected at autopsy not later
than 2 hours after death (males aged 50-60 years old) were
homogenized and splenocytes (SPL) were isolated in LSM.
Production of Geneticin (G-418) Resistant 653 Myeloma Cells.
[0105] Cells were washed in sterile phosphate buffered saline (PBS)
without Ca.sup.++ or Mg.sup.++. pBgl-neoR Plasmid DNA linearized by
BamH1 (constructed by P. Chumakov, Institute of Molecular Biology
of the Academy of Sciences of the USSR, Moscow, USSR) was added to
the cell suspension. Prior to adding the DNA to the cell
suspension, the DNA was twice phenol extracted using phenol-ether
at 4.degree. C., 96% ethanol precipitated and dried under sterile
conditions.
[0106] Transfection was performed by electroporation at 4.degree.
C. using a unit constructed by L. Chernomordik (Institute of
Electrical Chemistry of the Academy of Sciences of the USSR,
Moscow, USSR). Approximately 4.times.10.sup.6 653 myeloma cells and
3.5 .mu.g of plasmid DNA were combined in an 80 .mu.l
electroporation chamber. The final concentration of DNA was 44
.mu.g/ml). An electrical current impulse of 1.7 Kv/cm was pulsed
through the chamber for 100 .mu.sec. After resting for 10 minutes
the cells were transferred to 0.5 ml complete media in 16 mm.sup.2
wells at 5.times.10.sup.3 and 2.times.10.sup.4 cells/well. After 36
hours, 0.5 ml of media containing 1 mg/ml of Geneticin (G-418) was
added to a final concentration of 0.5 mg/ml. Subsequently, 50% of
the media volume was changed every 2 days for 12 days.
Production of Heteromyeloma.
[0107] G-418-resistant 653 cells were mixed with 8226 cells at a
1:1 ratio and pelleted. 50% (v/v) polyethylenglycol (PEG) 3350
(Sigma) was added (200-300 .mu.l per 4-5.times.10.sup.7 cells) for
1 min with constant stirring. Several portions of serum-free RPMI
1640 (RPMI-S.sup.-) were added for 5 minutes (first 10 ml), 1
minute (10 ml), and 1 minute (30 ml). Cells were pelletted
resuspended in regular RPMI 1640 with 20% FCS, hypoxanthine
(1.times.10.sup.4 M), aminopterine (4.times.10.sup.7 M), thymidine
(1.6.times.10.sup.5 M) (HAT, Flow Laboratories) and 500 .mu.g/ml
G-418 and seeded in 96-well plates (Linbro) at a density of
10.sup.5 cells per well. At two weeks the medium (1/2 volume) was
replaced with medium containing hypoxanthine (2.times.10.sup.4 M),
thymidine (3.2.times.10.sup.5 M) (HT, Flow laboratories) and G-418
(500 .mu.g/ml). The procedure was repeated after two weeks.
Production of Human Monoclonal Antibodies.
[0108] Fusion of B6B11 cells with human lymphocytes was
accomplished by the above-described method with following
modifications. Lymphocytes were mixed with B6B11 at a 1:1 or a 1:2
ratio, pelleted, washed with RPMI 1640-S- and incubated with PEG
(600 .mu.l per 10.sup.5 cells) for 3 minutes with constant
stirring. The portions of added RPMI-S- were as follows: 10 ml/10
minutes, 10 ml/10 5 minutes, 10 ml/l minute. Cells were pelleted,
re-suspended in regular RPMI supplemented with 15% FCS and seeded
in 96-well plates (1.5.times.10.sup.5 cells per well). HAT-medium
was added after 24 hours. The growth medium (1/2 volume) was
replaced with fresh HAT in 7-9 days. HAT-medium was replaced with
HT-medium at 15-18 days.
Cloning.
[0109] Parent heteromyeloma and hybridoma cells were cloned by the
limiting dilution method in medium conditioned by human umbilical
or aortic endothelial cells (Antonov A S, et al.,. 1986) (gift from
Dr. A. Antonov) (ECM). 100 .mu.l/well was incubated in 96-well
plates at 37.degree. C. overnight. Cells were planted at
approximately 1 to 2 cells per well. The culture medium was tested
for antibodies at 2.5-3 weeks.
Immunization In Vitro.
[0110] Freshly isolated lymphocytes were resuspended in
RPMI-S-containing 2.5 mM L-leucine methyl ester (Leu-OMe)
(Borrebaeck, C A K, et al., 1987) to a final concentration of 10
cells per ml. After 40 minutes of incubation at room temperature,
cells were washed 3 times with RPMI-S- and resuspended in regular
RPMI 1640 supplemented with 15% FCS. Medium conditioned by mouse
thymocytes (TCM) was used as a source of lymphokines (Reading C L.,
1982). Pokeweed mitogen (PWM) (Flow laboratories) to a final
concentration 5 .mu.g/ml, TCM (25%) and antigen in different
concentrations were added to the cell suspension. The cell
suspension (4-6.times.10.sup.6 cell/ml) was transferred to flasks
(30 ml/75 cm.sup.2 flask). Fusion was performed after 7-9 days of
cultivation. Concanavalin A (ConA) (Flow 5-10 .mu.g/ml),
Phytohemagglutinin (PHA) (Flow, 5-10 .mu.g/ml) and
lipopolysaccharide (LPS) (SIGMA, 10-15 .mu.g/ml) were used instead
of PWM. S. minnesota Re59S (gift of Dr. O. Luderitz, Max Plank
Institute fur Immunologie, Feiburg, Germany) was used as an
antigen. The bacteria were grown in medium containing 16 g/l
tryptic soy broth 10 (TSB), Difco), 16 g/l brain-heart infusion
(BHI) (Difco) and 4 g/l yeast extract (YE) (DIFCO) for 18 hours at
37.degree. C. with constant stirring and then heat inactivated. The
antigen concentration varied from 10.sup.7-10.sup.10 cells/ml.
