U.S. patent application number 10/850370 was filed with the patent office on 2004-11-25 for methods for generating genetically altered antibody-producing cell lines with improved antibody characteristics.
Invention is credited to Grasso, Luigi, Nicolaides, Nicholas C., Sass, Philip M..
Application Number | 20040237125 10/850370 |
Document ID | / |
Family ID | 27613794 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040237125 |
Kind Code |
A1 |
Nicolaides, Nicholas C. ; et
al. |
November 25, 2004 |
Methods for generating genetically altered antibody-producing cell
lines with improved antibody characteristics
Abstract
Dominant negative alleles of human mismatch repair genes can be
used to generate hypermutable cells and organisms. By introducing
these genes into cells and transgenic animals, new cell lines and
animal varieties with novel and useful properties can be prepared
more efficiently than by relying on the natural rate of mutation.
These methods are useful for generating genetic diversity within
immunoglobulin genes directed against an antigen of interest to
produce altered antibodies with enhanced biochemical activity.
Moreover, these methods are useful for generating
antibody-producing cells with increased level of antibody
production.
Inventors: |
Nicolaides, Nicholas C.;
(Boothwyn, PA) ; Grasso, Luigi; (Philadelphia,
PA) ; Sass, Philip M.; (Audubon, PA) |
Correspondence
Address: |
Felicity E. Groth
WOODCOCK WASHBURN LLP
One Liberty Place - 46th Floor
Philadelphia
PA
19119
US
|
Family ID: |
27613794 |
Appl. No.: |
10/850370 |
Filed: |
May 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10850370 |
May 19, 2004 |
|
|
|
09707468 |
Nov 7, 2000 |
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Current U.S.
Class: |
800/6 ;
435/69.1 |
Current CPC
Class: |
C07K 2317/567 20130101;
C07K 16/00 20130101 |
Class at
Publication: |
800/006 ;
435/069.1 |
International
Class: |
A01K 067/027 |
Claims
1-72. (Canceled)
73. A method for making a cell that produces a mutated
immunoglobulin, comprising the steps of: introducing into a cell
that expresses a nucleotide sequence encoding an immunoglobulin a
polynucleotide comprising a dominant negative allele of a mismatch
repair gene, wherein said dominant negative allele is a truncation
mutant of a PMS2, wherein said cell becomes hypermutable, and
selecting cells that comprise a mutation in said nucleotide
sequence encoding said immunoglobulin.
74. The method of claim 73 further comprising a step of expressing
a polynucleotide comprising said mutated nucleotide sequence in a
genetically stable cell.
75. The method of claim 73 or 74 wherein said polynucleotide
comprising said dominant negative allele is introduced by
transfection of a suspension of cells in vitro.
76. The method of claim 73 or 74 wherein said mismatch repair gene
is human PMS2.
77. The method of claim 73 or 74 wherein said allele comprises a
truncation mutation at codon 134.
78. The method of claim 77 wherein said truncation mutation is a
thymidine at nucleotide 424 of wild-type PMS2.
79. The method of claim 73 wherein said nucleotide sequence
encoding said immunoglobulin is co-introduced into said cell,
whereby said cell produces said immunoglobulin.
80. The method of claim 73 further comprising the step restoring
genetic stability of said hypermutable cell.
81. The method of claim 79 further comprising the step restoring
genetic stability of said hypermutable cell.
82. An isolated, hypermutable, immunoglobulin-producing cell
produced by the method of claim 73.
83. An isolated, hypermutable, immunoglobulin-producing cell
produced by the method of claim 74.
84. An isolated, genetically stable, mutated
immunoglobulin-producing cell produced by the method of claim 80,
wherein said isolated, genetically stable, mutated
immunoglobulin-producing cell produces an immunoglobulin having
increased affinity for antigen relative to a wild-type
immunoglobulin-producing cell.
85. A homogeneous culture of the isolated, genetically stable,
immunoglobulin-producing cells of claim 84.
86. An isolated, genetically stable, mutated
immunoglobulin-producing cell produced by the method of claim 85,
wherein said isolated, genetically stable, mutated
immunoglobulin-producing cell produces an immunoglobulin having
increased affinity for antigen relative to a wild-type
immunoglobulin-producing cell.
87. A homogeneous culture of the isolated, genetically stable,
mutated immunoglobulin-producing cells of claim 86.
88. An isolated, genetically stable, mutated
immunoglobulin-producing cell produced by the method of claim 80,
wherein said isolated, genetically stable, mutated
immunoglobulin-producing cell produces an increased titer of
immunoglobulin relative to a wild-type immunoglobulin-producing
cell.
89. A homogeneous culture of the isolated, genetically stable,
mutated immunoglobulin-producing cells of claim 88.
90. An isolated, genetically stable, mutated
immunoglobulin-producing cell produced by the method of claim 81,
wherein said isolated, genetically stable, mutated
immunoglobulin-producing cell produces an increased titer of
antibody relative to a wild-type immunoglobulin-producing cell.
91. A homogeneous culture of the isolated, genetically stable,
mutated immunoglobulin-producing cells of claim 90.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention is related to the area of antibody maturation
and cellular production. In particular, it is related to the field
of mutagenesis.
BACKGROUND OF THE INVENTION
[0002] The use of antibodies to block the activity of foreign
and/or endogenous polypeptides provides an effective and selective
strategy for treating the underlying cause of disease. In
particular is the use of monoclonal antibodies (MAb) as effective
therapeutics such as the FDA approved ReoPro (Glaser, V. (1996) Can
ReoPro repolish tarnished monoclonal therapeutics? Nat. Biotechnol.
14:1216-1217), an anti-platelet MAb from Centocor; Herceptin
(Weiner, L. M. (1999) Monoclonal antibody therapy of cancer. Semin.
Oncol. 26:43-51), an anti-Her2/neu MAb from Genentech; and Synagis
(Saez-Llorens, X. E., et al. (1998) Safety and pharmacokinetics of
an intramuscular humanized monoclonal antibody to respiratory
syncytial virus in premature infants and infants with
bronchopulmonary dysplasia. Pediat. Infect. Dis. J. 17:787-791), an
anti-respiratory syncytial virus MAb produced by Medimmune.
[0003] Standard methods for generating MAbs against candidate
protein targets are known by those skilled in the art. Briefly,
rodents such as mice or rats are injected with a purified antigen
in the presence of adjuvant to generate an immune response (Shield,
C. F., et al. (1996) A cost-effective analysis of OKT3 induction
therapy in cadaveric kidney transplantation. Am. J. Kidney Dis.
27:855-864). Rodents with positive immune sera are sacrificed and
splenocytes are isolated. Isolated splenocytes are fused to
melanomas to produce immortalized cell lines that are then screened
for antibody production. Positive lines are isolated and
characterized for antibody production. The direct use of rodent
MAbs as human therapeutic agents were confounded by the fact that
human anti-rodent antibody (HARA) responses occurred in a
significant number of patients treated with the rodent-derived
antibody (Khazaeli, M. B., et al., (1994) Human immune response to
monoclonal antibodies. J. Immunother. 15:42-52). In order to
circumvent the problem of HARA, the grafting of the complementarity
determining regions (CDRs), which are the critical motifs found
within the heavy and light chain variable regions of the
immunoglobulin (Ig) subunits making up the antigen binding domain,
onto a human antibody backbone found these chimeric molecules are
able to retain their binding activity to antigen while lacking the
HARA response (Emery, S. C., and Harris, W. J. "Strategies for
humanizing antibodies" In: ANTIBODY ENGINEERING C. A. K. Borrebaeck
(Ed.) Oxford University Press, N.Y. 1995. pp. 159-183. A common
problem that exists during the "humanization" of rodent-derived
MAbs (referred to hereon as HAb) is the loss of binding affinity
due to conformational changes in the 3 dimensional structure of the
CDR domain upon grafting onto the human Ig backbone (U.S. Pat. No.
5,530,101 to Queen et al.). To overcome this problem, additional
HAb vectors are usually needed to be engineeredby inserting or
deleting additional amino acid residues within the framework region
and/or within the CDR coding region itself in order to recreate
high affinity HAbs (U.S. Pat. No. 5,530,101 to Queen et al.). This
process is a very time consuming procedure that involves the use of
expensive computer modeling programs to predict changes that may
lead to a high affinity HAb. In some instances the affinity of the
HAb is never restored to that of the MAb, rendering them of little
therapeutic use.
[0004] Another problem that exists in antibody engineering is the
generation of stable, high yielding producer cell lines that is
required for manufacturing of the molecule for clinical materials.
Several strategies have been adopted in standard practice by those
skilled in the art to circumvent this problem. One method is the
use of Chinese Hamster Ovary (CHO) cells transfected with exogenous
Ig fusion genes containing the grafted human light and heavy chains
to produce whole antibodies or single chain antibodies, which are a
chimeric molecule containing both light and heavy chains that form
an antigen-binding polypeptide (Reff, M. E. (1993) High-level
production of recombinant immunoglobulins in mammalian cells. Curr.
Opin. Biotechnol. 4:573-576). Another method employs the use of
human lymphocytes derived from transgenic mice containing a human
grafted immune system or transgenic mice containing a human Ig gene
repertoire. Yet another method employs the use of monkeys to
produce primate MAbs, which have been reported to lack a human
anti-monkey response (Neuberger, M., and Gruggermann, M. (1997)
Monoclonal antibodies. Mice perform a human repertoire. Nature
386:25-26). In all cases, the generation of a cell line that is
capable of generating sufficient amounts of high affinity antibody
poses a major limitation for producing sufficient materials for
clinical studies. Because of these limitations, the utility of
other recombinant systems such as plants are currently being
explored as systems that will lead to the stable, high-level
production of humanized antibodies (Fiedler, U., and Conrad, U.
(1995) High-level production and long-term storage of engineered
antibodies in transgenic tobacco seeds. Bio/Technology
13:1090-1093).
[0005] A method for generating diverse antibody sequences within
the variable domain that results in HAbs and MAbs with high binding
affinities to antigens would be useful for the creation of more
potent therapeutic and diagnostic reagents respectively. Moreover,
the generation of randomly altered nucleotide and polypeptide
residues throughout an entire antibody molecule will result in new
reagents that are less antigenic and/or have beneficial
pharmacokinetic properties. The invention described herein is
directed to the use of random genetic mutation throughout an
antibody structure in vivo by blocking the endogenous mismatch
repair (MMR) activity of a host cell producing immunoglobulins that
encode biochemically active antibodies. The invention also relates
to methods for repeated in vivo genetic alterations and selection
for antibodies with enhanced binding and pharmacokinetic
profiles.
[0006] In addition, the ability to develop genetically altered host
cells that are capable of secreting increased amounts of antibody
will also provide a valuable method for creating cell hosts for
product development. The invention described herein is directed to
the creation of genetically altered cell hosts with increased
antibody production via the blockade of MMR.
[0007] The invention facilitates the generation of high affinity
antibodies and the production of cell lines with elevated levels of
antibody production. Other advantages of the present invention are
described in the examples and figures described herein.
SUMMARY OF THE INVENTION
[0008] The invention provides methods for generating genetically
altered antibodies (including single chain molecules) and antibody
producing cell hosts in vitro and in vivo, whereby the antibody
possess a desired biochemical property(s), such as, but not limited
to, increased antigen binding, increased gene expression, and/or
enhanced extracellular secretion by the cell host. One method for
identifying antibodies with increased binding activity or cells
with increased antibody production is through the screening of MMR
defective antibody producing cell clones that produce molecules
with enhanced binding properties or clones that have been
genetically altered to produce enhanced amounts of antibody
product.
[0009] The antibody producing cells suitable for use in the
invention include, but are not limited to rodent, primate, or human
hybridomas or lymphoblastoids; mammalian cells transfected and
expressing exogenous Ig subunits or chimeric single chain
molecules; plant cells, yeast or bacteria transfected and
expressing exogenous Ig subunits or chimeric single chain
molecules.
[0010] Thus, the invention provides methods for making hypermutable
antibody-producing cells by introducing a polynucleotide comprising
a dominant negative allele of a mismatch repair gene into cells
that are capable of producing antibodies. The cells that are
capable of producing antibodies include cells that naturally
produce antibodies, and cells that are engineered to produce
antibodies through the introduction of immunoglobulin encoding
sequences. Conveniently, the introduction of polynucleotide
sequences into cells is accomplished by transfection.
[0011] The invention also provides methods of making hypermutable
antibody producing cells by introducing a dominant negative
mismatch repair (MMR) gene such as PMS2 (preferably human PMS2),
MLH1, PMS1, MSH2, or MSH2 into cells that are capable of producing
antibodies. The dominant negative allele of a mismatch repair gene
may be a truncation mutation of a mismatch repair gene (preferably
a truncation mutation at codon 134, or a thymidine at nucleotide
424 of wild-type PMS2). The invention also provides methods in
which mismatch repair gene activity is suppressed. This may be
accomplished, for example, using antisense molecules directed
against the mismatch repair gene or transcripts.
[0012] Other embodiments of the invention provide methods for
making a hypermutable antibody producing cells by introducing a
polynucleotide comprising a dominant negative allele of a mismatch
repair gene into fertilized eggs of animals. These methods may also
include subsequently implanting the eggs into pseudo-pregnant
females whereby the fertilized eggs develop into a mature
transgenic animal. The mismatch repair genes may include, for
example, PMS2 (preferably human PMS2), MLH1, PMS1, MSH2, or MSH2.
The dominant negative allele of a mismatch repair gene may be a
truncation mutation of a mismatch repair gene (preferably a
truncation mutation at codon 134, or a thymidine at nucleotide 424
of wild-type PMS2).
[0013] The invention further provides homogeneous compositions of
cultured, hypermutable, mammalian cells that are capable of
producing antibodies and contain a dominant negative allele of a
mismatch repair gene. The mismatch repair genes may include, for
example, PMS2 (preferably human PMS2), MLH1, PMS1, MSH2, or MSH2.
The dominant negative allele of a mismatch repair gene may be a
truncation mutation of a mismatch repair gene (preferably a
truncation mutation at codon 134, or a thymidine at nucleotide 424
of wild-type PMS2). The cells of the culture may contain PMS2,
(preferably human PMS2), MLH1, or PMS1; or express a human mutL
homolog, or the first 133 amino acids of hPMS2.
[0014] The invention further provides methods for generating a
mutation in an immunoglobulin gene of interest by culturing an
immunoglobulin producing cell selected for an immunoglobulin of
interest wherein the cell contains a dominant negative allele of a
mismatch repair gene. The properties of the immunoglobulin produced
from the cells can be assayed to ascertain whether the
immunoglobulin gene harbors a mutation. The assay may be directed
to analyzing a polynucleotide encoding the immunoglobulin, or may
be directed to the immunoglobulin polypeptide itself.
[0015] The invention also provides methods for generating a
mutation in a gene affecting antibody production in an
antibody-producing cell by culturing the cell expressing a dominant
negative allele of a mismatch repair gene, and testing the cell to
determine whether the cell harbors mutations within the gene of
interest, such that a new biochemical feature (e.g.,
over-expression and/or secretion of immunoglobulin products) is
generated. The testing may include analysis of the steady state
expression of the immunoglobulin gene of interest, and/or analysis
of the amount of secreted protein encoded by the immunoglobulin
gene of interest. The invention also embraces prokaryotic and
eukaryotic transgenic cells made by this process, including cells
from rodents, non-human primates and humans.
