U.S. patent application number 10/813502 was filed with the patent office on 2004-08-12 for method for generating genetically altered antigens.
This patent application is currently assigned to Morphotek, Inc.. Invention is credited to Grasso, Luigi, Nicolaides, Nicholas C., Sass, Philip M..
Application Number | 20040158886 10/813502 |
Document ID | / |
Family ID | 32298537 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040158886 |
Kind Code |
A1 |
Nicolaides, Nicholas C. ; et
al. |
August 12, 2004 |
Method for generating genetically altered antigens
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
genes encoding for therapeutic antigens to produce altered
polypeptides with enhanced antigenic and immunogenic activity.
Moreover, these methods are useful for generating effective
vaccines.
Inventors: |
Nicolaides, Nicholas C.;
(Boothwyn, PA) ; Grasso, Luigi; (Philadelphia,
PA) ; Sass, Philip M.; (Audubon, PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Morphotek, Inc.
|
Family ID: |
32298537 |
Appl. No.: |
10/813502 |
Filed: |
March 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10813502 |
Mar 30, 2004 |
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09712691 |
Nov 14, 2000 |
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6737268 |
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Current U.S.
Class: |
800/21 ;
435/320.1; 435/325; 435/455; 435/69.3 |
Current CPC
Class: |
C12N 15/1024
20130101 |
Class at
Publication: |
800/021 ;
435/069.3; 435/455; 435/320.1; 435/325 |
International
Class: |
A01K 067/027; C12N
015/85 |
Claims
We claim:
1. A method for making a hypermutated antigen, comprising
introducing into a mammalian cell that expresses a preselected
antigen a polynucleotide comprising a dominant negative allele of a
mismatch repair gene.
2. The method of claim 1 wherein the polynucleotide is introduced
by transfection of a suspension of cells in vitro.
3. The method of claim 1 wherein the mismatch repair gene is
PMS2.
4. The method of claim 1 wherein the mismatch repair gene is human
PMS2.
5. The method of claim 1 wherein the mismatch repair gene is
MLH1.
6. The method of claim 1 wherein the mismatch repair gene is
PMS1.
7. The method of claim 1 wherein the mismatch repair gene is
MSH2.
8. The method of claim 1 wherein the mismatch repair gene is
MSH2.
9. The method of claim 4 wherein the allele comprises a truncation
mutation.
10. The method of claim 4 wherein the allele comprises a truncation
mutation at codon 134.
11. The method of claim 10 wherein the truncation mutation is a
thymidine at nucleotide 424 of wild-type PMS2.
12. The method of claim 1 wherein the polynucleotide is introduced
into a fertilized egg of an animal.
13. The method of claim 12 wherein the fertilized egg is
subsequently implanted into a pseudo-pregnant female whereby the
fertilized egg develops into a mature transgenic animal.
14. The method of claim 12 wherein the mismatch repair gene is
PMS2.
15. The method of claim 12 wherein the mismatch repair gene is
human PMS2.
16. The method of claim 12 wherein the mismatch repair gene is
human MLH1.
17. The method of claim 12 wherein the mismatch repair gene is
human PMS1.
18. The method of claim 11 wherein the mismatch repair gene is
human a human mutL homolog.
19. The method of claim 15 wherein the allele comprises a
truncation mutation.
20. The method of claim 15 wherein the allele comprises a
truncation mutation at codon 134.
21. The method of claim 19 wherein the truncation mutation is a
thymidine at nucleotide 424 of wild-type PMS2.
22. A homogeneous composition of cultured, hypermutable, mammalian
cells which comprise a preselected antigen and a dominant negative
allele of a mismatch repair gene.
23. The isolated hypermutable cell of claim 22 wherein the mismatch
repair gene is PMS2.
24. The isolated hypermutable cell of claim 23 wherein the mismatch
repair gene is human PMS2.
25. The isolated hypermutable cell of claim 22 wherein the mismatch
repair gene is MLH1.
26. The isolated hypermutable cell of claim 22 wherein the mismatch
repair gene is PMS1.
27. The isolated hypermutable cell of claim 22 wherein the mismatch
repair gene is a human mutL homolog.
28. The isolated hypermutable cell of claim 22 wherein the cells
express a protein consisting of the first 133 amino acids of
hPMS2.
29. A method for generating a mutation in a gene encoding an
antigen of interest comprising growing a mammalian cell comprising
said gene encoding an antigen of interest and a dominant negative
allele of a mismatch repair gene, and determining whether said gene
encoding an antigen of interest harbors a mutation.
30. The method of claim 29 wherein determining whether said gene
encoding an antigen of interest harbors a mutation is accomplished
by analyzing the nucleotide sequence of said gene.
31. The method of claim 30 wherein said nucleotide sequence is an
mRNA transcribed from said gene.
32. The method of claim 29 wherein determining whether said gene
encoding an antigen of interest harbors a mutation is accomplished
by analyzing a protein encoded by said gene.
33. The method of claim 30 wherein determining whether said gene
encoding an antigen of interest harbors a mutation is accomplished
by analyzing the phenotype of said gene.
34. The method of claim 32 wherein analyzing of said protein
comprises analyzing the antigenicity and immunogenicity of said
protein.
35. A method for generating a mutation in a gene encoding an
antigen of interest comprising growing a cell comprising said gene
and a polynucleotide encoding a dominant negative allele of a
mismatch repair gene; and testing the cell to determine whether the
cell harbors a mutation in said gene yielding at least one new
biochemical feature of said antigen.
36. The method of claim 35 wherein said new biochemical feature is
selected from the group consisting of increased antigenicity and
increased immunogenicity.
37. The method of claim 35 wherein said testing comprises analyzing
primary structure of said gene.
38. The method of claim 35 wherein said testing comprises analyzing
the secondary structure of said gene.
39. The method of claim 35 wherein said testing comprises analyzing
the antigenicity and immunogenicity of the polypeptide encoded by
said gene.
40. The method of claim 1 wherein said introduction of said
polynucleotideis in the presence of at least one DNA mutagen.
41. The method of claim 35 wherein said testing comprises analyzing
a nucleotide sequence said gene.
42. The method of claim 35 wherein said testing comprises analyzing
mRNA transcribed from said gene.
43. The method of claim 35 wherein said testing comprises analyzing
the antigen protein encoded by the gene of interest.
44. The method of claim 35 wherein said testing comprises analyzing
the biochemical activity of the protein encoded by said gene.
45. A hypermutable transgenic mammalian cell made by the method of
claim 35.
46. The transgenic mammalian cell of claim 45 wherein the cell is
from primate.
47. The transgenic mammalian cell of claim 45 wherein the cell is
from rodent.
48. The transgenic mammalian cell of claim 45 wherein the cell is
from human.
49. The transgenic mammalian cell of claim 45 wherein the cell is
eucaryotic.
50. The transgenic mammalian cell of claim 45 wherein the cell is
prokaryotic
51. A method for making randomly altered forms of a secreted
antigen comprising introducing a polynucleotide encoding a tagged
antigen into an MMR defective cell.
52. The method of claim 51 wherein said tagged antigen is screened
for increased antigenicity.
53. The method of claim 51 wherein said tagged antigen is screened
for increased immunogenicity.