Determination of Antibodies and Non-Specific Ig Production.
[0111] Enzyme linked immunoassay (ELISA) was used to test hybridoma
supernatants for the presence of antibodies against Salmonella
minnesota Re595 and LPS.
Screening for mAbs Reactive Against Bacteria.
[0112] 96-well plates were covered with glutaraldehyde (1%, 100
.mu.l per well) for 2 hours at room temperature. The plates were
washed with distilled water 3 times. Bacteria were resuspended in
50 mM ammonium carbonate buffer (pH 9.6) and transferred to plates
(5.times.10.sup.7 cells in 100 .mu.l per well), centrifuged at
780.times.g for 30 minutes and washed with distilled water 4 times.
The supernatants tested (100 .mu.l) were supplemented with 0.1%
Tween 20 (Fluka), put into bacteria-containing wells and incubated
for 1 hour at room temperature. The media was then removed and the
wells were washed with distilled water. Affinity purified rabbit
anti-human immunoglobulin conjugated to alkaline phosphatase
(RAH-AP), diluted in tris-buffered solution (TBS, 50 mM, pH 7.4),
containing 0.1% Tween 20 was added to 1 .mu.g in 100 .mu.l per
well. After 1 hour of incubation at room temperature and 6 washes
with distilled water 100 .mu.l of 4-nitrophenyl-phosphate (1 mg/ml,
Sigma) in diethanolamine buffer (10% diethanolamine, 0.5 mM MgCl2,
pH 9.8) was added. After 1 hour, the results were read using a
Multiscan (Flow Laboratories) at 405 nm. The negative control was
culture medium RPMI 1640 supplemented with 15% FCS.
Screening for mAbs Reactive Against Lipopolysaccharide.
[0113] LPS was purified from Salmonella Minnesota Re595 as
described (Galanos G, et al., 1969). The LPS preparation was
sonicated and transferred to the plates at 2.5 .mu.g per well in 5
mM ammonium carbonate buffer (pH 9.6). After overnight incubation
at room temperature, the above described procedures for determining
mAb reactive against bacteria were performed.
Screening for Non-Specific Production of mAbs.
[0114] Non-specific production of immunoglobulin and separate
chains was assessed after the addition of 100 .mu.l of rabbit
anti-human immunoglobulin (10 .mu.g/ml in phosphate buffer, PBS, pH
7.2) or 100 .mu.l/well (10 ng/ml in PBS) of mouse monoclonal
antibodies to light and heavy chains of human immunoglobulin
(MAH-L, H) (Rokhlin O V, 1989) (gift of O. Rochlin, CRC, Moscow).
Subsequent procedures were performed as described above.
Determination of the Isotype of Secreted Antibodies.
[0115] The isotype of human antibodies was determined by ELISA
using murine anti-human light and heavy chains (MAH-L, H) and goat
anti-mouse immunoglobulin (25 ug/ml) conjugated to peroxidase and
absorbed with human immunoglobulin.
Determination of Cytoplasmic Light or Heavy Chains Production.
[0116] Production of cytoplasmic light and/or heavy chains in
hybridomas, B6B11 and the parental cell lines was estimated
immunocytochemically using the peroxidase-anti-peroxidase system
(PAP)-Cell smears were air-dried, fixed for 45 seconds with 10%
formaldehyde (v/v) and 45% acetone (v/v) in phosphate buffered
saline (PBS, 10 mM NaH.sub.2PO.sub.4, pH 6.6) and incubated with
MAH-L,H (200 .mu.l, 5-10 mg/ml). Then 1 ml rabbit anti-mouse
immunoglobulin (38 mg/ml in PBS) was added. All incubations were 30
minutes. Washings were performed using PBS for 10 minutes.
Chromosomal Analysis.
[0117] Preparations of metaphase chromosomes were obtained by the
following technique. Colchicine was added to cells during
exponential growth (1.5-2 hours to parental lines and B6B11 cells).
Cells were then trypsinized and stained for G-banding as described
(Seabright S., 1971) (10-15 plates from each line). To count
chromosome number, at least 50 metaphase figures were analyzed for
each cell line.
DNA Analysis by Flow Cytometry.
[0118] To estimate the DNA content the cells (1.times.10.sup.6)
were fixed with 1 ml 70% ethanol, washed, incubated for 2-3 hours
with 0.3 mg/ml Ribonuclease A (Serva) in Hank's solution (pH 7.4)
and stained for 2 hours with propidium iodide (0.05 mg/ml, Sigma)
in Hank's solution. The DNA content was measured in a FACS-II
cytofluorometer (Becton Dickinson). Fluorescence. was excited by an
argon ion laser at 488 nm (164-05 Model, Spectra-Physics) at a
power of 400 mW and registered behind a 600 nm long pass
interference filter (Ditric Optica).
Parental Lines.
[0119] The myeloma line 653 was maintained in DMEM supplemented
with 10 FCS, 20 ng/ml 8-Azaguanine and 500 .mu.g/ml G-418. The
myeloma line 8226 producing lambda chains of human Ig was cultured
in RPMI-C containing 10% FCS. In order to create a heteromyeloma, a
non-producing clone of 8226 line was selected by cloning in ECM (2
cells per well). Lambda chain production was estimated at 2-2.5
weeks using MAH-L, H. The frequency of non-secreting clones was
1.times.10.sup.-3.