[0016] Other aspects of the invention encompass methods of
reversibly altering the hypermutability of an antibody producing
cell, in which an inducible vector containing a dominant negative
allele of a mismatch repair gene operably linked to an inducible
promoter is introduced into an antibody-producing cell. The cell is
treated with an inducing agent to express the dominant negative
mismatch repair gene (which can be PMS2 (preferably human PMS2),
MLH1, or PMS1). Alternatively, the cell may be induced to express a
human mutL homolog or the first 133 amino acids of hPMS2. In
another embodiment, the cells may be rendered capable of producing
antibodies by co-transfecting a preselected immunoglobulin gene of
interest. The immunoglobulin genes of the hypermutable cells, or
the proteins produced by these methods may be analyzed for desired
properties, and induction may be stopped such that the genetic
stability of the host cell is restored.
[0017] The invention also embraces methods of producing genetically
altered antibodies by transfecting a polynucleotide encoding an
immunoglobulin protein into a cell containing a dominant negative
mismatch repair gene (either naturally or in which the dominant
negative mismatch repair gene was introduced into the cell),
culturing the cell to allow the immunoglobulin gene to become
mutated and produce a mutant immunoglobulin, screening for a
desirable property of said mutant immunoglobulin protein, isolating
the polynucleotide molecule encoding the selected mutant
immunoglobulin possessing the desired property, and transfecting
said mutant polynucleotide into a genetically stable cell, such
that the mutant antibody is consistently produced without further
genetic alteration. The dominant negative mismatch repair gene may
be PMS2 (preferably human PMS2), MLH1, or PMS1. Alternatively, the
cell may express a human mutL homolog or the first 133 amino acids
of hPMS2.
[0018] The invention further provides methods for generating
genetically altered cell lines that express enhanced amounts of an
antigen binding polypeptide. These antigen-binding polyeptides may
be, for example, immunoglobulins. The methods of the invention also
include methods for generating genetically altered cell lines that
secrete enhanced amounts of an antigen binding polypeptide. The
cell lines are rendered hypermutable by dominant negative mismatch
repair genes that provide an enhanced rate of genetic hypermutation
in a cell producing antigen-binding polypeptides such as
antibodies. Such cells include, but are not limited to hybridomas.
Expression of enhanced amounts of antigen binding polypeptides may
be through enhanced transcription or translation of the
polynucleotides encoding the antigen binding polypeptides, or
through the enhanced secretion of the antigen binding polypeptides,
for example.
[0019] Methods are also provided for creating genetically altered
antibodies in vivo by blocking the MMR activity of the cell host,
or by transfecting genes encoding for immunoglobulin in a MMR
defective cell host.
[0020] Antibodies with increased binding properties to an antigen
due to genetic changes within the variable domain are provided in
methods of the invention that block endogenous MMR of the cell
host. Antibodies with increased binding properties to an antigen
due to genetic changes within the CDR regions within the light
and/or heavy chains are also provided in methods of the invention
that block endogenous MMR of the cell host.
[0021] The invention provides methods of creating genetically
altered antibodies in MMR defective Ab producer cell lines with
enhanced pharmacokinetic properties in host organisms including but
not limited to rodents, primates, and man.
[0022] These and other aspects of the invention are provided by one
or more of the embodiments described below. In one embodiment of
the invention, a method for making an antibody producing cell line
hypermutable is provided. A polynucleotide encoding a dominant
negative allele of a MMR gene is introduced into an
antibody-producing cell. The cell becomes hypermutable as a result
of the introduction of the gene.
[0023] In another embodiment of the invention, a method is provided
for introducing a mutation into an endogenous gene encoding for an
immunoglobulin polypeptide or a single chain antibody. A
polynucleotide encoding a dominant negative allele of a MMR gene is
introduced into a cell. The cell becomes hypermutable as a result
of the introduction and expression of the MMR gene allele. The cell
further comprises an immunoglobulin gene of interest. The cell is
grown and tested to determine whether the gene encoding for an
immunoglobulin or a single chain antibody of interest harbors a
mutation. In another aspect of the invention, the gene encoding the
mutated immunoglobulin polypeptide or single chain antibody may be
isolated and expressed in a genetically stable cell. In a preferred
embodiment, the mutated antibody is screened for at least one
desirable property such as, but not limited to, enhanced binding
characteristics.
[0024] In another embodiment of the invention, a gene or set of
genes encoding for Ig light and heavy chains or a combination
therein are introduced into a mammalian cell host that is MMR
defective. The cell is grown, and clones are analyzed for
antibodies with enhanced binding characteristics.
[0025] In another embodiment of the invention, a method will be
provided for producing new phenotypes of a cell. A polynucleotide
encoding a dominant negative allele of a MMR gene is introduced
into a cell. The cell becomes hypermutable as a result of the
introduction of the gene. The cell is grown. The cell is tested for
the expression of new phenotypes where the phenotype is enhanced
secretion of a polypeptide.
[0026] These and other embodiments of the invention provide the art
with methods that can generate enhanced mutability in cells and
animals as well as providing cells and animals harboring
potentially useful mutations for the large-scale production of high
affinity antibodies with beneficial pharmacokinetic profiles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1. Hybridoma cells stably expressing PMS2 and PMS134
MMR genes. Shown is steady state mRNA expression of MMR genes
transfected into a murine hybridoma cell line. Stable expression
was found after 3 months of continuous growth. The (-) lanes
represent negative controls where no reverse transcriptase was
added, and the (+) lanes represent samples reverse transcribed and
PCR amplified for the MMR genes and an internal housekeeping gene
as a control.
[0028] FIG. 2. Creation of genetically hypermutable hybridoma
cells. Dominant negative MMR gene alleles were expressed in cells
expressing a MMR-sensitive reporter gene. Dominant negative alleles
such as PMS134 and the expression of MMR genes from other species
results in antibody producer cells with a hypermutable phenotype
that can be used to produce genetically altered immunoglobulin
genes with enhanced biochemical features as well as lines with
increased Ig expression and/or secretion. Values shown represent
the amount of converted CPRG substrate which is reflective of the
amount of function .quadrature.-galactosidase contained within the
cell from genetic alterations within the pCAR-OF reporter gene.
Higher amounts of .beta.-galactosidase activity reflect a higher
mutation rate due to defective MMR.
[0029] FIG. 3. Screening method for identifying antibody-producing
cells containing antibodies with increased binding activity and/or
increased expression/secretion
[0030] FIG. 4. Generation of a genetically altered antibody with an
increased binding activity. Shown are ELISA values from 96-well
plates, screened for antibodies specific to hIgE. Two clones with a
high binding value were found in HB134 cultures.
[0031] FIG. 5. Sequence alteration within variable chain of an
antibody (a mutation within the light chain variable region in
MMR-defective HB134 antibody producer cells). Arrows indicate the
nucleotide at which a mutation occurred in a subset of cells from a
clone derived from HB134 cells. Panel A: The change results in a
Thr to Ser change within the light chain variable region. The
coding sequence is in the antisense direction. Panel B: The change
results in a Pro to His change within the light chain variable
region.
[0032] FIG. 6. Generation of MMR-defective clones with enhanced
steady state Ig protein levels. A Western blot of heavy chain
immunglobulins from HB134 clones with high levels of MAb (>500
ngs/ml) within the conditioned medium shows that a subset of clones
express higher steady state levels of immunoglobulins (Ig). The H36
cell line was used as a control to measure steady state levels in
the parental strain. Lane 1: fibroblast cells (negative control);
Lane 2: H36 cell; Lane 3: HB134 clone with elevated MAb levels;
Lane 4: HB134 clone with elevated MAb levels; Lane 5: HB134 clone
with elevated MAb levels.
[0033] Methods have been discovered for developing hypermutable
antibody-producing cells by taking advantage of the conserved
mismatch repair (MMR) process of host cells. Dominant negative
alleles of such genes, when introduced into cells or transgenic
animals, increase the rate of spontaneous mutations by reducing the
effectiveness of DNA repair and thereby render the cells or animals
hypermutable. Hypermutable cells or animals can then be utilized to
develop new mutations in a gene of interest. Blocking MMR in
antibody-producing cells such as but not limited to: hybridomas;
mammalian cells transfected with genes encoding for Ig light and
heavy chains; mammalian cells transfected with genes encoding for
single chain antibodies; eukaryotic cells transfected with Ig
genes, can enhance the rate of mutation within these cells leading
to clones that have enhanced antibody production and/or cells
containing genetically altered antibodies with enhanced biochemical
properties such as increased antigen binding. The process of MMR,
also called mismatch proofreading, is carried out by protein
complexes in cells ranging from bacteria to mammalian cells. A MMR
gene is a gene that encodes for one of the proteins of such a
mismatch repair complex. Although not wanting to be bound by any
particular theory of mechanism of action, a MMR complex is believed
to detect distortions of the DNA helix resulting from
non-complementary pairing of nucleotide bases. The
non-complementary base on the newer DNA strand is excised, and the
excised base is replaced with the appropriate base, which is
complementary to the older DNA strand. In this way, cells eliminate
many mutations that occur as a result of mistakes in DNA
replication.
[0034] Dominant negative alleles cause a MMR defective phenotype
even in the presence of a wild-type allele in the same cell. An
example of a dominant negative allele of a MMR gene is the human
gene hPMS2-134, which carries a truncating mutation at codon 134
(SEQ ID NO: 15). The mutation causes the product of this gene to
abnormally terminate at the position of the 134th amino acid,
resulting in a shortened polypeptide containing the N-terminal 133
amino acids. Such a mutation causes an increase in the rate of
mutations, which accumulate in cells after DNA replication.
Expression of a dominant negative allele of a mismatch repair gene
results in impairment of mismatch repair activity, even in the
presence of the wild-type allele. Any allele which produces such
effect can be used in this invention. Dominant negative alleles of
a MMR gene can be obtained from the cells of humans, animals,
yeast, bacteria, or other organisms. Such alleles can be identified
by screening cells for defective MMR activity. Cells from animals
or humans with cancer can be screened for defective mismatch
repair. Cells from colon cancer patients may be particularly
useful. Genomic DNA, cDNA, or mRNA from any cell encoding a MMR
protein can be analyzed for variations from the wild type sequence.
Dominant negative alleles of a MMR gene can also be created
artificially, for example, by producing variants of the hPMS2-134
allele or other MMR genes. Various techniques of site-directed
mutagenesis can be used. The suitability of such alleles, whether
natural or artificial, for use in generating hypermutable cells or
animals can be evaluated by testing the mismatch repair activity
caused by the allele in the presence of one or more wild-type
alleles, to determine if it is a dominant negative allele.
[0035] A cell or an animal into which a dominant negative allele of
a mismatch repair gene has been introduced will become
hypermutable. This means that the spontaneous mutation rate of such
cells or animals is elevated compared to cells or animals without
such alleles. The degree of elevation of the spontaneous mutation
rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold,
100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell
or animal. The use of chemical mutagens such as but limited to
methane sulfonate, dimethyl sulfonate, O6-methyl benzadine, MNU,
ENU, etc. can be used in MMR defective cells to increase the rates
an additional 10 to 100 fold that of the MMR deficiency itself.
[0036] According to one aspect of the invention, a polynucleotide
encoding for a dominant negative form of a MMR protein is
introduced into a cell. The gene can be any dominant negative
allele encoding a protein, which is part of a MMR complex, for
example, PMS2, PMS1, MLH1, or MSH2. The dominant negative allele
can be naturally occurring or made in the laboratory. The
polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a
chemically synthesized polynucleotide.
[0037] The polynucleotide can be cloned into an expression vector
containing a constitutively active promoter segment (such as but
not limited to CMV, SV40, Elongation Factor or LTR sequences) or to
inducible promoter sequences such as the steroid inducible pIND
vector (Invitrogen), where the expression of the dominant negative
MMR gene can be regulated. The polynucleotide can be introduced
into the cell by transfection.
[0038] According to another aspect of the invention, an
immunoglobulin (Ig) gene, a set of Ig genes or a chimeric gene
containing whole or parts of an Ig gene can be transfected into MMR
deficient cell hosts, the cell is grown and screened for clones
containing genetically altered Ig genes with new biochemical
features. MMR defective cells may be of human, primates, mammals,
rodent, plant, yeast or of the prokaryotic kingdom. The mutated
gene encoding the Ig with new biochemical features may be isolated
from the respective clones and introduced into genetically stable
cells (i.e., cells with normal MMR) to provide clones that
consistently produce Ig with the new biochemical features. The
method of isolating the Ig gene encoding Ig with new biochemical
features may be any method known in the art. Introduction of the
isolated polynucleotide encoding the Ig with new biochemical
features may also be performed using any method known in the art,
including, but not limited to transfection of an expression vector
containing the polynucleotide encoding the Ig with new biochemical
features. As an alternative to transfecting an Ig gene, a set of Ig
genes or a chimeric gene containing whole or parts of an Ig gene
into an MMR deficient host cell, such Ig genes may be transfected
simultaneously with a gene encoding a dominant negative mismatch
repair gene into a genetically stable cell to render the cell
hypermutable.
[0039] Transfection is any process whereby a polynucleotide is
introduced into a cell. The process of transfection can be carried
out in a living animal, e.g., using a vector for gene therapy, or
it can be carried out in vitro, e.g., using a suspension of one or
more isolated cells in culture. The cell can be any type of
eukaryotic cell, including, for example, cells isolated from humans
or other primates, mammals or other vertebrates, invertebrates, and
single celled organisms such as protozoa, yeast, or bacteria.
[0040] In general, transfection will be carried out using a
suspension of cells, or a single cell, but other methods can also
be applied as long as a sufficient fraction of the treated cells or
tissue incorporates the polynucleotide so as to allow transfected
cells to be grown and utilized. The protein product of the
polynucleotide may be transiently or stably expressed in the cell.
Techniques for transfection are well known. Available techniques
for introducing polynucleotides include but are not limited to
electroporation, transduction, cell fusion, the use of calcium
chloride, and packaging of the polynucleotide together with lipid
for fusion with the cells of interest. Once a cell has been
transfected with the MMR gene, the cell can be grown and reproduced
in culture. If the transfection is stable, such that the gene is
expressed at a consistent level for many cell generations, then a
cell line results.
[0041] An isolated cell is a cell obtained from a tissue of humans
or animals by mechanically separating out individual cells and
transferring them to a suitable cell culture medium, either with or
without pretreatment of the tissue with enzymes, e.g., collagenase
or trypsin. Such isolated cells are typically cultured in the
absence of other types of cells. Cells selected for the
introduction of a dominant negative allele of a mismatch repair
gene may be derived from a eukaryotic organism in the form of a
primary cell culture or an immortalized cell line, or may be
derived from suspensions of single-celled organisms.
[0042] A polynucleotide encoding for a dominant negative form of a
MMR protein can be introduced into the genome of an animal by
producing a transgenic animal. The animal can be any species for
which suitable techniques are available to produce transgenic
animals. For example, transgenic animals can be prepared from
domestic livestock, e.g., bovine, swine, sheep, goats, horses,
etc.; from animals used for the production of recombinant proteins,
e.g., bovine, swine, or goats that express a recombinant
polypeptide in their milk; or experimental animals for research or
product testing, e.g., mice, rats, guinea pigs, hamsters, rabbits,
etc. Cell lines that are determined to be MMR defective can then be
used as a source for producing genetically altered immunoglobulin
genes in vitro by introducing whole, intact immunoglobulin genes
and/or chimeric genes encoding for single chain antibodies into MMR
defective cells from any tissue of the MMR defective animal.
