U.S. patent application number 10/159006 was filed with the patent office on 2003-07-31 for fapalpha -specific antibody with improved producibility.
This patent application is currently assigned to Boehringer Ingelheim International GmbH. Invention is credited to Bamberger, Uwe, Garin-Chesa, Pilar, Leger, Olivier, Park, John Edward, Rettig, Wolfgang J., Saldanha, Jose William.
Application Number | 20030143229 10/159006 |
Document ID | / |
Family ID | 27239064 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143229 |
Kind Code |
A1 |
Park, John Edward ; et
al. |
July 31, 2003 |
FAPalpha -specific antibody with improved producibility
Abstract
Recombinant antibody proteins are provided that specifically
bind fibroblast activation protein alpha (FAP.alpha.) and comprise
framework modifications resulting in the improved producibility in
host cells. The invention also relates to the use of said
antibodies for diagnostic and therapeutic purposes and methods of
producing said antibodies.
Inventors: |
Park, John Edward;
(Biberach/Riss, DE) ; Garin-Chesa, Pilar;
(Biberach/Riss, DE) ; Bamberger, Uwe;
(Ochsenhausen, DE) ; Rettig, Wolfgang J.;
(Biberach/Riss, DE) ; Leger, Olivier; (Annemasse,
FR) ; Saldanha, Jose William; (Middlesex,
GB) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Assignee: |
Boehringer Ingelheim International
GmbH
|
Family ID: |
27239064 |
Appl. No.: |
10/159006 |
Filed: |
June 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10159006 |
Jun 3, 2002 |
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09301593 |
Apr 29, 1999 |
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6455677 |
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60086049 |
May 18, 1998 |
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Current U.S.
Class: |
424/145.1 ;
435/7.23; 530/388.25 |
Current CPC
Class: |
C12N 2799/026 20130101;
C07K 2319/00 20130101; C07K 2317/24 20130101; A61K 2039/505
20130101; A61K 38/00 20130101; C07K 2317/567 20130101; C07K 16/40
20130101 |
Class at
Publication: |
424/145.1 ;
530/388.25; 435/7.23 |
International
Class: |
G01N 033/574; A61K
039/395; C07K 016/22 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1998 |
EP |
98107925.4 |
Claims
What is claimed is:
1. An antibody protein having the complementary determining regions
of the monoclonal antibody F19 (ATCC Accession No. HB 8269), said
antibody protein specifically binding to fibroblast activation
protein, characterised in that it has framework modifications
resulting in the improved producibility in host cells as compared
to a chimeric antibody having the variable regions of F19 and
foreign constant regions.
2. An antibody protein characterized in that it has a variable
light chain region and a variable heavy chain region according to
claim 1, each joined to a human constant region.
3. The antibody protein of claim 2, wherein said human constant
region of the light chain is a human kappa constant region.
4. The antibody protein of claim 2, wherein said human constant
region of the heavy chain is a human gamma-1 constant region.
5. An antibody protein according to any one of claims 1 to 4,
characterised in that its expression level in crude media samples
as determined by ELISA and/or purified antibody yield exceeds the
expression levels and/or purification yield of the chimeric
antibody without framework modifications by at least a factor of
10.
6. An antibody protein according to any one of claims 1 to 4,
characterised in that its expression levels in crude media samples
as determined by ELISA and/or purified antibody yield exceeds the
expression levels and/or purification yield of the chimeric
antibody without framework modifications by at least a factor of
20.
7. An antibody protein according to any one of claims 1 to 4,
characterised in that its expression level in crude media samples
as determined by ELISA and/or purified antibody yield exceeds the
expression levels and/or purification yield of the chimeric
antibody without framework modifications by at least a factor of
100.
8. An antibody protein according to any one of claims 1 to 7,
characterised in that it displays improved producibility in
eukaryotic cells.
9. The antibody protein according to claim 8 wherein said
eukaryotic cell is a Chinese hamster ovary cell (CHO cell).
10. An antibody protein according to any one of claims 1 to 9,
wherein the amino acid in Kabat position 87 of the light chain
region is not asparagine.
11. The antibody protein of claim 10, wherein the amino acid in
Kabat position 87 of the light chain region is selected from
aromatic or aliphatic amino acids.
12. The antibody protein of claim 11, wherein said aromatic amino
acid in Kabat position 87 of the light chain region is a tyrosine
or phenylalanine.
13. The antibody protein according to any one of claims 1 to 12,
wherein the amino acid in Kabat position 36 of the light chain
region is selected from aromatic amino acids.
14. An antibody protein according to any one of claims 1 to 13 that
contains the variable region of the light chain as set forth in SEQ
ID NO:2.
15. An antibody protein of claim 14 characterized in that the
variable region of the light chain is encoded by a nucleotide
sequence as set forth in SEQ ID NO:1.
16. An antibody protein according to any one of claims 1 to 13 that
contains the variable region of the light chain as set forth in SEQ
ID NO:6.
17. An antibody protein of claim 16 characterized in that the
variable region of the light chain is encoded by a nucleotide
sequence as set forth in SEQ ID NO:5.
18. An antibody protein according to any one of claims 1 to 17
containing a variable region of the heavy chain as set forth in any
one of SEQ ID NOs: 8, 10, 12, 14.
19. An antibody protein according to claim 18 characterised in that
the variable region of the heavy chain is encoded by a nucleotide
sequence as set forth in SEQ ID NOs:7, 9, 11, 13.
20. An antibody protein according to any one of claims 1 to 14
containing the variable region of the light chain as set forth in
SEQ ID NO:2 and the variable region of the heavy chain as set forth
in SEQ ID NOs:12.
21. The antibody protein of claim 20 characterised in that the
variable region of the light chain is encoded by a nucleotide
sequence as set forth in SEQ ID NO:1 and the variable region of the
heavy chain is encoded by a nucleotide sequence as set forth in SEQ
ID NO:11.
22. An antibody protein according to claim 20 or claim 21
containing the constant region of the light chain as set forth in
SEQ ID NO:20 and the constant region of the heavy chain as set
forth in SEQ ID NO:22.
23. An antibody protein according to any one of claims 1 to 13, 18
or 19 containing the variable region of the light chain as set
forth in SEQ ID NO:2 and the variable region of the heavy chain as
set forth in SEQ ID NOs:8.
24. The antibody protein of claim 23 characterised in that the
variable region of the light chain is encoded by a nucleotide
sequence as set forth in SEQ ID NO:1 and the variable region of the
heavy chain is encoded by a nucleotide sequence as set forth in SEQ
ID NO:7.
25. A nucleotide sequence encoding an antibody protein according to
any one of claims 1 to 24.
26. A recombinant DNA vector that contains a nucleotide sequence of
claim 25.
27. The recombinant DNA vector of claim 26, said vector being an
expression vector.
28. A host cell carrying a vector according to claims 26 or 27.
29. The host cell of claim 28, wherein said host cell is a
eukaryotic cell.
30. The host cell of claim 29, wherein said eukaryotic host cell is
a mammalian cell.
31. The host cell of claim 30, wherein said eukaryotic host cell is
a CHO or a COS cell.
32. A method of producing antibody proteins according to any one of
claims 1 to 24, said method comprising the steps of: (a)
cultivating a host cell according to any one of claims 28 to 31
under conditions where said antibody protein is expressed by said
host cell, and (b) isolating said antibody protein.
33. The method of claim 32, wherein said host cell is a mammalian
cell, preferably a CHO or COS cell.
34. The method of claim 32 or 33, wherein said host cell is
cotransfected with two plasmids carrying the expression units for
light and heavy chains respectively.
35. An antibody protein according to any one of claims 1 to 24,
wherein said antibody protein is conjugated to a therapeutic
agent.
36. The antibody protein of claim 35, wherein said therapeutic
agent is a therapeutic agent selected from the group consisting of
radioisotopes, toxins, toxoids, inflammatory agents and
chemotherapeutic agents.
37. The antibody protein of claim 36, wherein said radioisotope is
a .beta.-emitting radioisotope.
38. The antibody protein of claim 37, wherein said radioisotope is
selected from the group consisting of .sup.186Rhenium,
.sup.188Rhenium, .sup.131Iodine and .sup.90Yttrium.
39. An antibody protein according to any one of claims 1 to 24,
characterised in that it is labelled.
40. The antibody protein of claim 39, wherein said label is a
detectable marker.
41. The antibody protein of claim 40, wherein the detectable marker
is a detectable marker selected from the group consisting of
enzymes, dyes, radioisotopes, digoxygenin, and biotin.
42. An antibody protein according to any one of claims 1 to 24
conjugated to an imageable agent.
43. The antibody protein of claim 42, wherein the imageable agent
is a radioisotope.
44. The antibody protein of claim 43, wherein said radioisotope is
a .gamma.-emitting radioisotopes.
45. The antibody protein of claim 44, wherein said radioisotope is
.sup.125I.
46. A pharmaceutical composition containing an antibody protein
according to any one of claims 1 to 24 and a pharmaceutically
acceptable carrier.
47. A pharmaceutical composition containing an antibody protein
according to any one of claims 35 to 38 and a pharmaceutically
acceptable carrier.
48. A pharmaceutical composition containing an antibody protein
according to any one of claims 42 to 45 and a pharmaceutically
acceptable carrier.
49. The pharmaceutical composition of claims 46 to 48, for use in
the treatment or imaging of tumors, wherein said tumors are
associated with activated stromal fibroblasts, preferably wherein
said tumors are tumors selected from the cancer group consisting of
colorectal cancers, non-small cell lung cancers, breast cancers,
head and neck cancer, ovarian cancers, lung cancers, bladder
cancers, pancreatic cancers and metastatic cancers of the
brain.
50. Use of an antibody protein according to anyone of claims 1 to
24 for the treatment of cancer.
51. Use of an antibody protein according to anyone of claims 35 to
38 for the treatment of cancer.
52. Use of an antibody protein according to anyone of claims 42 to
45 imaging activated stromal fibroblasts.
53. Use of an antibody protein according to anyone of claims 39 to
41 for detecting the presence of activated stromal fibroblasts in a
sample.
54. A method of treating tumors, wherein the tumor is associated
with activated stromal fibroblasts capable of specifically forming
a complex with antibody proteins according to any one of claims 1
to 24 or 35 to 38, which comprises contacting the tumor with an
amount of said antibody proteins effective to treat the tumor.
55. The method of claim 54, wherein the tumor is a tumor having
cancer cells selected from the cancer group consisting of
colorectal cancers, non-small cell lung cancers, breast cancers,
head and neck cancer, ovarian cancers, lung cancers, bladder
cancers, pancreatic cancers and metastatic cancers of the
brain.
56. The method of claim 54, wherein the contacting is effected in
vitro.
57. The method of claim 54, wherein the contacting is effected in
vivo.
58. A method of detecting the presence of activated stromal
fibroblasts in wound healing, inflammation or a tumor,
characterised in that (a) a sample, possibly containing activated
stromal fibroblasts, is contacted with an antibody protein
according to any one of claims 1 to 24 or 39 to 41 under conditions
suitable for the formation of a complex between said antibody and
antigen, (b) detecting the presence of said complex, thereby
detecting the presence of activated stromal fibroblasts in wound
healing, inflammation or a tumor.
59. The method of claim 58, wherein the tumor is a tumor having
cancer cells selected from the cancer group consisting of
colorectal cancers, non-small cell lung cancers, breast cancers,
head and neck cancer, ovarian cancers, lung cancers, bladder
cancers, pancreatic cancers and metastatic cancers of the
brain.
60. The method of claim 58 or 59, wherein the antibody protein is a
protein according to any one of claims 39 to 41.
61. A method of imaging the presence of activated stromal
fibroblasts in a healing wound, inflamed skin or a tumor, in a
human patient, characterised in that (a) an antibody protein
according to any one of claims 1 to 24 conjugated to an imageable
agent is administered to a human patient under conditions suitable
for the formation of an antibody-antigen complex, (b) imaging any
complex formed in this manner.
62. The method of claim 61, wherein the tumor is a tumor having
cancer cells selected from the cancer group consisting of
colorectal cancers, non-small cell lung cancers, breast cancers,
head and neck cancer, ovarian cancers, lung cancers, bladder
cancers, pancreatic cancers and metastatic cancers of the
brain.
63. A method of detecting tumor-stroma, characterised in that (a) a
suitable sample is contacted with an antibody protein according to
any one of claims 1 to 24, under conditions suitable for the
formation of an antibody-antigen complex, (b) detecting the
presence of any complex so formed, (c) relating the presence of
said complex to the presence of tumor-stroma.
64. The method of claim 62, wherein said antibody is labelled with
a detectable marker.
65. A method of imaging tumor-stroma in a human patient, which
comprises (a) administering to the patient an antibody protein
according to any one of claims 42 to 45, under conditions suitable
for the formation of an antibody-antigen complex, (b) imaging any
complex so formed, and thereby imaging the presence of tumor-stroma
in a human patient.
66. An antibody protein containing an amino acid sequence as set
forth in SEQ ID NO:2.
67. An antibody protein according to claim 66 further containing an
amino acid sequence as set forth in SEQ ID NO:12.
68. An antibody protein according to claim 66 or 67 further
containing an amino acid sequence as set forth in SEQ ID NO:20 and
an amino acid sequence as set forth in SEQ ID NO:22.
69. A DNA molecule coding for an antibody protein according to any
one of claims 66 to 68.
70. A host cell carrying a DNA molecule according to claim 69.
71. A method of producing an antibody protein of any one of claims
66 to 68, said method comprising the steps of (a) cultivating the
host cell of claim 70 under conditions where said antibody protein
is expressed by said host cell, and (b) isolating said protein.
72. An antibody protein according to any one of claims 66 to 68
which is conjugated to a radioisotope, preferably .sup.131I,
.sup.125I, .sup.186Re, .sup.188Re, or .sup.90Y.
73. A pharmaceutical composition comprising an antibody protein
according to any one of claims 66 to 68, or 72, and a
pharmaceutically acceptable carrier.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to antibody proteins that
specifically bind fibroblast activation protein alpha (FAP.alpha.).
The invention also relates to the use of said antibodies for
diagnostic and therapeutic purposes and methods of producing said
antibodies.
[0003] 2. Related Art
[0004] The invasive growth of epithelial cancers is associated with
a number of characteristic cellular and molecular changes in the
supporting stroma. A highly consistent molecular trait of the
reactive stroma of many types of epithelial cancer is induction of
the fibroblast activation protein alpha (from now on referred to as
FAP), a cell surface molecule of reactive stromal fibroblasts
originally identified with monoclonal antibody F19 (Garin-Chesa,
P., et al., "Cell surface glycoprotein of reactive stromal
fibroblasts as a potential antibody target in human epithelial
cancers," Proc. Natl. Acad. Sci. 87:7235 (1990)). Since the FAP
antigen is selectively expressed in the stroma of a range of
epithelial carcinomas, independent of location and histological
type, a FAP-targeting concept has been developed for imaging,
diagnosis and treatment of epithelial cancers and certain other
conditions. For this purpose a monoclonal antibody termed F19 that
specifically binds to FAP was developed and described in U.S. Pat.
No. 5,059,523 and WO 93/05804, which are hereby incorporated by
reference in their entirety.
[0005] One serious problem that arises when using non-human
antibodies for applications in vivo in humans is that they quickly
raise a human anti-non-human response that reduces the efficacy of
the antibody in patients and impairs continued administration.
Humanization of non-human antibodies is commonly achieved in one of
two ways: (1) by constructing non-human/human chimeric antibodies,
wherein the non-human variable regions are joined to human constant
regions (Boulianne, G. L., et al., "Production of functional
chimeric mouse/human antibody," Nature 312:643 (1984)) or (2) by
grafting the complementarity determining regions (CDRs) from the
non-human variable regions to human variable regions and then
joining these "reshaped human" variable regions to human constant
regions (Riechmann L., et al., "Reshaping human antibodies for
therapy," Nature 332:323 (1988)). Chimeric antibodies, although
significantly better than mouse antibodies, can still elicit an
anti-mouse response in humans (LoBuglio, A. F., et al.,
"Mouse/human chimeric monoclonal antibody in man: Kinetics and
immune response," Proc. Natl. Acad. Sci. 86:4220 (1989)).
CDR-grafted or reshaped human antibodies contain little or no
protein sequences that can be identified as being derived from
mouse antibodies. Although an antibody humanized by CDR-grafting
may still be able to elicit some immune reactions, such as an
anti-allotype or an anti-idiotypic response, as seen even with
natural human antibodies, the CDR-grafted antibody will be
significantly less immunogenic than a mouse antibody thus enabling
a more prolonged treatment of patients.
[0006] Another serious limitation relating to the commercial use of
antibodies for diagnosis, imaging and therapy is their
producibility in large amounts. In many instances recombinant
expression of native, chimeric and/or CDR-grafted antibodies in
cell culture systems is poor. Factors contributing to poor
producibility may include the choice of leader sequences and the
choice of host cells for production as well as improper folding and
reduced secretion. Improper folding can lead to poor assembly of
heavy and light chains or a transport incompetent conformation that
forbids secretion of one or both chains. It is generally accepted
that the L-chain confers the ability of secretion of the assembled
protein. In some instances multiple or even single substitutions
can result in the increased producibility of antibodies.