Example 2
Trioma MFP-2, a Fusion Partner for Generating Human Monoclonal
Antibodies
INTRODUCTION
[0120] A precursor hybridoma cell line was obtained by
hybridization of the commercially available human myeloma cell line
RPMI 8226 and mouse myeloma X63.Ag8.653 resistant to both
8-Azaguanine (8-Ag) and Geneticin 418 (G-418). One of the resulting
clones, B6B11, was selected in the presence of G-418. B6B11 was
grown in the presence of increased concentrations of 8-Ag and is
resistant to both G-418 and 8-Ag (See Example 1).
[0121] Although B6B11 can be used to make human hybridomas by
fusing with human lymph node-derived lymphocytes or spleen-derived
lymphocytes, B6B11 was not capable of fusing with human peripheral
blood lymphocytes (PBL) or resulted in a very low yield of hybrids
(see example 1).
[0122] In order to overcome this problem, B6B11 was fused with
human lymph node lymphocytes and several hybrids were obtained. The
resulting cells were analyzed for human immunoglobulin production
or production of separate immunoglobulin chains. Those clones,
which did not synthesize immunoglobulin or immunoglobulin chains
were selected for further evaluation in terms of fusion capability
and antibody secretion potential. These hybrids were determined to
be quite stable. These fusion products were designated "modified
fusion partner" (MFP) cells. These MFP cells as the product of the
fusion of the B6B11 hybridoma and lymphocytes are referred to
herein as "trioma" cells because they are, in essence, the product
of a three fused cells. One of the clones, MFP-2, exhibited a very
high efficiency for fusing with peripheral blood lymphocytes as
well as for fusing with human lymphocytes of any varied origin
(i.e. lymph nodes, spleen, Peyer's patches etc). MFP-2 was selected
on the basis of its superior characteristics and stability as a
fusion partner and was used in the experiments described herein
below.
[0123] The products of fusions between the MFP trioma cells and
lymphocytes are referred to herein as "tetroma" cells because they
are, in essence, the product of four fused cells.
Results
[0124] Immunoglobulin Production. In order to demonstrate that
human hybrioma (trioma) fusion partner cell line, MFP-2, is capable
of fusing with human lymphocytes and producing high yields of
hybrids with stable immunoglobulin production, experiments were
performed using human lymphocytes from different sources.
[0125] The heteromyeloma cell line, B6B11 (precursor to MFP-2), can
be fused with high efficiency with lymph node and spleen
lymphocytes. (See, Example 1). Up to 90% of the resulting hybrids
produced IgG or IgM. However, B6B11 was incapable of fusing to
lymphocytes derived from peripheral blood (PBLs). The trioma cell
line, MFP-2, (resulting from a fusion between B6B11 and human lymph
node lymphocytes) overcame this problem and exhibited high fusion
efficiency with PBL, yielding a high rate of immunoglobulin
production by the resulting tetroma hybrids. The capability of
MFP-2 to fuse with PBL was tested in two ways: (1) by fusion with
freshly isolated lymphocytes in suspension, and (2) by fusion with
thawed lymphocytes which had been stored frozen for various periods
of time. (See Experimental Procedures). The results of these
experiments are shown in FIG. 5.
[0126] The fusion efficiency was 10.sup.5 (1 hybrid per
10.sup.5lymph-mphocytes). Thirty primary hybridoma (tetroma)
populations were obtained and analyzed for capacity to secrete
immunoglobulin. (A primary hybridoma population is likely to be a
mixture of two or more individual clones). Twenty-seven populations
(90%) produced IgM at a level 5-fold greater than background.
Twenty-four populations (80%) secreted IgE at a level 5-fold
greater than background. The fusion of MFP-2 with lymphocyte
suspensions which had been frozen and thawed also resulted in
immunoglobulin-producing hybrids. Nineteen percent and 11% of these
hybridoma populations produced human IgM and IgG respectively. The
efficiency of fusion, itself, was not effected by the freeze-thaw
procedure. These results demonstrate that both freshly isolated as
well as frozen PBLs can be used to generate human hybridomas
capable of producing antibody.
Identification of Tumor-Associated Antigens and Production of
Specific Antibodies Using the MFP-2 Fusion Partner: Human
Monoclonal Antibodies Against Thyroglobulin.
[0127] In this experiment, human anti-thyroglobulin antibodies were
generated by MFP-2 fusion using lymph nodes from patients diagnosed
with thyroid adenocarcinoma. A periclavicular lymph node was
excised during lymphadenectomy surgery from a female thyroid cancer
patient and lymphocytes were isolated and fused with MFP-2,
generating tetroma cells.
[0128] The resulting hybridomas (tetromas) were tested for
production of human antibodies reactive against thyroglobulin using
an enzyme linked immunoassay (ELISA) procedure. Purified human
thyroglobulin was used to coat a microtitre plate. Results are
shown in FIG. 6. Thirty-three of 144 tetromas exhibited a response
against the thyroglobulin antigen. Eight of these were particularly
strong. (See FIG. 6). Thus, lymph node-derived tetromas from this
thyroid cancer patient were producing anti-thyroglobulin
antibodies. This was an unexpected and surprising result because
the patient had no known history of autoimmune (i.e. anti-thyroid
antibodies) disease. This suggests that the antibodies produced in
this patient to thyroglobulin were induced by the presence of
cancerous thyroid adenocarcinoma cells. Cancerous thyroid
adenocarcinoma cells are known to secrete thyroglobulin. This
experiment demonstrates that tumor cells can induce a humoral
immune response to tumor-associated antigens and that the
antibody-producing cells can be identified and immortalized through
the techniques described herein using the MFP-2 fusion partner in
order to produce human anti-tumor monoclonal antibodies.