[0043] Once a transfected cell line or a colony of transgenic
animals has been produced, it can be used to generate new mutations
in one or more gene(s) of interest. A gene of interest can be any
gene naturally possessed by the cell line or transgenic animal or
introduced into the cell line or transgenic animal. An advantage of
using such cells or animals to induce mutations is that the cell or
animal need not be exposed to mutagenic chemicals or radiation,
which may have secondary harmful effects, both on the object of the
exposure and on the workers. However, chemical mutagens may be used
in combination with MMR deficiency, which renders such mutagens
less toxic due to an undetermined mechanism. Hypermutable animals
can then be bred and selected for those producing genetically
variable B-cells that may be isolated and cloned to identify new
cell lines that are useful for producing genetically variable
cells. Once a new trait is identified, the dominant negative MMR
gene allele can be removed by directly knocking out the allele by
technologies used by those skilled in the art or by breeding to
mates lacking the dominant negative allele to select for offspring
with a desired trait and a stable genome. Another alternative is to
use a CRE-LOX expression system, whereby the dominant negative
allele is spliced from the animal genome once an animal containing
a genetically diverse immunoglobulin profile has been established.
Yet another alternative is the use of inducible vectors such as the
steroid induced pIND (Invitrogen) or pMAM (Clonetech) vectors which
express exogenous genes in the presence of corticosteroids.
[0044] Mutations can be detected by analyzing for alterations in
the genotype of the cells or animals, for example by examining the
sequence of genomic DNA, cDNA, messenger RNA, or amino acids
associated with the gene of interest. Mutations can also be
detected by screening for the production of antibody titers. A
mutant polypeptide can be detected by identifying alterations in
electrophoretic mobility, spectroscopic properties, or other
physical or structural characteristics of a protein encoded by a
mutant gene. One can also screen for altered function of the
protein in situ, in isolated form, or in model systems. One can
screen for alteration of any property of the cell or animal
associated with the function of the gene of interest, such as but
not limited to Ig secretion.
[0045] Examples of mismatch repair proteins and nucleic acid
sequences include the following:
1 PMS2 (mouse) (SEQ ID NO: 5) MEQTEGVSTE CAKAIKPTDG KSVHQICSGQ
VILSLSTAVK ELIENSVDAG ATTIDLRLKD 60 YGVDLIEVSD NGCGVEEENF
EGLALKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV 120 TISTCHGSAS
VGTRLVFDHN GKITQKTPYP RPKGTTVSVQ HLFYTLPVRY KEFQRNIKKE 180
YSKMVQVLQA YCIISAGVRV SCTNQLGQGK RHAVVCTSGT SGMKENIGSV FGQKQLQSLI
240 PFVQLPPSDA VCEEYGLSTS GRHKTFSTFR ASFHSARTAP GGVQQTGSFS
SSIRGPVTQQ 300 RSLSLSMRFY HMYNRHQYPF VVLNVSVDSE CVDINVTPDK
RQILLQEEKL LLAVLKTSLI 360 GMFDSDANKL NVNQQPLLDV EGNLVKLHTA
ELEKPVPGKQ DNSPSLKSTA DEKRVASISR 420 LREAFSLHPT KEIKSRGPET
AELTRSFPSE KRGVLSSYPS DVISYRGLRG SQDKLVSPTD 480 SPGDCMDREK
IEKDSGLSST SAGSEEEFST PEVASSFSSD YNVSSLEDRP SQETINCGDL 540
DCRPPGTGQS LKPEDHGYQC KALPLARLSP TNAKRFKTEE RPSNVNISQR LPGPQSTSAA
600 EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQ HLKAQNKHEL SYRKFRAKIC
PGENQAAEDE 660 LRKEISKSMF AEMEILGQFN LGFIVTKLKE DLFLVDQHAA
DEKYNFEMLQ QHTVLQAQRL 720 ITPQTLNLTA VNEAVLIENL EIFRKNGFDF
VIDEDAPVTE RAKLISLPTS KNWTFGPQDI 780 DELIFMLSDS PGVMCRPSRV
RQMFASRACR KSVMIGTALN ASEMKKLITH MGEMDHPWNC 840 PHGRPTMRHV
ANLDVISQN 859 PMS2 (mouse cDNA) (SEQ ID NO: 6) gaattccggt
gaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga 60
taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc
120 gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg
aggagtcctg 180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc
taaggccatc aagcctattg 240 atgggaagtc agtccatcaa atttgttctg
ggcaggtgat actcagttta agcaccgctg 300 tgaaggagtt gatagaaaat
agtgtagatg ctggtgctac tactattgat ctaaggctta 360 aagactatgg
ggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa 420
actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca
480 cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt
gcactaagtg 540 atgtcactat atctacctgc cacgggtctg caagcgttgg
gactcgactg gtgtttgacc 600 ataatgggaa aatcacccag aaaactccct
acccccgacc taaaggaacc acagtcagtg 660 tgcagcactt attttataca
ctacccgtgc gttacaaaga gtttcagagg aacattaaaa 720 aggagtattc
caaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780
gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg
840 gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag
ttgcaaagcc 900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg
tgaagagtac ggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgt
ttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag
acaggcagtt tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc
tctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140
catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag
1200 ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta
aagacctcct 1260 tgataggaat gtttgacagt gatgcaaaca agcttaatgt
caaccagcag ccactgctag 1320 atgttgaagg taacttagta aagctgcata
ctgcagaact agaaaagcct gtgccaggaa 1380 agcaagataa ctctccttca
ctgaagagca cagcagacga gaaaagggta gcatccatct 1440 ccaggctgag
agaggccttt tctcttcatc ctactaaaga gatcaagtct aggggtccag 1500
agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc
1560 cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg
gtgagtccca 1620 cggacagccc tggtgactgt atggacagag agaaaataga
aaaagactca gggctcagca 1680 gcacctcagc tggctctgag gaagagttca
gcaccccaga agtggccagt agctttagca 1740 gtgactataa cgtgagctcc
ctagaagaca gaccttctca ggaaaccata aactgtggtg 1800 acctggactg
ccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc 1860
aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag
1920 aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag
agcacctcag 1980 cagctgaggt cgatgtagcc ataaaaatga ataagagaat
cgtgctcctc gagttctctc 2040 tgagttctct agctaagcga atgaagcagt
tacagcacct aaaggcgcag aacaaacatg 2100 aactgagtta cagaaaattt
agggccaaga tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcag
aaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220
ttaacctggg atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg
2280 ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc
caggcgcaga 2340 ggctcatcac accccagact ctgaacttaa ctgctgtcaa
tgaagctgta ctgatagaaa 2400 atctggaaat attcagaaag aatggctttg
actttgtcat tgatgaggat gctccagtca 2460 ctgaaagggc taaattgatt
tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatga
actgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580
gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc
2640 tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac
cacccctgga 2700 actgccccca cggcaggcca accatgaggc acgttgccaa
tctggatgtc atctctcaga 2760 actgacacac cccttgtagc atagagttta
ttacagattg ttcggtttgc aaagagaagg 2820 ttttaagtaa tctgattatc
gttgtacaaa aattagcatg ctgctttaat gtactggatc 2880 catttaaaag
cagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg 2940
tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg
3000 agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa
3056 PMS2 (human) (SEQ ID NO: 7) MERAESSSTE PAKAIKPIDR KSVHQICSGQ
VVLSLSTAVK ELVENSLDAG ATNIDLKLKD 60 YGVDLIEVSD NGCGVEEENF
EGLTLKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV 120 TISTCHASAK
VGTRLMFDRN GKIIQKTPYP RPRGTTVSVQ QLFSTLPVRH KEFQRNIKKE 180
YAKMVQVLHA YCIISAGIRV SCTNQLGQGK RQPVVCTGGS PSIKENIGSV FGQKQLQSLI
240 PFVQLPPSDS VCEEYGLSCS DALHNLFYIS GFISQCTHGV GRSSTDRQFF
FINRRPCDPA 300 KVCRLVNEVY HMYNRHQYPF VVLNISVDSE CVDINVTPDK
RQILLQEEKL LLAVLKTSLI 360 GMFDSDVNKL NVSQQPLLDV EGNLIKMHAA
DLEKPMVEKQ DQSPSLRTGE EKKDVSISRL 420 REAFSLRHTT ENKPHSPKTP
EPRRSPLGQK RGMLSSSTSG AISDKGVLRP QKEAVSSSHG 480 PSDPTDRAEV
EKDSGHGSTS VDSEGFSIPD TGSHCSSEYA ASSPGDRGSQ EHVDSQEKAP 540
ETDDSFSDVD CHSNQEDTGC KFRVLPQPTN LATPNTKRFK KEEILSSSDI CQKLVNTQDM
600 SASQVDVAVK INKKVVPLDF SMSSLAKRIK QLHHEAQQSE GEQNYRKFRA
KICPGENQAA 660 EDELRKEISK TMFAEMEIIG QFNLGFIITK LNEDIFIVDQ
HATDEKYNFE MLQQHTVLQG 720 QRLIAPQTLN LTAVNEAVLI ENLEIFRKNG
FDFVIDENAP VTERAKLISL PTSKNWTFGP 780 QDVDELIFML SDSPGVMCRP
SRVKQMFASR ACRKSVMIGT ALNTSEMKKL ITHMGEMDHP 840 WNCPHGRPTM
RHIANLGVIS QN 862 PMS2 (human cDNA) (SEQ ID NO: 8) cgaggcggat
cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60
aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta
120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc
tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatctta
ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc
ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac
tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg
cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420
actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc
480 agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg
ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc aaaatggtcc
aggtcttaca tgcatactgt 600 atcatttcag caggcatccg tgtaagttgc
accaatcagc ttggacaagg aaaacgacag 660 cctgtggtat gcacaggtgg
aagccccagc ataaaggaaa atatcggctc tgtgtttggg 720 cagaagcagt
tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt 780
gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc
840 atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt
tttctttatc 900 aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg
tgaatgaggt ctaccacatg 960 tataatcgac accagtatcc atttgttgtt
cttaacattt ctgttgattc agaatgcgtt 1020 gatatcaatg ttactccaga
taaaaggcaa attttgctac aagaggaaaa gcttttgttg 1080 gcagttttaa
agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc 1140
agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg
1200 gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg
agaagaaaaa 1260 aaagacgtgt ccatttccag actgcgagag gccttttctc
ttcgtcacac aacagagaac 1320 aagcctcaca gcccaaagac tccagaacca
agaaggagcc ctctaggaca gaaaaggggt 1380 atgctgtctt ctagcacttc
aggtgccatc tctgacaaag gcgtcctgag acctcagaaa 1440 gaggcagtga
gttccagtca cggacccagt gaccctacgg acagagcgga ggtggagaag 1500
gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc
1560 agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc
gcaggaacat 1620 gtggactctc aggagaaagc gcctgaaact gacgactctt
tttcagatgt ggactgccat 1680 tcaaaccagg aagataccgg atgtaaattt
cgagttttgc ctcagccaac taatctcgca 1740 accccaaaca caaagcgttt
taaaaaagaa gaaattcttt ccagttctga catttgtcaa 1800 aagttagtaa
atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat 1860
aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta
1920 catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag
ggcaaagatt 1980 tgtcctggag aaaatcaagc agccgaagat gaactaagaa
aagagataag taaaacgatg 2040 tttgcagaaa tggaaatcat tggtcagttt
aacctgggat ttataataac caaactgaat 2100 gaggatatct tcatagtgga
ccagcatgcc acggacgaga agtataactt cgagatgctg 2160 cagcagcaca
ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact 2220
gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat
2280 tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc
cttgccaact 2340 agtaaaaact ggaccttcgg accccaggac gtcgatgaac
tgatcttcat gctgagcgac 2400 agccctgggg tcatgtgccg gccttcccga
gtcaagcaga tgtttgcctc cagagcctgc 2460 cggaagtcgg tgatgattgg
gactgctctt aacacaagcg agatgaagaa actgatcacc 2520 cacatggggg
agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac 2580
atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt
2640 tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt
ttgttttaaa 2700 atgaaacctg ctacttaaaa aaaatacaca tcacacccat
ttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 PMS1 (human) (SEQ ID
NO:9) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD
KIEVRDNGEG 60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI
CCIAEVLITT RTAADNFSTQ 120 YVLDGSGHIL SQKPSHLGQG TTVTALRLFK
NLPVRKQFYS TAKKCKDEIK KIQDLLMSFG 180 ILKPDLRIVF VHNKAVIWQK
SRVSDHKMAL MSVLGTAVMN NMESFQYHSE ESQIYLSGFL 240 PKCDADHSFT
SLSTPERSFI FINSRPVHQK DILKLIRHHY NLKCLKESTR LYPVFFLKID 300
VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNK TDVSAADIVL
360 SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG KNTDDCLNHQ
ISIGDFGYGH 420 CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS
SVKHTQSENG NKDHIDESGE 480 NEEEAGLENS SEISADEWSR GNILKNSVGE
NIEPVKILVP EKSLPCKVSN NNYPIPEQMN 540 LNEDSCNKKS NVIDNKSGKV
TAYDLLSNRV IKKPMSASAL FVQDHRPQFL IENPKTSLED 600 ATLQIEELWK
TLSEEEKLKY EEKATKDLER YNSQMKRAIE QESQMSLKDG RKKIKPTSAW 660
NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP FSMKNLKINF KKQNKVDLEE
720 KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR LLENHKLPAE
PLEKPIMLTE 780 SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI
KLIPGVSITE NYLEIEGMAN 840 CLPFYGVADL KEILNAILNR NAKEVYECRP
RKVISYLEGE AVRLSRQLPM YLSKEDIQDI 900 IYRMKHQFGN EIKECVHGRP
FFHHLTYLPE TT 932 PMS1 (human) (SEQ ID NO: 10) ggcacgagtg
gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60
ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa
120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa
aactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta
tggatttgat aaaattgagg 240 tgcgagataa cggggagggt atcaaggctg
ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagt
catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt
ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420
ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac
480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag
aatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga
tgaaataaaa aagatccaag 600 atctcctcat gagctttggt atccttaaac
ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa
agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc
tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780
tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa
840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa
gatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga
atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag
ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa
aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta
tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140
ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg
1200 aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc
caaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt
aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg
aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttca
atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg
ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500
atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt
1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag
aatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa
agtaagtaat aataattatc 1680 caatccctga acaaatgaat cttaatgaag
attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt
acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc
aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860
ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg
1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga
tacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact
aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc
agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa
ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt
acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220
acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg
2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat
agagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact
tcctgcagag ccactggaaa 2400 agccaattat gttaacagag agtcttttta
atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaa
agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg
tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580
aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc
2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct
cgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca
attacccatg tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa
tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca
ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt
taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940
tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca
3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa
ataaactaat 3060 aac 3063 MSH2 (human) (SEQ ID NO: 11) MAVQPKETLQ
LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT 60
QGVIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA
120 YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ
RKLGLCEFPD 180 NDQFSNLEAL LIQIGPKECV LPGGETAGDM GKLRQIIQRG
GILITERKKA DFSTKDIYQD 240 LNRLLKGKKG
EQMNSAVLPE MENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM 300
KLDIAAVPAL NLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWTKQPL MDKNRIEERL
360 NLVEAFVEDA ELRQTLQEDL LRRFPDLNRL AKKFQRQAAN LQDCYRLYQG
INQLPNVIQA 420 LEKHEGKHQK LLLAVFVTPL TDLRSDFSKF QEMIETTLDM
DQVENHEFLV KPSFDPNLSE 480 LREIMNDLEK KMQSTLISAA RDLGLDPGKQ
IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS 540 TVDIQKNGVK FTNSKLTSLN
EEYTKNKTEY EEAQDAIVKE IVNISSGYVE PMQTLNDVLA 600 QLDAVVSFAH
VSNGAPVPYV RPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD 660
KQMFHIITGP NMGGKSTYIR QTGVIVLMAQ IGCFVPCESA EVSIVDCILA RVGAGDSQLK
720 GVSTFMAEML ETASILRSAT KDSLIIIDEL GRGTSTYDGF GLAWAISEYI
ATKIGAFCMF 780 ATHFHELTAL ANQIPTVNNL HVTALTTEET LTMLYQVKKG
VCDQSFGIHV AELANFPKHV 840 IECAKQKALE LEEFQYIGES QGYDIMEPAA
KKCYLEREQG EKIIQEFLSK VKQMPFTEMS 900 EENITIKLKQ LKAEVIAKNN
SFVNEIISRI KVTT 934 MSH2 (human cDNA) (SEQ ID NO: 12) ggcgggaaac
agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 60
gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg
120 gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg
cgccttttcg 180 accggggcga cttctatacg gcgcacggcg aggacgcgct
gctggccgcc cgggaggtgt 240 tcaagaccca gggggtgatc aagtacatgg
ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttag taaaatgaat
tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360 atagagttga
agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt 420
atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta
480 acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca
gttgatggcc 540 agagacaggt tggagttggg tatgtggatt ccatacagag
gaaactagga ctgtgtgaat 600 tccctgataa tgatcagttc tccaatcttg
aggctctcct catccagatt ggaccaaagg 660 aatgtgtttt acccggagga
gagactgctg gagacatggg gaaactgaga cagataattc 720 aaagaggagg
aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt 780
atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat
840 tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc
aagtttttag 900 aactcttatc agatgattcc aactttggac agtttgaact
gactactttt gacttcagcc 960 agtatatgaa attggatatt gcagcagtca
gagcccttaa cctttttcag ggttctgttg 1020 aagataccac tggctctcag
tctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagact
tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg 1140
agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag
1200 aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt
caaagacaag 1260 cagcaaactt acaagattgt taccgactct atcagggtat
aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacat gaaggaaaac
accagaaatt attgttggca gtttttgtga 1380 ctcctcttac tgatcttcgt
tctgacttct ccaagtttca ggaaatgata gaaacaactt 1440 tagatatgga
tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc 1500
tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa
1560 gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat
tccagtgcac 1620 agtttggata ttactttcgt gtaacctgta aggaagaaaa
agtccttcgt aacaataaaa 1680 actttagtac tgtagatatc cagaagaatg
gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga agagtatacc
aaaaataaaa cagaatatga agaagcccag gatgccattg 1800 ttaaagaaat
tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860
tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc
1920 catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta
aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat
tcctaatgac gtatactttg 2040 aaaaagataa acagatgttc cacatcatta
ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca aactggggtg
atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga
agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc 2220
aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt
2280 ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact
tctacctacg 2340 atggatttgg gttagcatgg gctatatcag aatacattgc
aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt catgaactta
ctgccttggc caatcagata ccaactgtta 2460 ataatctaca tgtcacagca
ctcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt
ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta 2580
agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg
2640 gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat
ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc tgtccaaggt
gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc acaataaagt
taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaat
gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga
atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt 2940
atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag
3000 atatttagta atattttact ttgaggacat tttcaaagat ttttattttg
aaaaatgaga 3060 gctgtaactg aggactgttt gcaattgaca taggcaataa
taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg 3145 MLH1
(human) (SEQ ID NO: 13) MSFVAGVIRR LDETVVNRIA AGEVIQRPAN AIKEMIENCL
DAKSTSIQVI VKEGGLKLIQ 60 IQDNGTGIRK EDLDIVCERF TTSKLQSFED
LASISTYGFR GEALASISHV AHVTITTKTA 120 DGKCAYRASY SDGKLKAPPK
PCAGNQGTQI TVEDLFYNIA TRRKALKNPS EEYGKILEVV 180 GRYSVHNAGI
SFSVKKQGET VADVRTLPNA STVDNIRSIF GNAVSRELIE IGCEDKTLAF 240
KMNGYISNAN YSVKKCIFLL FINHRLVEST SLRKAIETVY AAYLPKNTHP FLYLSLEISP
300 QNVDVNVHPT KHEVHFLHEE SILERVQQHI ESKLLGSNSS RMYFTQTLLP
GLAGPSGEMV 360 KSTTSLTSSS TSGSSDKVYA HQMVRTDSRE QKLDAFLQPL
SKPLSSQPQA IVTEDKTDIS 420 SGRARQQDEE MLELPAPAEV AAKNQSLEGD
TTKGTSEMSE KRGPTSSNPR KRHREDSDVE 480 MVEDDSRKEM TAACTPRRRI
INLTSVLSLQ EEINEQGHEV LREMLHNHSF VGCVNPQWAL 540 AQHQTKLYLL
NTTKLSEELF YQILIYDFAN FGVLRLSEPA PLFDLAMLAL DSPESGWTEE 600
DGPKEGLAEY IVEFLKKKAE MLADYFSLEI DEEGNLIGLP LLIDNYVPPL EGLPIFILRL
660 ATEVNWDEEK ECFESLSKEC AMFYSIRKQY ISEESTLSGQ QSEVPGSIPN
SWKWTVEHIV 720 YKALRSHILP PKHFTEDGNI LQLANLPDLY KVFERC 756 MLH1
(human) (SEQ ID NO: 14) cttggctctt ctggcgccaa aatgtcgttc gtggcagggg
ttattcggcg gctggacgag 60 acagtggtga accgcatcgc ggcgggggaa
gttatccagc ggccagctaa tgctatcaaa 120 gagatgattg agaactgttt
agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180 ggaggcctga
agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg 240
gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt
300 atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt
ggctcatgtt 360 actattacaa cgaaaacagc tgatggaaag tgtgcataca
gagcaagtta ctcagatgga 420 aaactgaaag cccctcctaa accatgtgct
ggcaatcaag ggacccagat cacggtggag 480 gacctttttt acaacatagc
cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540 gggaaaattt
tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca 600
gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg
660 gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga
aattggatgt 720 gaggataaaa ccctagcctt caaaatgaat ggttacatat
ccaatgcaaa ctactcagtg 780 aagaagtgca tcttcttact cttcatcaac
catcgtctgg tagaatcaac ttccttgaga 840 aaagccatag aaacagtgta
tgcagcctat ttgcccaaaa acacacaccc attcctgtac 900 ctcagtttag
aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa 960
gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag
1020 ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc
aggacttgct 1080 ggcccctctg gggagatggt taaatccaca acaagtctga
cctcgtcttc tacttctgga 1140 agtagtgata aggtctatgc ccaccagatg
gttcgtacag attcccggga acagaagctt 1200 gatgcatttc tgcagcctct
gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260 gaggataaga
cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320
ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag
1380 gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag
aaagagacat 1440 cgggaagatt ctgatgtgga aatggtggaa gatgattccc
gaaaggaaat gactgcagct 1500 tgtacccccc ggagaaggat cattaacctc
actagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagg gacatgaggt
tctccgggag atgttgcata accactcctt cgtgggctgt 1620 gtgaatcctc
agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680
aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt
1740 ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt
agatagtcca 1800 gagagtggct ggacagagga agatggtccc aaagaaggac
ttgctgaata cattgttgag 1860 tttctgaaga agaaggctga gatgcttgca
gactatttct ctttggaaat tgatgaggaa 1920 gggaacctga ttggattacc
ccttctgatt gacaactatg tgcccccttt ggagggactg 1980 cctatcttca
ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040
gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag
2100 gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa
ctcctggaag 2160 tggactgtgg aacacattgt ctataaagcc ttgcgctcac
acattctgcc tcctaaacat 2220 ttcacagaag atggaaatat cctgcagctt
gctaacctgc ctgatctata caaagtcttt 2280 gagaggtgtt aaatatggtt
atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340 cgatacaaag
tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400
cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata
2460 aataaataga tgtgtcttaa cata 2484 hPMS2-134 (human) (SEQ ID NO:
15) MERAESSSTE PAKAIKPIDR KSVHQICSGQ VVLSLSTAVK ELVENSLDAG
ATNIDLKLKD 60 YGVDLIEVSD NGCGVEEENF EGLTLKHHTS KIQEFADLTQ
VETFGFRGEA LSSLCALSDV 120 TISTCHASAK VGT 133 hPMS2-134 (human cDNA)
(SEQ ID NO: 16) cgaggcggat cgggtgttgc atccatggag cgagctgaga
gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca
gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt
aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc
taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240
tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt
300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga
agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc
acgcatcggc gaaggttgga 420 acttga 426
[0046] For further information on the background of the invention
the following references may be consulted, each of which is
incorporated herein by reference in its entirety:
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[0080] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
EXAMPLE 1
Stable Expression of Dominant Negative MMR Genes in Hybridoma
Cells
[0081] It has been previously shown by Nicolaides et al.
(Nicolaides et al. (1998) A Naturally Occurring hPMS2 Mutation Can
Confer a Dominant Negative Mutator Phenotype Mol. Cell: Biol.
18:1635-1641) that the expression of a dominant negative allele in
an otherwise MMR proficient cell could render these host cells MMR
deficient. The creation of MMR deficient cells can lead to the
generation of genetic alterations throughout the entire genome of a
host organisms offspring, yielding a population of genetically
altered offspring or siblings that may produce biochemicals with
altered properties. This patent application teaches of the use of
dominant negative MMR genes in antibody-producing cells, including
but not limited to rodent hybridomas, human hybridomas, chimeric
rodent cells producing human immunoglobulin gene products, human
cells expressing immunoglobulin genes, mammalian cells producing
single chain antibodies, and prokaryotic cells producing mammalian
immunoglobulin genes or chimeric immunoglobulin molecules such as
those contained within single-chain antibodies. The cell expression
systems described above that are used to produce antibodies are
well known by those skilled in the art of antibody
therapeutics.
[0082] To demonstrate the ability to create MMR defective
hybridomas using dominant negative alleles of MMR genes, we first
transfected a mouse hybridoma cell line that is known to produce
and antibody directed against the human IgE protein with an
expression vector containing the human PMS2 (cell line referred to
as HBPMS2), the previously published dominant negative PMS2 mutant
referred herein as PMS134 (cell line referred to as HB134), or with
no insert (cell line referred to as HBvec). The results showed that
the PMS134 mutant could indeed exert a robust dominant negative
effect, resulting in biochemical and genetic manifestations of MMR
deficiency. Unexpectedly was the finding that the full length PMS2
also resulted in a lower MMR activity while no effect was seen in
cells containing the empty vector. A brief description of the
methods is provided below.
[0083] The MMR proficient mouse H36 hybridoma cell line was
transfected with various hPMS2 expression plasmids plus reporter
constructs for assessing MMR activity. The MMR genes were cloned
into the pEF expression vector, which contains the elongation
factor promoter upstream of the cloning site followed by a
mammalian polyadenylation signal. This vector also contains the
NEOr gene that allows for selection of cells retaining this
plasmid. Briefly, cells were transfected with 1 .mu.g of each
vector using polyliposomes following the manufacturer's protocol
(Life Technologies). Cells were then selected in 0.5 mg/ml of G418
for 10 days and G418 resistant cells were pooled together to
analyze for gene expression. The pEF construct contains an intron
that separates the exon 1 of the EF gene from exon 2, which is
juxtaposed to the 5' end of the polylinker cloning site. This
allows for a rapid reverse transcriptase polymerase chain reaction
(RT-PCR) screen for cells expressing the spliced products. At day
17, 100,000 cells were isolated and their RNA extracted using the
trizol method as previously described (Nicolaides N. C., Kinzler,
K. W., and Vogelstein, B. (1995) Analysis of the 5' region of PMS2
reveals heterogeneous transcripts and a novel overlapping gene.
Genomics 29:329-334). RNAs were reverse transcribed using
Superscript II (Life Technologies) and PCR amplified using a sense
primer located in exon 1 of the EF gene (5'-ttt cgc aac ggg ttt gcc
g-3') and an antisense primer (5'-gtt tca gag tta agc ctt cg-3')
centered at nt 283 of the published human PMS2 cDNA, which will
detect both the full length as well as the PMS134 gene expression.
Reactions were carried out using buffers and conditions as
previously described (Nicolaides, N. C., et al. (1995) Genomic
organization of the human PMS2 gene family. Genomics 30:195-206),
using the following amplification parameters: 94.degree. C. for 30
sec, 52.degree. C. for 2 min, 72.degree. C. for 2 min, for 30
cycles. Reactions were analyzed on agarose gels. FIG. 1 shows a
representative example of PMS expression in stably transduced H36
cells.
[0084] Expression of the protein encoded by these genes were
confirmed via western blot using a polyclonal antibody directed to
the first 20 amino acids located in the N-terminus of the protein
following the procedures previously described (data not shown)
(Nicolaides et al. (1998) A Naturally Occurring hPMS2 Mutation Can
Confer a Dominant Negative Mutator Phenotype. Mol. Cell. Biol.
18:1635-1641.
EXAMPLE 2
hPMS134 Causes a Defect in MMR Activity and Hypermutability in
Hybridoma Cells
[0085] A hallmark of MMR deficiency is the generation of unstable
microsatellite repeats in the genome of host cells. This phenotype
is referred to as microsatellite instability (MI) (Modrich, P.
(1994) Mismatch repair, genetic stability, and cancer Science
266:1959-1960; Palombo, F., et al. (1994) Mismatch repair and
cancer Nature 36:417). MI consists of deletions and/or insertions
within repetitive mono-, di- and/or tri nucleotide repetitive
sequences throughout the entire genome of a host cell. Extensive
genetic analysis eukaryotic cells have found that the only
biochemical defect that is capable of producing MI is defective MMR
(Strand, M., et al. (1993) Destabilization of tracts of simple
repetitive DNA in yeast by mutations affecting DNA mismatch repair
Nature 365:274-276; Perucho, M. (1996) Cancer of the microsatellite
mutator phenotype. Biol Chem. 377:675-684; Eshleman J. R., and
Markowitz, S. D. (1996) Mismatch repair defects in human
carcinogenesis. Hum. Mol. Genet. 5:1489-494). In light of this
unique feature that defective MMR has on promoting MI, it is now
used as a biochemical marker to survey for lack of MMR activity
within host cells (Perucho, M. (1996) Cancer of the microsatellite
mutator phenotype. Biol Chem. 377:675-684; Eshleman J. R., and
Markowitz, S. D. (1996) Mismatch repair defects in human
carcinogenesis. Hum. Mol. Genet. 5:1489-494; Liu, T., et al. (2000)
Microsatellite instability as a predictor of a mutation in a DNA
mismatch repair gene in familial colorectal cancer Genes
Chromosomes Cancer 27:17-25).