54. The method of claim 51 wherein the cells are made MMR defective
by introducing at least one dominant negative allele of an MMR
gene.
55. The method of claim 51 wherein the cells are naturally MMR
defective.
56. A method of producing a mutated antigen in a reversibly
unstable cell comprising introducing into a cell containing a
preselected antigen of interest, an inducible expression vector
comprising a polynucleotide encoding a dominant negative allele of
a mismatch repair gene; inducing said cell to express said dominant
negative mismatch repair gene; and detecting at least one new
biochemical feature of said antigen.
57. The method of claim 56 wherein said new biochemical feature is
selected from the group consisting of a nucleotide mutation,
increased antigenicity and increased immunogenicity.
58. The method of claim 56 wherein said preselected antigen of
interest is encoded on a polynucleotide previously transfected into
said cell.
59. The method of claim 56 further comprising ceasing induction of
said dominant negative allele of a mismatch repair gene, thereby
stabilizing said cell.
60. The method of claim 58 further comprising isolating the
polynucleotide previously transfected into said cell after
detection of said new biochemical feature.
61. An polynucleotide molecule for expressing an antigen in a
hypermutable cell comprising an expression cassette wherein said
cassette comprises a 3' sequence encoding a plurality of histidine
residues, a 5' leader sequence of an expressed gene, and a
polylinker to allow cloning of a nucleotide sequence encoding a
preselected antigen.
62. The polynucleotide molecule of claim 61 wherein said cell is a
mammalian cell.
63. The polynucleotide molecule of claim 61 wherein said cell is a
human cell.
64. The method of claim 63 wherein said 5' leader sequence is a 5'
leader sequence of IL-2.
65. A method of producing a mutated antigen comprising introducing
a polynucleotide encoding a preselected antigen in the expression
cassette said polynucleotide molecule of claim 61, and introducing
said polynucleotide molecule into a cell comprising a dominant
negative allele of a mismatch repair gene.
66. A hypermutated antigen produced by the method of claim 65.
67. A method of eliciting an immune response in an animal
comprising administering to said animal an immunogenic amount of at
least one hypermutated antigen of claim 66.
68. The method of claim 67 wherein said antigen is a mutated form
of an antigen derived from a pathogenic organism selected from the
group consisting of, bacteria, fungi, protozoa, helminths, and
viruses.
69. An immunogenic composition comprising at least one hypermutated
antigen of claim 66 and a pharmaceutically acceptable carrier.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention is related to the area of genetic alterations
of antigens as potent vaccines. In particular it is related to the
field of mutagenesis.
BACKGROUND OF THE INVENTION
[0002] The use of vaccines to build immunity against foreign and/or
endogenous polypeptides provides an effective and selective
strategy for treating the underlying cause of disease. In
particular is the use of killed viruses such as the polio mellitus
and the Hepatitis B virus (John T. J. (2000) New Engl. J. Med.
14:806-807). Standard methods for generating vaccines against
candidate pathogenic organisms or molecules are known by those
skilled in the art. Vaccines for human use are developed in animal
models to survey for the ability of killed or defective whole
agents such as parasites, viruses or recombinant polypeptides to
cause immunity against infection of the pathogenic agent (Boyce T.
G. et al. (2000) Vaccine 19:217-226). Briefly, rodents such as mice
or rats are injected with a purified antigen in the presence of
adjuvant to generate an immune response (Boyce T. G. et al. (2000)
Vaccine 19:217-226). Unfortunately, not all antigens are capable of
eliciting a strong immune response when injected into a host
organism (Hoshino Y. and A. Z. Kapikian (2000) J. Health Popul.
Nutr. 18:5-14; Orenstein W. A. et al. (2000) Am. J. Public Health
90:1521-525; Lechmann M. and T. J. Liang (2000) Semin. Liver Dis.
20:211-226). While the reasons for the lack of immune response are
not clear, some factors, such as the lack of T-cell epitopes which
are important for stimulating cellular-mediated immune responses,
may be absent within a given antigen (Ausiello C. M. et al. (1999)
Infect. Immun.67:40644071; Brosstoff S. (1995) Adv. Exp. Med. Biol.
383:249-254). In the case of parasitic infections, the development
of effective vaccines has been hampered by the presence of many
different developmental stages that occur within an infected host
and that a diverse array of allelic forms occurs within genes
encoding for prominent surface antigens (MALARIA OBSTACLES AND
OPPORTUNITIES, Oaks, S. C. et al., Eds., National Academy Press, p
1, 1991; Anders, R. F. "Vaccines Against Asexual Blood Stages of
Plasmodium falciparum" NEW GENERATION VACCINES, 2.sup.nd Ed.,
Anders, R. F., pp. 1035-1055, 1997). It is believed by many skilled
in the art that the generation of highly antigenic polypeptides may
overcome these limitations and produce a protective immune response
to pathogens (McLeod R. et al. (1995) Curr. Opin.
Immunol.7:539-552).
[0003] A method for generating diverse sequences within a
polypeptide would be useful for the creation of more potent
therapeutic agents. Moreover, the generation of randomly altered
nucleotides and encoded polypeptide residues throughout an entire
antigen molecule may result in new reagents that are: 1) more
antigenic; 2) more immunogenic; and 3) have beneficial
pharmacokinetic properties.
SUMMARY OF THE INVENTION
[0004] The invention described herein is directed to the use of
random genetic mutation of a polypeptide in vivo by blocking the
endogenous mismatch repair (MMR) activity of a host cell yielding
structurally altered antigens that can be screened for antigenicity
and immunogenicity in comparison to the wild type molecule. The use
of mammalian cell-based high throughput screens as taught by this
application will facilitate identification of randomly altered
antigens that may serve as effective vaccines. Moreover, the
invention describes methods for repeated in vivo genetic
alterations and selection for antigens with enhanced immunogenicity
and pharmacokinetic profiles.
[0005] The ability to develop and screen genetically altered
mammalian cells that secrete structurally altered polypeptides in a
high throughput manner provides a valuable method for creating
vaccines for therapeutic development. A potential problem in
generating potent vaccine antigens against endogenous to the
mammalian host is the source of antigen production. In many
instances recombinant polypeptides that are naturally produced by
mammalian cells are generated recombinantly using insect, yeast or
bacterial expression systems. These sources typically produce large
amounts of proteins that are distinct from the mammalian-produced
polypeptides, and may differ from the natural protein due to
altered folding or altered post-translational modifications such as
hyperglycosylation. The invention described herein is directed to
the creation of genetically altered mammalian cell hosts that
produce structurally altered polypeptides as vaccine agents via the
blockade of MMR.
[0006] The present invention facilitates the generation of highly
antigenic polypeptides as vaccines. The advantages of the present
invention are further described in the examples and figures
described herein.
[0007] The present invention provides methods for generating
genetically altered antigens in vivo, whereby the antigen possesses
desired biochemical property(s), such as, but not limited to,
increased antigenicity and immunogenicity. One method for
identifying antigens with increased antigenicity is through the
screening of mismatch repair ("MMR") defective cell clones that
produce desired antigens.