[0007] Because of the clinical importance of specific immunological
targeting in vitro and in vivo of specific disease-related antigens
for diagnosis and therapy in humans, there is a growing need for
antibodies that combine the features of antigen specificity, low
immunogenicity and high producibility.
[0008] Therefore, the problem underlying the present invention was
to provide antibody proteins that combine the properties of
specific binding to FAP, low immunogenicity in humans, and high
producibility in recombinant systems.
SUMMARY OF THE INVENTION
[0009] The technical problem is solved by the embodiments
characterized in the claims.
[0010] The present invention provides new antibody proteins having
the complementary determining regions of the monoclonal antibody
F19 (ATCC Accession No. HB 8269), said new antibody proteins
specifically binding to fibroblast activation protein (FAP),
characterized in that they have framework modifications resulting
in the improved producibility in host cells as compared to a
chimeric antibody having the variable regions of F19 and foreign
constant regions.
[0011] As used herein, an "antibody protein" is a protein with the
antigen binding specificity of a monoclonal antibody.
[0012] "Complementarity determining regions of a monoclonal
antibody" are understood to be those amino acid sequences involved
in specific antigen binding according to Kabat (Kabat, E. A., et
al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH
Publication No. 91-3242. U.S. Department of Health and Human
Services, Public Health Service, National Institutes of Health,
Bethesda, Md. (1991)) in connection with Chothia and Lesk (Chothia
and Lesk, J. Mol. Biol., 196:901-917 (1987)).
[0013] As used herein, the term "framework modifications" refers to
the exchange, deletion or addition of single or multiple amino
acids in the variable regions surrounding the individual
complementarity determining regions. Framework modifications may
have an impact on the immunogenicity, producibility or binding
specificity of an antibody protein.
[0014] "Fibroblast activation protein (FAP)", also designated
fibroblast activation protein alpha (FAP.alpha.), is a
membrane-bound glycoprotein belonging to the serine protease gene
family (WO 97/34927). No shed or secreted form of FAP is known. FAP
can be characterized by its binding to the monoclonal antibody F19
(F19 is obtainable from the hybridoma cell line with the accession
No. HB 8269 deposited at the ATCC).
[0015] The term "fibroblast activation protein specific binding" of
an antibody protein is defined herein by its ability to
specifically recognize and bind FAP-expressing human cells. The
binding specificity of the proteins of the invention can be
determined by standard methods for the evaluation of binding
specificity such as described in an exemplary fashion in examples
6, 8 and 12.
[0016] The term "chimeric antibody" refers to an antibody protein
having the light and heavy chain variable regions as described in
FIGS. 17 and 18 and foreign constant regions. "Foreign constant
regions" as defined herein are constant regions which are different
from the constant regions of F19. For comparing an antibody protein
of the invention to a chimeric antibody it is to be understood that
such a chimeric antibody must contain the same constant regions as
said antibody protein. For the purpose of demonstration and
comparison alone the human constant heavy and light chains as
described in FIGS. 19 to 22 are used in an exemplary fashion.
[0017] To provide the antibody proteins of the present invention,
the nucleic acid sequences of the heavy and light chain genes of
the murine antibody designated F19 were determined from RNA
extracted from F19 hybridoma cells (ATCC Accession No. HB
8269).
[0018] In one embodiment the present invention relates to antibody
proteins having the complementary determining regions of the
monoclonal antibody F19 (ATCC Accession No. HB 8269), said new
antibody proteins specifically binding to fibroblast activation
protein (FAP), characterized in that they have framework
modifications resulting in the improved producibility in host cells
as compared to a chimeric antibody having the variable regions of
F19 and foreign constant regions, wherein said antibody protein is
derived from the murine antibody designated F19 (ATCC Accession No.
HB 8269).
[0019] To generate humanized FAP-specific antibody proteins a
chimeric antibody was constructed, having variable regions of the
light and heavy chains of F19 and human light and heavy constant
regions, respectively. The construction and production of chimeric
mouse/human antibodies is well known (Boulianne et al. (1984),
referenced above) and demonstrated in an exemplary fashion in
examples 1 and 2.
[0020] The variable regions of the antibody proteins of the present
invention are typically linked to at least a portion of the
immunoglobulin constant region (F.sub.C), typically that of a human
immunoglobulin. Human constant region DNA sequences can be isolated
in accordance with well-known procedures from a variety of human
cells, but preferably immortalized B cells (see Kabat et al.,
supra, and WO 87/02671). Hence the antibody proteins of the
invention may contain all or only a portion of the constant region
as long as they exhibit specific binding to the FAP antigen. The
choice of the type and extent of the constant region depends on
whether effector functions like complement fixation or antibody
dependent cellular toxicity are desired, and on the desired
pharmacological properties of the antibody protein. The antibody
protein of the invention will typically be a tetramer consisting of
two light chain/heavy chain pairs, but may also be dimeric, i.e.,
consisting of a light chain/heavy chain pair, e.g., a Fab or Fv
fragment.
[0021] Therefore, in a further embodiment the invention relates to
antibody proteins according to the invention, characterized in that
they have a variable light chain region and a variable heavy chain
region, each joined to a human constant region.
[0022] In particular, the variable region of the light chain was
joined to a human kappa constant region and the variable region of
the heavy chain was joined to a human gamma-1 constant region.
Other human constant regions for humanizing light and heavy chains
are also available to the expert.
[0023] Therefore, in one particular embodiment the antibody
proteins of the invention contain a human kappa constant
region.
[0024] Also, in another particular embodiment the antibody proteins
of the invention contain a human gamma-1 constant region.
[0025] One particular "chimeric F19 antibody" protein (cF19)
consists of the light and heavy chain variable and constant regions
described in FIGS. 17 to 22. cF19 demonstrates specific binding and
high avidity to the FAP antigen. As demonstrated in example 2, the
expression of cF19 in COS cells (cells derived from the kidney of
an African green monkey) is poor, ranging from about 10 to 60
ng/ml, which is at least 10 fold less than most antibodies.
[0026] In an attempt to increase expression levels of cF19, the
leader sequence of the F19 V.sub.L region was changed by
substitution of proline to leucine at position 9. This single
change in amino acid in the leader sequence resulted in at least
doubling the amount of chimeric antibody produced in COS cells. For
the expression of this particular chimeric antibody in COS cells
the following mutated leader sequence of the light chain:
MDSQAQVMLLLLWVSGTCG, and the following leader sequence of the heavy
chain: MGWSWVFLFLLSGTAGVS were used.
[0027] According to the invention the term "improved producibility"
in host cells refers to the substantial improvement of expression
levels and/or purified antibody yields when compared with the
expression levels and/or antibody yields of a chimeric antibody
without framework modifications as defined above. Two particular
but not limiting examples for demonstrating improved producibility
are exemplified for the COS cell expression system (in examples 2
and 5) and for the CHO cell expression system (in examples 10 and
11).
[0028] While the mutation of the leader sequence only leads to a
doubling of the expression yield of the chimeric F19 antibody, a
substantial improvement as defined herein refers to an improvement
in expression level and/or purification yield of at least a factor
of 10.
[0029] In a preferred embodiment, the invention refers to antibody
proteins, characterized in that their expression levels in crude
media samples as determined by ELISA and/or purified antibody
yields exceed the expression levels and/or purification yields of
the chimeric antibodies without framework modifications by at least
a factor of 10.
[0030] In more preferred embodiment, the invention refers to
antibody proteins, characterized in that their expression levels in
crude media samples as determined by ELISA and/or purified antibody
yields exceed the expression levels and/or purification yields of
the chimeric antibodies without framework modifications by at least
a factor of 20.
[0031] In a most preferred embodiment, antibody proteins,
characterized in that their expression levels in crude media
samples as determined by ELISA and/or purified antibody yields
exceed the expression levels and/or purification yields of the
chimeric antibodies without framework modifications by at least a
factor of 100.
[0032] Improved producibility of the recombinant antibody proteins
of the invention can be demonstrated for eukaryotic cells in
general as shown for COS and CHO (Chinese hamster ovary derived
cells) eukaryotic cells (see examples 5 and 11). In a further
embodiment, the present invention relates to recombinant antibody
proteins characterized in that they display improved producibility
in eukaryotic cells.
[0033] In a preferred embodiment the present invention relates to
antibody proteins, wherein said eukaryotic cell is a Chinese
hamster ovary cell (CHO cell).
[0034] It was unexpectedly found that certain framework
modifications of the light chain variable regions determine the
improved producibility of the antibody proteins of the invention.
Three versions of reshaped light chain variable regions, designated
version A, B and C, as described in FIGS. 1 to 6, were
prepared.
[0035] Light chain variable region versions A, B, and C demonstrate
substantially improved producibility in CHO cells (see example 11).
While light chain variable region versions A and C differ from
light chain variable region version B by only two common amino acid
residues they display an even further substantial improvement in
producibility. There is at least another 10 fold difference in
antibody secretion levels between the human reshaped F19 light
chain version B and versions A or C. Reshaped human F19 light chain
version A and B only differ in their amino acid sequences by two
residues at positions 36 (Tyr to Phe mutation) and 87 (Tyr to Asp
mutation) (nomenclature according to Kabat). This negative effect
on the secretory capability of antibodies containing the light
chain variable region version B could have been indirect if the Tyr
to Asp and Tyr to Phe mutations, considered individually or
together, merely caused improper folding of the protein. But this
is unlikely to be the case since antigen binding assays show that
immunoglobulins containing F19 light chain version B have similar
avidities to those paired with F19 light chain version A or C,
suggesting that they were not grossly misfolded.
[0036] Residue 87 in reshaped human F19 light chain version B seems
particularly responsible for the reduction of secretion when
compared to versions A and C.
[0037] In a preferred embodiment, the present invention relates to
antibody proteins according to the invention, wherein the amino
acid in Kabat position 87 of the light chain region is not
asparagine.
[0038] In a more preferred embodiment, the invention relates to
antibody proteins according to the invention, wherein the amino
acid in Kabat position 87 of the light chain region is selected
from aromatic or aliphatic amino acids.
[0039] In a most preferred embodiment, the present invention
relates to antibody proteins according to the invention, wherein
the aromatic amino acid in Kabat position 87 of the light chain
region is a tyrosine or phenylalanine.
[0040] In a further embodiment, the present invention also pertains
to antibody proteins according to the invention, wherein the amino
acid in Kabat position 36 of the light chain region is selected
from aromatic amino acids.
[0041] In a particular embodiment the invention relates to the
specific antibody proteins that may be prepared from the
individually disclosed reshaped variable regions of the light and
heavy chains.
[0042] Especially light chain variable region versions A and C are
particularly suitable to practice the invention because of their
exceptionally high producibility, while retaining full FAP-binding
specificity and achieving low immunogenicity. This holds especially
true when compared to the chimeric antibody having the variable
regions of F19 and the same constant regions but also when compared
to light chain version B.
[0043] Therefore, in one embodiment the present invention relates
to antibody proteins that contain the variable region of the light
chain as set forth in SEQ ID NO:2.
[0044] In a further embodiment the invention also relates to
antibody proteins, characterized in that the variable region of the
light chain is encoded by a nucleotide sequence as set forth in SEQ
ID NO:1.
[0045] In one embodiment the present invention relates to antibody
proteins that contain the variable region of the light chain as set
forth in SEQ ID NO:6.
[0046] In a further embodiment the invention also relates to
antibody proteins characterized in that the variable region of the
light chain is encoded by a nucleotide sequence as set forth in SEQ
ID NO:5.
[0047] The present invention also discloses several different
variable regions of the heavy chain that work particularly well
with the variable regions of the light chain versions A and C in
terms of improved producibility.
[0048] In one embodiment the invention relates to antibody proteins
containing a variable region of the heavy chain as set forth in any
one of SEQ ID NOS:8, 10, 12 and 14.
[0049] In another embodiment the invention relates to antibody
proteins characterized in that the variable region of the heavy
chain is encoded by a nucleotide sequence as set forth in any one
of SEQ ID NOS:7, 9, 11 and 13.
[0050] In a very particular embodiment the invention relates to
antibody proteins containing the variable region of the light chain
as set forth in SEQ ID NO:2 and the variable region of the heavy
chain as set forth in SEQ ID NO:12. Most preferably, this antibody
protein additionally contains the constant region of the light
chain as set forth in SEQ ID NO:20 and the constant region of the
heavy chain as set forth in SEQ ID NO:22.
[0051] Thus a further aspect of the present invention is an
antibody protein containing an amino acid sequence as set forth in
SEQ ID NO:2. More preferably, such an antibody protein further
contains an amino acid sequence as set forth in SEQ ID NO:12. More
preferably, said antibody protein further contains an amino acid
sequence as set forth in SEQ ID NO:20 and an amino acid sequence as
set forth in SEQ ID NO:22. A further aspect of the invention is an
antibody protein as described in this paragraph which is conjugated
to a radioisotope, preferably .sup.131I, .sup.125I, .sup.186Re,
.sup.188Re, or .sup.90Y. An additional aspect of the present
invention is a DNA molecule coding for an antibody protein as
described in this paragraph. A further aspect of the invention is a
host cell carrying such a DNA molecule. Accordingly, a further
aspect of the invention is a method of producing an antibody
protein as described in this paragraph, said method comprising the
steps of cultivating such a host cell under conditions where said
antibody protein is expressed by said host cell, and isolating said
protein. A further aspect of the invention is a pharmaceutical
composition comprising an antibody protein of the present invention
and a pharmaceutically acceptable carrier.
[0052] In a further particular embodiment the invention relates to
antibody proteins characterized in that the variable region of the
light chain is encoded by a nucleotide sequence as set forth in SEQ
ID NO:1 and the variable region of the heavy chain is encoded by a
nucleotide sequence as set forth in SEQ ID NO:11.
[0053] In a further particular embodiment the invention relates to
antibody proteins containing the variable region of the light chain
as set forth in SEQ ID NO:2 and the variable region of the heavy
chain as set forth in SEQ ID NO:8.
[0054] In a further particular embodiment the invention relates to
antibody proteins characterized in that the variable region of the
light chain is encoded by a nucleotide sequence as set forth in SEQ
ID NO:1 and the variable region of the heavy chain is encoded by a
nucleotide sequence as set forth in SEQ ID NO:7.
[0055] Humanization of the variable region of a murine antibody may
be achieved employing methods known in the art. EP 0230400
discloses grafting of the CDRs of a murine variable region into the
framework of a human variable region. WO 90/07861 discloses methods
of reshaping a CDR-grafted variable region by introducing
additional framework modifications. WO 92/11018 discloses methods
of producing humanized Ig combining donor CDRs with an acceptor
framework that has a high homology to the donor framework. WO
92/05274 discloses the preparation of framework mutated antibodies
starting from a murine antibody. Further prior art references
related to humanization of murine monoclonal antibodies are EP
0368684; EP 0438310; WO 92/07075 or WO 92/22653. Thus, the expert
can produce the antibodies of the present invention starting from
the publicly available murine monoclonal antibody F19 and employing
techniques known in the art, e.g., from WO 92/05274; DNA molecules
coding for the antibody proteins of the present invention may of
course also be obtained by state-of-the-art synthetic procedures,
e.g., by chemical synthesis of appropriate oligonucleotides and
subsequent ligation and amplification procedures (see e.g., Frank
et al., Methods Enzymol 154:221-249 (1987)).
[0056] In a further aspect, the present invention relates to
nucleic acid molecules containing the coding information for the
antibody proteins according to the invention as disclosed above.
Preferably, a nucleic acid molecule according to the present
invention is a nucleic acid molecule containing a nucleotide
sequence selected from SEQ ID NOS:1, 3, 5, 7, 9, 11, 13 or 15.
[0057] A further aspect of the present invention is a recombinant
DNA vector containing the nucleotide sequence of any one of the
above-mentioned nucleic acids, especially when said nucleotide
sequence is operationally linked to an expression control sequence
as in expression vectors. Preferred is a recombinant DNA vector,
said vector being an expression vector.
[0058] A further aspect of the present invention is a host cell
carrying a vector as described, especially an expression vector.
Such a host cell can be a prokaryotic or eukaryotic cell.
Preferably, such a host cell is a eukaryotic cell, a yeast cell, or
a mammalian cell. More preferably, said host cell is a CHO (Chinese
hamster ovary) cell or a COS cell.
[0059] Accordingly, a still further aspect of the present invention
is a method of producing antibody proteins according to the
invention. Such a method comprises the steps of:
[0060] (a) cultivating a host cell as described above under
conditions where said antibody protein is expressed by said host
cell, and
[0061] (b) isolating said antibody protein.
[0062] Mammalian host cells, preferably CHO or COS cells are
preferred. Host cells for producing the antibody proteins of the
invention may be transfected with a single vector containing the
expression units for both, the light and the heavy chain (see,
e.g., WO 94/11523). In one particular embodiment the method of
producing antibody proteins according to the invention pertains to
host cells, wherein said host cells are cotransfected with two
plasmids carrying the expression units for the light and heavy
chains respectively (see, e.g., EP 0481790).
[0063] The antibody proteins of the invention provide a highly
specific tool for targeting therapeutic agents to the FAP antigen.