[0086] A method used to detect MMR deficiency in eukaryotic cells
is to employ a reporter gene that has a polynucleotide repeat
inserted within the coding region that disrupts its reading frame
due to a frame shift. In the case where MMR is defective, the
reporter gene will acquire random mutations (i.e. insertions and/or
deletions) within the polynucleotide repeat yielding clones that
contain a reporter with an open reading frame. We have employed the
use of an MMR-sensitive reporter gene to measure for MMR activity
in HBvec, HBPMS2, and HBPMS134 cells. The reporter construct used
the pCAR-OF, which contains a hygromycin resistance (HYG) gene plus
a .beta.-galactosidase gene containing a 29 bp out-of-frame poly-CA
tract at the 5' end of its coding region. The pCAR-OF reporter
would not generate .beta.-galactosidase activity unless a
frame-restoring mutation (i.e., insertion or deletion) arose
following transfection. HBvec, HBPMS2, and HB134 cells were each
transfected with pCAR-OF vector in duplicate reactions following
the protocol described in Example 1. Cells were selected in 0.5
mg/ml G418 and 0.5 mg/ml HYG to select for cells retaining both the
MMR effector and the pCAR-OF reporter plasmids. All cultures
transfected with the pCAR vector resulted in a similar number of
HYG/G418 resistant cells. Cultures were then expanded and tested
for .beta.-galactosidase activity in situ as well as by biochemical
analysis of cell extracts. For in situ analysis, 100,000 cells were
harvested and fixed in 1% gluteraldehyde, washed in phosphate
buffered saline solution and incubated in 1 ml of X-gal substrate
solution [0.15 M NaCl, 1 mM MgCl.sub.2, 3.3 mM K.sub.4Fe(CN).sub.6,
3.3 mM K.sub.3Fe(CN).sub.6, 0.2% X-Gal] in 24 well plates for 2
hours at 37.degree. C. Reactions were stopped in 500 mM sodium
bicarbonate solution and transferred to microscope slides for
analysis. Three fields of 200 cells each were counted for blue
(.beta.-galactosidase positive cells) or white
(.beta.-galactosidase negative cells) to assess for MMR
inactivation. Table 1 shows the results from these studies. While
no .beta.-galactosidase positive cells were observed in HBvec
cells, 10% of the cells per field were .beta.-galactosidase
positive in HB134 cultures and 2% of the cells per field were
.beta.-galactosidase positive in HBPMS2 cultures.
[0087] Cell extracts were prepared from the above cultures to
measure .beta.-galactosidase using a quantitative biochemical assay
as previously described (Nicolaides et al. (1998) A Naturally
Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator
Phenotype Mol. Cell. Biol. 18:1635-1641; Nicolaides, N. C., et al.
(1992) The Jun family members, c-JUN and JUND, transactivate the
human c-myb promoter via an Ap1 like element. J. Biol. Chem.
267:19665-19672). Briefly, 100,000 cells were collected,
centrifuged and resuspended in 200 .mu.ls of 0.25M Tris, pH 8.0.
Cells were lysed by freeze/thawing three times and supernatants
collected after microfugation at 14,000 rpms to remove cell debris.
Protein content was determined by spectrophotometric analysis at
OD.sup.280. For biochemical assays, 20 .mu.g of protein was added
to buffer containing 45 mM 2-mercaptoethanol, 1 mM MgCl.sub.2, 0.1
M NaPO.sub.4 and 0.6 mg/ml Chlorophenol
red-.beta.-D-galactopyranoside (CPRG, Boehringer Mannheim).
Reactions were incubated for 1 hour, terminated by the addition of
0.5 M Na2CO.sub.3, and analyzed by spectrophotometry at 576 nm. H36
cell lysates were used to subtract out background. FIG. 2 shows the
.beta.-galactosidase activity in extracts from the various cell
lines. As shown, the HB134 cells produced the highest amount of
.beta.-galactosidase, while no activity was found in the HBvec
cells containing the pCAR-OF. These data demonstrate the ability to
generate MMR defective hybridoma cells using dominant negative MMR
gene alleles.
[0088] Table 1. .beta.-galactosidase expression of HBvec, HBPMS2
and HB134 cells transfected with pCAR-OF reporter vectors. Cells
were transfected with the pCAR-OF .beta.-galactosidase reporter
plasmid. Transfected cells were selected in hygromycin and G418,
expanded and stained with X-gal solution to measure for
.beta.-galactosidase activity (blue colored cells). 3 fields of 200
cells each were analyzed by microscopy. The results below represent
the mean.+-.standard deviation of these experiments.
2 TABLE 1 CELL LINE # BLUE CELLS HBvec 0 +/- 0 HBPMS2 4 +/- 1 HB134
20 +/- 3
EXAMPLE 3
Screening Strategy to Identify Hybridoma Clones Producing
Antibodies with Higher Binding Affinities and/or Increased
Immunoglobulin Production
[0089] An application of the methods presented within this document
is the use of MMR deficient hybridomas or other immunoglobulin
producing cells to create genetic alterations within an
immunoglobulin gene that will yield antibodies with altered
biochemical properties. An illustration of this application is
demonstrated within this example whereby the HB134 hybridoma (see
Example 1), which is a MMR-defective cell line that produces an
anti-human immunoglobulin type E (hIgE) MAb, is grown for 20
generations and clones are isolated in 96-well plates and screened
for hIgE binding. FIG. 3 outlines the screening procedure to
identify clones that produce high affinity MAbs, which is presumed
to be due to an alteration within the light or heavy chain variable
region of the protein. The assay employs the use of a plate Enzyme
Linked Immunosorbant Assay (ELISA) to screen for clones that
produce high-affinity MAbs. 96-well plates containing single cells
from HBvec or HB134 pools are grown for 9 days in growth medium
(RPMI 1640 plus 10% fetal bovine serum) plus 0.5 mg/ml G418 to
ensure clones retain the expression vector. After 9 days, plates
are screened using an hIgE plate ELISA, whereby a 96 well plate is
coated with 50 .mu.ls of a 1 .mu.g/ml hIgE solution for 4 hours at
4.degree. C. Plates are washed 3 times in calcium and magnesium
free phosphate buffered saline solution (PBS.sup.-/-) and blocked
in 100 .mu.ls of PBS.sup.-/- with 5% dry milk for 1 hour at room
temperature. Wells are rinsed and incubated with 100 .mu.ls of a
PBS solution containing a 1:5 dilution of conditioned medium from
each cell clone for 2 hours. Plates are then washed 3 times with
PBS.sup.-/- and incubated for 1 hour at room temperature with 50
.mu.ls of a PBS.sup.-/- solution containing 1:3000 dilution of a
sheep anti-mouse horse radish peroxidase (HRP) conjugated secondary
antibody. Plates are then washed 3 times with PBS.sup.-/- and
incubated with 50 .mu.ls of TMB-HRP substrate (BioRad) for 15
minutes at room temperature to detect amount of antibody produced
by each clone. Reactions are stopped by adding 50 .mu.ls of 500 mM
sodium bicarbonate and analyzed by OD at 415 nm using a BioRad
plate reader. Clones exhibiting an enhanced signal over background
cells (H36 control cells) are then isolated and expanded into 10 ml
cultures for additional characterization and confirmation of ELISA
data in triplicate experiments. ELISAs are also performed on
conditioned (CM) from the same clones to measure total Ig
production within the conditioned medium of each well. Clones that
produce an increased ELISA signal and have increased antibody
levels are then further analyzed for variants that over-express
and/or over-secrete antibodies as described in Example 4. Analysis
of five 96-well plates each from HBvec or HB134 cells have found
that a significant number of clones with a higher Optical Density
(OD) value is observed in the MMR-defective HB134 cells as compared
to the HBvec controls. FIG. 4 shows a representative example of
HB134 clones producing antibodies that bind to specific antigen (in
this case IgE) with a higher affinity. FIG. 4 provides raw data
from the analysis of 96 wells of HBvec (left graph) or HB134 (right
graph) which shows 2 clones from the HB134 plate to have a higher
OD reading due to 1) genetic alteration of the antibody variable
domain that leads to an increased binding to IgE antigen, or 2)
genetic alteration of a cell host that leads to
over-production/secretion of the antibody molecule. Anti-Ig ELISA
found thatthe two clones, shown in FIG. 4 have Ig levels within
their CM similar to the surrounding wells exhibiting power OD
values. These data suggest that a genetic alteration occurred
within the antigen binding domain of the antibody which in turn
allows for higher binding to antigen.
[0090] Clones that produced higher OD values as determined by ELISA
were further analyzed at the genetic level to confirm that
mutations within the light or heavy chain variable region have
occurred that lead to a higher binding affinity hence yielding to a
stronger ELISA signal. Briefly, 100,000 cells are harvested and
extracted for RNA using the Triazol method as described above. RNAs
are reverse transcribed using Superscript II as suggested by the
manufacturer (Life Technology) and PCR amplified for the antigen
binding sites contained within the variable light and heavy chains.
Because of the heterogeneous nature of these genes, the following
degenerate primers are used to amplify light and heavy chain
alleles from the parent H36 strain.
3 Light chain sense: 5'-GGA TTT TCA GGT GCA (SEQ ID NO: 1) GAT TTT
CAG-3' Light chain antisense: 5'-ACT GGA TGG TGG GAA (SEQ ID NO: 2)
GAT GGA-3' Heavy chain sense: 5'-A(G/T) GTN (A/C)AG (SEQ ID NO: 3)
CTN CAG (C/G)AG TC-3' Heavy chain antisense: 5'-TNC CTT G(A/G)C CCC
(SEQ ID NO: 4) AGT A(G/A)(A/T)C-3'
[0091] PCR reactions using degenerate oligonucleotides are carried
out at 94.degree. C. for 30 sec, 52.degree. C. for 1 min, and
72.degree. C. for 1 min for 35 cycles. Products are analyzed on
agarose gels. Products of the expected molecular weights are
purified from the gels by Gene Clean (Bio 101), cloned into
T-tailed vectors, and sequenced to identify the wild type sequence
of the variable light and heavy chains. Once the wild type sequence
has been determined, non-degenerate primers were made for RT-PCR
amplification of positive HB134 clones. Both the light and heavy
chains were amplified, gel purified and sequenced using the
corresponding sense and antisense primers. The sequencing of RT-PCR
products gives representative sequence data of the endogenous
immunoglobulin gene and not due to PCR induced mutations. Sequences
from clones were then compared to the wild type sequence for
sequence comparison. An example of the ability to create in vivo
mutations within an immunoglobulin light or heavy chain is shown in
FIG. 5, where HB134 clone92 was identified by ELISA to have an
increased signal for hIgE. The light chain was amplified using
specific sense and antisense primers. The light chain was RT-PCR
amplified and the resulting product was purified and analyzed on an
automated ABI377 sequencer. As shown in clone A, a residue -4
upstream of the CDR region 3 had a genetic change from ACT to TCT,
which results in a Thr to Ser change within the framework region
just preceding the CDR#3. In clone B, a residue -6 upstream of the
CDR region had a genetic change from CCC to CTC, which reslts in a
Pro to His change within framework region preceeding CDR#2.
[0092] The ability to generate random mutations in immunoglobulin
genes or chimeric immunoglobulin genes is not limited to
hybridomas. Nicolaides et al. (Nicolaides et al. (1998) A Naturally
Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator
Phenotype Mol. Cell. Biol. 18:1635-1641) has previously shown the
ability to generate hypermutable hamster cells and produce
mutations within an endogenous gene. A common method for producing
humanized antibodies is to graft CDR sequences from a MAb (produced
by immunizing a rodent host) onto a human Ig backbone, and
transfection of the chimeric genes into Chinese Hamster Ovary (CHO)
cells whih in turn produce a functional Ab that is secreted by the
CHO cells (Shields, R. L., et al. (1995) Anti-IgE monoclonal
antibodies that inhibit allergen-specific histamine release. Int.
Arch. Allergy Immunol. 107:412-413). The methods described within
this application are also useful for generating genetic alterations
within Ig genes or chimeric Igs transfected within host cells such
as rodent cell lines, plants, yeast and prokaryotes (Frigerio L, et
al. (2000) Assembly, secretion, and vacuolar delivery of a hybrid
immunoglobulin in plants. Plant Physiol. 123:1483-1494).
[0093] These data demonstrate the ability to generate hypermutable
hybridomas, or other Ig producing host cells that can be grown and
selected, to identify structurally altered immunoglobulins yielding
antibodies with enhanced biochemical properties, including but not
limited to increased antigen binding affinity. Moreover,
hypermutable clones that contain missense mutations within the
immunoglobulin gene that result in an amino acid change or changes
can be then further characterized for in vivo stability, antigen
clearance, on-off binding to antigens, etc. Clones can also be
further expanded for subsequent rounds of in vivo mutations and can
be screened using the strategy listed above.
[0094] The use of chemical mutagens to produce genetic mutations in
cells or whole organisms are limited due to the toxic effects that
these agents have on "normal" cells. The use of chemical mutagens
such as MNU in MMR defective organisms is much more tolerable
yielding to a 10 to 100 fold increase in genetic mutation over MMR
deficiency alone (Bignami M, (2000) Unmasking a killer: DNA
O(6)-methylguanine and the cytotoxicity of methylating agents.
Mutat. Res. 462:71-82). This strategy allows for the use of
chemical mutagens to be used in MMR-defective Ab producing cells as
a method for increasing additional mutations within immunoglobulin
genes or chimeras that may yield functional Abs with altered
biochemical properties such as enhanced binding affinity to
antigen, etc.
EXAMPLE 4
Generation of Antibody Producing Cells with Enhanced Antibody
Production
[0095] Analysis of clones from H36 and HB134 following the
screening strategy listed above hasidentified a significant number
of clones that produce enhanced amounts of antibody into the
medium. While a subset of these clones gave higher Ig binding data
as determined by ELISA as a consequence of mutations within the
antigen binding domains contained in the variable regions, others
were found to contain "enhanced" antibody production. A summary of
the clones producing enhanced amounts of secreted MAb is shown in
TABLE 2, where a significant number of clones from HB134 cells were
found to produce enhanced Ab production within the conditioned
medium as compared to H36 control cells.
[0096] TABLE 2. Generation of hybridoma cells producing high levels
of antibody. HB134 clones were assayed by ELISA for elevated Ig
levels. Analysis of 480 clones showed that a significant number of
clones had elevated MAb product levels in their CM. Quantification
showed that several of these clones produced greater than 500
ngs/ml of MAb due to either enhanced expression and/or secretion as
compared to clones from the H36 cell line.