Production of Human Monoclonal Antibodies Against Breast Cancer
Associated Antigens.
[0129] In another experiment, human monoclonal antibodies were
produced against cancer associated antigens using lymph node and
peripheral blood lymphocytes from breast cancer patients. Axillary
lymph nodes were excised from breast cancer patients who underwent
mastectomy or lumpectomy. Lymphocytes isolated from these lymph
nodes were fused to MFP-2 and the resulting tetromas were screened
against breast cancer cell lines MCF7, SK-BR-3, ZR-75-1. Nearly all
the tetromas were producing IgG or IgM (approximately 85% and 10%
respectively). Surprisingly, nearly 15% of the tetromas assayed
against breast cancer cell lines produced antibodies specifically
directed against cancer cells. The tetroma supernatants were tested
in two ways: (1) on a live cells in the CELISA (cellular ELISA)
assay and (2) by Western blotting using cell lysates. The molecular
weight range of the specific antigens recognized by human
monoclonal antibodies was 25 to 160 kDA. In order to delineate the
nature of the antigenic target, immunoprecipitation followed by
microsequencing is performed. In addition, random peptide
combinatorial libraries are used to identify the molecular targets
of the cancer-specific antibodies.
[0130] In one patient with Stage IV breast cancer, lymph nodes were
not available so PBLs were fused to MFP-2 and 156 tetromas were
obtained. The tetromas were analyzed for immunoglobulin production
as well as for cancer-specific antibody production. IgM was
produced by 28 tetromas; 87 tetromas produced IgG. Four of the IgM
antibodies and seven of the IgG antibodies were identified as
reactive against cellular antigens; three IgM anti-bodies and four
IgG antibodies were specific for breast cancer cells. The rest of
the tetromas exhibited immunoreactivity against other cell types
including human prostate cancer cell lines, human diploid
fibroblasts and human skin fibroblasts. These latter antibodies
were probably directed to common antigens (common for normal and
cancerous cells).
[0131] The PBLs were isolated from the blood of a patient who
received 77 cycles of chemotherapy which would reasonably be
expected to have a depressing effect on the patients immune system.
None-the-less, this patient still produced anti-cancer antibodies
suitable for fusing with MFP-2.
[0132] Human tetromas generated, from fusing MFP-2 and prostate
cancer lymphocytes are tested for the presence of PSA-specific
antibodies as well as antibodies directed to prostate cancer cell
lines LNCaP, DU-145, and PC-3.
Production of Human Antibodies Against Infectious
Disease-Associated Antigens.
[0133] Infectious diseases are commonly accompanied by a
well-developed humoral and cellular immune response. Patients with
certain infections often contain large numbers of specific antibody
producing cells. One important application of the antibody
immunotherapy described by the present invention, is the production
of human monoclonal antibodies to proinflammatory cytokines which
are involved in septic shock. Among these targets are cytokines
such as tumor necrosis factor .alpha. (TNF-.alpha.) and
interleukin-1a (IL-1a). Additional targets include other cytokines
and lymphokines, infectious agents and their toxins, including
tetanus toxin, anthrax toxin, botulinum toxin, and lipid A. The
peripheral blood of patients infected with bacteria, fungi,
protozoa or viruses typically contains circulating
antibody-producing cells which can be isolated and used as a source
for fusion with MFP-2. For example, PBLs from patients with septic
shock, Hanta virus infection, HIV, HTLV-I, HTLV-II, influenza,
hepatitis, or herpes virus can be fused with MFP-2 and the
resulting tetroma cells can be screened against the respective
antigens. In AIDS, in particular, patient lymphocytes can be
immortalized using the techniques described herein in order to
generate bulk quantities of anti-HIV antibodies for use in passive
immunotherapy in an autologous or heterologous manner.
Production of Human Antibodies Against Autoimmune Disease.
[0134] A general consideration for the use of human monoclonal
antibodies in autoimmune disease is to block autoantibodies, or to
block CD4.sup.+ T cells which are involved in autoimmune cellular
cytotoxicity. In one approach, human monoclonal antibodies against
CD4.sup.+ cells are generated following fusion with the MFP-2
trioma cell. Resulting tetroma cells which produce anti-CD4
antibodies are used to reduce or deplete CD4.sup.+ T cells, thereby
relieving autoimmune cellular attack. In another approach MFP-2 is
used to generate tetroma cells capable of producing anti-idiotypic
antibodies directed to specific autoantibodies. For example,
autoimmune thyroiditis is an autoimmune dysfunction in which there
is a high titer of anti-thyroglobulin antibodies in a patient's
plasma. PBL-derived lymphocytes are isolated from such patients for
fusion with MFP-2. The resultant tetroma cells are screened for
those capable of producing antibodies with a substantial
anti-idiotypic immune response directed against the autoantibodies
reactive with thyroglobulin. These anti-idiotypic antibodies are
then used to modulate the autoimmune disease by reducing or
depleting the anti-thyroglobulin antibodies. Such an approach may
be used autologously or heterologously. In an autologous approach,
the anti-idiotypic antibody-producing cells are identified in
peripheral blood of the patient to be treated, then isolated and
fused with MFP-2 and following selection for specific
anti-anti-thyroglobulin antibodies, passively administered to the
original patient. In a heterologous approach, the
anti-anti-thyroglobulin antibodies are administered to a different
patient.