[0008] The invention also provides methods for rendering cells
expressing a target antigen hypermutable. The cells include, but
are not limited to rodent, primate, human, plant, yeast or
bacterial cells. The antigens can be generated from endogenous
genes or from introduced transgenes.
[0009] The invention also provides methods for generating
genetically altered cell lines that express antigenic
polypeptides.
[0010] In some embodiments, the invention provides methods for
generating genetically altered cell lines that produce immunogenic
polypeptides.
[0011] In other embodiments, the invention provides methods for
producing an antigen expression cassette for high throughput
screening of altered polypeptides in vivo.
[0012] In other embodiments, the invention provides methods of
mutating a gene of interest in a mismatch repair defective
cell.
[0013] In some embodiments, the invention provides methods of
creating genetically altered antigens in vivo by blocking the MMR
activity of the cell host.
[0014] Still other embodiments of the invention provide methods of
creating genetically altered polypeptides in vivo by transfecting
genes encoding for an antigen in a MMR defective cell host.
[0015] The invention also embraces methods of creating antigens
with increased immunogencity due to genetic alterations within the
antigen-encoding gene by blocking endogenous MMR of the cell
host.
[0016] In some embodiments, the invention provides methods of
creating a library of randomly altered antigens from mammalian
cells by blockade of MMR of the cell host.
[0017] In other embodiments, the invention provides methods of
creating antigens with enhanced pharmacokinetic profiles due to
genetic changes within the encoding gene by blocking endogenous MMR
of the cell host.
[0018] The invention also provides methods of creating genetically
altered antigens in MMR defective cells as vaccine agents.
[0019] In some embodiments, the invention provides methods for high
throughput screening of antigens produced by MMR defective
cells.
[0020] These and other objects of the invention are provided by one
or more of the embodiments described below. In one embodiment of
the invention, a method for making MMR defective cell lines
expressing a target antigen will be provided. A polynucleotide
encoding a dominant negative allele of an MMR gene is introduced
into a target antigen-producing cell. The cell becomes hypermutable
as a result of the introduction of the gene.
[0021] In another embodiment of the invention, an isolated
hypermutable cell producing antigenic peptides is provided. The
cell is defective for mismatch repair and exhibits an enhanced rate
of hypermutation. The cell produces a polypeptide from a mutated
gene encoding for the polypeptide.
[0022] In another embodiment of the invention, a method is provided
for introducing a mutation into an endogenous gene encoding for a
target polypeptide. 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 a gene of interest. The
cell is grown and tested to determine whether the gene encoding for
a polypeptide of interest harbors a mutation.
[0023] In another embodiment of the invention, a method is provided
for producing a cell-based screening assay to identify antigenic
proteins as vaccines. A polynucleotide encoding a dominant negative
allele of a MMR gene is introduced into a cell expressing a
secreted antigen. The cell becomes hypermutable as a result of the
introduction of the gene. The cell is grown and conditioned medium
from the cell is tested for the expression of antigenic
polypeptides.
[0024] In another embodiment of the invention, a gene, or set of
genes encoding for polypeptides or a combination therein, are
introduced into a mammalian cell host that is defective in MMR. The
cell is grown and clones are analyzed for antigens with enhanced
antigenicity.
[0025] In another embodiment of the invention, a method is provided
for producing a cell-based screening assay to identify antigenic
proteins as vaccines. A polynucleotide encoding a secreted antigen
is introduced into a naturally MMR defective cell. The gene is
hypermutable as a result of the introduction of MMR deficiency. The
cell is grown and conditioned medium from the cell is tested for
the expression of antigenic polypeptides.
[0026] In another embodiment of the invention, a method will be
provided for restoring genetic stability in a cell containing a
polynucleotide encoding for a dominant negative allele of a MMR
gene. The expression of the dominant negative MMR gene is
suppressed and the cell restores its genetic stability including
but not limited to genetic stability within the antigen-encoding
genes.
[0027] In another embodiment of the invention, a method will be
provided for restoring genetic stability in a cell containing a
polynucleotide encoding a dominant negative allele of an MMR gene
and a newly selected phenotype. The expression of the dominant
negative mismatch repair gene is suppressed and the cell restores
its genetic stability and the new phenotype is stable.
[0028] 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
highly antigenic polypeptides as potent vaccines.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1: In situ .beta.-galactosidase staining of
TKPMS134/pCAR-OF or TKvect/pCAR-OF cells to assay for MMR defective
cells containing genetically altered .beta.-galactosidase genes.
Arrows indicate Blue (.beta.-galactosidase positive) cells.
[0030] FIG. 2: Schematic representation of sequence alterations of
the .beta.-galactosidase gene produced by MMR defective host
cells
[0031] FIG. 3: Schematic representation of sec-hist secretion
proteins for screening of structurally altered antigenic
polypeptides and the sec-hist expression cassette (SEQ ID NO:17).
Panel A: Schematic representation of sec-hist protein; Panel B:
Sequence of sec-hist expression cassette. In Panel B, the italic
bold sequence represents a HindIII site for subcloning; the double
underlined sequence on the 5' end represents leader sequence from
the human IL-2; the underlined sequence on the 3' end represents
the poly histidine sequence followed by 2 termination codons;
sequence in capital letters represents sequence from the polylinker
region of pUC 18; the polylinker contains the following restriction
enzymes for cloning cDNAs: SacI-SacII-NotI-XbaI-SpeI-BamHI-Sm-
aI-PstI-EcoRI.
[0032] FIG. 4: Schematic diagram for high throughput screening of
conditioned medium from TK clones for the identification of
antigenic sec-hist polypeptides. Assays employ an in vitro
antigenicity test using splenocytes from naive mice (non-primed)
and antigen-exposed (primed) mice. Clones exhibiting positive CM
are then genetically analyzed to confirm structural alterations
within the sec-hist sequence, followed by protein purification and
retesting of purified proteins. Purified proteins with the best
stimulatory activity are then screened in vivo for immunogenicity.
The screening assay can be repeated for several rounds to add
additional alterations within the antigen (long arrow).
[0033] The inventors have discovered a method for developing
hypermutable cells producing therapeutic antigens 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 or
genes of interest. Blocking MMR in cells producing antigens
(including, but not limited to, mammalian cells, plant cells, yeast
cells, and prokaryotic cells) can enhance the rate of mutation
within the gene encoding for the antigen that can be screened to
identify clones producing structurally altered polypeptides with
enhanced antigenicitiy and immunogenicity.
[0034] In one aspect of the invention, the methods are useful for
the production of antigens that have increased antigenicity and/or
immunogenicity. Such antigens may be used as immunogens to elicit
immune responses in animals against these antigens.
[0035] The antigens may be derived from, for example, pathogenic
organisms or cancer cells such that an immune response is directed
against the pathogenic organism or cancer cell and exerts an effect
on the organism or cancer cell. The effect may be, for example, to
prevent, inhibit or terminate the growth of the pathogenic organism
or cancer cell when an immunogenic amount of the antigen is
administered to an animal.