Therefore, in a further aspect, the invention relates to antibody
proteins according to the invention, wherein said antibody protein
is conjugated to a therapeutic agent. Of the many therapeutic
agents known in the art, therapeutic agents selected from the group
consisting of radioisotopes, toxins, toxoids, inflammatogenic
agents, enzymes, antisense molecules, peptides, cytokines, and
chemotherapeutic agents are preferred. Among the radioisotopes,
gamma, beta and alpha-emitting radioisotopes may be used as a
therapeutic agent. .beta.-emitting radioisotopes are preferred as
therapeutic radioisotopes. .sup.186Rhenium, .sup.188Rhenium,
.sup.131Iodine and .sup.90Yttrium have been proven to be
particularly useful .beta.-emitting isotopes to achieve localized
irradiation and destruction of malignant tumor cells. Therefore,
radioisotopes selected from the group consisting of
.sup.186Rhenium, .sup.188Rhenium, .sup.131Iodine and .sup.90Yttrium
are particularly preferred as therapeutic agents conjugated to the
antibody proteins of the invention. For example, for the
radioiodination of an antibody of the invention, a method as
disclosed in WO 93/05804, may be employed.
[0064] A further aspect of the present invention pertains to
antibody proteins according to the invention, characterized in that
they are labelled. Such an FAP-specific labelled antibody allows
for the localization and/or detection of the FAP antigen in vitro
and/or in vivo. A label is defined as a marker that may be directly
or indirectly detectable. An indirect marker is defined as a marker
that cannot be detected by itself but needs a further directly
detectable marker specific for the indirect marker. Preferred
labels for practicing the invention are detectable markers. From
the large variety of detectable markers, a detectable marker
selected from the group consisting of enzymes, dyes, radioisotopes,
digoxygenin, and biotin is most preferred.
[0065] A further aspect of the present invention relates to
antibody proteins according to the invention, characterized in that
they are conjugated to an imageable agent. A large variety of
imageable agents, especially radioisotopes, are available from the
state of the art. For practicing the invention gamma-emitting
isotopes are more preferred. Most preferred is .sup.125Iodine.
[0066] One aspect of the present invention relates to
pharmaceutical compositions containing an antibody protein
according to the present invention as described above and a
pharmaceutically acceptable carrier. Such pharmaceutical
compositions are useful for treating tumors, wherein said tumors
are associated with activated stromal fibroblasts. There are two
possible effector principles for an anti-tumor stroma immunotherapy
that may act synergistically: (a) an unmodified (unconjugated,
"naked") antibody according to the invention may induce immune
destruction or inflammatory reactions in the tumor stroma while (b)
an antibody conjugated to a therapeutic agent, such as for example,
a radioisotope or other toxic substance, may achieve localized
irradiation and destruction of the malignant tumor cells.
Accordingly, a further aspect of the present invention is the use
of an antibody protein as described for the manufacture of a
pharmaceutical composition, especially for the treatment of
tumors.
[0067] One further embodiment are pharmaceutical compositions
containing an antibody protein according to the invention
conjugated to a therapeutic agent as described above and a
pharmaceutically acceptable carrier useful for treating tumors,
wherein said tumors are associated with activated stromal
fibroblasts. Another embodiment pertains to pharmaceutical
compositions containing an antibody protein according to the
present invention conjugated to an imageable agent as described
above and a pharmaceutically acceptable carrier useful for imaging
the presence of activated stromal fibroblasts in a healing wound,
inflamed skin or a tumor, in a human patient. A most preferred
embodiment relates to the pharmaceutical compositions mentioned
above, wherein said tumors are tumors selected from the cancer
group consisting of colorectal cancers, non-small cell lung
cancers, breast cancers, head and neck cancer, ovarian cancers,
lung cancers, invasive bladder cancers, pancreatic cancers and
cancers metastatic of the brain.
[0068] In an animal or human body, it can prove advantageous to
apply the pharmaceutical compositions as described above via an
intravenous or other route, e.g., systemically, locally or
topically to the tissue or organ of interest, depending on the type
and origin of the disease or problem treated, e.g., a tumor. For
example, a systemic mode of action is desired when different organs
or organ systems are in need of treatment as in e.g., systemic
autoimmune diseases, or allergies, or transplantations of foreign
organs or tissues, or tumors that are diffuse or difficult to
localize. A local mode of action would be considered when only
local manifestations of neoplastic or immunologic action are
expected, such as, for example local tumors.
[0069] The antibody proteins of the present invention may be
applied by different routes of application known to the expert,
notably intravenous injection or direct injection into target
tissues. For systemic application, the intravenous, intravascular,
intramuscular, intraarterial, intraperitoneal, oral, or intrathecal
routes are preferred. A more local application can be effected
subcutaneously, intracutaneously, intracardially, intralobally,
intramedullarly, intrapulmonarily or directly in or near the tissue
to be treated (connective-, bone-, muscle-, nerve-, epithelial
tissue). Depending on the desired duration and effectiveness of the
treatment, pharmaceutical antibody compositions may be administered
once or several times, also intermittently, for instance on a daily
basis for several days, weeks or months and in different
dosages.
[0070] For preparing suitable antibody preparations for the
applications described above, the expert may use known injectable,
physiologically acceptable sterile solutions. For preparing a
ready-to-use solution for parenteral injection or infusion, aqueous
isotonic solutions, such as, e.g., saline or corresponding plasma
protein solutions are readily available. The pharmaceutical
compositions may be present as lyophylisates or dry preparations,
which can be reconstituted with a known injectable solution
directly before use under sterile conditions, e.g., as a kit of
parts. The final preparation of the antibody compositions of the
present invention are prepared for injection, infusion or perfusion
by mixing purified antibodies according to the invention with a
sterile physiologically acceptable solution, that may be
supplemented with known carrier substances or/and additives (e.g.,
serum albumin, dextrose, sodium bisulfite and EDTA).
[0071] The amount of the antibody applied depends on the nature of
the disease. In cancer patients, the applied dose of a `naked`
antibody may be between 0.1 and 100 mg/m.sup.2, preferably between
5 and 50 mg/m.sup.2 per application. For radiolabeled antibodies,
e.g., with iodine-131, the maximally tolerated dose (MTD) has to be
determined which must not be exceeded in therapeutic settings.
Application of radiolabeled antibody to cancer patients may then be
carried out by repeated (monthly or weekly) intravenous infusion of
a dose which is below the MTD (see, e.g., Welt et al., J. Clin.
Oncol. 12:1193-1203 (1994)).
[0072] Furthermore, one aspect of the present invention relates to
the use of the antibody proteins according to the invention for the
treatment of cancer. In a preferred embodiment the present
invention relates to the use of antibody proteins according to the
invention conjugated to a therapeutic agent as described above for
the treatment of cancer. In another preferred embodiment the
present invention relates to the use of antibody proteins according
to the invention conjugated to an imageable agent for imaging
activated stromal fibroblasts. In a further preferred embodiment
the present invention relates to the use of labelled antibody
proteins according to the invention for detecting the presence of
activated stromal fibroblasts in a sample.
[0073] One aspect of the invention relates to a method of treating
tumors, wherein the tumor is associated with activated stromal
fibroblasts capable of specifically forming a complex with antibody
proteins according to the invention, present as naked/unmodified
antibodies, modified antibody proteins, such as, e.g., fusion
proteins, or antibody proteins conjugated to a therapeutic agent,
which comprises contacting the tumor with an effective amount of
said antibodies. In a preferred embodiment the present invention
relates to a method of treating tumors as mentioned above, wherein
the tumor is a tumor having cancer cells selected from the cancer
group consisting of colorectal cancers, non-small cell lung
cancers, breast cancers, head and neck cancer, ovarian cancers,
lung cancers, invasive bladder cancers, pancreatic cancers and
metastatic cancers of the brain. The method of treating tumors as
described above may be effected in vitro or in vivo.
[0074] A further aspect of the invention relates to a method of
detecting the presence of activated stromal fibroblasts in wound
healing, inflammation or in tumors, characterized in that
[0075] (a) a sample, possibly containing activated stromal
fibroblasts, is contacted with an antibody protein according to the
invention under conditions suitable for the formation of a complex
between said antibody and antigen,
[0076] (b) detecting the presence of said complex, thereby
detecting the presence of activated stromal fibroblasts in wound
healing, inflammation or a tumor.
[0077] In a preferred embodiment, the present invention relates to
a method of detecting the presence of activated stromal fibroblasts
in a tumor, wherein the tumor is a tumor having cancer cells
selected from the cancer group consisting of colorectal cancers,
non-small cell lung cancers, breast cancers, head and neck cancer,
ovarian cancers, lung cancers, bladder cancers, pancreatic cancers
and metastatic cancers of the brain. Most preferred antibody
proteins of the invention are those which are characterized in that
they are labelled as mentioned above.
[0078] A further aspect of the invention relates to a method of
imaging the presence of activated stromal fibroblasts in a healing
wound, inflamed tissue (rheumatoid arthritis and cirrhosis are also
positive) or a tumor, in a human patient, characterized in that
[0079] (a) an antibody protein according to the present invention
conjugated to an imageable agent is administered to a human patient
under conditions suitable for the formation of an antibody-antigen
complex,
[0080] (b) imaging any complex formed in this manner,
[0081] (c) thereby imaging the presence of activated stromal
fibroblasts in a human patient.
[0082] In a preferred embodiment the present invention relates to a
method of imaging the presence of activated stromal fibroblasts as
described above in tumors, wherein the tumor is a tumor having
cancer cells selected from the cancer group consisting of
colorectal cancers, non-small cell lung cancers, breast cancers,
head and neck cancer, ovarian cancers, lung cancers, bladder
cancers, pancreatic cancers and metastatic cancers of the
brain.
[0083] In a further aspect the present invention relates to a
method of detecting tumor-stroma, characterized in that
[0084] (a) a suitable sample is contacted with an antibody protein
according to the present invention, under conditions suitable for
the formation of an antibody-antigen complex,
[0085] (b) detecting the presence of any complex so formed,
[0086] (c) relating the presence of said complex to the presence of
tumor-stroma.
[0087] Antibody proteins for practicing the invention are
preferably labeled with a detectable marker.
[0088] In a further aspect the present invention relates to a
method of imaging tumor-stroma in a human patient, which
comprises
[0089] (a) administering to the patient an antibody according to
the invention conjugated to an imageable agent as described above
under conditions suitable for the formation of an antibody-antigen
complex,
[0090] (b) imaging any complex so formed, and thereby imaging the
presence of tumor-stroma in a human patient.
BRIEF DESCRIPTION OF THE FIGURES
[0091] FIG. 1. DNA sequence of F19 human reshaped light chain
variable region version A (hF19L.sub.A) SEQ ID NO:1.
[0092] FIG. 2. Amino acid sequence of F19 human reshaped light
chain variable region version A (hF19L.sub.A) SEQ ID NO:2.
[0093] FIG. 3. DNA sequence of F19 human reshaped light chain
variable region version B (hF19L.sub.B) SEQ ID NO:3. Nucleotides
differing from version A are underlined and in bold type.
[0094] FIG. 4. Amino acid sequence of F19 human reshaped light
chain variable region version B (hF19L.sub.B) SEQ ID NO:4. Amino
acids differing from version A are underlined and in bold type.
[0095] FIG. 5. DNA sequence of F19 human reshaped light chain
variable region version C (hF19L.sub.C) SEQ ID NO:5. Nucleotides
differing from version A are underlined and in bold type.
[0096] FIG. 6. Amino acid sequence of F19 human reshaped light
chain variable region version C (hF19L.sub.C) SEQ ID NO:6. Amino
acids differing from version A are underlined and in bold type.
[0097] FIG. 7. DNA sequence of F19 human reshaped variable region
heavy chain version A (hF19H.sub.A) SEQ ID NO:7.
[0098] FIG. 8. Amino acid sequence of F19 human reshaped heavy
chain variable region version A (hF19H.sub.A) SEQ ID NO:8
[0099] FIG. 9. DNA sequence of F19 human reshaped heavy chain
variable region version B (hF19H.sub.B) SEQ ID NO:9. Nucleotides
differing from version A are underlined and in bold type.
[0100] FIG. 10. Amino acid sequence of F19 human reshaped heavy
chain variable region version B (hF19H.sub.B) SEQ ID NO:10. Amino
acids differing from version A are underlined and in bold type.
[0101] FIG. 11. DNA sequence of F19 human reshaped heavy chain
variable region version C (hF19H.sub.C) SEQ ID NO:11. Nucleotides
differing from version A are underlined and in bold type.
[0102] FIG. 12. Amino acid sequence of F19 human reshaped heavy
chain variable region version C (hF19H.sub.C) SEQ ID NO:12. Amino
acids differing from version A are underlined and in bold type.
[0103] FIG. 13. DNA sequence of F19 human reshaped heavy chain
variable region version D (hF19H.sub.D) SEQ ID NO:13. Nucleotides
differing from version A are underlined and in bold type.
[0104] FIG. 14. Amino acid sequence of F19 human reshaped heavy
chain variable region version D (hF19H.sub.D) SEQ ID NO:14. Amino
acids differing from version A are underlined and in bold type.
[0105] FIG. 15. DNA sequence of F19 human reshaped heavy chain
variable region version E (hF19H.sub.E) SEQ ID NO:15. Nucleotides
differing from version A are underlined and in bold type.
[0106] FIG. 16. Amino acid sequence of F19 human reshaped heavy
chain variable region version E (hF19H.sub.E) SEQ ID NO:16. Amino
acids differing from version A are underlined and in bold type.
[0107] FIG. 17. Amino acid sequence of F19 chimeric light chain
variable region (chF19LC) SEQ ID NO:17.
[0108] FIG. 18. Amino acid sequence of F19 chimeric heavy chain
variable region (chF19HC) SEQ ID NO:18.
[0109] FIG. 19. DNA sequence of human kappa light constant chain
SEQ ID NO:19.
[0110] FIG. 20. Amino acid sequence of human light constant chain
SEQ ID NO:20.
[0111] FIG. 21. DNA sequence of human heavy constant chain SEQ ID
NO:21.
[0112] FIG. 22. Amino acid sequence of human heavy constant chain
SEQ ID NO:22.
[0113] FIG. 23. Mammalian cell expression vectors used to produce
chimeric and reshaped human antibodies with human kappa light
chains and human gamma-1 heavy chains.
[0114] A. Light chain expression vector: pKN100
[0115] B. Heavy chain expression vector: pG1D105
[0116] FIG. 24. DNA and amino acid sequences of mouse F19 light
chain variable region as modified for use in the construction of
chimeric F19 light chain. Restriction sites are indicated by bold
letters. The Kozak sequence, CDRs 1 to 3 and the splice donor site
are underlined.
[0117] FIG. 25. DNA and amino acid sequences of mouse F19 heavy
chain variable region as modified for use in the construction of
chimeric F19 heavy chain. Restriction sites are indicated by bold
letters. The Kozak sequence and the splice donor site are
underlined.
[0118] FIG. 26. DNA sequence of F19 chimeric antibody cloned into
pKN100 mammalian expression vector. Restriction sites are indicated
by bold letters and underlined. CDRs 1 to 3 and the splice donor
site are underlined. This is the DNA sequence of the mouse F19
light chain inside the pKN100 eukaryotic expression vector. This
vector has a cDNA version of the human kappa constant region gene
(allotype Km(3)) terminated by a strong artificial termination
sequence. In addition, the Neo selection gene is also terminated by
this artificial sequence and is also in the same orientation as the
kappa light chain expression cassette.
[0119] The essential components of the pKN100 eukaryotic expression
vector are:
1 1-6 EcoRI site 7-1571 HCMVi promoter/enhancer 583-587 TATAA box
610 Start of transcription 728-736 Splice donor site 731 Beginning
of intron 1557 End of intron 1544-1558 Splice acceptor site
1590-1598 Kozak sequence 1599-1658 peptide leader sequence
1659-1997 mouse F19 light chain 1996-2004 splice donor site
2011-2657 cDNA copy of human Kappa constant region (Km(3)) gene
2664-2880 Artificial spaC2 termination sequence 2887-7845 This is
the pSV2neo vector DNA fragment comprising of the Amp-resistance
gene (in the opposite orientation), the ColEI and SV40 origins of
replication and the Neo-resistance gene (in the same orientation as
the HCMVi-KCT cassette) 7852-8068 Artificial spaC2 termination
signal
[0120] This sequence ends immediately upstream of the EcoRI site
(position 1-6) at the beginning of the sequence. As a vector this
DNA sequence would be circular.
[0121] FIG. 27. DNA sequence of F19 chimeric antibody cloned into
pg1d105 mammalian expression vector. Restriction sites are
indicated by bold letters and underlined. CDRs 1 to 3 and the
splice donor site are underlined. This is the DNA sequence of the
eukaryotic expression vector pG1D105 containing the mouse F19 heavy
chain variable region. This vector contains a cDNA version of the
human gamma-1 constant region (allotype G1m (non-a, -z, -x) also
known as Gm1(17) allotype).
[0122] The essential components of the construct are:
2 1-2501 pBR322 based sequence including Ampicillin resistance gene
and ColEI origin plus the SV40 origin and the crippled SV40 early
promoter 2502-3226 dhfr gene 3233-4073 SV40 poly A sequence etc.