Other Applications: Preventing Rejection of Transplanted Organs,
Blood Clotting.
[0135] Among other applications of human monoclonal antibodies, is
prevention of organ transplant rejection by blocking T cells
through the OKT-3 (anti-CD3) marker. Antibodies to adhesion
molecules (anti-integrin antibodies) also prevent migration of
immune cells, which is important, for example in rheumatoid
arthritis. Blood clotting may be modulated, for example, in acute
cardiac ischemia following coronary angioplasty, using human
monoclonal antibodies against GPIIb/IIIa of platelet. Intravenous
infusion of immunoglobulins helps to neutralize the Fc-receptor
mediated cell aggregation of platelet or other blood cells (e.g.
thromobytopenic purpura).
[0136] In addition, this approach may be used to detoxify or
neutralize toxin or venom exposure. Such exposures include, but are
not limited to snake, spider or poison toad bites or yellow jacket
or scorpion stings. The horse anti-serum currently used to
neutralize rattle snake venom causes serum sickness disease in 30%
of cases.
[0137] There is a shortage of natural human immunoglobulin required
for these kinds of treatments. The human monoclonal antibody
production system described herein facilitates production, in
vitro, of unlimited quantities of human immunoglobulins which can
be selected to fit particular need. For example, in the case of
immunoglobulin which blocks Fc receptors, instead of treating the
patient with the pooled preparation of immunoglobulins where only a
small fraction of molecules possess the required qualities, the
immunoglobulin preparation of the molecules with the required
properties can be produced using the fusion partner described
herein.
Discussion
[0138] There has long been a need for human monoclonal antibodies
for diagnosis, treatment, and monitoring of cancer. Attempts to
employ xenoantibodies in clinical trials have not produced
promising results. Non-human antibodies from mice, for example,
cause development of a human anti-mouse immune response,
sensitization to foreign protein which may eventually result in
anaphylactic reaction, and lack of biological effect since the
effector properties of the xenoantibodies may mismatch the
components of the human immune system. Human monoclonal antibodies
have numerous advantages. One is that human monoclonal antibodies
can identify those tumor-associated antigens (TAA) which are
immunogenic only in humans, while xenoantibodies in most cases
recognize those antigens and antigenic epitopes which express
immunodominance in a host and are often the tissue specific
epitopes. Another advantage is the well-developed interaction of
human monoclonal antibodies with the effector components (such as
complement) of the host immune system. In addition, allergic and/or
anaphylactic reaction to the injectible human monoclonal antibodies
is less of a concern since human monoclonal antibodies are syngenic
in human subjects. Alternative attempts have been made to develop
antibodies such as chimeric antibodies (partially human, partially
murine), where the Fc part of the murine immunoglobulin was
substituted with the human IgG-Fc. Humanized antibodies, are human
immunoglobulins grafted with the CDR regions of the specific murine
antibodies. Single chain (Fc) human antibodies have been developed
in phage using phage display libraries. A downside of these
approaches is that the resulting antibodies are not natural; they
have not emerged as part of a natural immune response to cancer or
infectious agent.
[0139] Use of the hybridoma techniques described herein and the
availability of the MFP-2 trioma fusion partner cell line described
herein, facilitates identification, immortalization, and ex-vivo
expansion of antibody-producing cells which emerge in vivo as a
result of natural humoral immune responses to an antigen. Since
such cells are a part of the natural immune system response, the
antibodies produced by these cells dovetail with the other
components of the immune system and are able to provide an
effective and specific biological response.
[0140] A number of breast cancer specific antigens have been
described which are potential targets for the immunotherapy of
cancer, including HER2/neu, Mucin 1 and Mucin 2, p53, c-myc, blood
antigens T, Tn and sialyl-Tn, tuncated form of EGF, Lewis-Y antigen
and others. The presence of circulating antibodies to these
antigens have also been described in cancer patients. (G. Moller,
1995). Lymph nodes are important sites of such antibody-producing
cells. By isolating lymph node (or peripheral blood) lymphocytes
and immortalizing them by fusing them with human hybridoma fusion
partner MFP-2, hybrids (tetromas), which produce antibodies
directed against cancer-associated antigens may be obtained. As
described above, specific monoclonal antibody producing cells are
identified and may be produced in unrestricted fashion, ex-vivo
(using bioreactors, SCID mice, etc). The antibodies may be used
therapuetically as passive immunotherapy either autologously in the
same subject or heterologously in a different subject. Even another
cancer may be treated, provided there is an overlapping tumor
antigen.
[0141] Syngenic or allogenic use of human monoclonal antibody can
be highly effective since such an antibody can be infused many
times without the risk or threat of developing an anti-xenogenic
immune response. The infused antibodies, depending on their
effector functions, can initialize complement dependent cytolysis
of the target tumor cells, or antibody-dependent cellular
cytotoxicity antibody dependent cellular cytotoxicity (ADCC) (by NK
or CTL cells), or provide direct cytotoxic effect through
apoptosis.
SUMMARY
[0142] A unique fusion partner cell line, MFP, was obtained which
can be used to generate specific human monoclonal antibodies. These
monoclonal antibodies may be in vivo based on a natural immune
response to infectious agents, cancer cells or an autoimmune
dysfunction, or can be in vitro based by immunization of human
lymphoid cells in vitro.
[0143] The methods described herein for generating specific
monoclonal antibodies may be used to provide adoptive humoral
immunotherapy either as an autologous procedure or as a
heterologous procedure. Lymphocytes isolated from a patient with a
cancer or infectious disease are immortalized by fusion with MFP-2.