4TABLE 2 Production of MAb in CM from H36 and HB134 clones. Cell
Line % clones > 400 ng/ml % clones > 500 ng/ml H36 1/480 =
0.2% 0/480 = 0% HB134 50/480 = 10% 8/480 = 1.7%
[0097] Cellular analysis of HB134 clones with higher MAb levels
within the conditioned medium (CM) were analyzed to determine if
the increased production was simply due to genetic alterations at
the Ig locus that may lead to over-expression of the polypeptides
forming the antibody, or due to enhanced secretion due to a genetic
alteration affecting secretory pathway mechanisms. To address this
issue, we expanded three HB134 clones that had increased levels of
antibody within their CM. 10,000 cells were prepared for western
blot analysis to assay for intracellular steady state Ig protein
levels (FIG. 6). In addition, H36 cells were used as a standard
reference (Lane 2) and a rodent fibroblast (Lane 1) was used as an
Ig negative control. Briefly, cells were pelleted by centrifugation
and lysed directly in 300 .mu.l of SDS lysis buffer (60 mM Tris, pH
6.8, 2% SDS, 10% glycerol, 0.1 M 2-mercaptoethanol, 0.001%
bromophenol blue) and boiled for 5 minutes. Lysate proteins were
separated by electrophoresis on 4-12% NUPAGE gels (for analysis of
Ig heavy chain. Gels were electroblotted onto Immobilon-P
(Millipore) in 48 mM Tris base, 40 mM glycine, 0.0375% SDS, 20%
methanol and blocked at room temperature for 1 hour in
Tris-buffered saline (TBS) plus 0.05% Tween-20 and 5% condensed
milk. Filters were probed with a 1:10,000 dilution of sheep
anti-mouse horseradish peroxidase conjugated monoclonal antibody in
TBS buffer and detected by chemiliuminescence using Supersignal
substrate (Pierce). Experiments were repeated in duplicates to
ensure reproducibility. FIG. 6 shows a representative analysis
where a subset of clones had enhanced Ig production which accounted
for increased Ab production (Lane 5) while others had a similar
steady state level as the control sample, yet had higher levels of
Ab within the CM. These data suggest a mechanism whereby a subset
of HB134 clones contained a genetic alteration that in turn
produces elevated secretion of antibody.
[0098] The use of chemical mutagens to produce genetic mutations in
cells or whole organisms are limited due to the toxic effects that
these agents have on "normal" cells. The use of chemical mutagens
such as MNU in MMR defective organisms is much more tolerable
yielding to a 10 to 100 fold increase in genetic mutation over MMR
deficiency alone (Bignami M, (2000) Unmasking a killer: DNA
O(6)-methylguanine and the cytotoxicity of methylating agents.
Mutat. Res. 462:71-82). This strategy allows for the use of
chemical mutagens to be used in MMR-defective Ab producing cells as
a method for increasing additional mutations within immunoglobulin
genes or chimeras that may yield functional Abs with altered
biochemical properties such as enhanced binding affinity to
antigen, etc.
EXAMPLE 5
Establishment of Genetic Stability in Hybridoma Cells with New
Output Trait
[0099] The initial steps of MMR are dependent on two protein
complexes, called MutS.alpha. and MutL.alpha. (Nicolaides et al.
(1998) A Naturally Occurring hPMS2 Mutation Can Confer a Dominant
Negative Mutator Phenotype. Mol. Cell. Biol. 18:1635-1641).
Dominant negative MMR alleles are able to perturb the formation of
these complexes with downstream biochemicals involved in the
excision and polymerization of nucleotides comprising the
"corrected" nucleotides. Examples from this application show the
ability of a truncated MMR allele (PMS134) as well as a full length
human PMS2 when expressed in a hybridoma cell line is capable of
blocking MMR resulting in a hypermutable cell line that gains
genetic alterations throughout its entire genome per cell division.
Once a cell line is produced that contains genetic alterations
within genes encoding for an antibody, a single chain antibody,
over expression of immunoglobulin genes and/or enhanced secretion
of antibody, it is desirable to restore the genomic integrity of
the cell host. This can be achieved by the use of inducible vectors
whereby dominant negative MMR genes are cloned into such vectors,
introduced into Ab producing cells and the cells are cultured in
the presence of inducer molecules and/or conditions. Inducible
vectors include but are not limited to chemical regulated promoters
such as the steroid inducible MMTV, tetracycline regulated
promoters, temperature sensitive MMR gene alleles, and temperature
sensitive promoters.
[0100] The results described above lead to several conclusions.
First, expression of hPMS2 and PMS134 results in an increase in
microsatellite instability in hybridoma cells. That this elevated
microsatellite instability is due to MMR deficiency was proven by
evaluation of extracts from stably transduced cells. The expression
of PMS134 results in a polar defect in MMR, which was only observed
using heteroduplexes designed to test repair from the 5' direction
(no significant defect in repair from the 3' direction was observed
in the same extracts) (Nicolaides et al. (1998) A Naturally
Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator
Phenotype. Mol. Cell. Biol. 18:1635-1641). Interestingly, cells
deficient in hMLH1 also have a polar defect in MMR, but in this
case preferentially affecting repair from the 3' direction
(Drummond, J. T, et al. (1996) Cisplatin and adriamycin resistance
are associated with MutLa and mismatch repair deficiency in an
ovarian tumor cell line. J. Biol. Chem. 271:9645-19648). It is
known from previous studies in both prokaryotes and eukaryotes that
the separate enzymatic components mediate repair from the two
different directions. Our results, in combination with those of
Drummond et al. (Shields, R. L., et al. (1995) Anti-IgE monoclonal
antibodies that inhibit allergen-specific histamine release. Int.
Arch Allergy Immunol. 107:412-413), strongly suggest a model in
which 5' repair is primarily dependent on hPMS2 while 3' repair is
primarily dependent on hMLH1. It is easy to envision how the
dimeric complex between PMS2 and MLH1 might set up this
directionality. The combined results also demonstrate that a defect
in directional MMR is sufficient to produce a MMR defective
phenotype and suggests that any MMR gene allele is useful to
produce genetically altered hybridoma cells, or a cell line that is
producing Ig gene products. Moreover, the use of such MMR alleles
will be useful for generating genetically altered Ig polypeptides
with altered biochemical properties as well as cell hosts that
produce enhanced amounts of antibody molecules.
[0101] Another method that is taught in this application is that
ANY method used to block MMR can be performed to generate
hypermutablility in an antibody-producing cell that can lead to
genetically altered antibodies with enhanced biochemical features
such as but not limited to increased antigen binding, enhanced
pharmacokinetic profiles, etc. These processes can also to be used
to generate antibody producer cells that have increased Ig
expression as shown in Example 4, FIG. 6 and/or increased antibody
secretion as shown in Table 2.
[0102] In addition, we demonstrate the utility of blocking MMR in
antibody producing cells to increase genetic alterations within Ig
genes that may lead to altered biochemical features such as, but
not limited to, increased antigen binding affinities (FIGS. 5A and
5B). The blockade of MMR in such cells can be through the use of
dominant negative MMR gene alleles from any species including
bacteria, yeast, protozoa, insects, rodents, primates, mammalian
cells, and man. Blockade of MMR can also be generated through the
use of antisense RNA or deoxynucleotides directed to any of the
genes involved in the MMR biochemical pathway. Blockade of MMR can
be through the use of polypeptides that interfere with subunits of
the MMR complex including but not limited to antibodies. Finally,
the blockade of MMR may be through the use chemicals such as but
not limited to nonhydrolyzable ATP analogs, which have been shown
to block MMR (Galio, L, et al. (1999) ATP hydrolysis-dependent
formation of a dynamic ternary nucleoprotein complex with MutS and
MutL. Nucl. Acids Res. 27:2325-23231).
Sequence CWU 1
1
23 1 24 DNA Artificial Sequence Oligonucleotide primer 1 ggattttcag
gtgcagattt tcag 24 2 21 DNA Artificial Sequence Oligonucleotide
primer 2 actggatggt gggaagatgg a 21 3 19 DNA Artificial Sequence
Oligonucleotide primer 3 akgtnmagct ncagsagtc 19 4 19 DNA
Artificial Sequence Oligonucleotide primer 4 tnccttgrcc ccagtarwc
19 5 859 PRT Mus musculus 5 Met Glu Gln Thr Glu Gly Val Ser Thr Glu
Cys Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Gly Lys Ser Val His
Gln Ile Cys Ser Gly Gln Val Ile 20 25 30 Leu Ser Leu Ser Thr Ala
Val Lys Glu Leu Ile Glu Asn Ser Val Asp 35 40 45 Ala Gly Ala Thr
Thr Ile Asp Leu Arg Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile
Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 75 80
Glu Gly Leu Ala Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85
90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu
Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys
His Gly Ser 115 120 125 Ala Ser Val Gly Thr Arg Leu Val Phe Asp His
Asn Gly Lys Ile Thr 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro Lys
Gly Thr Thr Val Ser Val Gln 145 150 155 160 His Leu Phe Tyr Thr Leu
Pro Val Arg Tyr Lys Glu Phe Gln Arg Asn 165 170 175 Ile Lys Lys Glu
Tyr Ser Lys Met Val Gln Val Leu Gln Ala Tyr Cys 180 185 190 Ile Ile
Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205
Gly Lys Arg His Ala Val Val Cys Thr Ser Gly Thr Ser Gly Met Lys 210
215 220 Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu
Ile 225 230 235 240 Pro Phe Val Gln Leu Pro Pro Ser Asp Ala Val Cys
Glu Glu Tyr Gly 245 250 255 Leu Ser Thr Ser Gly Arg His Lys Thr Phe
Ser Thr Phe Arg Ala Ser 260 265 270 Phe His Ser Ala Arg Thr Ala Pro
Gly Gly Val Gln Gln Thr Gly Ser 275 280 285 Phe Ser Ser Ser Ile Arg
Gly Pro Val Thr Gln Gln Arg Ser Leu Ser 290 295 300 Leu Ser Met Arg
Phe Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 305 310 315 320 Val
Val Leu Asn Val Ser Val Asp Ser Glu Cys Val Asp Ile Asp Val 325 330
335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu
340 345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp
Ala Asn 355 360 365 Lys Leu Asn Val Asn Gln Gln Pro Leu Leu Asp Val
Glu Gly Asn Leu 370 375 380 Val Lys Leu His Thr Ala Glu Leu Glu Lys
Pro Val Pro Gly Lys Gln 385 390 395 400 Asp Asn Ser Pro Ser Leu Lys
Ser Thr Ala Asp Glu Lys Arg Val Ala 405 410 415 Ser Ile Ser Arg Leu
Arg Glu Ala Phe Ser Leu His Pro Thr Lys Glu 420 425 430 Ile Lys Ser
Arg Gly Pro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro 435 440 445 Ser
Glu Lys Arg Gly Val Leu Ser Ser Tyr Pro Ser Asp Val Ile Asp 450 455
460 Tyr Arg Gly Leu Arg Gly Ser Gln Asp Lys Leu Val Ser Pro Thr Asp
465 470 475 480 Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys
Asp Ser Gly 485 490 495 Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu
Phe Ser Thr Pro Glu 500 505 510 Val Ala Ser Ser Phe Ser Ser Asp Tyr
Asn Val Ser Ser Leu Glu Asp 515 520 525 Arg Pro Ser Gln Glu Thr Ile
Asn Cys Gly Asp Leu Asp Cys Arg Pro 530 535 540 Pro Gly Thr Gly Gln
Ser Leu Lys Pro Glu Asp His Gly Tyr Gln Cys 545 550 555 560 Lys Ala
Leu Pro Leu Ala Arg Leu Ser Pro Thr Asn Ala Lys Arg Phe 565 570 575
Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Ile Ser Gln Arg Leu Pro 580
585 590 Gly Pro Gln Ser Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys
Met 595 600 605 Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln Asn Lys
His Glu Leu 610 615 620 Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln
Asn Lys His Glu Leu 625 630 635 640 Ser Tyr Arg Lys Phe Arg Ala Lys
Ile Cys Pro Gly Glu Asn Gln Ala 645 650 655 Ala Glu Asp Glu Leu Arg
Lys Glu Ile Ser Lys Ser Met Phe Ala Glu 660 665 670 Met Glu Ile Leu
Gly Gln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu 675 680 685 Lys Glu
Asp Leu Phe Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr 690 695 700
Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu 705
710 715 720 Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala
Val Leu 725 730 735 Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe
Asp Phe Val Ile 740 745 750 Asp Glu Asp Ala Pro Val Thr Glu Arg Ala
Lys Leu Ile Ser Leu Pro 755 760 765 Thr Ser Lys Asn Trp Thr Phe Gly
Pro Gln Asp Ile Asp Glu Leu Ile 770 775 780 Phe Met Leu Ser Asp Ser
Pro Gly Val Met Cys Arg Pro Ser Arg Val 785 790 795 800 Arg Gln Met
Phe Ala Ser Arg Ala Cys Arg Lys Ser Val Met Ile Gly 805 810 815 Thr
Ala Leu Asn Ala Ser Glu Met Lys Lys Leu Ile Thr His Met Gly 820 825
830 Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg
835 840 845 His Val Ala Asn Leu Asp Val Ile Ser Gln Asn 850 855 6
3056 DNA Mus musculus 6 gaattccggt gaaggtcctg aagaatttcc agattcctga
gtatcattgg aggagacaga 60 taacctgtcg tcaggtaacg atggtgtata
tgcaacagaa atgggtgttc ctggagacgc 120 gtcttttccc gagagcggca
ccgcaactct cccgcggtga ctgtgactgg aggagtcctg 180 catccatgga
gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg 240
atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg
300 tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat
ctaaggctta 360 aagactatgg ggtggacctc attgaagttt cagacaatgg
atgtggggta gaagaagaaa 420 actttgaagg tctagctctg aaacatcaca
catctaagat tcaagagttt gccgacctca 480 cgcaggttga aactttcggc
tttcgggggg aagctctgag ctctctgtgt gcactaagtg 540 atgtcactat
atctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc 600
ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg
660 tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg
aacattaaaa 720 aggagtattc caaaatggtg caggtcttac aggcgtactg
tatcatctca gcaggcgtcc 780 gtgtaagctg cactaatcag ctcggacagg
ggaagcggca cgctgtggtg tgcacaagcg 840 gcacgtctgg catgaaggaa
aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttt
tgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca 960
cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg
1020 cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc
cctgtgaccc 1080 agcaaaggtc tctaagcttg tcaatgaggt tttatcacat
gtataaccgg catcagtacc 1140 catttgtcgt ccttaacgtt tccgttgact
cagaatgtgt ggatattaat gtaactccag 1200 ataaaaggca aattctacta
caagaagaga agctattgct ggccgtttta aagacctcct 1260 tgataggaat
gtttgacagt gatgcaaaca agcttaatgt caaccagcag ccactgctag 1320
atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa
1380 agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggta
gcatccatct 1440 ccaggctgag agaggccttt tctcttcatc ctactaaaga
gatcaagtct aggggtccag 1500 agactgctga actgacacgg agttttccaa
gtgagaaaag gggcgtgtta tcctcttatc 1560 cttcagacgt catctcttac
agaggcctcc gtggctcgca ggacaaattg gtgagtccca 1620 cggacagccc
tggtgactgt atggacagag agaaaataga aaaagactca gggctcagca 1680
gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagt agctttagca
1740 gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata
aactgtggtg 1800 acctggactg ccgtcctcca ggtacaggac agtccttgaa
gccagaagac catggatatc 1860 aatgcaaagc tctacctcta gctcgtctgt
cacccacaaa tgccaagcgc ttcaagacag 1920 aggaaagacc ctcaaatgtc
aacatttctc aaagattgcc tggtcctcag agcacctcag 1980 cagctgaggt
cgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc 2040
tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg
2100 aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa
gcagcagaag 2160 atgaactcag aaaagagatt agtaaatcga tgtttgcaga
gatggagatc ttgggtcagt 2220 ttaacctggg atttatagta accaaactga
aagaggacct cttcctggtg gaccagcatg 2280 ctgcggatga gaagtacaac
tttgagatgc tgcagcagca cacggtgctc caggcgcaga 2340 ggctcatcac
accccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa 2400
atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca
2460 ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt
ggaccccaag 2520 atatagatga actgatcttt atgttaagtg acagccctgg
ggtcatgtgc cggccctcac 2580 gagtcagaca gatgtttgct tccagagcct
gtcggaagtc agtgatgatt ggaacggcgc 2640 tcaatgcgag cgagatgaag
aagctcatca cccacatggg tgagatggac cacccctgga 2700 actgccccca
cggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga 2760
actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg
2820 ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat
gtactggatc 2880 catttaaaag cagtgttaag gcaggcatga tggagtgttc
ctctagctca gctacttggg 2940 tgatccggtg ggagctcatg tgagcccagg
actttgagac cactccgagc cacattcatg 3000 agactcaatt caaggacaaa
aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056 7 862 PRT Homo sapiens