4074-4079 ligated BamHI and BglII site (BstYI) 4080-4302 SPA site
plus C2 termination signal 4303-5867 HCMVi promoter 5879-5885
unique HindIII restriction site for cloning of immunoglobulin
variable genes 5886-5894 Kozak sequence 5895-5951 signal peptide
5952-6323 mouse F19 heavy chain 6323-6330 splice donor site
6331-6336 unique BamHI restriction site for cloning of
immunoglobulin variable genes 6337-7388 cDNA copy of human gamma-1
constant regions preceded by a 62 bp intron 7389-7709 Arnie
termination sequence
[0123] The human gamma-1 constant region used in this construct has
a G1m (non-a, -z, -x) also known as Gm1(17) allotype which is
defined by a glutamic acid (E) residue at position 356 (according
to Eu numbering), and a methionine (M) residue at position 358
(according to Eu numbering) and a lysine (K) residue at position
214 (according to Eu numbering). These three residues are
underlined in the sequence above.
[0124] FIG. 28. PCR-based method for the construction of human
reshaped F19 light chain. This figure provides a schematic overview
of the strategy of construction. The dotted lines indicate a
complementary sequence of at least 21 bases between the
primers.
[0125] FIG. 29. Nucleotide and deduced amino acid sequences of
reshaped human F19 light chain variable regions version A, B and C.
Nucleotide and deduced amino acid sequences are aligned and
compared with that of version A, dashes indicate nucleotide
identity, dots indicate amino acid identity with this sequence.
Amino acids are numbered according to Kabat et al. (1991). The
locations of CDRs are indicated in boxes.
[0126] FIG. 30. DNA sequence of F19 LA (human reshaped light chain
version A) cloned into pKN100 mammalian expression vector.
Restriction sites are indicated by bold letters and underlined.
CDRs 1 to 3 and the splice donor site are underlined. This is the
DNA sequence of the reshaped F19 light chain version. A cloned into
pKN100 eukaryotic expression vector. This vector has a cDNA version
of the human kappa constant region gene (allotype Km(3)) terminated
by a strong artificial termination sequence. In addition, the Neo
selection gene is also terminated by this artificial sequence and
is also in the same orientation as the kappa light chain expression
cassette.
[0127] The components of the vector are:
3 7-1571 HCMVi promoter/enhancer 583-587 TATAA box. 610 Start of
transcription. 728-736 Splice donor site. 731 Beginning of intron.
1557 End of intron. 1544-1558 Splice acceptor site. 1590-1598 Kozak
sequence 1599-1658 peptide leader sequence 1659-1997 reshaped F19
light chain version A 1996-2004 splice donor site 2011-2657 cDNA
copy of human kappa constant region (Km(3)) gene. 2664-2880
Artificial spaC2 termination sequence. 2887-7845 This is the
pSV2neo vector DNA fragment comprising of the Amp-resistance gene
(in the opposite orientation), the ColEI and SV40 origins of
replication and the Neo-resistance gene (in the same orientation as
the HCMVi-KCT cassette). 7852-8068 Artificial spaC2 termination
signal.
[0128] This sequence ends immediately upstream of the EcoRI site
(position 1-6) at the beginning of the sequence below. As a vector
this DNA sequence would be circular.
[0129] FIG. 31. PCR-based method for the construction of human
reshaped F19 heavy chain. This figure provides a schematic overview
of the strategy of construction. The dotted lines indicate a
complementary sequence of at least 21 bases between the
primers.
[0130] FIG. 32. Nucleotide and deduced amino acid sequences of
reshaped human F19 heavy chain variable region versions a to e.
Nucleotide and deduced amino acid sequences are aligned and
compared with that of version A, dashes indicate nucleotide
identity, dots indicate amino acid identity with this sequence.
Amino acids are numbered according to Kabat et al. (1991). The
location of CDRs is indicated by boxes.
[0131] FIG. 33. DNA sequence of F19Ha (human reshaped heavy chain
version a) cloned into pg1d105 mammalian expression vector.
Restriction sites are indicated by bold letters and underlined.
CDRs 1 to 3 and the splice donor site are underlined. This is the
DNA sequence of the eukaryotic expression vector pG1D105 containing
the reshaped version A of F19 heavy chain variable region. This
vector contains a cDNA version of the human gamma-1 constant region
(allotype G1m (non-a,- z, -x) also known as Gm1(17) allotype).
[0132] The essential components of the construct are:
4 1-2501 pBR322 based sequence including Ampicillin resistance gene
ColEI origin plus the SV40 origin and the crippled SV40 early
promoter 2502-3226 dhfr gene 3233-4073 SV40 poly A sequence etc.
4080-4302 SPA site plus C2 termination signal 4303-5867 HCMVi
promoter/enhancer 5879-5885 unique HindIII restriction site for
cloning of immunoglobulin variable genes 5886-5894 Kozak sequence
5895-5951 signal peptide 5952-6323 reshaped F19 heavy chain version
A 6323-6330 splice donor site 6331-6336 unique BamHI restriction
site for cloning of immunoglobulin variable genes 6337-7388 cDNA
copy of human gamma-1 constant regions preceded by a 62 bp intron
7389-7709 Arnie termination sequence
[0133] The human gamma-1 constant region used in this construct has
a G1m (non-a, -z, -x) also known as Gm1(17) allotype which is
defined by a glutamic acid (E) residue at position 356 (according
to Eu numbering), a methionine (M) residue at position 358
(according to Eu numbering) and a lysine (K) residue at position
214 (according to Eu numbering). These three residues are
underlined in the sequence above.
[0134] FIG. 34. Heavy (panel A) and light (panel B) chains RNA
splicing events taking place during antibody F19 expression in
mammalian cells--schematic overview.
[0135] A. Heavy chain RNA splicing
[0136] B. Kappa light chain RNA splicing
[0137] FIG. 35. Concentration dependence of L.sub.AH.sub.C
supernatant binding to CD8-FAP.
[0138] FIG. 36. Binding of biotinylated L.sub.AH.sub.C to human
FAP.
[0139] FIG. 37. CD8-FAP carries the F19 epitope as detected with
cF19.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLES
Example 1
Construction of Mouse-human Chimeric Genes
[0140] The chimeric F19 (cF19) antibody was designed to have the
mouse F19 V.sub.L and V.sub.H regions linked to human kappa and
gamma-1 constant regions, respectively. PCR primers were used to
modify the 5'- and 3'- sequences flanking the cDNA sequences coding
for the mouse F19 V.sub.L and V.sub.H regions (Table 1). PCR
primers specific for F19 light chain V-region were designed. These
adapted mouse F19 variable regions were then subcloned into
mammalian cell expression vectors already containing the human
kappa (pKN100 vector) or gamma-1 (pG1D105 vector) constant regions
(FIG. 23). These vectors employ the human cytomegalovirus (HCMV)
promoter/enhancer to efficiently transcribe the light and heavy
chains. The vectors also contain the SV40 origin of replication to
permit efficient DNA replication and subsequent protein expression
in COS cells. The expression vectors were designed to have the
variable regions inserted as HindIII-BamHII DNA fragments. PCR
primers were designed to introduce these restrictions sites at the
5'-(HindIII) and 3'-(BamHI) ends of the cDNAs coding for the
V-regions. In addition the PCR primers were designed to introduce
the Kozak sequence (GCCGCCACC) at the 5'-ends of both the light and
heavy chain cDNAs to allow efficient translation (Kozak, M., "At
least six nucleotides preceding the AUG initiator codon enhance
translation in mammalian cells," J. Mol. Biol. 196:947 (1987)), and
to introduce splice donor sites at the 3'-ends of both the light
and heavy chain cDNAs for the variable regions to be spliced to the
constant regions. The PCR primers used in the construction of the
chimeric F19 light and heavy chains are shown in Table 1. The DNA
and amino acid sequences of the mouse F19 V.sub.L and V.sub.H
regions as adapted for use in the construction of chimeric F19
light and heavy chains are shown in FIGS. 24 and 25. The DNA
sequences of mouse F19 light and heavy chains cloned into the
eukaryotic expression vectors pKN100 and pG1D105, respectively, are
shown in FIGS. 26 and 27.
5TABLE 1 PCR primers for the construction of chimeric F19 antibody
A. Light chain variable region 1. Primer for the construction of
the 5'-end (37 mer) 5' CAGA AAGCTT GCCGCCACC ATG GAT TCA CAG GCC
CAG 3' HindIII Kozak sequence M D S Q A Q 2. Primer for the
construction of the 3'-end (35 mer) 5' CCGA GGATCC ACTCACG TTT CAG
CTC CAG CTT GGT 3' BamHI Splice donor site B. Heavy chain variable
region 1. Primer for the construction of the 5'-end (37 mer) 5'
CAGA AAGCTT GCCGCCACCATG GGA TGG AGC TGG GTC 3' HindIII Kozak
sequence M G W S W V 2. Primer for the construction of the 3'-end
(35 mer) 5' CCGA GGATCC ACTCACC TGA GGA GAC GGT GAC TGA 3' BamHI
Splice donor site
Example 2
Expression and Binding Activity of Chimeric F19 Antibody
[0141] The two plasmid DNAs coding for the chimeric F19 light and
heavy chains (see example 1) were co-transfected into COS cells to
look for transient expression of chimeric F19 antibody as described
below. After incubation for 72 hours, the medium was collected,
centrifuged to remove cellular debris, and analyzed by ELISA for
the production of a human IgG1-like antibody. The COS cell
supernatant containing the chimeric F19 antibody was analyzed for
its ability to bind to HT 1080 cells (see example 13) expressing
the FAP antigen on their surface.
Transfection of COS Cells Using Electroporation
[0142] The mammalian expression vectors pg1d105 and pKN100
containing the chimeric or reshaped human heavy and light chains
versions, respectively, were tested in COS cells to look for
transient expression of F19 antibodies. COS-7 cells were passaged
routinely in DMEM (Gibco BRL cat. #41966) containing penicillin (50
IU/ml), streptomycin (50 mg/ml), L-glutamine and 10%
heat-inactivated gamma globulin-free foetal calf serum (FCS, Harlan
Sera-Lab cat. #D0001). The DNA was introduced into the COS cells by
electroporation using the Gene Pulsar apparatus (BioRad). DNA (10
mg of each vector) was added to a 0.8 ml aliquot of
1.times.10.sup.7 cells/ml in Phosphate-buffered saline (PBS,
Ca.sup.2+ and Mg.sup.2+ free). A pulse was delivered at 1,900
volts, 25 mF capacitance. After a 10 minutes recovery period at
ambient temperature the electroporated cells were added to 8 ml of
DMEM containing 5% FCS. After incubation at 37.degree. C. for 72
hours, the medium was collected, centrifuged to remove cellular
debris, and stored under sterile conditions at 4.degree. C. for
short periods of time, or at -20.degree. C. for longer periods.
ELISA Method for Measuring Assembled IgG1/kappa Antibody
Concentrations in COS Cell Supernatants
[0143] Samples of antibodies produced in transfected COS cells were
assayed by ELISA to determine how much chimeric or reshaped human
antibody had been produced. For the detection of antibody, plates
were coated with goat anti-human IgG (Fcg fragment specific)
antibody (Jackson ImmunoResearch Laboratories Inc., #109-005-098).
The samples from COS cells were serially diluted and added to each
well. After incubation for 1 h at 37.degree. C. and washing,
horseradish peroxidase conjugated goat anti-human kappa light chain
(Sigma, A-7164) was added. After incubation for 30 minutes at
37.degree. C. and washing, K-blue substrate (a mixture of 3,3',5,5'
tetramethylbenzidine and hydrogen peroxide, Bionostics Limited, #KB
175) was added for 30 minutes at room temperature. The reaction was
stopped using Red Stop solution (Bionostics Limited, #RS20) and the
optical density read on a microplate reader at 650 nm. Purified
human IgG1/Kappa antibody (Sigma, I-3889) of known concentration
was used as a standard.
[0144] The expression of chimeric F19 antibody in COS cells was
poor (Table 2), between 10 and 60 ng/ml, which is at least 10 fold
less than most antibodies.
[0145] In an attempt to increase expression levels of the chimeric
F19 antibody, the leader sequence of F19 V.sub.L region was changed
by substitution of leucine to proline at position-9. This single
change in amino acid in the leader sequence resulted in at least
doubling the amount of chimeric antibody produced in COS cells.
[0146] Cell binding results show that chimeric F19 binds
specifically and with the expected avidity to the FAP target.
6TABLE 2 Chimeric F19 antibody concentrations in COS cell
supernatants (These are the results of three independent
transfections) Transfected Antibody components Human .gamma.1/K
Heavy chain Kappa light chain [in .mu.g/ml] cF19 cF19 (F19 leader
sequence) 0.060 cF19 cF19 (mutated leader sequence) 0.212 cF19 cF19
(F19 leader sequence) 0.056 cF19 cF19 (mutated leader sequence)
0.108 cF19 cF19 (F19 leader sequence) 0.011 cF19 cF19 (mutated
leader sequence) 0.087
Example 3
Construction of the Reshaped Human F19 Light Chain Versions A to C
(L.sub.A-L.sub.B)
[0147] The construction of the first version of reshaped human F19
V.sub.L region (L.sub.A) was carried out using overlapping PCR
fragments in a method similar to that described by Daugherty B. L.,
et al., "Polymerase chain reaction (PCR) facilitates the cloning,
CDR-grafting, and rapid expression of a murine monoclonal antibody
directed against the CD18 component of leukocyte integrins," Nucl.
Acids Res. 19:2471 (1991). Ten oligonucleotides were synthesized
that consisted of five primer pairs, APCR1-vla1, vla2-vla3,
vla4-vla5, vla6-vla7, and vla8-APCR4 (Table 3 and FIG. 28). There
was an overlapping sequence of at least 21 bases between adjacent
pairs (FIG. 28). APCR1 and APCR4 hybridized to the flanking pUC19
vector sequences. The mutagenic primers were designed such that
their 5' end immediately followed the wobble position of a codon.
This strategy was used to counteract the gratuitous addition of one
nucleotide to the 3' end of the strand complementary to the
mutagenic primer by the DNA polymerase during PCR (Sharrocks, A.
D., and Shaw, P. E., "Improved primer design for PCR-based,
site-directed mutagenesis," Nucl. Acids Res. 20:1147 (1992)). The
appropriate primer pairs (0.2 .mu.M of each) were combined with 10
ng of version "B" of reshaped human L25VL region cDNA, and 1 unit
of AmpliTaq (Perkin Elmer Cetus) DNA polymerase in 50 .mu.l of PCR
buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 .mu.M
dNTPs, and 1.5 mM MgCl.sub.2. This was overlaid with mineral oil
and PCR was performed for 25 cycles, each cycle consisting of a
denaturation step at 94.degree. C. for 1 minute, a primer annealing
step at 55.degree. C. for 1 minute, and an extension step at 72
.degree. C. for 2 minutes. This was followed by a single cycle
consisting of a further elongation step at 72.degree. C. for
10-minutes followed by cooling to 4.degree. C. The ramp time
between the primer-annealing and extension steps was 2.5 minutes.
The PCR products of the five reactions (A, B, C, D and E) were then
purified by gel electrophoresis followed by DNA elution using
Wizard PCR preps (Promega). PCR products A, B, C, D, and E were
assembled by their complementarity to one another. In the second
set of PCR reactions, PCR products B and C, and D and E, (50 ng of
each) were added to 50 ml PCR reactions (as described above) each
containing 1 unit of AmpliTaq (Perkin Elmer Cetus) DNA polymerase.
The reactions were cycled for 20 cycles as described above with the
exception that the annealing temperature was raised to 60.degree.
C. In the third set of PCR reactions, PCR products F and G were
PCR-amplified using 1 ml of each prior PCR reaction and the
appropriate pair of PCR primers (vla2-vla5 or vla6-APCR4). The PCR
reactions contained 1 unit of AmpliTaq DNA polymerase in 50 ml PCR
reaction (as described above) and were amplified for 25 cycles as
in the first stage. In the fourth set of PCR reactions, the PCR
product H was PCR-amplified using 1 ml of each prior PCR reaction
and the vla2-APCR4 pair of PCR primers. Finally, PCR products A and
H were assembled by their own complementarity in a two step-PCR
reaction similar to that described above using RSP and UP as the
terminal primers. The fully assembled fragment representing the
entire reshaped human F19 V.sub.L region including a leader
sequence was digested with HindIII and BamHI and cloned into pUC19
for sequencing. A clone having the correct DNA sequence was
designated reshF19La (FIG. 29) and was then subcloned into the
eukaryotic expression vector pKN100. The DNA sequence of reshF19La
cloned into pKN100 is shown in FIG. 30.
[0148] The second version of reshaped human F19 V.sub.L region
(L.sub.B) was constructed using the same scheme as that described
for La but where vla4 and vla7 primers were substituted by vlb4 and
vlb7 respectively (Table 3). The DNA sequence of L.sub.B is shown
in FIG. 29.
[0149] The third version of reshaped human F19 V.sub.L region
(L.sub.C) was constructed using the QuikChange.TM. site-directed
mutagenesis kit from Stratagene. The QuikChange site-directed
mutagenesis method was performed according to the manufacturer's
instructions, using reshF19La in pKN100 vector as double stranded
DNA template. The mutagenic oligonucleotide primers F19Lc-sense and
F19Lc-antisense (Table 3) for use in this protocol were designed
according to the manufacturer's instructions. Briefly, both the
mutagenic primers contained the desired point mutation (codon TTT
at Kabat residue position 49 (Phe) changed to TAT coding for Tyr)
and annealed to the same sequence on opposite strands of LA in
pKN100 vector. The point mutation was verified by DNA sequencing
the entire V.sub.L region. The DNA sequence of L.sub.C is shown in
FIG. 29. To eliminate the possibility that random mutations
occurred in the pKN100 during the PCR reaction, the V.sub.L region
was cut out of the pKN100 vector as an HindIII/BamHI fragment and
re-subcloned into an unmodified pKN100 vector cut with the same two
restriction enzymes beforehand.