[0036] The pathogenic organisms-from which antigens may be derived
include bacteria, fungi, parasitic protozoa, helminths, and
viruses. Non-limiting examples include species of the following
genera: Staphylococcus, Streptococcus, Bacillus, Bordetella,
Clostridium, Escherichia, Haemophilus, Helicobacter, Klebsiella,
Listeria, Salmonella, Vibrio, Yersinia, Neisseria, Treponema,
Borrelia, Corynebacterium, Mycobacterium, Mycoplasma, Chlamydia,
Acremonium, Aspergillus, Blastomyces, Candida, Acanthamoeba,
Ascaris, Babesia, Cryptosporidium, Echinococcus, Entamoeba,
Giardia, Necator, Ancylostoma, Unicinaria, Leishmania, Onchocerca,
Plasmodium, Schistosoma, Strongyloides, Taenia, Toxoplasma,
Trichinella, Trichomonas, Trichuris, Trypanosoma, Dirofilaria,
Brugia, Wuchereria, and Eimeria. Non-limiting examples of viruses
include adenovirus, arborviruses, coronavirus, cytomegalovirus,
enteroviruses, Epstein-Barr virus, hepatitis viruses, herpes
viruses, immunodeficiency viruses (e.g., HIV, FIV SIV), papilloma
viruses, T-cell leukemia viruses, influenza viruses, mumps viruses,
parainfluenzae viruses, parvoviruses, poxviruses, Rabies virus,
respiratory syncytial virus, rhinoviruses, rotaviruses, Rubella
viruses, and varicella-zoster viruses.
[0037] The antigens derived from the pathogenic organisms, for
example, may be antigens known to elicit an immune response, for
which an enhanced immune response is desired, or the antigen may be
one that is known to generate a weak response for which an enhanced
response is desired. It is also possible that some antigens that
did not previously elicit an immune response will become antigenic
as a result of the methods of the invention and the phenomenon of
hypermutability of cells which contain dominant negative alleles of
mismatch repair genes.
[0038] The antigens produced by the method of the invention are
novel immunogens that may be administered in an appropriate
pharmaceutical carrier, such as an adjuvant, for administration to
animals as a vaccine. The antigens of the invention may be
administered to animals in immunogenic amounts such that an
antibody and/or a cell-mediated immune response is elicited. The
administration of the antigens of the invention may be administered
as a single dose, or, preferably as a plurality of doses to effect
a boosted immune response. The route of administration may be any
accepted route of immunization including, for example, oral,
intrmuscular, intrperitoneal, subcutaneous, intradermal,
intranasal, or transdermal.
[0039] Doses for humans can readily be extrapolated from animal
studies as taught by Katocs et al., Chapter 27 of REMINGTON'S
PHARMACEUTICAL SCIENCES, 18.sup.th Edition, Gennaro (Ed.) Mack
Publishing Co., Easton, Pa., 1990. Immunogenic dosages can be
adjusted by one skilled in the art, and may vary depending on
several factors, including the age, health, physical condition,
weight, type and extent of the disease or disorder of the
recipient, frequency of treatment, the nature of concurrent
therapy, if required, and the nature and scope of the desired
effect(s) (Nies et al., Chapter 3, GOODMAN & GILMAN'S THE
PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th Ed., Hardman et al.,
Eds., McGraw-Hill, New York, N.Y., 1996). Typically, an immunogenic
amount of the antigens of the invention will be in the range of
about 5 to about 100 .mu.g.
[0040] The antigens of the present invention may be administered as
single antigens or may be administered as combinations of antigens.
As a non-limiting example, the antigen combinations may be antigens
of the same pathogenic organism, or may be antigens of different
pathogenic organisms, such that immune responses are elicited to
more than one pathogenic organism.
[0041] The antigens of the present invention are hypermutated by
the methods of the invention which take advantage of the mismatch
repair system. 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.
[0042] 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.
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 that produces such effect can be used in this
invention.
[0043] Dominant negative alleles of a MMR gene can be obtained from
the cells of humans, animals, yeast, bacteria, or other organisms
(Prolla T. A. et al. (1994) Science 264:1091-1093; Strand M. et al.
(1993) Nature 365:274-276; Su, S. S. et al. (1988) J. Biol. Chem.
263:6829-6835). 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.
[0044] 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.
[0045] 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.
[0046] 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 (EF) or LTR sequences]
or to inducible promoter sequences such as the steroid inducible
pIND vector (InVitrogen), tetracycline, or MMTV, where the
expression of the dominant negative MMR gene can be regulated. The
polynucleotide can be introduced into the cell by transfection.
[0047] According to another aspect of the invention, a gene, a set
of genes or a chimeric gene encoding for whole or parts of a
therapeutic antigen can be transfected into MMR deficient cell
hosts, the cell is grown and screened for clones containing
genetically altered genes encoding for antigens with new
biochemical features including but not limited to increased
antigenicity. MMR defective cells may be of human, primates,
mammals, rodent, plant, yeast or of the prokaryotic kingdom.
[0048] 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.
[0049] 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 dominant negative 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.
[0050] 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.
[0051] 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 genes encoding
for therapeutic antigens in vitro by introducing whole, intact
genes and/or chimeric genes encoding for a therapeutic antigen(s)
into MMR defective cells from any tissue of the MMR defective
animal.
[0052] 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 cells that may be isolated and cloned to identify new cell
lines that are useful for producing structurally altered
polypeptides. Once an altered polypeptide 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 protein
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.
[0053] 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 antigenicity. 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 antigenicity.
[0054] According to another aspect of the invention, a high
throughput mammalian cell-based assay is presented. A MMR defective
cell line is transfected with a secretion cassette containing a
leader sequence for secretion at the N-terminus fused to the target
antigen. Cells are grown and clones are plated by limiting dilution
into microtitre plates and conditioned medium are screened for
antigenic peptides. The advantage of such an approach is that the
antigen is more similar to the natural polypeptide than it would be
if produced by bacterial, yeast or baculovirus systems which tend
to cause misfolding and/or distorted post-translational
modifications.
[0055] Examples of mismatch repair proteins and nucleic acid
sequences include the following:
1 PMS2 (mouse) (SEQ ID NO:5) MEQTEGVSTE CAKAIKPIDG 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
VGTRLMFDHN 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 GEQNYRKFPA
KICPGENQAA 660 EDELRKEISK TMFAEMEIIG QFNLGFIITK LNEDIFIVDQ
HATDEKYNFE MLQQHTVLQG 720 QRLIAPQTLN LTAVNEAVLI ENLEIFRKNG
FDFVIDENAP VTERAKLISL PTSKNWTFGP 780 QDVDELIFML SDSPGVMCRP
SRVKQMFASR ACRKSVMIGI 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 VHNKAVTWQK
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
KLDIAAVRAL NLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWIKQPL 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
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the following references may be consulted, each of which is
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[0097] 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 Cells Results
in Widespread Mutations of a Reporter Gene and its Encoded
Polypeptide
[0098] Expression of a dominant negative allele in an otherwise MMR
proficient cell could render these host cells MMR deficient
(Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641).
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 antigen-producing cells, including but not
limited to rodent, human, primate, yeast, insect, and prokaryotic
cells producing proteins that may serve as therapeutic antigens for
vaccination. The cell expression systems described above that are
used to produce antigens are well known by those skilled in the art
of vaccine therapeutics.