7 Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1
5 10 15 Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val
Val 20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn
Ser Leu Asp 35 40 45 Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys
Asp Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys
Gly Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Thr Leu Lys His
His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val
Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys
Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125 Ala
Lys Val Gly Thr Arg Leu Met Phe Asp His Asn Gly Lys Ile Ile 130 135
140 Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val Ser Val Gln
145 150 155 160 Gln Leu Phe Ser Thr Leu Pro Val Arg His Lys Glu Phe
Gln Arg Asn 165 170 175 Ile Lys Lys Glu Tyr Ala Lys Met Val Gln Val
Leu His Ala Tyr Cys 180 185 190 Ile Ile Ser Ala Gly Ile Arg Val Ser
Cys Thr Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg Gln Pro Val Val
Cys Thr Gly Gly Ser Pro Ser Ile Lys 210 215 220 Glu Asn Ile Gly Ser
Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 225 230 235 240 Pro Phe
Val Gln Leu Pro Pro Ser Asp Ser Val Cys Glu Glu Tyr Gly 245 250 255
Leu Ser Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe 260
265 270 Ile Ser Gln Cys Thr His Gly Val Gly Arg Ser Ser Thr Asp Arg
Gln 275 280 285 Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys
Val Cys Arg 290 295 300 Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg
His Gln Tyr Pro Phe 305 310 315 320 Val Val Leu Asn Ile Ser Val Asp
Ser Glu Cys Val Asp Ile Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln
Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350 Ala Val Leu Lys
Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Val Asn 355 360 365 Lys Leu
Asn Val Ser Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 370 375 380
Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu Lys Gln 385
390 395 400 Asp Gln Ser Pro Ser Leu Arg Thr Gly Glu Glu Lys Lys Asp
Val Ser 405 410 415 Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu Arg His
Thr Thr Glu Asn 420 425 430 Lys Pro His Ser Pro Lys Thr Pro Glu Pro
Arg Arg Ser Pro Leu Gly 435 440 445 Gln Lys Arg Gly Met Leu Ser Ser
Ser Thr Ser Gly Ala Ile Ser Asp 450 455 460 Lys Gly Val Leu Arg Pro
Gln Lys Glu Ala Val Ser Ser Ser His Gly 465 470 475 480 Pro Ser Asp
Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His 485 490 495 Gly
Ser Thr Ser Val Asp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly 500 505
510 Ser His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly
515 520 525 Ser Gln Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr
Asp Asp 530 535 540 Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu
Asp Thr Gly Cys 545 550 555 560 Lys Phe Arg Val Leu Pro Gln Pro Thr
Asn Leu Ala Thr Pro Asn Thr 565 570 575 Lys Arg Phe Lys Lys Glu Glu
Ile Leu Ser Ser Ser Asp Ile Cys Gln 580 585 590 Lys Leu Val Asn Thr
Gln Asp Met Ser Ala Ser Gln Val Asp Val Ala 595 600 605 Val Lys Ile
Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser 610 615 620 Leu
Ala Lys Arg Ile Lys Gln Leu His His Glu Ala Gln Gln Ser Glu 625 630
635 640 Gly Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly
Glu 645 650 655 Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser
Lys Thr Met 660 665 670 Phe Ala Glu Met Glu Ile Ile Gly Gln Phe Asn
Leu Gly Phe Ile Ile 675 680 685 Thr Lys Leu Asn Glu Asp Ile Phe Ile
Val Asp Gln His Ala Thr Asp 690 695 700 Glu Lys Tyr Asn Phe Glu Met
Leu Gln Gln His Thr Val Leu Gln Gly 705 710 715 720 Gln Arg Leu Ile
Ala Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu 725 730 735 Ala Val
Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp 740 745 750
Phe Val Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile 755
760 765 Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val
Asp 770 775 780 Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met
Cys Arg Pro 785 790 795 800 Ser Arg Val Lys Gln Met Phe Ala Ser Arg
Ala Cys Arg Lys Ser Val 805 810 815 Met Ile Gly Thr Ala Leu Asn Thr
Ser Glu Met Lys Lys Leu Ile Thr 820 825 830 His Met Gly Glu Met Asp
His Pro Trp Asn Cys Pro His Gly Arg Pro 835 840 845 Thr Met Arg His
Ile Ala Asn Leu Gly Val Ile Ser Gln Asn 850 855 860 8 2771 DNA Homo
sapiens 8 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac
agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcaga
tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta
gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaa
ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag
aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300
caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc
360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc
gaaggttgga 420 actcgactga tgtttgatca caatgggaaa attatccaga
aaacccccta cccccgcccc 480 agagggacca cagtcagcgt gcagcagtta
ttttccacac tacctgtgcg ccataaggaa 540 tttcaaagga atattaagaa
ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt 600 atcatttcag
caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag 660
cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg
720 cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga
ctccgtgtgt 780 gaagagtacg gtttgagctg ttcggatgct ctgcataatc
ttttttacat ctcaggtttc 840 atttcacaat gcacgcatgg agttggaagg
agttcaacag acagacagtt tttctttatc 900 aaccggcggc cttgtgaccc
agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg 960 tataatcgac
accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt 1020
gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg
1080 gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa
gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt aacttaataa
aaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa gcaggatcaa
tccccttcat taaggactgg agaagaaaaa 1260 aaagacgtgt ccatttccag
actgcgagag gccttttctc ttcgtcacac aacagagaac 1320 aagcctcaca
gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt 1380
atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa
1440 gaggcagtga gttccagtca cggacccagt gaccctacgg acagagcgga
ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat tctgaggggt
tcagcatccc agacacgggc 1560 agtcactgca gcagcgagta tgcggccagc
tccccagggg acaggggctc gcaggaacat 1620 gtggactctc aggagaaagc
gcctgaaact gacgactctt tttcagatgt ggactgccat 1680 tcaaaccagg
aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca 1740
accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa
1800 aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt
gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg agttctttag
ctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag tgaaggggaa
cagaattaca ggaagtttag ggcaaagatt 1980 tgtcctggag aaaatcaagc
agccgaagat gaactaagaa aagagataag taaaacgatg 2040 tttgcagaaa
tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat 2100
gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg
2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct
caacttaact 2220 gctgttaatg aagctgttct gatagaaaat ctggaaatat
ttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc tccagtcact
gaaagggcta aactgatttc cttgccaact 2340 agtaaaaact ggaccttcgg
accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400 agccctgggg
tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc 2460
cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc
2520 cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac
catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt
cactgtatgg aataattggt 2640 tttatcgcag atttttatgt tttgaaagac
agagtcttca ctaacctttt ttgttttaaa 2700 atgaaacctg ctacttaaaa
aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760 cttttcaaac c 2771
9 932 PRT Homo sapiens 9 Met Lys Gln Leu Pro Ala Ala Thr Val Arg
Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile Thr Ser Val Val Ser Val
Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu Asp Ala Gly Ala Thr
Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45 Phe Asp Lys Ile
Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 50 55 60 Asp Ala
Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 65 70 75 80
His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 85
90 95 Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg
Thr 100 105 110 Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly
Ser Gly His 115 120 125 Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln
Gly Thr Thr Val Thr 130 135 140 Ala Leu Arg Leu Phe Lys Asn Leu Pro
Val Arg Lys Gln Phe Tyr Ser 145 150 155 160 Thr Ala Lys Lys Cys Lys
Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 165 170 175 Met Ser Phe Gly
Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 180 185 190 Asn Lys
Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys Met 195 200 205
Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser 210
215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr Leu Ser Gly Phe
Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser Leu
Ser Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro
Val His Gln Lys Asp Ile 260 265 270 Leu Lys Leu Ile Arg His His Tyr
Asn Leu Lys Cys Leu Lys Glu Ser 275 280 285 Thr Arg Leu Tyr Pro Val
Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 290 295 300 Asp Val Asp Val
Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 305 310 315 320 Asn
Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys 325 330
335 Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp
340 345 350 Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr
Asp Val 355 360 365 Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr
Ser Asn Val Asp 370 375 380 Thr Ser Val Ile Pro Phe Gln Asn Asp Met
His Asn Asp Glu Ser Gly 385 390 395 400 Lys Asn Thr Asp Asp Cys Leu
Asn His Gln Ile Ser Ile Gly Asp Phe 405 410 415 Gly Tyr Gly His Cys
Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 420 425 430 Lys Asn Ala
Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn 435 440 445 Ser
Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val Lys His 450 455
460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu Ser Gly Glu
465 470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile
Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser
Val Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys Ile Leu Val Pro Glu
Lys Ser Leu Pro Cys Lys Val 515 520 525 Ser Asn Asn Asn Tyr Pro Ile
Pro Glu Gln Met Asn Leu Asn Glu Asp 530 535 540 Ser Cys Asn Lys Lys
Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 545 550 555 560 Thr Ala
Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met Ser 565 570 575
Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln Phe Leu Ile Glu 580
585 590 Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu
Leu 595 600 605 Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu
Glu Lys Ala 610 615 620 Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met
Lys Arg Ala Ile Glu 625 630 635 640 Gln Glu Ser Gln Met Ser Leu Lys
Asp Gly Arg Lys Lys Ile Lys Pro 645 650 655 Thr Ser Ala Trp Asn Leu
Ala Gln Lys His Lys Leu Lys Thr Ser Leu 660 665 670 Ser Asn Gln Pro
Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys 675 680 685 Arg Arg
Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser Met Lys 690 695 700
Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp Leu Glu Glu 705
710 715 720 Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp
Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu Asn
Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu
Glu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro Leu Glu Lys Pro Ile
Met Leu Thr Glu Ser Leu Phe Asn 770 775 780 Gly Ser His Tyr Leu Asp
Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 785 790 795 800 Arg Tyr Ser
Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 805 810 815 Gly
Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 820 825
830 Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala
835 840 845 Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala
Lys Glu 850 855 860 Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr
Leu Glu Gly Glu 865 870 875 880 Ala Val Arg Leu Ser Arg Gln Leu Pro
Met Tyr Leu Ser Tyr Glu Asp 885 890 895 Ile Gln Asp Ile Ile Tyr Arg
Met Lys His Gln Phe Gly Asn Glu Ile 900 905 910 Lys Glu Cys Val His
Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 915 920 925 Pro Glu Thr
Thr 930 10 3063 DNA Homo sapiens 10 ggcacgagtg gctgcttgcg
gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60 ctgctctgtt
aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa 120
gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg
180 atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat
aaaattgagg 240 tgcgagataa cggggagggt atcaaggctg ttgatgcacc
tgtaatggca atgaagtact 300 acacctcaaa aataaatagt catgaagatc
ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt ggggtcaatt
tgttgtatag ctgaggtttt aattacaaca agaacggctg 420 ctgataattt
tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac 480
cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg
540 taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa
aagatccaag 600 atctcctcat gagctttggt atccttaaac ctgacttaag
gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa agcagagtat
cagatcacaa gatggctctc atgtcagttc 720 tggggactgc tgttatgaac
aatatggaat cctttcagta ccactctgaa gaatctcaga 780 tttatctcag
tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa 840
caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa
900 agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt
ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag ctgatgttga
tgtaaattta acaccagata 1020 aaagccaagt attattacaa aataaggaat
ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta tggaccatta
cctagtacaa attcttatga aaataataaa acagatgttt 1140 ccgcagctga
catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg 1200
aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata
1260 tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag
ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg aaatttctaa
cattgataaa aacactaaga 1380 atgcatttca ggacatttca atgagtaatg
tatcatggga gaactctcag acggaatata 1440 gtaaaacttg ttttataagt
tccgttaagc acacccagtc agaaaatggc aataaagacc 1500 atatagatga
gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt 1560
ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac
1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat
aataattatc 1680 caatccctga acaaatgaat cttaatgaag attcatgtaa
caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt acagcttatg
atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc aagtgctctt
tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860 ctaagactag
tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg 1920
aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc
1980 aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc
agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc agaagcacaa
gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa ctccttcagt
cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt acagatcccc
ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220 acaaagttga
cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg 2280
atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag
2340 aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag
ccactggaaa 2400 agccaattat gttaacagag agtcttttta atggatctca
ttatttagac gttttatata 2460 aaatgacagc agatgaccaa agatacagtg
gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg tttcaagata
aaattgatac caggagtttc aattactgaa aattacttgg 2580 aaatagaagg
aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc 2640
ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga
2700 taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg
tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa tgaagcacca
gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca ttttttcatc
atttaaccta tcttccagaa actacatgat 2880 taaatatgtt taagaagatt
agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940 tctggtttta
aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca 3000
ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat
3060 aac 3063 11 934 PRT Homo sapiens 11 Met Ala Val Gln Pro Lys
Glu Thr Leu Gln Leu Glu Ser Ala Ala Glu 1 5 10 15 Val Gly Phe Val
Arg Phe Phe Gln Gly Met Pro Glu Lys Pro Thr Thr 20 25 30 Thr Val
Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu 35 40 45
Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly Val Ile 50
55 60 Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln Ser Val Val
Leu 65 70 75 80 Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp Leu Leu