7TABLE 3 PCR primers for the construction of reshaped human F19
light chain variable regions 1. Primers for the synthesis of
version "A" F19vla1 (36 mer): 5'
GTCATCACAATGTCTCCGGAGGAACCTGGAACCCAG 3' F19vla2 (29 mer): 5'
CTCCGGAGACATTGTGATGACCCAATCTC 3' F19vla3 (45 mer): 5'
GAATATAAAAGGCTCTGACTGGACTTGCAGTTGATGGTGGCCCTC 3' F19vla4 (72 mer):
5' CAGTCAGAGCCTTTTATATTCTAGAAATCAAAAGAACT-
ACTTGGCCTGGTATCAGCAGAAACCAGGACAGCC 3' F19vla5 (44 mer): 5'
ACCCCAGATTCCCTAGTGCTAGCCCAAAAGATGAGGAGTTTGGG 3' F19vla6 (67 mer):
5' TAGCACTAGGGAATCTGGGGTACCTGATAGGTTCAGTGGCAGTGG-
GTTTGGGACAGACTTCACCCTC 3' F19vla7 (53 mer): 5'
GTCCCTTGTCCGAACGTGAGCGGATAGCTAAAATATTGCTGACAGTAATAAAC 3' F19vla8
(33 mer): 5' GCTCACGTTCGGACAAGGGACCAAGGTGGAAAT 3' 2. Primers for
the synthesis of version "B" F19vlb4 (72 mer): 5'
CAGTCAGAGCCTTTTATATTCTAGAAATCAAAAGAACTACTTGGCCTGGTTCCAGCA-
GAAACCAGGACAGCC 3' F19vlb7 (57 mer): 5'
TCCCTTGTCCGAACGTGAGCGGATAGCTAAAATATTGCTGACAGTCATAAACTGCC 3' 3.
Primers for the synthesis of version "C" F19Lc-sense (34 mer): 5'
CCCAAACTCCTCATCTATTGGGCTAGCACTAGGG 3' F19Lc-antisense (34 mer): 5'
CCCTAGTGCTAGCCCAATAGATGAGGAGTTTGGG 3' 4. Primers hybridizing to the
flanking PUC19 vector sequences APCR1 (17 mer, sense primer): 5'
TACGCAAACCGCCTCTC 3' APCR4 (18 mer, anti-sense primer): 5'
GAGTGCACCATATGCGGT 3' RSP (-24) (16 mer, sense primer): 5'
AACAGCTATGACCATG 3' UP (-40) (17 mer, anti-sense primer): 5'
GTTTTCCCAGTCACGAC 3'+TZ,149
Example 4
Construction of the Reshaped Human F19 Heavy Chain Versions A to E
(H.sub.A-H.sub.E)
[0150] Version "A" of reshaped human F19 V.sub.H regions (H.sub.A)
was constructed using the same PCR methods as described for the
construction of version "A" of reshaped human F19 V.sub.L region
(L.sub.A) (FIG. 31). The template DNA was version "A" of reshaped
human 226 V.sub.H (Lger, O. J. P., et al., "Humanization of a mouse
antibody against human alpha-4 integrin: a potential therapeutic
for the treatment of multiple sclerosis," Hum. Antibod. 8:3
(1997)). Six PCR primers were designed and synthesized for the
construction of version "A" of reshaped human F19 V.sub.H region
(Table 4). PCR products A, B, C, and D were obtained using
APCR1-Vha1, Vha2-Vha3, Vha4-Vha5 and Vha6-APCR4 as PCR primer
pairs, respectively. The PCR conditions were essentially as
described for the construction of reshaped human F19 V.sub.L
region. A clone having the correct DNA sequence was designated
reshF19Ha (FIG. 32) and was then subcloned into the eukaryotic
expression vector pG1D105. The DNA sequence of reshF19Ha cloned
into pG1D105 is shown in FIG. 33.
[0151] The third version of reshaped human F19 V.sub.H region
(H.sub.C) was constructed using the same scheme as that described
for H.sub.A but where Vha4 primer was substituted by Vhc4 (Table
4). The DNA sequence of H.sub.C is shown in FIG. 32. The second
(H.sub.B) and fourth (H.sub.D) version of reshaped human F19
V.sub.H region were constructed based on the PCR-mutagenesis
methods of Kamman et al. (Kamman, M., et al, "Rapid insertional
mutagenesis of DNA by polymerase chain reaction (PCR)," Nucl. Acids
Res. 17:5404 (1989)). For H.sub.B and H.sub.D, a mutagenic primer
F19VHbd6 (Tyr-91 to Phe-91, Table 4) was used paired with APCR4 in
PCR reactions with H.sub.A and H.sub.C as the template DNA,
respectively. The PCR products VHb and VHd were restriction enzyme
digested with PstI and BamHI and subcloned into reshF19Ha and
reshF19Hc, respectively, previously digested with the same two
restriction enzymes. The DNA sequences of H.sub.B and H.sub.D are
shown in FIG. 32.
[0152] Version "E" of reshaped human F19 V.sub.H region (H.sub.E)
was constructed based on the PCR-mutagenesis methods of Kamman et
al. (1989) already mentioned above:
[0153] For reshF19He mutagenic primer F19MscIHe (Table 5) was used
paired with primer F19HindIII (Table 5) in PCR reactions with
H.sub.C cloned in pg1d105 mammalian expression vector as the
template DNA. The appropriate primer pairs (0.2 mM of each) were
combined with 10 ng of cDNA of version "A" of reshaped human 226 VH
region in 100 ml of PCR buffer containing 10 mM KCl, 10 mM
(NH.sub.4)2SO.sub.4, 20 mM Tris-HCl (pH 8.8) 2 mM MgSO.sub.4, 0.1%
Triton X-100 and 200 mM dNTPs. Reaction mixtures were overlaid with
mineral oil and kept at 94.degree. C. for 5 minutes. Then 1 unit of
Deep Vent DNA polymerase (New England Biolabs) was added ("Hot
Start" PCR; Chou Q., Russell, M., et al., "Prevention of pre-PCR
mis-priming and primer dimerization improves low-copy-number
amplifications," Nucl. Acids Res. 20:1717 (1992)) and PCR was
performed for 25 cycles on a TRIO-Thermoblock Thermal Cycler
(Biometra, Gottingen, Germany). Each cycle consisting of a
denaturation step at 94.degree. C. for 1 minute, a primer-annealing
step at 70.degree. C. for 1 minute, and an extension step at
72.degree. C. for 2 minutes. This was followed by a single cycle
consisting of a further elongation step at 72.degree. C. for 10
minutes followed by cooling at 4.degree. C. The PCR products were
then extracted and purified from a TAE 1.4% standard agarose gel
using a QIAquick.TM. gel extraction kit, following the protocol
supplied by the manufacturer (QIAGEN Ltd., UK). The PCR product
V.sub.He was then restriction enzyme digested with MscI and HindIII
and ligated into reshF19Hc cloned in pg1d105 previously digested
with the same two restriction enzymes. The MscI restriction
recognition site is unique to all the reshaped human F19 V.sub.H
region versions and is not present in the pg1d105 expression
vector. The HindIII restriction recognition site is a unique site
in pg1d105 for cloning of V.sub.H immunoglobulin genes.
Electroporation-competent XL-1 Blue E. coli cells were transformed
with 1 .mu.l of the ligated DNA and plated on agarose plates
containing Ampicillin. Colonies were then screened for the presence
and correct size of inserts by direct PCR on colonies (Guissow, D.,
and Clackson, T., "Direct clone characterization from plaques and
colonies by the polymerase chain reaction," Nucl. Acids Res.
17:4000 (1989)) with primers HCMi and Hucg1 hybridizing to the
flanking pg1d105 vector sequences (Table 5). DNA from positive
colonies was prepared using a Plasmid Midi kit, following the
protocol supplied by the manufacturer (QIAGEN Ltd., UK). DNA
sequencing was performed by the dideoxy chain termination method
(Sanger, F., et al., "DNA sequencing with chain-terminating
inhibitors," Proc. Natl. Acad. Sci. U.S.A. 74:5463 (1977)) directly
from circular vector DNA using conventional heat denaturation
(Andersen, A., et al., "A fast and simple technique for sequencing
plasmid DNA with sequenase using heat denaturation," Biotechniques
13:678 (1992)) and Sequenase 2.0 (USB, Cleveland, Ohio). The DNA
sequences of reshF19He is shown in FIG. 32.
8TABLE 4 PCR primers for the construction of reshaped human F19
heavy chain variable regions versions A to D 1. Primers for the
synthesis of version "A" F19vha1 (47 mer): 5'
GTGTATTCAGTGAAGGTGTATCTACTAGTTTTACAGCTGACTTTC- AC 3' F19vha2 (53
mer): 5' TAGTAGATACACCTTCACTGAATA- CACCATACACTGGGTTAGACAGGCCCTG 3'
F19vha3 (71 mer): 5'
CCCTTGAACTTCTGGTTGTAGTTAGGAATACCATTGTTAGGATTAATACCTCCTATCCACTCCAGCCTTT-
G 3' F19vha4 (71 mer): 5' TAACTACAACCAGAAGTTCAAGGGC-
CGGGCCACCTTGACCGTAGGCAAGTCTGCCAGCACCGCCTACATGG 3' F19vha5 (63 mer):
5' GCATGGCCCTCGTCGTAACCATAGGCGATTCTTCTTCTGGCGCAGTAGTAGAC-
TGCAGTGTCC 3' F19vha6 (48 mer): 5'
CTATGGTTACGACGAGGGCCATGCTATGGACTACTGGGGTCAAGGAAC 3' 2. Primers for
the synthesis of version "C" F19vhc4 (71 mer): 5'
TAACTACAACCAGAAGTTCAAGGGCCGGGTCACCATCACCGTAGACACCTCTGCCAGCACCGCCTACATG-
G 3' 3. Primers for the synthesis of version "B" and "D" F19vhbd6
(27 mer): 5' GGACACTGCAGTCTACTTCTGCGCCAG 3' 4. Primers hybridizing
to the flanking PUC19 vector sequences APCR1 (17 mer, sense
primer): 5' TACGCAAACCGCCTCTC 3' APCR4 (18 mer, anti-sense primer):
5' GAGTGCACCATATGCGGT 3'
[0154]
9TABLE 5 PCR primer for the construction of reshaped human F19
heavy chain variable regions version E 1. Primer for the synthesis
of version "E" F19MsclHe (65 mer, anti-sense): 5'
CCTTTGGCCAGGGGCCTGTCTAACCCAGTGT-
ATGGTGTATTCAGTGAAGGTGTATCCACTAGTTTCCACTAGTTT 3' MscI 2. Primers
hybridizing to the flanking pg1d105 mammalian expression vector
sequences HCMi (28 mer, sense): 5' GTCACCGTCCTTGACACGCGTCTCGGGA 3'
Hucg1 (17 mer, anti-sense): 5' TTGGAGGAGGGTGCCAG 3'
Example 5
Reshaped Human F19 Antibody Concentrations in COS Cell
Supernatants
[0155] COS cells were transfected with one pair of a series of
reshaped human F19 antibody constructs and the human antibody
concentration was measured using the IgG1/Kappa ELISA as described
in example 2.
10TABLE 6 Reshaped human F19 antibody concentrations in COS cell
supernatants Transfected Antibody components Human .gamma.1/K Heavy
chain Kappa light chain Concentration [.mu.g/ml] H.sub.A L.sub.A
2.50 H.sub.A L.sub.B 0.18 H.sub.B L.sub.A 1.25 H.sub.B L.sub.B 0.10
H.sub.D L.sub.A 1.15 H.sub.D L.sub.B 0.18 H.sub.A L.sub.A 1.50
H.sub.A L.sub.C 1.56 H.sub.C L.sub.A 1.47 H.sub.C L.sub.C 1.97 cF19
L.sub.A 1.54 cF19 L.sub.B 0.07 cF19 L.sub.C 2.14
[0156]
11TABLE 7 Reshaped human F19 antibody concentrations in COS cell
supernatants Transfected Antibody components Human .gamma.1/K Heavy
chain Kappa light chain concentration [.mu.g/ml] H.sub.A L.sub.A
2.00 H.sub.A L.sub.C 2.50 H.sub.C L.sub.A 2.90 H.sub.C L.sub.C 3.00
H.sub.E L.sub.A 2.80 H.sub.E L.sub.C 3.50
RNA Splicing Events Required for the Expression of Immunoglobulin
Genes in Mammalian Cells
[0157] Both mammalian expression vectors pKN100 and pg1d105 have an
intron between the variable and the constant regions which is
removed during the process of gene expression to give rise to an
messenger RNA. The splicing event which consists of a DNA
recombination between the heavy or light chain splice donor sites
and the immunoglobulin splice acceptor site is described in FIG.
34.
Example 6
Flow Cytometric Analysis of the Binding of cF19 and L.sub.AH.sub.C
to FAP-expressing Human Cells
[0158] The ability of L.sub.AH.sub.C to bind to both recombinant
and endogenously expressed FAP on cell surface was tested.
[0159] The example was conducted to determine the binding of
L.sub.AH.sub.C to cellular FAP. Both naturally FAP expressing MF-SH
human tumor cells (Shirasuma, K., et al., Cancer 55:2521-2532
(1985)) and FAP-transfected human tumor cell lines were used as
cellular targets. L.sub.AH.sub.C was studied in cytofluorometric
assays evaluating direct binding to target cells as well as by the
inhibitory effect on the binding of either murine F19 or chimeric
cF19 anti-FAP antibodies.
[0160] Antibodies and cell lines used were F19 (murine monoclonal
anti-human FAP antibody, IgG1 subclass), mIgG (murine
immunoglobulin, IgG class), cF19 (chimeric monoclonal anti-human
FAP antibody, IgG1 subclass), L.sub.AH.sub.C (reshaped monoclonal
anti-human FAP antibody, IgG1 subclass), hIgG1 (human
immunoglobulin, IgG1 subclass), MF-SH (human malignant fibrous
histiocytoma cell line), HT-1080 (human fibrosarcoma cell line),
HT-1080FAP clone 33 (HT-1080 cell line transfected with cDNA
encoding human FAP). Antibodies were biotinylated as described in
examples 8 and 12.
Direct Binding of L.sub.AH.sub.C to FAP on the Surface of Human
Tumor Cell Lines
[0161] 5.times.10.sup.5 cells of the tumor cell line under
investigation were incubated with the indicated concentration of
test or control antibody in a total volume of 0.2 ml
phosphate-buffered saline (PBS) supplemented with 1% bovine serum
albumin (BSA) for 30 minutes on ice. Subsequently, cells were
washed twice with 2 ml of PBS, resuspended in 0.2 ml of PBS
supplemented with 1% BSA, a 1:20 dilution of mouse anti-human IgG
FITC-labelled (Dianova) as secondary reagent was added and
incubated for another 30 minutes on ice.
[0162] Alternatively, 5.times.10.sup.5 cells of the tumor cell line
under investigation were incubated with the indicated concentration
of biotin-labelled cF19 in a total volume of 0.2 ml PBS
supplemented with 1% BSA for 30 minutes on ice. Subsequently, cells
were washed twice with 2 ml of PBS, resuspended in 0.2 ml of PBS
supplemented with 1% BSA, and incubated for another 30 minutes on
ice with 1:40 dilution of streptavidin-FITC (Dianova) as secondary
reagent.
[0163] Cells were again washed twice with 2 ml of PBS, resuspended
in a total volume of 0.5 ml of PBS supplemented with 1%
paraformaldehyde (PFA) and kept on ice. Single cell fluorescence
was determined cytofluorometrically by analysing the cellular green
fluorescence at 488 nm in an EPICS XL (Coulter)
fluorescence-activated cell analyzer.
Competition of L.sub.AH.sub.C for Binding of Biotinylated cF19 to
Cell-surface FAP on FAP-expressing Human Cell Lines
[0164] 5.times.10.sup.5 cells of the tumor cell line under
investigation were incubated with the indicated amounts of
unlabeled test or control antibody added together with 1 .mu.g/ml
biotin-labelled cF19 antibody. Subsequently, cells were washed
twice with 2 ml of PBS, resuspended in 0.2 ml of PBS supplemented
with 1% BSA, 1:40 diluted streptavidin-FITC (Dianova) as secondary
reagent and incubated for another 30 minutes on ice.
[0165] Cells were then washed twice with 2 ml of PBS, resuspended
in a total volume of 0.5 ml PBS supplemented with 1% PFA and kept
on ice. Single cell fluorescence was determined
cytofluorometrically by analysing the cellular green fluorescence
at 488 nm in an EPICS XL (Coulter) fluorescence-activated cell
analyzer.
[0166] Both, cF19 and L.sub.AH.sub.C bind in a concentration
dependent manner specifically to to FAP-transfected HT-1080FAP
clone33 human tumor cells (Table 8). No binding to FAP-negative
HT-1080 cells was detectable (Table 9). Both cF19 and
L.sub.AH.sub.C bound in a concentration dependent manner to human
MF-SH cells endogenously expressing FAP (Table 10).