The resulting tetromas, producing antibodies directed to the
respective antigens, are selected in vitro. Following selection,
these antibody-producing cells are expanded and antibodies may be
produced using a bioreactor or immune-deficient mice (e.g., nude
mice or SCID mice). Such antibodies may then be used for the
treatment of the original donor as an autologous adoptive
immunotherapy procedure or for the treatment of a different subject
as a heterologous, adoptive immunology procedure.
[0144] The developed antibodies may also be applied both to
invasive diagnostics (imaging, immunoscintigraphy) or therapy (drug
targeting, radioimmunotherapy, complement-dependent cytolysis,
ADCC, apoptotic cytolysis etc.)
[0145] This approach also provides a method for identification of
novel tumor markers or novel infectious agent antigens. The immune
system responds to cancer cells or infectious agents by producing
antibodies directed to different components of the foreign
formation and can recognize different neo-epitopes. Fusing tumor
reactive or infectious agent antigen reactive immunoglobulin with
MFP-2 can be used to identify novel tumor markers or infectious
antigens. Such antibodies are important in treatment against
specific cancers or infectious agents, and in the generation of
specific imaging and diagnostic techniques. Previous attempts to
generate human anti-tumor or anti-infectious antibodies required
forced or artificial immunization of a subject with purified or
isolated antigen. In the present invention, the antigen may be
unknown; the starting material for developing antibodies is the
pool of immunocompetent lymphocytes which evolved as a part of
natural immune response to the foreign antigens presented in their
natural form and in natural environment in vivo. In an autologous
application, selection can be conducted using an autologous tissue
of interest (e.g. tumor biopsies) which will increase the chances
to select the right antibody. Also, autologous blood plasma and
white blood cells can be used to select for cytotoxic antibodies
from the same donor.
[0146] Thus, the MFP fusion partner (1) allows fusion with
peripheral blood lymphocytes yielding high levels of hybrids; (2)
allows consideration of an adoptive humoral immunotherapy on an
individual basis (selection of the antibodies against tumor cells
or infectious agents derived from the same donor the lymphocytes
were obtained from and the autologous treatment of the patient);
(3) fusion with the donor's lymphocytes undergoing immunization in
vitro; (4) allows use of frozen lymphocytes or lymphocytes derived
from plasmapheresis as a source of antibody-producing cells.
Experimental Procedures
[0147] Hybridoma fusion partner MFP-2 was developed as a trioma
cell line by fusing non-producing heteromyeloma B6B11 with human
lymphocytes isolated from the paraclavicular lymph node.
Isolation of Lymphocytes.
[0148] Paraclavicular lymph nodes from a patient diagnosed with
metastatic thyroid cancer were excised during the surgery and
placed into sterile conservation media RPMI1640 supplemented with
L-glutamine (4 mM), non essential amino acids (100.times. stock),
vitamins (100.times. stock), sodium pyruvate (1 mM) and Gentamicin
(2.times. concentration). Lymph node tissue was transferred to a
100 mm tissue culture TC dish in the same media and gently
disrupted with forceps and scissors. The disrupted tissue was
passed through a metal sieve (50 mesh) using a glass pestle. The
suspension was transferred into 15 ml sterile conical tubes
containing lymphocyte separation media (Histopaque 1.077 Sigma) as
an underlying layer at a ratio of 2:1 (lymphocytes suspension:
Histopaque). Following centrifugation at 400.times.g for 20
minutes, an opaque ring formed at the border between layers. Red
blood cells (RBC) were present as a pellet at the bottom of the
tube. If RBC are not present in the starting lymphocyte suspension
(which is a quite normal situation for lymph nodes) the separation
step can be skipped. The opaque ring containing lymphocytes was
carefully collected using a Pasteur pipette and was diluted 10-fold
diluted with regular serum-free RPMI 1640. Cells were spun at
300.times.g for 10 minutes and washed twice with media.
[0149] The final lymphocyte suspension was diluted with media and
cells were counted using 0.05% Trypan Blue. Cell viability after
isolation was usually 95%. Total yield was approximately
4.times.10.sup.7 cells.
[0150] Preparation of B6B11. Heteromyeloma B6B11 was grown in RPMI
1640 with 10% cosmic calf serum (Hyclone), standard set of
supplements (L-Glu, 4mM non-essential amino acids, vitamins, Sodium
Pyruvate) without antibiotics. Before fusion, cells were cultured
in the presence of 8-Ag (20 .mu.g/ml) to avoid reversion of
HAT-sensitive cells to wildtype. Cells were grown to a density of
10% in logarithmic growth phase.
Cell Fusion.
[0151] Both B6B11 cells and lymph node lymphocytes were washed 3
times by centrifugation at 300.times.g for 5 minutes in order to
remove any residential protein in the media. Cells were mixed at a
ratio of 5:1 (lymphocyte: myeloma) and spun at 300.times.g for 10
minutes. The supernatant was carefully and completely removed the
pellet was "puffed" gently and 100 .mu.l of PEG/DMSO solution
warmed to room temperature was added to the cell mixture which was
gently tapped for 3 minutes. Then 15 ml of Hank's Balanced Salt
Solution (HBSS) and PBS (1:1) (from a 10.times. stock, Cellgro)
were added as follows: 10 ml slowly in 10 minutes, then 5 ml over 5
minutes, then 10 ml of complete media (media for cell culturing)
over 5 minutes and finally S ml over 1 minute. The total volume was
30 ml. Then 600 p1 of HT solution (of 10.times. stock) and 1 drop
(about 20-30 p1) of DMSO were added to the tube. The cell
suspension was mixed in a tube, transferred to Petri dish
(100.times.15) and incubated in a 37.degree. C. CO.sub.2 incubator
overnight. The cells were then harvested, pelleted at 300 .times.g
for 10 minutes and resuspended in complete media supplemented with
HAT-solution and HT-solution (both from 50.times. stock) and then
plated into 96-well plates in a 200 .mu.l volume at about 250,000
cells per well. Twice a week, 50% of the media was replaced with
fresh media. Cells were cultured in the presence of HAT and HT for
14-20 days before screening for antibody production.