[0099] To demonstrate the ability to create MMR defective mammalian
cells using dominant negative alleles of MMR genes, we first
transfected a MMR proficient rodent cell line with an expression
vector containing the human the previously published dominant
negative PMS2 mutant referred herein as PMS134 (cell line referred
to as TKPMS134), or with no insert (cell line referred to as
TKvec). A fragment containing the PMS134 cDNA was cloned into the
pEF expression vector which contains the constitutively active
elongation factor promoter along with the neomycin resistance gene
as selectable marker. The results showed that the PMS134 mutant
could exert a robust dominant negative effect, resulting in
biochemical and genetic manifestations of MMR deficiency. A brief
description of the methods are provided below.
[0100] 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). 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. 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.
[0101] 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 polynucelotide repeat yielding clones that
contain a finctional reporter gene. An example of the ability to
alter desired genes via defective MMR comes from experiments using
Syrian Hamster fibroblasts (TK) cells (described above), where a
mammalian expression construct containing a defective
.beta.-galactosidase gene (referred to as pCAR-OF) was transfected
into TKPMS134 or TKvect cells as described above. The pCAR-OF
vector consists of a .beta.-galactosidase gene containing a
29-basepair poly-CA tract inserted at the 5' end of its coding
region, which causes the wild-type reading frame to shift
out-of-frame. This chimeric gene is cloned into the pCEP4, which
contains the constitutively cytomegalovirus (CMV) promoter upstream
of the cloning site and also contains the hygromycin-resistance
(HYG) gene that allows for selection of cells containing this
vector. The pCAR-OF reporter cannot generate .beta.-galactosidase
activity unless a frame-restoring mutation (i.e., insertion or
deletion) arises following transfection into a host. Another
reporter vector called pCAR-IF contains a .beta.-galactosidase in
which a 27-bp poly-CA repeat was cloned into the same site as the
pCAR-OF gene, but it is biologically active because the removal of
a single repeat restores the open reading frame and produces a
functional chimeric .beta.-galactosidase polypeptide (not shown).
The PCAR vectors also contain the neomycin resistance gene as
selectable marker. In these proof-of-concept studies, TKPMS134 and
TKvect cells were transfected with the pCAR-OF reporter vector and
selected for 17 days in neomycin plus hygromycin selection medium.
After the 17.sup.th day, resistant colonies were stained for
.beta.-galactosidase production to determine the number of clones
containing a genetically altered .beta.-galactosidase gene. All
conditions produced a relatively equal number of
neomycin/hygromycin resistant cells, however, only the cells
expressing the PMS134 dominant negative allele (TKPMS134) contained
a subset of clones that were positive for .beta.-galactosidase
activity (Table 1). This result was also observed using a similar
experimental strategy with a MMR proficient human cell line (data
not shown). Table 1 shows the data from these experiments, where
cell colonies were stained in situ for .beta.-galactosidase
activity and scored for activity. Cells were scored positive if the
colonies turned blue in the presence of X-gal substrate and scored
negative if colonies remained white. Analysis of triplicate
experiments showed a significant increase in the number of
.beta.-galactosidase positive cells in the TKPMS134 cultures, while
no .beta.-galactosidase cells were seen in the control TKvect
cells.
2TABLE 1 Number of TKPMS134 and TKvect cells containing functional
.beta.-galactosidase gene as a result of MMR deficiency. % Clones
Cells White Colonies Blue Colonies with altered B-gal TKvect 65 +/-
9 0 0/65 = 0% TKPMS134 40 +/- 12 28 +/- 4 28/68 = 41%
[0102] Table 1. .beta.-galactosidase expression of HBvec, HBPMS2
and HB 134 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.-galaciosidase activity (blue colored cells). 3 plates each
were analyzed by microscopy. The results below represent the
mean.+-.standard deviation of these experiments.
[0103] TKPMS134/pCAR-OF clones that were pooled and expanded also
showed a number of cells that contained a functional
.beta.-galactosidase gene. No .beta.-galactosidase positive cells
were observed in TKvect cells transfected with the pCAR-OF vector.
These data are shown in FIG. 1 where the dark staining in panel B
represent .beta.-galactosidase positive cells present in the
TKPMS134/pCAR-OF cultures while none are found in the TKvect cells
grown under similar conditions (panel A). These data demonstrate
the ability of dominant negative alleles of MMR genes to generate
in vivo gene alterations, which allows for the rapid screening of
clones with altered polypeptides exhibiting new biochemical
features.
[0104] To confirm that alterations within the nucleotide sequences
of the P-galactosidase gene was indeed responsible for the in vivo
.beta.-galactosidase activity present in TKPMS134 clones, RNA was
isolated from TKPMS134/pCAR-OF and TKvect/pCAR-OF and the
.beta.-galactosidase mRNA primary structure was examined by reverse
transcriptase polymerase chain reaction (RT-PCR) amplification and
sequencing. Sequence analysis of .beta.-galactosidase message from
TKvect cells found no structural alterations in the input gene
sequence. Analysis of the .beta.-galactosidase message from
TKPMS134 cells found several changes within the coding sequences of
the gene. These sequence alterations included insertion and
deletions of the poly-CA tract in the amino terminus as expected.
Other alterations included insertions of sequences outside of the
poly-CA repeat as well as a series of single base alterations
(transversions and transitions) contained throughout the length of
the gene.
[0105] In Situ X-Gal Staining
[0106] For in situ analysis, 100,000 cells are 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 are stopped in 500 mM sodium bicarbonate
solution and transferred to microscope slides for analysis. Three
plates each are 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.
EXAMPLE 2
Generation of an Expression Cassette for Screening of Structurally
Altered Polypeptides in MMR Defective Cells
[0107] In order to produce recombinant proteins for screening of
highly antigenic polypeptides, a fusion gene cassette was
engineered that encodes for a secreted polypeptide containing a six
polyhistidine domain at the C-terminus, which is useful for
purification. This gene cassette is referred to as sec-hist. This
gene was constructed by PCR using DNA from the pUC18 plasmid as
template. The sense primers contained nucleotide sequences
corresponding to the leader sequence of human interleukin-2 (ref
32), which has been found to produce robust amounts of secreted
polypeptides from TK cells (personal observation). This domain was
introduced at the 5' end of the pUC18 polylinker. Antisense primers
containing nucleotide sequences encoding for 6 histidines were used
to position these residues at the 3' end of the pUC18 polylinker.
The nucleotide sequence of these primers are listed below.
3
5'aagcttccatgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaa-
acagtgca (SEQ ID NO:1) CAAAAGCTGGAGCTC-3'
[0108] The italic sequence represents a HindIII site for
subcloning. The underlined sequence represents leader sequence from
the human IL-2. Sequence in capital letters represents sequence
from the start of the polylinker region of pUC18.
[0109] Antisense Primer:
[0110] 5'ccggatccctactagtggtgatggtgatggtgGCTTGATATCGAATTCCTG-3'
(SEQ ID NO:2)
[0111] The italic sequence represents a HindIII site for
subcloning. The underlined sequence represents 6 codons encoding
for histidine residues followed by 2 termination codons. Sequence
in capital letters represents sequence to the 3' end of the pUC 18
polylinker.
[0112] Amplified products were obtained using buffer conditions as
previously described. Amplification reactions were carried out at
94.degree. C. for 30 sec, 52.degree. C. for 2 min, and 72.degree.