[0167] Biotinylated cF19 bound to human HT-1080FAP clone 33 (Table
11) in a concentration dependent manner. No binding was detectable
to FAP-negative HT-1080 cells (Table 12).
[0168] Binding of biotinylated cF19 to HT-1080FAP clone 33 cells
was inhibited by both unlabelled cF19 and unlabelled L.sub.AH.sub.C
(Table 13).
[0169] Chimeric anti-human FAP monoclonal antibody cF19 as well as
reshaped human anti-human FAP monoclonal antibody L.sub.AH.sub.C
(example 10) were shown to bind directly to FAP expressed on human
cell lines either endogenously expressing this protein or
transfected with cDNA encoding for it. This binding was shown to be
concentration dependent. Binding of biotinylated cF19 could be
inhibited by both unlabelled cF19 and unlabelled
L.sub.AH.sub.C.
[0170] Using cytofluorometric technology, direct binding as well as
inhibition of specifically binding reagents showed specificity of
chimeric cF19 and reshaped L.sub.AH.sub.C human monoclonal
antibodies to cell surface expressed FAP.
12TABLE 8 Binding of anti-FAP antibodies to HT-1080FAP clone 33
cells Concentration of antibody Mean fluorescence intensity [ng/ml]
hIgG1 cF19 L.sub.AH.sub.C 500 0.12 6.65 2.76 100 0.12 1.63 0.66 20
0.12 0.43 0.22 4.0 0.12 0.17 0.15 0.8 0.12 0.14 0.13
[0171]
13TABLE 9 Binding of anti-FAP antibodies to non-transfected HT-1080
cells Concentration of antibody Mean fluorescence intensity [ng/ml]
hIgG1 cF19 L.sub.AH.sub.C 500 0.11 0.11 0.12 100.0 0.11 0.11 0.11
20.0 0.11 0.11 0.12 4.0 0.11 0.11 0.12 0.8 0.11 0.11 0.11
[0172]
14TABLE 10 Binding of anti-FAP antibodies to MF-SH cells
Concentration of antibody Mean fluorescence intensity [ng/ml] hIgG1
cF19 L.sub.AH.sub.C 4,000 0.6 3.6 2.8 2,000 n.d. 3.3 2.5 1,000 n.d.
2.4 1.9 500 n.d. 1.8 1.3 n.d.: not done
[0173]
15TABLE 11 Binding of biotinylated cF19 antibody to HT-1080FAP
clone 33 cells Concentration of antibody Mean fluorescence
intensity [ng/ml] Biotinylated hIgG1 Biotinylated cF19 5,000.0 0.2
36.5 1,000.0 0.2 18.1 200.0 0.2 4.5 40.0 0.2 1.3 8.0 0.2 0.5 1.6
0.3 0.3
[0174]
16TABLE 12 Binding of biotinylated cF19 antibody to non-transfected
HT-1080 cells Concentration of antibody Mean fluorescence intensity
[ng/ml] Biotinylated hIgG1 Biotinylated cF19 5,000.0 0.1 0.1
1,000.0 0.1 0.1 200.0 0.1 0.1 40.0 0.1 0.1 8.0 0.1 0.1 1.6 0.1
0.1
[0175]
17TABLE 13 Competition of anti-FAP antibodies with the binding of
biotinylated cF19 to HT-1080FAP clone 33 cells Concentration of
competitor antibody Mean fluorescence Competitor antibody
[.mu.g/ml] concentration No 0.00 11.2 hIgG1 1.00 9.0 hIgG1 3.16
11.3 hIgG1 10.00 9.8 hIgG1 31.66 10.3 cF19 1.00 7.5 cF19 3.16 4.8
cF19 10.00 1.3 cF19 31.66 1.2 L.sub.AH.sub.C 1.00 8.0
L.sub.AH.sub.C 3.16 5.5 L.sub.AH.sub.C 10.00 2.9 L.sub.AH.sub.C
31.66 1.7 Biotinylated cF19 was used at a concentration of 1
.mu.g/ml in all tests shown in Table 13.
Example 7
In Vitro Immune Effector Functions of Monoclonal Antibody
L.sub.AH.sub.C
[0176] This experiment was conducted to determine the potential of
the monoclonal antibody (mAb) L.sub.AH.sub.C with specificity for
fibroblast activation antigen (FAP) to lyse FAP-expressing targets
in the presence of human complement or human mononuclear
leukocytes, respectively.
[0177] In particular, the ability of L.sub.AH.sub.C to mediate
cytotoxic effects against HT-1080FAP clone 33 cells, which
expressed human FAP on the surface, was studied. Cytotoxicity was
determined in vitro using the following approach:
.sup.51Cr-labelled target cells were incubated in the presence of
L.sub.AH.sub.C with human serum as source of complement or human
MNC (peripheral blood mononuclear cells) as effectors. Release of
.sup.51Cr was measured as measure of target-cell lysis.
[0178] Antibodies and cell lines used were L.sub.AH.sub.C (reshaped
human anti-human FAP IgG1 antibody), hIgG1 (human IgG1 isotype
control), 3S193 (murine monoclonal anti-Lewis.sup.y IgG3 antibody),
mIgG (murine IgG control), HT-1080 (human fibrosarcoma), HT-1080FAP
clone 33, (HT1080 transfected with cDNA encoding human FAP), MCF-7
(human breast adenocarcinoma cell line).
Complement-mediated Lysis of Target Cells by L.sub.AH.sub.C
[0179] Tumor cells were radiolabelled by incubation in RPMI 1640
medium with 100 .mu.l-Ci.sup.51Cr (NEN) at 37.degree. C. for one
hour. Subsequently, cells were washed twice in .sup.51Cr-free
medium and resuspended at a concentration of 2.times.10.sup.5 cells
per ml.
[0180] Human serum as source of complement was freshly prepared
from blood of different volunteers. Blood was taken by puncturing
the arm vein, remained at room temperature for one hour to allow
clotting to occur, and was kept at 4.degree. C. over night. Serum
was separated by centrifugation and taken off from the
sediment.
[0181] The antibody under study was diluted from the stock solution
to the appropriate concentration in RPMI1640 cell culture
medium.
[0182] 1.times.10.sup.5 radiolabelled tumor cells of the indicated
cell line were incubated for 2 h at 37.degree. C. in an incubator
(95% air and 5% CO.sub.2) in the presence of different
concentrations of test or control antibody and 25% (v/v) human
serum as the source of human complement. Incubations were performed
in U-shaped 96-well plates in a total volume of 200 .mu.l RPMI1640
and done in triplicate. After the incubation period, plates were
centrifuged, 100 .mu.l of the supernatant was removed and
radioactivity was counted in a gamma-counter. The total amount of
incorporated radioactivity was determined by measuring 10.sup.4
target cells. Spontaneous release was defined as activity released
from the target cells in the absence of both antibody and
complement during the described incubation period.
[0183] Specific lysis was calculated as follows: 1 Specific lysis (
in % ) } = [ activity sample ] - [ activity spontaneous release ] [
maximum activity ] - [ activity spontaneous release ] .times.
100
Antibody-dependent Cellular Cytotoxicity (ADCC) of
L.sub.AH.sub.C
[0184] Tumor cells were radiolabelled by incubation in RPMI1640
medium with 100 .mu.l-Ci.sup.51Cr at 37.degree. C. for one hour.
Subsequently, cells were washed twice in .sup.51Cr-free medium and
resuspended at a concentration of 2.times.10.sup.5 cells per
ml.
[0185] MNC (peripheral blood mononuclear cells) were prepared from
peripheral blood taken by puncturing the arm vein of different
healthy human volunteers. Clotting was prevented by the addition of
20% citrate buffer. MNC from 4 ml of this blood preparation were
purified by centrifugation (30 minutes at 400 G and room
temperature) on 3 ml of lymphocyte preparation medium (Boehringer
Mannheim, Germany). MNC (peripheral blood mononuclear cells) were
taken off from the gradient, washed three times and diluted with
RPMI1640 to the appropriate concentration. Lymphocyte activated
killer (LAK) cells were derived from MNC (peripheral blood
mononuclear cells) by incubation for 5 days at 37.degree. C. in an
95% air and 5% CO.sub.2 incubator at an initial density of
1.3.times.10.sup.6 cells per ml in the presence of 100 U
recombinant human Interleukin-2 (IL-2). The antibody under study
was diluted from the stock solution to the appropriate
concentration in RPMI1640 cell culture medium.
[0186] 1.times.10.sup.4 radiolabelled tumor cells of the indicated
cell line were incubated for 5 h at 37.degree. C. and 5% CO.sub.2
in the presence of different concentrations of test or control
antibody and MNC. MNC were added in amounts to reach the indicated
effector:target cell ratio. Incubation was performed in U-shaped
96-well plates in a total volume of 200 .mu.L RPMI1640 and done in
duplicate.
[0187] After the incubation period, plates were centrifugated, 100
.mu.l of the supernatant were taken off and radioactivity was
determined in a gamma-counter. The total amount of incorporated
radioactivity was determined by measuring 10.sup.4 target cells.
Spontaneous release was defined as activity released from the
target cells in the absence of both antibody and effector cells
during the described incubation period.
[0188] Specific lysis was calculated as follows: 2 Specific lysis (
in % ) } = [ activity sample ] - [ activity spontaneous release ] [
maximum activity ] - [ activity spontaneous release ] .times.
100
Antibody-mediated Complement Lysis of Tumor Cells
[0189] No L.sub.AH.sub.C-specific complement-mediated lysis (above
that seen with an isotype control) was observed in HT-1080FAP clone
33 cells treated with L.sub.AH.sub.C at concentrations up to 50
.mu.g/ml (Table 14, Table 15a).
[0190] Lytic activity of human serum used as source of complement
was shown by lysis of MCF-7 human breast carcinoma cells in the
presence of 12.5 .mu.g/ml 3S193, a murine monoclonal
anti-Lewis.sup.y antibody with known complement activating ability
(Table 15b).
Antibody-mediated Cellular Lysis of Tumor Cells
[0191] In the presence of L.sub.AH.sub.C at concentrations up to 10
.mu.g/ml, no ADCC (antibody-dependent cellular toxicity) mediated
by human MNC (Table 16) or human LAK cells (lymphokine activated
killer cells, Table 17) of L.sub.AH.sub.C on HT-1080FAP clone 33 as
measured by lysis was detectable above that seen with an isotype
control at an effector:target ratio of 50:1.
[0192] In appropriate in vitro assays with either human complement
or with human MNC as effector mechanisms, human anti-FAP monoclonal
antibody L.sub.AH.sub.C revealed no detectable cytotoxic effects
above isotype controls on FAP-expressing tumor cell line HT-1080FAP
clone 33.
18TABLE 14 Specific complement lysis (in %) of HT-1080FAP clone 33
tumor cell targets mediated by L.sub.AH.sub.C Source of human
serum: HT-1080 clone 33: Concentration of antibody hIgG1 isotype
control L.sub.AH.sub.C A 50 .mu.g/ml 5 4 A 10 .mu.g/ml 5 3 B 50
.mu.g/ml 7 5 B 10 .mu.g/ml 6 5 0 .mu.g/ml 0 0 Incubation: 2 hours
at 37.degree. C., 25% serum from human volunteers A or B,
respectively, as source of complement.
[0193]
19TABLE 15a Specific complement lysis (in %) of HT-1080FAP clone 33
tumor cell targets mediated by human anti-FAP monoclonal antibody
L.sub.AH.sub.C Source of human serum: HT-1080 clone 33:
Concentration of antibody hIgG1 L.sub.AH.sub.C A 10.00 .mu.g/ml 2 1
A 2.50 .mu.g/ml 2 2 A 0.60 .mu.g/ml 1 1 A 0.15 .mu.g/ml 1 2 A 0.00
.mu.g/ml 2 2 B 10.00 .mu.g/ml 2 2 B 2.50 .mu.g/ml 2 2 B 0.60
.mu.g/ml 2 2 B 0.15 .mu.g/ml 2 2 B 0.00 .mu.g/ml 2 2 C 10.00
.mu.g/ml 2 2 C 2.50 .mu.g/ml 1 1 C 0.60 .mu.g/ml 1 1 C 0.15
.mu.g/ml 2 1 C 0.00 .mu.g/ml 3 3 Incubation: 2 hours at 37.degree.
C., 25% serum from human volunteers A, B or C, respectively, as
source of complement.
[0194]
20TABLE 15b Specific complement lysis (in %) of MCF-7 tumor cell
targets mediated by murine anti-Lewis.sup.y monoclonal antibody
3S193 Source of human serum: MCF-7: Concentration of antibody mIgG
3S193 A 10.00 .mu.g/ml 0 21 A 2.50 .mu.g/ml 1 21 A 0.60 .mu.g/ml 0
21 A 0.15 .mu.g/ml 1 18 A 0.00 .mu.g/ml 0 0 B 10.00 .mu.g/ml 1 13 B
2.50 .mu.g/ml 0 17 B 0.60 .mu.g/ml 1 18 B 0.15 .mu.g/ml 1 15 B 0.00
.mu.g/ml 0 0 C 10.00 .mu.g/ml 1 22 C 2.50 .mu.g/ml 0 23 C 0.60
.mu.g/ml 1 26 C 0.15 .mu.g/ml 1 20 C 0.00 .mu.g/ml 1 1 Incubation:
2 hours at 37.degree. C., 25% serum from human volunteers A, B or
C, as source of complement.
[0195]
21TABLE 16 ADCC (antibody-dependant cellular cytotoxicity)
(specific lysis in %) of HT-1080FAP clone 33 target cells by human
MNC (peripheral blood mononuclear cells) mediated by L.sub.AH.sub.C
HT-1080FAP clone 33: Concentration of antibody: HT-1080FAP clone
33: [in .mu.g/ml] hIgG1 L.sub.AH.sub.C 10 2 2 2.5 2 2 0.625 2 2
0.156 3 3 0 3 3 Incubation: 5 hours at 37.degree. C., 10.sup.4
target cells and an effector:target cell ration of 50:1.
[0196]
22TABLE 17 ADCC (antibody-dependent cellular cytotoxicity, specific
lysis in %) of HT-1080FAP clone 33 target cells by LAK cells
(lymphokine activated killer cells) mediated by L.sub.AH.sub.C
Concentration of antibody: HT-1080FAP clone 33: [in .mu.g/ml] hIgG1
L.sub.AH.sub.C 10 12 14 2.5 14 17 0.625 14 21 0.156 15 21 0 14 14
Incubation: 5 hours at 37.degree. C., 10.sup.4 target cells and an
effector:target cell ration of 50:1.
Example 8
Immunohistochemical Analysis of Monoclonal Antibody L.sub.AH.sub.C
Binding to Normal and Neoplastic Human Tissues
[0197] This experiment was performed to determine the binding
characteristics of the humanized mAb L.sub.AH.sub.C to normal and
neoplastic human tissues.
[0198] The following antibodies were used: L.sub.AH.sub.C, cF19,
and the negative control hIgG1 were directly biotinylated according
to methods of the state of the art and used at concentrations of
2.5 to 0.25 mg/ml in 2% BSA/PBS (bovine serum albumin in
phosphate-buffered saline). Murine mAb F19 was used as tissue
culture supernatant of the F19 hybridoma, at dilutions of 1:5 to
1:10 in 2% BSA/PBS.
[0199] The following reagents were used for immunochemical assays:
Streptavidin peroxidase complex (Vector Labs., Burlingame, Calif.,
USA), Avidin-biotin peroxidase complex (Vector Labs.), Biotinylated
horse anti-mouse (Vector Labs.), DAB (diaminobenzidine, Sigma
Chemical Co., St. Louis, Mo., USA), Harris' hematoxylin.
[0200] Fresh frozen tissue samples examined included the following:
Normal colon, breast, lung, stomach, pancreas, skin, larynx,
urinary bladder, smooth and skeletal muscle. Among the tumors
tested were carcinomas from breast, colon, lung, esophagus, uterus,
ovary, pancreas, stomach, and head and neck.
[0201] An indirect immunoperoxidase method was carried out
according to state of the art methods (Garin-Chesa, P., et al.,
"Cell surface glycoprotein of reactive stromal fibroblasts as a
potential antibody target in human epithelial cancers," Proc. Natl.
Acad. Sci. USA 87:7235-7239 (1990)) on five micrometer thickness
fresh frozen sections. DAB was used as a substrate for the final
reaction product. The sections were counterstained with
Harris'hematoxylin and examined for antigen expression.
L.sub.AH.sub.C Expression in Normal Human Tissues
[0202] The normal tissues tested were negative for L.sub.AH.sub.C
expression, except for the normal pancreas in which a subset of
positive endocrine cells in the islets of Langerhans (A cells) were
identified with L.sub.AH.sub.C, cF19 and F19. (Table 18). No
immunoreactivity was observed with the hlgG1 (human immunoglobulin
IgG1 subclass) used as a negative control.
L.sub.AH.sub.C Expression in Tumors
[0203] In the tumor samples, L.sub.AH.sub.C, cF19 and F19 showed an
indistinguishable pattern of expression in the tumor stromal
fibroblasts. A strong and homogeneous expression was found in the
majority of the cases examined, especially in the cancer samples
derived from breast, colon, lung, pancreas and in the squamous cell
carcinomas (SQCC) of the head and neck tested (Table 18). No
immunoreactivity was observed with the hlgG1 used as negative
control.