ELISA Screening for Nonspecific Imoglobulin.
[0152] ELISA plates were coated with polyclonal goat-anti-human IgG
(Fc-specific) (Sigma), goat-anti-human IgM (.mu.-specific) (Sigma)
or goat-anti-human Ig(G+M+A) H-chains (Sigma) in 100 .mu.l of
plating buffer (0.1 M Sodium Carbonate, pH 9.0) at 100 ng per well.
The plates were sealed with Parafilm or sealing covers and
incubated overnight at 4.degree. C. The antigen was washed out with
distilled water twice. Residual drops of water were removed and 200
.mu.l of blocking solution (0.4% dry non-fat milk in PBS) was added
to the wells. Complete cell culture media served as a negative
control. Human serum (1:2000) was used as a positive control.
Plates were incubated for 2 hours at room temperature or overnight
at 4.degree. C. The plates were washed 4 times with distilled water
and secondary antibodies (same as capture antibodies but conjugated
to HRP) diluted in 0.4% milk/PBS at 1:2000 were added to the wells.
After 1 hour incubation at room temperature the wells were washed 4
times with H.sub.2O and peroxidase substrate (ortophenylendiamine
in phosphate-citrate buffer with peroxide) was added to the plates.
The color reaction was stopped by adding 20 .mu.l of 10% sulfuric
acid. Colorimetric reading was performed on a Multiscan reader at
A.sub.492. Samples which exhibited at least a 3-fold increase over
background were considered to be immunoglobulin-producing
cells.
Assay for the Intracellular (Non-Secreted) Presence of
Immunoglobulins or Their Individual Chains.
[0153] Cells which did not secrete immunoglobulin in the
supernatant culture media were tested for the presence of
intracellular immunoglobulin-immunoreactive material. ELISA plates
were coated with goat-anti-human kappa chain (Sigma),
goat-anti-human lambda chain (Sigma) and goat-anti-human IgH
(G,M,A) as described above. Cells were grown in 75 cm.sup.2 flasks
to the density 10.sup.6 cells per ml, harvested and washed 3 times
with HBSS. Cells were resuspended in PBS and disrupted by
sonication (8.times.15 seconds at 25 MHz on ice). The suspension
was spun for 15 minutes at 10,000 .times.g and the supernatant was
used for immunoglobulin testing. An equivalent of 2.times.10.sup.6
cells was used. As a negative control mouse fibroblasts 3T3 were
used at the same protein amount equivalent. The rest of the
protocol was the same as described above for the hybridoma
supernatant testing. Clones which showed the signal equal to the
control cells or lower were chosen as potential candidates for
fusion with human peripheral blood lymphocytes. These trioma clones
were designated as modified fusion partner series (MFP-S) and
numbered sequentially (MFP-1, MFP-2, MFP-3, etc.) Six
non-producing, non-secreting triomas were selected for further
analysis.
Selection for 8-Ag Resistant MFP Mutants.
[0154] To use MFP trioma cells as fusion partners, the MFP cells
were placed in complete media containing an increasing amounts of
8-Ag. Resistance to 8-Ag is determined by the impaired enzyme HGPRT
or its absence. Selection was therefore focused on cells which
survived in the presence of 8-Ag. After 5 to 10 passages at the
lower concentrations of 8-Ag (5 .mu.g/ml) the survivors were
cultured in media with a higher concentration (10 .mu.g/ml). This
was repeated until a concentration of 20 .mu.g/ml was reached.
After 5-6 passages in the presence of 8-Ag (20 .mu.g/ml) cells were
tested for their viability in HAT-media. None of the cells grown on
8-Ag survived after 3 days of culture in the presence of HAT.
Fusion Efficiency.
[0155] The MFP clones were tested for ability to fuse with lymph
node lymphocytes and PBL. MFP-2 yielded approximately 2-3 hybrids
per 10.sup.5 lymph node lymphocytes and 0.7-1.5 hybrids per
10.sup.5 of PBL. The immunoglobulin secretion rate for the hybrids
developed using MFP-2 ranged between 0.5 to 15 ug/ml with no
decrease over 7 months.
Second Series of Experiments
[0156] 1. The trioma cell line MFP-2 used for fusion with human
peripheral blood B-lymphocytes and human lymph node B-lymphocytes
can be also used for fusion with human peripheral blood and lymph
node T-cells and yield stable hybrids. [0157] 2. The trioma cell
line MFP-2 can be used for fusion with peripheral blood and lymph
node lymphocytes from two primate species: rhesus monkey (Macaque
mulatta) and baboon (Papio hamadryas) yielding monkey
immunoglobulin-producing hybrids. This has a potential application
for the development of monkey monoclonal antibodies to different
infectious agents to test them in primate models. [0158] 3. Trioma
fusion partner cell line MFP-2 was adapted to the growth in
protein-free media with the growth characteristics not different
from those when cultured in serum containing or serum-free (protein
supplemented-media). [0159] 4. It was inferred that, since MFP-2
can be cultured in protein-free media, the deriving hybridomas
would be relatively easy to adapt to the same protein-free media.