C. for 2 min for 25 cycles. Products were run on 1% agarose gels
containing ethidium bromide, and products of the expected molecular
weight were excised and purified by Gene Clean (Bio101). Products
were then cloned into T-tailed vectors (InVitrogen) as suggested by
the manufacturer. Recombinant clones were analyzed by restriction
analysis and by DNA sequencing. Several clones contained fragments
with the expected genomic sequence. The parental clone is referred
to as TAsec-hist.
[0113] A schematic diagram of the sec-hist fusion protein is shown
in FIG. 3A In order to generate TK cells that secrete the sec-hist
polypeptide, the TAsec-hist plasmid is digested with HindIII to
release the sec-hist insert. The insert was cloned into the unique
HindIII site of the pCEP4 mammalian expression vector, which also
contains the Hygromycin resistance gene as selectable marker.
Recombinant clones were analyzed by restriction digest and
sequencing to assure the authenticity of the construct.
[0114] Inserts can now be designed via PCR or direct cloning using
the restricition sites contained within the polylinker (see FIG.
3B).
[0115] Recombinant pCEPsec-hist plasmid will then be transfected
into TK cells as previously described using cationic lipids. Cells
will be cotransfected along with the pEFPMS134, which is a
mammalian expression vector containing the PMS134 dominant negative
MMR gene allele under control of the constitutive elongation factor
(EF) promoter. This vector contains the neomycin resistance gene
and allows for double selection of TK cells for both the sec-hist
and pEFPMS134 vectors. TK cells will also be cotransfected with the
sec-hist and pEF empty vector as a control.
[0116] Cells are co-selected for 14 days in 0.6 mg/ml G418 and 0.8
mg/ml hygromycin B (these concentrations have been previously
determined for double transfection of TK cells). After 14 days,
macroscopic colonies will be isolated and subcloned into 24 well
dishes (Nunc) as 1 ml cultures. Clones will then be analyzed for
secreted sec-hist protein using both ELISA and western blot
analysis of conditioned supernatants from sec-hist/pEFPMS134,
sec-hist/pEFempty vector, and parental TK cells. A monoclonal
anti-HIS antibody (Santa Cruz), which has been successfully used
for other western and ELISA studies, will be used for both assays.
Analysis of PMS134 expression will be determined by western blot
using a PMS2-specific polyclonal antibody (Morphotek, personal
communication).
[0117] ELISA will be performed on conditioned medium (CM) from TK
cells transfected with pCEP4sec-hist to screen for high producers
of the sec-hist polypeptide. ELISAs are carried out as follows.
Twohundred microliter aliquots of conditioned medium are taken from
pCEP4sec-hist transfected and control cells. Aliquots are placed
into 1.5 ml eppendorf tubes and centrifuged at 14,00.times.g for 3
minutes to pellet cell debris. Supernates are then collected and 50
.mu.ls are placed into triplicate wells of a 96-well polystyrene
microtiter plate (Nunc). Plates are incubated at room temperature
for 4 hours, washed twice with 200 .mu.ls of 1.times. Phosphate
Buffered Saline (PBS) solution, pH 7.0 (Life Technologies), and
blocked with 100 .mu.ls of 5% milk in 1.times.PBS for 1 hour.
Plates are then incubated with a monoclonal anti-HIS antibody
(diluted 1:1000 in 1.times.PBS) (Santa Cruz) for 2 hours at room
temperature and then washed twice with 200 .mu.ls of 1.times.PBS,
and probed with an anti-mouse-horse radish peroxidase (HRP)
conjugated secondary antibody diluted 1:3000 in PBS. Plates are
then incubated at room temperature for 1 hour, washed three times
with 200 .mu.ls of 1.times.PBS and incubated with TMB substrate
(BioRad) for 15-30 minutes. After incubation is completed,
reactions are stopped using 0.1N H.sub.2SO.sub.4 and plates are
read using a BioRad microplate reader at 415 nm. Clones are
determined to be positive for secreted sec-hist if expected cells
are found to produce a significant signal over control cells.
Conditioned medium from positive cultures will then be analyzed by
western blot using the anti-HIS antibody as probe to confirm ELISA
data.
[0118] Western blot analysis will be carried out as follows.
Briefly, 50 .mu.s of CM or 50,000 cells from each culture is
directly lysed in 2.times. lysis buffer (60 mM Tris, pH 6.8, 2%
SDS, 10% glycerol, 0.1 M 2-mercaptoethanol, 0.001% bromophenol
blue) and samples are boiled for 5 minutes. Lysate proteins are
separated by electrophoresis on 4-20% Tris glycine gels (Novex) and
then electroblotted onto Immobilon-P (Millipore) in 48 mM Tris
base, 40 mM glycine, 0.0375% SDS, 20% methanol and blocked
overnight at 4.degree. C. in Tris-buffered saline plus 0.05%
Tween-20 and 5% condensed milk. Filters are then probed with an
antibody generated against the PMS134 or the polyHIS tag (Santa
Cruz), followed by a secondary HRP-conjugated antibody. After
incubation with the secondary antibody, blots are developed using
chemiluminescence (Pierce) and exposed to film to determine PMS134
and sec-hist expression. Clones exhibiting expression of both genes
will then be used in experiments described above.
[0119] A potential technical problem may exist in expressing the
sec-hist protein due to toxicity that it may have on the growth of
TK cells. If the production of no or low amounts of sec-hist
polypeptide is found to occur in the above analysis, a HindIII
sec-hist fragment from the TAsec-hist plasmid will be subcloned
into the unique HindIII site of the pND/V5 steroid inducible vector
(Invitrogen). This vector has been found to produce robust protein
expression in TK cells upon in steroid induction. This application
teaches the use of employing an inducible vector containing the
sec-hist expression cassette to express polypeptides in TK cells
that may be toxic under constitutively expressed conditions. Cells
that are found to co-express the PMS134 and the sec-hist genes or
the control cell expressing the pEF empty vector and the sec-hist
gene are cultured under high growth conditions in media containing
neomycin, hygromycin and vitamins, which has been shown to increase
the doubling time of TK cells and enhance the genetic alteration of
.beta.-galactosidase reporter plasmids in vivo (data not shown).
Briefly, cells are grown in vitamin enriched medium for 20
doublings (.about.17 days), a time at which it has been found that
20-40% of clones contain sequence alterations within a particular
genetic locus. After selection, cells will be subcloned in 96-well
microtiter plates by limiting dilutions. Clones will be grown for 5
days in the presence of neomycin/hygromycin-free medium containing
heat inactivated serum to remove complement for in vitro antigenic
assays that will be performed using murine lymphocytes as described
in section below.
[0120] The sec-hist expression vector cassette can also be
transfected into cell lines that are "naturally" defective for MMR
such as the human cell lines derived from colon cancer tumors such
as but not limited to HCT116 and DLD-1. The vector can be in the
constitutive backbone pCEP4 or under control of the
steroid-inducible vectors pIND or pMAM.
[0121] The techniques described above teach us the use of producing
structurally altered antigens from mammalian cells to ensure proper
folding or post-translation modifications of the polypeptide. This
approach gives an advantage over others that employ the use of
prokaryotic, yeast or baculovirus produced antigens that have been
found to produce weak antigenic responses due to misfolding or
improper post-translational modifications.