[0204] L.sub.AH.sub.C, cF19 and F19 showed immunoreactivity with
the tumor stromal fibroblasts in the epithelial cancer samples
tested. No L.sub.AH.sub.C or F19 immuno-reactivity was seen with
either the fibrocytes of the normal organ mesenchyme or the
parenchymal cells of normal adult organs. Anti-FAP immunoreactivity
was only observed in a subset of endocrine cells in the pancreatic
islets, presumably glucagon-producing A cells, and in four of nine
uterine samples tested, representing subsets of stromal fibroblasts
in these tissues.
[0205] Immunohistochemical analysis of L.sub.AH.sub.C in normal
human tissues and FAP-expressing human carcinomas showed
indistinguishable patterns of binding for L.sub.AH.sub.C, cF19 and
murine mAb F19.
23TABLE 18 Immunoreactivity of mAbs L.sub.AH.sub.C, cF19 and F19
with normal human tumor samples Tissue type No. L.sub.AH.sub.C cF19
F19 Breast 4 Epithelial cell ducts/acini - - - Myoepithelial cells
- - - Connective tissue - - - Blood vessels - - - Colon 6 Crypts of
Lieberkuhn - - - Connective tissue - - - Lymphoid tissue - - -
Smooth muscle - - - Blood vessels - - - Myenteric plexus - - - Lung
4 Bronchus: - - - Bronchial epithelium - - - Hyaline cartilage - -
- Connective tissue - - - Mucous glands - - - Alveolus: Pneumocytes
(type I/II) - - - Alveolar phagocytes - - - Alveolar capillaries -
- - Stomach 3 Surface epithelium - - - Gastric glands - - - Chief
cells - - - Parietal (oxyntic) cells - - - Mucous cells - - -
Neuroendocrine cells - - - Connective tissue - - - Blood vessels -
- - Smooth muscle - - - Esophagus 1 Surface epithelium - - -
Connective tissue - - - Small intestine 1 Epithelium of villi &
crypts - - - Connective tissue - - - Smooth muscle - - - Blood
vessels - - - Lymphoid tissue - - - Urinary bladder 2 Urothelium -
- - Connective tissue - - - Smooth muscle - - - Blood vessels - - -
Pancreas 3 Duct epithelium - - - Acinar epithelium - - - Islets of
Langerhans: - - - B-cells - - - A-cells +* +* +* D-cells - - -
Connective tissue - - - Blood vessels - - - Nerves - - - Larynx 1
Squamous epithelium - - - Mucous glands - - - Connective tissue - -
- Hyaline cartilage - - - Blood vessels - - - Skeletal muscle - - -
Lymph node 1 Lymphoid cells - - - Lymph sinuses - - - Connective
tissue - - - Blood vessels - - - Spleen 1 Red & white pulp - -
- Sinuses - - - Connective tissue - - - Liver 1 Hepatocytes - - -
Bile ducts - - - Portal triad - - - Thyroid gland 2 Follicular
epithelium - - - Parafollicular cells - - - Connective tissue - - -
Prostate gland 1 Glandular epithelium - - - Stroma - - - Testicle 1
Seminiferous tubules - - - Stroma - - - Ovary 3 Follicles - - -
Stroma - - - Uterine cervix 1 Epithelium - - - Stroma - - - Uterus
9 Endometrium: - - - glands - - - stroma +** +** +** blood vessels
- - - Myometrium - - - Cerebral cortex 1 Neurons - - - Neurological
cells - - - Blood vessels - - - Cerebellum 1 Molecular layer - - -
Granular cell layer - - - Purkinje cells - - - Blood vessels - - -
Skin 3 Squamous epithelium - - - Melanocytes - - - Skin appendages
- - - Connective tissue - - - Blood vessels - - - Acetone-fixed
frozen sections were tested by the avidin-biotin complex
immunoperoxidase procedure. No number of tissue samples derived
from different individuals tested. *Identification of A-cells,
based on morphology and location within the islets. **Positive
immunoreactivity in the stroma in 4 out of 9 samples tested. The
positive samples represent early (x2) and intermediate (x2) phase
proliferative endometrium.
Example 9
Species Specificity of L.sub.AH.sub.C Binding in Tissue
Sections
[0206] This experiment was conducted to assess the reactivity of
L.sub.AH.sub.C with tissues from mouse, rat, rabbit and cynomolgus
monkeys by immunohistochemical methods.
[0207] Also used in these tests were cF19 and human IgG1 (hIgG1) as
negative controls. The reagents used for immunohistochemistry were
Streptavidin peroxidase complex (Vector Labs., Burlingame, Calif.,
USA), DAB (Sigma Chemical Co., St. Louis, Mo., USA) and Harris'
hematoxylin.
[0208] The following fresh frozen tissue samples from mouse, rat,
rabbit and cynomolgus were tested: Brain, liver, lung, kidney,
stomach, pancreas, intestine, thymus, skin, muscle, heart, spleen,
ovary, uterus and testes. As positive control, sections from normal
human pancreas and a breast carcinoma sample were included in every
assay.
Immunohistochemistry
[0209] An indirect immunoperoxidase method was carried out as
described in the state of the art (Garin-Chesa, P., et al., "Cell
surface glycoprotein of reactive stromal fibroblasts as a potential
antibody target in human epithelial cancers," Proc. Natl. Acad.
Sci. USA 87:7235-7239 (1990)) on five micrometer thickness fresh
frozen sections. The antibodies L.sub.AH.sub.C, cF19 and hlgG1 (at
1 .mu.g/ml) were biotinylated according to the state of the art and
were detected with streptavidin peroxidase complex. DAB was used as
a substrate for the final reaction product. The sections were
counterstained with Harris' hematoxylin and examined for antigen
expression.
[0210] The normal tissues tested did not react with either
L.sub.AH.sub.C or cF19 in the experiments (Table 1).
[0211] The normal human pancreas used as positive control showed
L.sub.AH.sub.C and cF19 binding in a subset of endocrine cells in
the islets of Langerhans as previously described for F19. In
addition, binding of L.sub.AH.sub.C and cF19 was seen in the tumor
stromal fibroblasts in the breast carcinoma sample.
[0212] Immunohistochemical analysis of normal tissues from mouse,
rat, rabbit and cynomolgus failed to detect any binding of either
L.sub.AH.sub.C or cF19, in the experiments performed.
24TABLE 19 Binding of L.sub.AH.sub.C to tissue sections of
non-human species, as determined by immunohistochemistry
Organ/Tissue type Mouse Rat Rabbit Cynomolgus Brain Cerebral cortex
-- -- -- Cerebellum -- -- -- -- Liver Hepatocytes -- -- -- --
Portal triad -- -- -- -- Lung Bronchi -- -- -- -- Alveoli -- -- --
-- Kidney Glomeruli -- -- -- -- Tubular epithelium -- -- -- --
Stomach Glandular epithelium -- -- -- -- Smooth muscle -- -- -- --
Pancreas Exocrine acini -- -- -- -- Endocrine islets -- -- -- --
Intestine Glandular epithelium -- -- -- -- Smooth muscle -- -- --
-- Thymus Lymphocytes -- -- -- -- Skin Keratinocytes -- -- -- --
Sweat glands -- -- -- -- Hair follicles -- -- -- -- Skeletal muscle
-- -- -- -- Heart -- -- -- -- Spleen Lymphocytes -- -- -- -- Ovary
Follicular epithelium -- -- -- -- Stroma -- -- -- -- Uterus
Myometrium -- -- -- -- Cervix uteri -- -- -- -- Testis Tubular
epithelium nt nt nt -- Connective tissue -- -- -- -- nt = not
tested
Example 10
Construction of Cell Lines Producing Chimeric and Reshaped Anti-FAP
Monoclonal Antibodies
[0213] The objective of this experiment was to demonstrate stable
cell lines according to the invention expressing L.sub.AH.sub.C,
L.sub.AH.sub.A, L.sub.BH.sub.B, L.sub.BH.sub.D and cF19 in CHO DG44
cells. Stable cell lines transfected with humanized or chimeric F19
antibodies were produced and their identity was confirmed by PCR
amplification of heavy and light variable regions using genomic DNA
derived from each transfectant as template.
[0214] CHO DG44 cells maintained under serum-free conditions in
SFM-II medium. Lipofectin and SFM-II serum-free medium were
obtained from Gibco/BRL. Geneticin and all restriction enzymes were
obtained from Boehringer Mannheim. Pfu polymerase was obtained from
Stratagene.
[0215] DNA for transfections was purified from E. coli cells using
QiaFilter Maxi Cartridges (Qiagen) as directed by the manufacturer.
All DNA preparations were examined by restriction enzyme digestion.
Sequences of L.sub.AH.sub.C variable regions in their respective
vectors were confirmed using an ABI PRISM 310 Sequencer
(Perkin-Elmer).
[0216] Further information regarding the vectors and DNA sequences
employed is available in the prior examples.
Transfection of CHO DG44 Cells
[0217] Cells in logarithmic growth were plated into 6 well plates
containing 1 ml fresh SFM-II medium. Plasmids encoding heavy and
light chains of humanized or chimeric F19 versions were
cotransfected into CHO DG44 cells using liposomal transfection.
Liposomes were prepared using 6 .mu.l lipofectin reagent and 0.5
.mu.g of each vector (one for the desired heavy chain and one for
the light) as described for LipofectAMINE transfections except that
SFM-II medium was used to dilute all reagents. Twenty-four hours
later, cells were diluted 1:10 into SFM-II medium containing 300
.mu.g/ml Geneticin. After the initial phase of cell killing was
over (10-14 days), the concentration of Geneticin was reduced to
200 mg/ml and methotrexate was added to a final concentration of 5
nM. Methotrexate concentrations were increased after 10-14 days to
a final concentration of 20 nM.
PCR Amplification of Transfectant DNA
[0218] 10.sup.7 CHO DG44 cells were centrifuged in an Eppendorf
microcentrifuge briefly at full speed, washed once with PBS, and
pelleted once again. Genomic DNA was prepared by ethanol
precipitation after SDS lysis and Proteinase K treatment of the
cell pellets.
[0219] A mixture containing one of the following primer pairs,
dNTPs, buffer, and Pfu polymerase was used to amplify either the
heavy or light chain variable region using genomic DNA as template.
The resulting PCR products were digested with the appropriate
restriction enzyme and analyzed by agarose gel electrophoresis to
confirm their identity.
25 Light chain primer set: 5'-GAG ACA TTG TGA CCC AAT CTC C-3' PKN
1690 5'-GAC AGT CAT AAA CTG CCA CAT CTT C-3' PKN.1930.R Heavy chain
primer set: 5'-TTG ACA CGC GTC TCG GGA AGC TT-3' PG 5863 5'-GGC GCA
GAG GAT CCA CTC ACC T-3' PG 6332.R
[0220] The undigested heavy chain PCR product has a predicted size
of 469 bp while the light chain PCR product has a predicted size of
286 bp. Verification of identity was determined by restriction
enzyme digest with BstEII (heavy chain) or NlaIV (light chain).
[0221] CHO cell lines were transfected with L.sub.AH.sub.C,
L.sub.AH.sub.A, L.sub.BH.sub.B and L.sub.BH.sub.D, as well as cF19.
Geneticin-resistant cells were obtained and these cells were
further selected for resistance to methotrexate. PCR amplification,
followed by restriction enzyme cleavage of the light and heavy
chain DNA produced the expected bands and confirmed the identity of
L.sub.AH.sub.C, L.sub.BH.sub.B, L.sub.AH.sub.A and L.sub.BH.sub.D
transfectants.
[0222] The cells described were maintained under serum-free
conditions at all times and were not treated with animal-derived
products such as trypsin.
[0223] Producer cell lines transfected with expressing monoclonal
L.sub.AH.sub.C, L.sub.AH.sub.A, L.sub.BH.sub.B, L.sub.BH.sub.D and
cF19 antibodies were produced. Their identities were confirmed
using PCR amplification and restriction enzyme cleavage of the
resulting PCR products of both their heavy and light chain variable
regions.
Example 11
Expression of Antibody Proteins in Chinese Hamster Ovary DG 44
Cells and their Purification
[0224] The objective of this experiment was to express and purify
L.sub.AH.sub.C, L.sub.AH.sub.A, L.sub.BH.sub.B, and L.sub.BH.sub.D
mAbs to enable their characterization. Other goals included the
establishment of a quantitative ELISA to permit measurement of
antibody concentrations in both crude media samples as well as
purified Ig samples and determination of relative expression levels
of various humanized F19 constructs using this assay.
[0225] Serum-free CHO DG44 cells and USP-grade methotrexate were
obtained from the Biotechnical Production Unit of the Dr. Karl
Thomae GmbH, Biberach, Germany; both products are also commercially
available. Cells were maintained under serum-free conditions at all
times. SFM-II serum-free medium was obtained from Gibco/BRL.
Protein A agarose was from Pierce Chemical (Indianapolis, Ind.,
USA). Human IgG1 standards (Cat. No. 13889), p-Nitrophenyl
phosphate tablets (N 2640), bovine serum albumin (BSA) (A 7906),
and goat anti-human kappa chain specific alkaline
phosphatase-conjugated antibody (A 3813) were obtained from Sigma
Chemical (St. Louis, Mo., USA). Goat anti-human gamma-chain
specific alkaline phosphatase-conjugated antibody was obtained from
Jackson Immunoresearch Laboratories (through Stratech Scientific).
Tris-buffered saline (TBS) consisted of 150 mM NaCl, 50 mM Tris, pH
7.5.
Cell Culture Conditions for Antibody Expression
[0226] Cells were cultured and maintained in T-175 flasks in SFM-II
serum-free medium without agitation. The medium contained 200
.mu.g/ml Geneticin and 20 nM methotrexate without antibiotics.
Cells were passaged by dilution, were not adherent, and grew in
small clusters. When the cells reached stationary phase, the medium
was collected and centrifuged to remove cells and frozen at
-20.degree. C. until needed.
Purification of L.sub.AH.sub.C
[0227] All purification steps were carried out at 4.degree. C. A
C10/10 column (Pharmacia Fine Chemicals) was packed with Protein A
agarose (3 ml bed volume). The column was washed with TBS and
preeluted once with 0.1 M Na citrate, pH 3.0 to insure that no
loosely bound material remained on the column. The column was then
immediately reequilibrated with TBS and stored at 4.degree. C.
Spent culture supernatants were thawed and centrifuged at
10,000.times.g for 30 minutes prior to Protein A chromatography to
remove debris and diluted with an equal volume of TBS. This
material was loaded onto the Protein A column at 0.5 ml/minute
using a P-1 peristaltic pump (Pharmacia) and washed with TBS until
the absorbance at 280 nm was undetectable. Elution of the antibody
was initiated with 0.1 M Na citrate pH 3.0 at approximately 0.2
ml/minute. The elution was monitored at 280 nm and one ml fractions
of the eluted material were collected into tubes containing
sufficient Tris base pH 9 to neutralize the citrate buffer.
Protein-containing fractions were pooled and concentrated using an
Amicon filtration apparatus with a YM-30 filter and dialyzed
against PBS. The column was immediately regenerated with TBS.
Protein dye-binding assays were performed with the BioRad
(Hercules, Calif.) protein determination kit, according to the
manufacturer's instructions, using bovine serum albumin as a
standard.
Human IgG (Gamma Immunoglobulin) ELISA
[0228] ELISA plates were coated overnight with 100 .mu.l of goat
anti-human gamma-chain specific alkaline phosphatase-conjugated
antibody at 0.4 mg/ml in coating buffer at 4.degree. C. Coating
antibody was removed and plates were blocked with 2% BSA in PBS for
2 hours. All subsequent steps were performed at 37.degree. C.
Blocking buffer was replaced with antibody samples or human IgG1
standard diluted in dilution buffer, serially diluted in a 200 ml
volume, and incubated for one hour. Negative controls included
dilution buffer and/or culture medium of nontransfected cells.
Wells were washed and 100 .mu.l of goat anti-human kappa chain
specific alkaline phosphatase-conjugated antibody diluted 1:5000
was added and incubated for one hour. Wells were washed and 100
.mu.l reaction buffer was added and incubated for 30 minutes. The
reaction was stopped by addition of 1 M NaOH and absorbance read at
405 nm in an ELISA plate reader. Results were analyzed by
four-parameter iterative curve fitting.
[0229] Amino acid analysis was performed according to methods
available in the state of the art.
[0230] Monoclonal antibody L.sub.AH.sub.C was produced and purified
to homogeneity using Protein A affinity chromatography. ELISA
assays using human IgG1 as standard indicated L.sub.AH.sub.C
recoveries exceeding 70%. The purity of the material was estimated
to be >90% by SDS-polyacrylamide gel electrophoresis.
Representative expression data and typical purification yields are
shown in Table 20.