[0160] 5. Four out of 6 hybridomas were successfully adapted to
protein-free media without changing the growth characteristics and
loosing the antibody production. This feature of MFP-2 adds to the
advantage of this cell line in developing hybridomas capable of
growing in protein-free media. [0161] 6. 27 human hybridomas,
producing human monoclonal antibodies to breast and
prostate-associated antigens have been developed using MFP-2 and
peripheral blood and lymph node B-lymphocytes from breast and
prostate cancer patients. [0162] 7. 23 human hybridomas derive from
breast cancer patient and 4 derive from prostate cancer patients.
[0163] 8. Prostate cancer-derived hybridomas: [0164] 1. hyridoma
(32-B8) produces IgM, lambda antibody which reacts specifically
with 2 human prostate adenocarcinoma cell lines and with one human
breast adenocarcinoma cell line and is directed to an unknown
antigen most likely of a non-protein nature (western blot is
negative, although it well may be that the antigen is a protein but
the antigen determinant is conformational and labile) [0165] 2.
hybridoma (32-F6) also produces IgM, lambda antibody reactive with
both prostate and breast adenocarcinoma cells and recognizing the
proteinous antigen of 60-kDa molecular weight. [0166] 3. hybridoma
(39-A7) is also IgM, lambda antibody directed to an unknown protein
target specific for both breast and prostate adenocarcinoma. [0167]
4. hybridoma (50-1B3) produces IgM, kappa antibody directed to both
breast and prostate adenocarcinoma to a molecular target of unknown
nature [0168] 9. Breast cancer-associated hybridomas are the
following: [0169] 1. hybridoma (13-42), IgM, kappa recognizes
protein antigen of .sup..about.42 kDa molecular weight which is
present both on the surface and intracellularly of adenocarcinoma
cells (breast and prostate) but not in human normal fibroblasts.
[0170] 2. hyridoma (13-74), IgM, kappa reacts with protein antigen
of .sup..about.65 kDa specific for the breast adenocarcinoma cells
and expressed on the cell surface as well as intracellularly [0171]
3. hybridoma (13-82), IgM, kappa is reactive with intracellular
protein antigen specific only for breast and prostate
adenocarcinoma cells but not for human skin fibroblasts. [0172] 4.
hybridoma (13-2C1), IgM kappa is reactive with a protein of
.sup..about.100 kDa which is present both in adenocarcinoma and
normal fibroblast cells. [0173] 5. hybridoma (22-3E9) isotype is
not determined, recognizes several protein targets (which may be
all related) of molecular weight 35, 45 and 250 kDa which are
present. on both adenocarcinoma and fibroblasts. The antigen is
mostly on the surface of the cells. Reacts specifically with
primary cancerous lesions [0174] 6. hybridoma (22-6E7), IgM,
lambda, the antigen is unknown, the antibody is reactive only with
breast adenocarcinoma cells in culture. [0175] 7. hybridoma
(22-BD11), IgM, lambda, antigen is unknown, reacts with human
breast and prostate adenocarcinoma cells in culture. [0176] 8.
hybridoma (27-F7), IgM, kappa, reacts only with breast
adenocarcinoma cells in culture. The antigen is a TAX interacting
protein 2 of molecular weight .sup..about.35-40 kDa [0177] 9.
hybridoma (27-B1) same as 27-F7, shows high specific reactivity
with the cancerous lesions in primary tumors, no cross-reactivity
with the connective tissue or with normal mammary epithelial cells
[0178] 10. hybridoma (36-G7) antibody isotype is not determined;
specificity is the same as 27-B1 [0179] 11. hybridoma (27-F10),
IgG, lambda, reactive with the protein approx. 200 kDa on breast
adenocarcinoma cells [0180] 12. hybridoma (33-2F10), IgM, kappa,
antigen is not known, reactive with breast adenocarcinoma cells
[0181] 13. hybridoma (33-2H6), IgM, lambda, recognizes 65 kDa
protein on breast and prostate adenocarcinoma cells but not on
human skin fibroblasts [0182] 14. hybridoma (59-3G7), IgM, lambda,
is reactive with a 70 kDa protein lamin A or C in adenocarcinoma
cells. Cross-reactivity with other cells has not been Tested [0183]
15. hybridoma (59-2F6), IgG, lambda, reacts only with breast
adenocarcinoma cells with unknown antigen [0184] 16. hybridoma
(69-C12), IgM, kappa, reactive mostly with breast adenocarcinoma
cells directed to a protein, 50 kDa 17--hybridoma (76-2F6), IgM,
lambda, reactive with unknown antigen only on breast adenocarcinoma
cells [0185] 18. hybridoma (83-3A6), isotype not determined,
reactive only with breast adenocarcinoma cells [0186] 19. hyridoma
(85-E1), IgM, lambda, reactive only with breast adenocarcinoma
cells expressing Her2/neu; antigen is not identified yet [0187] 20.
hybridoma (88-1D8), isotype is not determined yet, recognizes
protein antigens on breast cancer cells; molecular weights vary
-70, 90 and 100 kDa [0188] 21. hybridoma (89) isotype is not
determined, reactive only with Her2/neu- negative adenocarcinoma
cells; antigen is not known [0189] 22. hybridoma (100-1F4), IgM,
kappa, only reactive with breast adenocarcinoma cells; antigen is
not known [0190] 23. hybridoma (100-2H3) similar to 100-1F4
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