EXAMPLE 3
Screening Strategy to Identify Cell Clones Producing Highly
Antigenic Polypeptides
[0122] In order to identify antigenic polypeptides produced by
TKPMS134 cell clones, the following in vitro assays will be
performed.
[0123] First, the lymphocyte stimulatory activity of sec-hist
polypeptides will be measured by adding CM of TKsec-hist cells with
or without the PMS134 to lymphocytes derived from naive or whole
antigen exposed Balb/C mice. Briefly, 2 mice will be infected with
whole antigen in the presence of Freund's Complete Adjuvant by
subcutaneous injection in the tail with a 100-.mu.l 1/1 mixture of
complete Freund's adjuvant (CFA) (Difco). Two subcutaneous boosts
will be performed with the same quantity of antigen, mixed 1/1 with
incomplete Freund's adjuvant (Difco), after 2 and 4 weeks. Two
control mice will receive adjuvant alone. Mice are sacrificed 5
days after the second boost (at day 33). Peripheral blood
mononuclear cells (PBMCs) from whole blood and splenocytes from
spleens of will be harvested following the previously described
procedures (Nicolaides, N. C. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:13175-13180). For splenocyte assays, whole spleens are
pressed through sterile wire mesh into RPMI medium (Life
Technology). Next, cells are washed twice in RPMI and incubated for
10 minutes in RBC lysis buffer. Cells are then washed again and
resuspended at 1.times.10.sup.5 cells/ml in RPMI-1640 medium plus
10% heat inactivated fetal bovine serum. One hundred microliters of
cells are aliquoted into twenty 96-well titer flat bottom
plates.
[0124] For PBMC isolation, whole blood is isolated by eye puncture
and collected into vacutainer tubes containing EDTA. An equal
volume of PBS (Mg.sup.2+/Ca.sup.2+-free) is added to whole blood.
PBMCs are isolated by centrifugation over Ficoll-Paque gradients
(Pharmacia Biotech 17-1440-02). Purified cells are seeded at
1.times.10.sup.5 cells/ml in RPMI 1640 containing 10%
heat-inactivated fetal bovine serum (Life Technologies, Inc.) and
100 .mu.ls are plated in 96 well flat bottom microtiter plates and
incubated at 37.degree. C. in 5% CO.sub.2.
[0125] To measure for T-cell activation, PBMCs and splenocytes from
primed and non-primed mice are incubated with 10% conditioned
medium (CM) from TKsec-hist cells with or without the PMS134 gene.
5 .mu.g/ml of concavalinA (ConA) is used as a positive control for
splenocyte culture assays, while 5 .mu.g/ml phorbol 12-myristate
13-acetate and 1 .mu.g/ml phytohemagglutinin (Sigma) are used as a
positive control for PBMC cultures. CM from parental TK cells grown
in the presence of RPMI with 10% heat inactivated medium will be
used as negative control. Previous studies using CM from TK cells
have found no stimulatory activity to be produced on PBMCs or
splenocytes (N. Nicolaides, personal observation). Cultures are
incubated at 37.degree. C. in 5% CO.sub.2 for 6 days and scored for
antigenic activity as determined by proliferation assay.
Proliferation is assayed using a modified protocol of the acid
phosphatase assay as described (Grasso, L. et al. (1998) J. Biol.
Chem. 273:24016-24024). Briefly, 50 .mu.ls of a buffer containing
0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100, and 10 mM
p-nitrophenyl phosphate (Sigma 104 phosphatase substrate) is added
directly to each well containing 0.2 ml of growth medium and
incubated for 1.5 h at room temperature. Reactions are terminated
by the addition of 0.05 N sodium hydroxide and quantified by
absorbance at 410 nm using a BioRad plate reader. Data is
represented as a stimulation index (SI), which is the proliferation
of experimental data points divided by the mean of 10 aliquots of
CM from TK parental cells. All experiments will be performed in at
least triplicate.
[0126] It is expected that several TKsec-hist clones co-expressing
the PMS134 protein will be found to have an enhanced antigenicity
on PBMCs and/or splenocytes due to conformational changes that will
occur within the coding region of the target antigen. These changes
may form secondary domains that serve as T and/or B cell epitopes
and in turn are responsible for stimulating their respective
activation. Clones that reproducibly produce enhanced antigenic
activation will be sequenced to confirm and identify that a
structural alteration(s) has indeed occurred within the coding
region of the gene. Sequence data may also shed additional light
into the importance of critical domains within this candidate
vaccine polypeptide for additional rounds of alteration that may
lead to the creation of a super-antigen that may serve as a potent
vaccine.
[0127] Sequence analysis of clones will be performed as follows.
First positive clones will be expanded from 96-well plates to
24-well plates. Confluent wells will be expanded and cells will be
harvested for RNA extraction. RNA extraction and reverse
transcription will be carried out using the Trizol method as
previously described (Nicolaides, N. C. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:13175-13180; Grasso, L. et al. (1998) J. Biol.
Chem. 273:24016-24024). Reverse transcription will be carried out
using Superscript II (Life Technology) as previously described
(Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641).
cDNAs will be amplified to isolate the sec-hist transcript using
the sense primer: 5'-catgtacaggatgcaactcctg-3' (SEQ ID NO:3), which
is located at the IL-2 leader sequence site (see FIG. 3), and the
antisense primer: 5'-tactagtggtgatggtgatggtg-3' (SEQ ID NO:4),
which is located at the C-terminal polyhis site. Amplification is
carried out at 94.degree. C. for 30 sec, 52.degree. C. for 2 min,
72.degree. C. for 2 min for 30 cycles. Reactions are analyzed on
agarose gels. If products of the expected molecular weight are
generated then samples will be cleaned using the QIAquick PCR
template kit (Qiagen) to remove PCR amplimers and sequenced using
the following primers that cover the entire coding region of the
sec-hist gene. Clones are then sequenced using primers specific to
the gene encoding the antigen.
[0128] Clones producing genetically altered sec-hist polypeptides
will then be expanded into T-75 flasks to a density that will
enable for the sufficient production of secreted sec-hist
polypeptide in the CM. Conditioned medium containing the sec-hist
polypeptide is then collected, and centrifuged at 3,500.times.g for
10 minutes to remove cellular debris. CM is then loaded onto a 10
ml-HiTrap Nickel column following the manufacturer's protocol
(Pharmacia). After absorption and washing, the column is treated
with 200 mM imidazole to elute the fusion protein. Recovered
polypeptides are then analyzed by SDS-PAGE using 4-12% NuPAGE gels
(Novex) and silver stained following the manufacturer's protocol
(Novex). Due to the random nature of defective MMR, the possibility
exists that sec-hist alleles may be generated by clones producing
enhanced antigenic polypeptides, which contain a nonsense or
frame-shift mutation, therefore forming polypeptides lacking a
polyHIS C-terminus. If a nonsense or frameshift mutation does occur
in clones producing polypeptides with increased antigenicity, then
the new allele is reengineered via PCR to contain a polyHIS tag at
the C-terminus, and this new fusion protein will be rescreened as
above.