Leu Val Arg 85 90 95 Gln Tyr Arg Val Glu Val Tyr Lys Asn Arg Ala
Gly Asn Lys Ala Ser 100 105 110 Lys Glu Asn Asp Trp Tyr Leu Ala Tyr
Lys Ala Ser Pro Gly Asn Leu 115 120 125 Ser Gln Phe Glu Asp Ile Leu
Phe Gly Asn Asn Asp Met Ser Ala Ser 130 135 140 Ile Gly Val Val Gly
Val Lys Met Ser Ala Val Asp Gly Gln Arg Gln 145 150 155 160 Val Gly
Val Gly Tyr Val Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys 165 170 175
Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu Ile 180
185 190 Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu Thr Ala
Gly 195 200 205 Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Arg Gly Gly
Ile Leu Ile 210 215 220 Thr Glu Arg Lys Lys Ala Asp Phe Ser Thr Lys
Asp Ile Tyr Gln Asp 225 230 235 240 Leu Asn Arg Leu Leu Lys Gly Lys
Lys Gly Glu Gln Met Asn Ser Ala 245 250 255 Val Leu Pro Glu Met Glu
Asn Gln Val Ala Val Ser Ser Leu Ser Ala 260 265 270 Val Ile Lys Phe
Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln 275 280 285 Phe Glu
Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile 290 295 300
Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu Asp Thr 305
310 315 320 Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys Cys Lys
Thr Pro 325 330 335 Gln Gly Gln Arg Leu Val Asn Gln Trp Ile Lys Gln
Pro Leu Met Asp 340 345 350 Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu
Val Glu Ala Phe Val Glu 355 360 365 Asp Ala Glu Leu Arg Gln Thr Leu
Gln Glu Asp Leu Leu Arg Arg Phe 370 375 380 Pro Asp Leu Asn Arg Leu
Ala Lys Lys Phe Gln Arg Gln Ala Ala Asn 385 390 395 400 Leu Gln Asp
Cys Tyr Arg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn 405 410 415 Val
Ile Gln Ala Leu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu 420 425
430 Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser
435 440 445 Lys Phe Gln Glu Met Ile Glu Thr Thr Leu Asp Met Asp Gln
Val Glu 450 455 460 Asn His Glu Phe Leu Val Lys Pro Ser Phe Asp Pro
Asn Leu Ser Glu 465 470 475 480 Leu Arg Glu Ile Met Asn Asp Leu Glu
Lys Lys Met Gln Ser Thr Leu 485 490 495 Ile Ser Ala Ala Arg Asp Leu
Gly Leu Asp Pro Gly Lys Gln Ile Lys 500 505 510 Leu Asp Ser Ser Ala
Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys 515 520 525 Glu Glu Lys
Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile 530 535 540 Gln
Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser Leu Asn 545 550
555 560 Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala Gln Asp
Ala 565 570 575 Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly Tyr Val
Glu Pro Met 580 585 590 Gln Thr Leu Asn Asp Val Leu Ala Gln Leu Asp
Ala Val Val Ser Phe 595 600 605 Ala His Val Ser Asn Gly Ala Pro Val
Pro Tyr Val Arg Pro Ala Ile 610 615 620 Leu Glu Lys Gly Gln Gly Arg
Ile Ile Leu Lys Ala Ser Arg His Ala 625 630 635 640 Cys Val Glu Val
Gln Asp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr 645 650 655 Phe Glu
Lys Asp Lys Gln Met Phe His Ile Ile Thr Gly Pro Asn Met 660 665 670
Gly Gly Lys Ser Thr Tyr Ile Arg Gln Thr Gly Val Ile Val Leu Met 675
680 685 Ala Gln Ile
Gly Cys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile 690 695 700 Val
Asp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys 705 710
715 720 Gly Val Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile
Leu 725 730 735 Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu
Leu Gly Arg 740 745 750 Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala
Trp Ala Ile Ser Glu 755 760 765 Tyr Ile Ala Thr Lys Ile Gly Ala Phe
Cys Met Phe Ala Thr His Phe 770 775 780 His Glu Leu Thr Ala Leu Ala
Asn Gln Ile Pro Thr Val Asn Asn Leu 785 790 795 800 His Val Thr Ala
Leu Thr Thr Glu Glu Thr Leu Thr Met Leu Tyr Gln 805 810 815 Val Lys
Lys Gly Val Cys Asp Gln Ser Phe Gly Ile His Val Ala Glu 820 825 830
Leu Ala Asn Phe Pro Lys His Val Ile Glu Cys Ala Lys Gln Lys Ala 835
840 845 Leu Glu Leu Glu Glu Phe Gln Tyr Ile Gly Glu Ser Gln Gly Tyr
Asp 850 855 860 Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Leu Glu Arg
Glu Gln Gly 865 870 875 880 Glu Lys Ile Ile Gln Glu Phe Leu Ser Lys
Val Lys Gln Met Pro Phe 885 890 895 Thr Glu Met Ser Glu Glu Asn Ile
Thr Ile Lys Leu Lys Gln Leu Lys 900 905 910 Ala Glu Val Ile Ala Lys
Asn Asn Ser Phe Val Asn Glu Ile Ile Ser 915 920 925 Arg Ile Lys Val
Thr Thr 930 12 3145 DNA Homo sapiens 12 ggcgggaaac agcttagtgg
gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 60 gtttcgacat
ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg 120
gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg
180 accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc
cgggaggtgt 240 tcaagaccca gggggtgatc aagtacatgg ggccggcagg
agcaaagaat ctgcagagtg 300 ttgtgcttag taaaatgaat tttgaatctt
ttgtaaaaga tcttcttctg gttcgtcagt 360 atagagttga agtttataag
aatagagctg gaaataaggc atccaaggag aatgattggt 420 atttggcata
taaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta 480
acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc
540 agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga
ctgtgtgaat 600 tccctgataa tgatcagttc tccaatcttg aggctctcct
catccagatt ggaccaaagg 660 aatgtgtttt acccggagga gagactgctg
gagacatggg gaaactgaga cagataattc 720 aaagaggagg aattctgatc
acagaaagaa aaaaagctga cttttccaca aaagacattt 780 atcaggacct
caaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat 840
tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag
900 aactcttatc agatgattcc aactttggac agtttgaact gactactttt
gacttcagcc 960 agtatatgaa attggatatt gcagcagtca gagcccttaa
cctttttcag ggttctgttg 1020 aagataccac tggctctcag tctctggctg
ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagact tgttaaccag
tggattaagc agcctctcat ggataagaac agaatagagg 1140 agagattgaa
tttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag 1200
aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag
1260 cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta
cctaatgtta 1320 tacaggctct ggaaaaacat gaaggaaaac accagaaatt
attgttggca gtttttgtga 1380 ctcctcttac tgatcttcgt tctgacttct
ccaagtttca ggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaa
aaccatgaat tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaatt
aagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa 1560
gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac
1620 agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt
aacaataaaa 1680 actttagtac tgtagatatc cagaagaatg gtgttaaatt
taccaacagc aaattgactt 1740 ctttaaatga agagtatacc aaaaataaaa
cagaatatga agaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatt
tcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctca
gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920
catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca
1980 ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac
gtatactttg 2040 aaaaagataa acagatgttc cacatcatta ctggccccaa
tatgggaggt aaatcaacat 2100 atattcgaca aactggggtg atagtactca
tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccatt
gtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg
agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt 2280
ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tctacctacg
2340 atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt
ggtgcttttt 2400 gcatgtttgc aacccatttt catgaactta ctgccttggc
caatcagata ccaactgtta 2460 ataatctaca tgtcacagca ctcaccactg
aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaa
agttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat
agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg 2640
gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag
2700 agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg
ccctttactg 2760 aaatgtcaga agaaaacatc acaataaagt taaaacagct
aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaat gaaatcattt
cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaa
tattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccc
tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag 3000
atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga
3060 gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt
gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg 3145 13 756 PRT Homo
sapiens 13 Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu Asp Glu Thr
Val Val 1 5 10 15 Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro
Ala Asn Ala Ile 20 25 30 Lys Glu Met Ile Glu Asn Cys Leu Asp Ala
Lys Ser Thr Ser Ile Gln 35 40 45 Val Ile Val Lys Glu Gly Gly Leu
Lys Leu Ile Gln Ile Gln Asp Asn 50 55 60 Gly Thr Gly Ile Arg Lys
Glu Asp Leu Asp Ile Val Cys Glu Arg Phe 65 70 75 80 Thr Thr Ser Lys
Leu Gln Ser Phe Glu Asp Leu Ala Ser Ile Ser Thr 85 90 95 Tyr Gly
Phe Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala His 100 105 110
Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys Cys Ala Tyr Arg Ala 115
120 125 Ser Tyr Ser Asp Gly Lys Leu Lys Ala Pro Pro Lys Pro Cys Ala
Gly 130 135 140 Asn Gln Gly Thr Gln Ile Thr Val Glu Asp Leu Phe Tyr
Asn Ile Ala 145 150 155 160 Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser
Glu Glu Tyr Gly Lys Ile 165 170 175 Leu Glu Val Val Gly Arg Tyr Ser
Val His Asn Ala Gly Ile Ser Phe 180 185 190 Ser Val Lys Lys Gln Gly
Glu Thr Val Ala Asp Val Arg Thr Leu Pro 195 200 205 Asn Ala Ser Thr
Val Asp Asn Ile Arg Ser Ile Phe Gly Asn Ala Val 210 215 220 Ser Arg
Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe 225 230 235
240 Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys
245 250 255 Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr
Ser Leu 260 265 270 Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu
Pro Lys Asn Thr 275 280 285 His Pro Phe Leu Tyr Leu Ser Leu Glu Ile
Ser Pro Gln Asn Val Asp 290 295 300 Val Asp Val His Pro Thr Lys His
Glu Val His Phe Leu His Glu Glu 305 310 315 320 Ser Ile Leu Glu Arg
Val Gln Gln His Ile Glu Ser Lys Leu Leu Gly 325 330 335 Ser Asn Ser
Ser Arg Met Tyr Phe Thr Gln Thr Leu Leu Pro Gly Leu 340 345 350 Ala
Gly Pro Ser Gly Glu Met Val Lys Ser Thr Thr Ser Leu Thr Ser 355 360
365 Ser Ser Thr Ser Gly Ser Ser Asp Lys Val Tyr Ala His Gln Met Val
370 375 380 Arg Thr Asp Ser Arg Glu Gln Leu Lys Asp Ala Phe Leu Gln
Pro Leu 385 390 395 400 Ser Lys Pro Leu Ser Ser Gln Pro Gln Ala Ile
Val Thr Glu Asp Lys 405 410 415 Thr Asp Ile Ser Ser Gly Arg Ala Arg
Gln Gln Asp Glu Glu Met Leu 420 425 430 Glu Leu Pro Ala Pro Ala Glu
Val Ala Ala Lys Asn Gln Ser Leu Glu 435 440 445 Gly Asp Thr Thr Lys
Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro 450 455 460 Thr Ser Ser
Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp Val Glu 465 470 475 480
Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro 485
490 495 Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu
Glu 500 505 510 Ile Asn Glu Gln Gly His Glu Val Leu Arg Glu Met Leu
His Asn His 515 520 525 Ser Phe Val Gly Cys Val Asn Pro Gln Trp Ala
Leu Ala Gln His Gln 530 535 540 Thr Lys Leu Tyr Leu Leu Asn Thr Thr
Lys Leu Ser Glu Glu Leu Phe 545 550 555 560 Tyr Gln Ile Leu Ile Tyr
Asp Phe Ala Asn Phe Gly Val Leu Arg Leu 565 570 575 Ser Glu Pro Ala
Pro Leu Phe Asp Leu Ala Met Leu Ala Leu Asp Ser 580 585 590 Pro Glu
Ser Gly Trp Thr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala 595 600 605
Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp 610
615 620 Tyr Phe Ser Leu Glu Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu
Pro 625 630 635 640 Leu Leu Ile Asp Asn Tyr Val Pro Pro Leu Glu Gly
Leu Pro Ile Phe 645 650 655 Ile Leu Arg Leu Ala Thr Glu Val Asn Trp
Asp Glu Glu Lys Glu Cys 660 665 670 Phe Glu Ser Leu Ser Lys Glu Cys
Ala Met Phe Tyr Ser Ile Arg Lys 675 680 685 Gln Tyr Ile Ser Glu Glu
Ser Thr Leu Ser Gly Gln Gln Ser Glu Val 690 695 700 Pro Gly Ser Ile
Pro Asn Ser Trp Lys Trp Thr Val Glu His Ile Val 705 710 715 720 Tyr
Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu 725 730
735 Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val
740 745 750 Phe Glu Arg Cys 755 14 2484 DNA Homo sapiens 14
cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag
60 acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa
tgctatcaaa 120 gagatgattg agaactgttt agatgcaaaa tccacaagta
ttcaagtgat tgttaaagag 180 ggaggcctga agttgattca gatccaagac
aatggcaccg ggatcaggaa agaagatctg 240 gatattgtat gtgaaaggtt
cactactagt aaactgcagt cctttgagga tttagccagt 300 atttctacct
atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt 360
actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga
420 aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat
cacggtggag 480 gacctttttt acaacatagc cacgaggaga aaagctttaa
aaaatccaag tgaagaatat 540 gggaaaattt tggaagttgt tggcaggtat
tcagtacaca atgcaggcat tagtttctca 600 gttaaaaaac aaggagagac
agtagctgat gttaggacac tacccaatgc ctcaaccgtg 660 gacaatattc
gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt 720
gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg
780 aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac
ttccttgaga 840 aaagccatag aaacagtgta tgcagcctat ttgcccaaaa
acacacaccc attcctgtac 900 ctcagtttag aaatcagtcc ccagaatgtg
gatgttaatg tgcaccccac aaagcatgaa 960 gttcacttcc tgcacgagga
gagcatcctg gagcgggtgc agcagcacat cgagagcaag 1020 ctcctgggct
ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct 1080
ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga
1140 agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga
acagaagctt 1200 gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc
agccccaggc cattgtcaca 1260 gaggataaga cagatatttc tagtggcagg
gctaggcagc aagatgagga gatgcttgaa 1320 ctcccagccc ctgctgaagt
ggctgccaaa aatcagagct tggaggggga tacaacaaag 1380 gggacttcag
aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat 1440
cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct
1500 tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca
ggaagaaatt 1560 aatgagcagg gacatgaggt tctccgggag atgttgcata
accactcctt cgtgggctgt 1620 gtgaatcctc agtgggcctt ggcacagcat
caaaccaagt tataccttct caacaccacc 1680 aagcttagtg aagaactgtt
ctaccagata ctcatttatg attttgccaa ttttggtgtt 1740 ctcaggttat
cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800
gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag
1860 tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat
tgatgaggaa 1920 gggaacctga ttggattacc ccttctgatt gacaactatg
tgcccccttt ggagggactg 1980 cctatcttca ttcttcgact agccactgag
gtgaattggg acgaagaaaa ggaatgtttt 2040 gaaagcctca gtaaagaatg
cgctatgttc tattccatcc ggaagcagta catatctgag 2100 gagtcgaccc
tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160
tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat
2220 ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata
caaagtcttt 2280 gagaggtgtt aaatatggtt atttatgcac tgtgggatgt
gttcttcttt ctctgtattc 2340 cgatacaaag tgttgtatca aagtgtgata
tacaaagtgt accaacataa gtgttggtag 2400 cacttaagac ttatacttgc
cttctgatag tattccttta tacacagtgg attgattata 2460 aataaataga
tgtgtcttaa cata 2484 15 133 PRT Homo sapiens 15 Met Glu Arg Ala Glu
Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp
Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20 25 30 Leu
Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40
45 Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp
50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu
Asn Phe 65 70 75 80 Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile
Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe
Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val
Thr Ile Ser Thr Cys His Ala Ser 115 120 125 Ala Lys Val Gly Thr 130
16 426 DNA Homo sapiens 16 cgaggcggat cgggtgttgc atccatggag
cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga
tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa
gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180
aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga
240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac
atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggct
ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt
tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 17 19 DNA
Artificial Sequence Oligonucleotide primer 17 tttcgcaacg ggtttgccg
19 18 20 DNA Artificial Sequence Oligonucleotide primer 18
gtttcagagt taagccttcg 20 19 13 DNA Human immunoglobulin E light
chain misc_feature (6)..(6) n is a, c, g, or t 19 tacgtngaat aat 13
20 13 DNA Human immunoglobulin E light chain 20 tacgttgaat aat 13
21 63 DNA Human immunoglobulin E light chain 21 aacgtgacca
tggtcgtctt cagtccgcga agggagtttg ggaactaagt atcctgtagg 60 ttg 63 22
63 DNA Human immunoglobulin E light chain 22 aacgtgacca tggtcgtctt
cagtccgcga agggggtttg ggaactaagt atcctgtagg 60 ttg 63 23 63 DNA
Human immunoglobulin E light chain 23 aacgtgacca tggtcgtctt
cagtccgcga agggrgtttg ggaactaagt atcctgtagg 60 ttg 63
* * * * *