[0129] Purified polypeptides will be rescreened at a final
concentration of 10 .mu.g/ml in the in vitro assays described above
to confirm that the antigenic activity is indeed coming from the
sec-hist protein. TK cells producing altered antigens with enhanced
activity on both PBMCs and splenocytes will then be tested along
with the non-altered sec-hist protein in vivo in Balb/C mice for
the ability to illicit an immune response in the absence of
adjuvant. A schematic diagram outlining the screening procedure is
given in FIG. 4.
EXAMPLE 4
Screening Strategy to Identify Cell Clones Producing Highly
Immunogenic Antigens
[0130] To test the immunogenic potential that the altered
polypeptides identified from EXAMPLE 3 have in vivo, Balb/C mice
will be injected with the 6 most antigenic polypeptides and the
wild type sec-hist polypeptide produced from TKEFempty/sec-hist
cells in the absence of adjuvant. Briefly, mice will be immunized
with 30 .mu.gs of purified sec-hist protein in sterile phosphate
buffered saline (PBS) without adjuvant by subcutaneous injection in
the tail. One group of mice will receive a 100 .mu.ls of a mixture
of polypeptides diluted 1/1 in complete Freund's adjuvant (CFA)
(Difco) as a positive control. Two subcutaneous boosts will be
performed with the same quantity of antigen, and the mouse
receiving the polypeptide mixture with adjuvant will be boosted
with a 1/1 mixture with incomplete Freund's adjuvant (Difco), at 2
and 4 weeks after the initial injection. Mice will be bled 5 and 15
days after the second boost (at day 33) to measure for antigen
titers. Control mice will receive PBS alone. Before the start of
immunization, a prebleed will be obtained from each mouse.
[0131] Immune responses will be measured by screening for whole Ig
and sec-hist specific antibody titers by ELISA following the
protocol described above. For antigen specific antisera titers,
96-well plates will be coated with a 50 uls of a solution
containing 5 .mu.g/ml each antigen used for vaccination in PBS.
Plates will then be probed with serial dilutions of prebleeds, 5
day and 15 day bleeds. Detection of antibodies will be done using a
sheep anti-mouse-HRP conjugated secondary antibody followed by
incubation with TMB substrate as described above.
[0132] Success of the strategy proposed in this program will be
demonstrated with the generation of antisera from mice immunized
with altered sec-hist protein that is able to cross react with the
native sec-hist protein. If no titers are found in the samples
without adjuvant, mice will be administrated antigen in complete
Freund's Adjuvant and titers analyzed 14 days later, followed by
immunization in Incomplete Freund's at day 17 if no titers are
found and analyzed at day 31.
[0133] A potential problem that may occur with the outlined
strategy is that antibody titers may be generated against the
sec-polyhistidine fusion domain. To determine if antisera is able
to identify authentic sec-his polypeptides, the antigen lacking the
polyhistidine domain will be generated by in vitro
transcription-translation (TNT) in the presence of radiolabelled
methionine. A template containing the sec-hist polypeptide will
also be made and the encoded protein used as a control. Templates
for the TNT reactions will be generated by PCR as described
(Nicolaides, N. C. et al. (1998) Mol. Cell. Biol. 18:1635-1641).
Briefly, the TAsec-hist plasmid will be used as template to amplify
gene segments that encode for the untqagged antigen or the sec-hist
tagged protein.
[0134] Translated polypeptides are first analyzed by SDS-PAGE gel
electrophoresis and autoradiography to ensure that the polypeptide
with the expected molecular weight are synthesized. Proteins are
then immunoprecipitated using antiserum from vaccinated mice to
determine if these antibodies recognize authentic antigen
sequences. Briefly, immunoprecipitations are performed on in
vitro-translated proteins by mixing the translation reaction
mixtures with 100 .mu.ls of mouse antiserum or 1 .mu.g of a
HIS-specific monoclonal antibody (MAB) (Santa Cruz) in 400 .mu.ls
of EBC buffer (50 mM Tris [pH 7.5], 0.1 M NaCl, 0.5% Nonidet P-40).
After incubation for 1 h at 4.degree. C., protein A-Sepharose
(Sigma) is added to a final concentration of 10% and the reaction
mixtures are incubated at 4.degree. C. for 1 h. Proteins bound to
protein A are washed five times in EBC and separated by
electrophoresis on 4-20% Tris-glycine gradient gels, which are then
dried and autoradiographed.
[0135] If antisera are able to react with authentic sequences
lacking HIS residues, the data from these studies will be continued
in further preclinical animal studies.
[0136] Discussion
[0137] The initial steps of MMR are dependent on two protein
complexes, called MutSc.alpha. and MutL.alpha.. The use of 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 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 antigen, 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 antigen-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.
[0138] The results described above lead to several conclusions.
First, expression of PMS134 results in an increase in
microsattelite instability in TK cells. That this elevated
microsattelite instability is due to MMR deficiency was proven by
evaluation of extracts from stably transduced cells and stability
of a tract contained within the pCAR-OF vector. 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). Interestingly, cells deficient in hMLH1 also
have a polar defect in MMR, but in this case preferentially
affecting repair from the 3' direction. It is known from previous
studies in both prokaryotes and eukaryotes that the separate
enzymatic components mediate repair from the two different
directions. These results 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
TK cells, or a cell line that is producing antigenic gene products.
Moreover, the use of such MMR alleles will be useful for generating
genetically altered polypeptides with altered structures as
effective vaccine agents.
[0139] This application also teaches us that ANY method used to
block MMR can be performed to generate hypermutablility in an
antigen-producing cell that can lead to genetically altered
proteins with enhanced biochemical features such as but not limited
to increased antigenicity, increased immunogenicity, and enhanced
pharmacokinetic profiles.
[0140] 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) Nucl. Acids Res.
27:2325-2331; Spampinato, C. and P. Modrich (2000) J. Biol. Chem.
275:9863-9869).
Sequence CWU 1
1
17 1 86 DNA Artificial Sequence Oligonucleotide primer 1 aagcttccat
gtacaggatg caactcctgt cttgcattgc actaagtctt gcacttgtca 60
caaacagtgc acaaaagctg gagctc 86 2 51 DNA Artificial Sequence
Oligonucleotide primer 2 ccggatccct actagtggtg atggtgatgg
tggcttgata tcgaattcct g 51 3 22 DNA Artificial Sequence
Oligonucleotide primer 3 catgtacagg atgcaactcc tg 22 4 23 DNA
Artificial Sequence Oligonucleotide primer 4 tactagtggt gatggtgatg
gtg 23 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 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 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 Ser
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 Asn Lys Arg Ile Val Leu Leu Glu Phe Ser Leu
Ser Ser Leu Ala Lys 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 Lys 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 Asn 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 Lys Leu 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 17 181 DNA Artificial Sequence
Sec-hist expression cassette 17 aagcttccat gtacaggatg caactcctgt
cttgcattgc actaagtctt gcacttgtca 60 caaacagtgc acaaaagctg
gagctccacc gcggtggcgg ccgctctaga actagtggat 120 cccccggggc
tgcaggaatt cgatatcaag ccaccatcac catcaccact agtagaagct 180 t
181
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