26TABLE 20 Expression data and purification yields FAP antibody
proteins in CHO cells Yield Expression levels in improvement crude
media Purified antibody [purified Antibody samples (ELISA) yields
antibody] L.sub.AH.sub.C 7-10 mg/l .about.5-7 mg/l 500-700
L.sub.AH.sub.A 5-7 mg/l .about.3-4 mg/l 300-400 L.sub.BH.sub.B
0.5-1 mg/l .about.0.2-0.5 mg/l 20-50 L.sub.BH.sub.D 0.8-1.5 mg/l
.about.0.3-0.8 mg/l 30-60 Chimeric F19 .about.0.02 mg/l <0.01
mg/l 1 Representative expression data for each of the anti-FAP
antibodies produced in this study are shown. Recoveries after
Protein A agarose affinity chromatography were based on protein
dye-binding measurements of the purified Ig using BSA as a
standard.
Example 12
Binding of Monoclonal Antibody L.sub.AH.sub.C to Isolated
Recombinant Human FAP
[0231] The objective of this study was to characterize binding of
L.sub.AH.sub.C to isolated recombinant human FAP.
CD8-FAP ELISA
[0232] ELISA plates were coated overnight with 100 .mu.l of mouse
anti-rat antibody (Sigma Chemical R0761) at 1:2000 in coating
buffer at 4.degree. C. Coating antibody was removed and plates were
blocked with 2% BSA in PBS for one hour. All subsequent steps were
performed at room temperature. Blocking buffer was replaced with
100 ml of 1 .mu.g/ml rat anti-CD8 antibody (Pharmingen 01041D) and
incubated for one hour. Plates were washed and 100 .mu.l CD8-FAP
culture supernatant (see example 14) (1:2 in PBS) was added and
allowed to bind for one hour. Plates were washed and antibody
samples were added (two-fold serial dilutions) in a 100 .mu.l
volume and incubated for one hour. Negative controls included human
IgG and/or culture medium of nontransfected cells. Wells were
washed and 100 .mu.l of horse radish peroxidase (HRP) conjugated
mouse anti-human IgG1 antibody (Zymed 05-3320) diluted 1:500 in
dilution buffer were added and incubated for one hour. Wells were
washed and 100 .mu.l HRP substrate, (azino-bis
(3-ethylbenzthiazoline 6-sulfonic) acid, Sigma Chemical A9941),
were added and incubated for 60 minutes. The reaction was stopped
by addition of 1 M NaOH and absorbance read at 405/490 nm in an
ELISA plate reader. Results were analyzed by four-parameter curve
iterative curve fitting.
[0233] Alternatively, plates were coated directly with cF19. FAP
(recombinant human FAP, see example 13) was allowed to bind to
these plates as above and biotinylated L.sub.AH.sub.C (.about.1
.mu.g/ml) was then added. Antibody binding was detected with
HRP-streptavidin conjugate as above.
Solubilization of Membrane-bound Human FAP
[0234] FAP-expressing 293FAP I/2 cells or control 293 cells were
washed with PBS and lysed with 1% Triton X-114 in Tris-buffered
saline. Nuclei and debris were removed by centrifugation at
10,000.times.g. The supernatant was phase-partitioned (Estreicher,
A., et al., "Characterization of the cellular binding site for the
urokinase-type plasminogen activator," J. Biol. Chem. 264:1180-1189
(1989)) to enrich membrane proteins. The detergent phase was
collected and diluted in buffer containing 1% Empigen BB
(Calbiochem) to prevent reaggregation of the Triton X-114. This
material was subjected to Concanavalin A agarose chromatography
(Rettig, W. J., et al., "Regulation and heteromeric structure of
the fibroblast activation protein in normal and transformed cells
of mesenchymal and neuroectodermal origin," Cancer Res 53:3327-3335
(1993)).
Biotinylation of L.sub.AH.sub.C
[0235] L.sub.AH.sub.C (1-2 mg) was dialyzed against 50 mM
bicarbonate buffer and biotinylated with a ten-fold molar excess of
sulfosuccinimidyl-6-biotinamido hexanoate (NHS-LC biotin, Pierce
Chemical, Rockford, Ill., USA) for 2 hours at room temperature.
Unreacted product was removed by repeated microdialysis in a
microconcentrator.
Transient Transfections
[0236] COS-7 cells (American Type Tissue Culture Collection,
reference number CRL 1651) were cotransfected by electroporation
with the heavy and light chain vectors encoding L.sub.AH.sub.C.
[0237] Anti-CD8 monoclonal antibody was immobilized onto microtiter
plates. CD8-FAP from medium of insect cells infected with CD8-FAP
baculovirus was allowed to bind to these plates. Spent medium from
COS-7 cell cultures transiently transfected with two separate
vectors encoding L.sub.AH.sub.C was serially diluted and added to
the wells containing the immobilized CD8-FAP. L.sub.AH.sub.C bound
to isolated immobilized CD8-FAP protein (FIG. 35). Culture
supernatants from mock-transfected COS-7 cells failed to
demonstrate binding.
[0238] Recombinant membrane-bound FAP from detergent extracts of
293FAP 1/2 cells or control extracts was serially diluted and
immobilized via chimeric F19 monoclonal antibody bound to
microtiter plates. Biotinylated L.sub.AH.sub.C bound recombinant
human FAP immobilized with cF19 (FIG. 36) in a
concentration-dependent manner.
[0239] L.sub.AH.sub.C recognized isolated immobilized recombinant
human FAP carrying the epitope for murine F19. L.sub.AH.sub.C bound
to both CD8-FAP produced in insect cells, as well as FAP protein
produced in 293FAP I/2 cells.
[0240] Culture supernatants from COS-7 cells transfected with
either heavy and light chain vectors encoding L.sub.AH.sub.C or
without DNA (Control) were collected three days posttransfection.
CD8-FAP was immobilized via an anti-CD8 antibody as described in
the text. Serial dilutions of the COS-7 supernatants were allowed
to bind to the immobilized CD8-FAP and subsequently detected with
an HRP-conjugated anti-human IgG1 antibody.
[0241] Detergent extracts of FAP-expressing 293FAP I/2 cells or
control 293 cells were serially diluted and added to cF19-coated
microtiter plates. Biotinylated L.sub.AH.sub.C was added and
binding of biotinylated L.sub.AH.sub.C was detected with
HRP-conjugated streptavidin.
Example 13
Characterization of HT-1080 Fibrosarcoma Cells and 293 Human
Embryonic Kidney Cells Transfected with cDNA for Human FAP
[0242] Fibroblast activation protein (FAP) is a cell-surface,
membrane-bound protein which carries the F19 epitope and is
expressed on tumor stromal fibroblasts. Cell lines expressing
recombinant FAP protein and matched controls lacking FAP were
generated for the characterization of anti-FAP monoclonal
antibodies.
[0243] Cells used were HT-1080 cells (reference number CCL 121) and
293 human embryonic kidney cells (reference number CRL 1573) were
obtained from the American Type Culture Collection (Maryland, USA).
Transfectam was obtained from Promega (Madison, Wis.). Geneticin
and all restriction enzymes were obtained from Boehringer Mannheim.
DNA for transfections was purified from E. coli cells using
QiaFilter Maxi Cartridges (Qiagen) as directed by the manufacturer.
All DNA preparations were examined by restriction enzyme digestion.
Vector sequences were confirmed using an ABI PRISM 310
Sequencer.
[0244] Further information regarding the vectors and DNA sequences
employed has been described in Scanlan, M. J., et al., "Molecular
cloning of fibroblast activation protein alpha, a member of the
serine protease family selectively expressed in stromal fibroblasts
of epithelial cancers," Proc. Natl. Acad. Sci. USA 89:10832-10836
(1992). The FAP cDNA sequence has been deposited in Genbank
(accession number HS09287).
Cell Culture and Immunoassays
[0245] HT-1080 cells were transfected with 1 mg DNA using
Transfectam according to the manufacturer's instructions. Human
embryonic kidney 293 cells were transfected by calcium phosphate
transfection (Brann, M. R., et al., "Expression of cloned
muscarinic receptor in A9 L cells," Mol. Pharmacol. 32:450-455
(1987)) with 10 mg DNA. Twenty-four hours later, cells were diluted
1:10 into fresh medium containing 200 mg/ml Geneticin. Colonies
were picked and examined by immunofluorescence for FAP expression
as described in Rettig, W. J., et al., "Cell-surface glycoproteins
of human sarcomas: differential expression in normal and malignant
tissues and cultured cells," Proc. Natl. Acad. Sci. USA
85:3110-3114 (1988).
[0246] Immunoprecipitations with cF19 were performed with
metabolically labelled cells as described in Rettig, W. J., et al.,
"Regulation and heteromeric structure of the fibroblast activation
protein in normal and transformed cells of mesenchymal and
neuroectodermal origin," Cancer Res. 53:3327-3335 (1993).
[0247] HT-1080 and 293 cells were tested for FAP antigen expression
in immunofluorescence assays with anti-FAP antibodies and were
found to be antigen-negative. Transfection of these cells with
FAP.38 vector resulted in the generation of Geneticin-resistant
colonies. Isolated colonies were picked and analyzed by
immunofluorescence for FAP expression. Two cell clones were
identified, designated HT-1080FAP clone 33 and 293FAP I/2, which
express cell surface-bound FAP protein, as recognized by cF19
antibody. Staining of nonpermeabilized HT-1080FAP clone 33 cells
and 293FAP I/2 with cF19 antibody confirmed the cell surface
localization of the FAP protein.
[0248] Immunoprecipitation of radiolabelled FAP protein with cF19
from extracts of 35S-methionine labelled HT-1080FAP clone 33 cells
or 293FAP I/2 cells resulted in the appearance of a 93 kilodalton
band after autoradiography. This band is not detectable in
immunoprecipitates of parental HT-1080 or 293 cell extracts.
[0249] Two stably transfected cell lines, HT-1080FAP clone 33 and
293FAP I/2, express FAP on the cell surface as determined in
immunological assays with anti-FAP mAbs. Neither parental HT-1080
cells nor parental 293 cells express detectable levels of FAP.
Example 14
Generation and Characterization of CD8-FAP Fusion Protein
[0250] A soluble form of human FAP (fibroblast activation protein)
in the form of a CD8-FAP fusion protein was produced in insect
cells for the characterization of L.sub.AH.sub.C containing the
binding site for anti-FAP mAbs. Murine CD8 was chosen to permit
secretion of the protein and to provide an additional epitope
tag.
[0251] The cDNA encoding the extracellular domain of CD8,
consisting of the first 189 amino acids of murine CD8.alpha.
(Genbank M12825), was linked to that of the extracellular domain of
FAP (amino acids 27 to 760), essentially as described by Lane, et
al. (Lane, P., et al., "Soluble CD40 ligand can replace the normal
T cell-derived CD40 ligand signal to B cells in T cell-dependent
activation," J. Exp. Med. 177:1209-1213 (1993)) using standard PCR
protocols. The authenticity of all clones was verified by DNA
sequencing. The resulting DNA was inserted into the pVL1393 vector
(Invitrogen) and transfection of Sf9 cells (Invitrogen) with this
vector and amplification of the resulting recombinant baculovirus
were performed as described (Baculovirus Expression Vectors. A
Laboratory Manual, O'Reilly, D. R., et al., eds., Oxford University
Press:, New York (1994)). The spent medium of High Five.TM. cells
(Invitrogen) infected with recombinant CD8-FAP baculovirus for four
days was collected and cleared by ultracentrifugation.
[0252] The CD8-FAP ELISA (enzyme-linked immunosorbent assay) has
been described above (example 12).
[0253] Insect cell cultures infected with CD8-FAP virus secreted a
fusion protein into the medium which carries the F19 epitope and is
recognized by an anti-FAP antibody (FIG. 1). Neither the cell
culture medium alone nor medium from insect cells infected with
CD8-CD40L fusion protein bound anti-FAP antibody.
[0254] Soluble CD8-FAP protein carrying the F19 epitope was
secreted into the medium of infected insect cell cultures. Culture
supernatant from cells infected with a control construct did not
contain antigen bearing the F19 epitope.
[0255] A soluble form of FAP, CD8-FAP, was produced in insect cells
and CD8-FAP was shown to carry the epitope recognized by cF19.
[0256] Supernatants from insect cells infected with recombinant
baculovirus encoding either CD8-FAP or CD8-CD40L fusion protein
were collected four days postinfection. Cell culture medium without
cells was used as an additional control (medium). Serial dilutions
of these materials were added to anti-CD8 antibody-coated
microtiter plates and allowed to bind. cF19 (1 mg/ml) was
subsequently added and allowed to bind. Bound cF19 was detected
with horseradish peroxidase-conjugated anti-human antibody.
Sequence CWU 1
1
108 1 339 DNA Homo sapiens 1 gacattgtga tgacccaatc tccagactct
ttggctgtgt ctctagggga gagggccacc 60 atcaactgca agtccagtca
gagcctttta tattctagaa atcaaaagaa ctacttggcc 120 tggtatcagc
agaaaccagg acagccaccc aaactcctca tcttttgggc tagcactagg 180
gaatctgggg tacctgatag gttcagtggc agtgggtttg ggacagactt caccctcacc
240 attagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata
ttttagctat 300 ccgctcacgt tcggacaagg gaccaaggtg gaaataaaa 339 2 113
PRT Homo sapiens 2 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Leu Leu Tyr Ser 20 25 30 Arg Asn Gln Lys Asn Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile
Phe Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe
Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95
Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100
105 110 Lys 3 339 DNA Homo sapiens 3 gacattgtga tgacccaatc
tccagactct ttggctgtgt ctctagggga gagggccacc 60 atcaactgca
agtccagtca gagcctttta tattctagaa atcaaaagaa ctacttggcc 120
tggttccagc agaaaccagg acagccaccc aaactcctca tcttttgggc tagcactagg
180 gaatctgggg tacctgatag gttcagtggc agtgggtttg ggacagactt
caccctcacc 240 attagcagcc tgcaggctga agatgtggca gtttatgact
gtcaacaata ttttagctat 300 ccgctcacgt tcggacaagg gaccaaggtg
gaaataaaa 339 4 113 PRT Homo sapiens 4 Asp Ile Val Met Thr Gln Ser
Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile
Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Arg Asn Gln
Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 Pro
Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55
60 Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr
65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Asp Cys
Gln Gln 85 90 95 Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110 Lys 5 339 DNA Homo sapiens 5 gacattgtga
tgacccaatc tccagactct ttggctgtgt ctctagggga gagggccacc 60
atcaactgca agtccagtca gagcctttta tattctagaa atcaaaagaa ctacttggcc
120 tggtatcagc agaaaccagg acagccaccc aaactcctca tctattgggc
tagcactagg 180 gaatctgggg tacctgatag gttcagtggc agtgggtttg
ggacagactt caccctcacc 240 attagcagcc tgcaggctga agatgtggca
gtttattact gtcagcaata ttttagctat 300 ccgctcacgt tcggacaagg
gaccaaggtg gaaataaaa 339 6 113 PRT Homo sapiens 6 Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30
Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35
40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe
Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln 85 90 95 Tyr Phe Ser Tyr Pro Leu Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys 7 372 DNA Homo sapiens
7 caggtgcaac tagtgcagtc cggcgccgaa gtgaagaaac ccggtgcttc cgtgaaagtc
60 agctgtaaaa ctagtagata caccttcact gaatacacca tacactgggt
tagacaggcc 120 cctggccaaa ggctggagtg gataggaggt attaatccta
acaatggtat tcctaactac 180 aaccagaagt tcaagggccg ggccaccttg
accgtaggca agtctgccag caccgcctac 240 atggaactgt ccagcctgcg
ctccgaggac actgcagtct actactgcgc cagaagaaga 300 atcgcctatg
gttacgacga gggccatgct atggactact ggggtcaagg aacccttgtc 360
accgtctcct ca 372 8 124 PRT Homo sapiens 8 Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Ile
His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Arg Ala Thr Leu Thr Val Gly Lys Ser Ala Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu
Gly His Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 9 372 DNA Homo sapiens 9 caggtgcaac tagtgcagtc
cggcgccgaa gtgaagaaac ccggtgcttc cgtgaaagtc 60 agctgtaaaa
ctagtagata caccttcact gaatacacca tacactgggt tagacaggcc 120
cctggccaaa ggctggagtg gataggaggt attaatccta acaatggtat tcctaactac
180 aaccagaagt tcaagggccg ggccaccttg accgtaggca agtctgccag
caccgcctac 240 atggaactgt ccagcctgcg ctccgaggac actgcagtct
acttctgcgc cagaagaaga 300 atcgcctatg gttacgacga gggccatgct
atggactact ggggtcaagg aacccttgtc 360 accgtctcct ca 372 10 124 PRT
Homo sapiens 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr
Thr Phe Thr Glu Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro
Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn
Gly Ile Pro Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr
Leu Thr Val Gly Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala
Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp 100 105
110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 11 372
DNA Homo sapiens 11 caggtgcaac tagtgcagtc cggcgccgaa gtgaagaaac
ccggtgcttc cgtgaaagtc 60 agctgtaaaa ctagtagata caccttcact
gaatacacca tacactgggt tagacaggcc 120 cctggccaaa ggctggagtg
gataggaggt attaatccta acaatggtat tcctaactac 180 aaccagaagt
tcaagggccg ggtcaccatc accgtagaca cctctgccag caccgcctac 240
atggaactgt ccagcctgcg ctccgaggac actgcagtct actactgcgc cagaagaaga
300 atcgcctatg gttacgacga gggccatgct atggactact ggggtcaagg
aacccttgtc 360 accgtctcct ca 372 12 124 PRT Homo sapiens 12 Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr 20
25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Thr Ser
Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Ile Ala Tyr
Gly Tyr Asp Glu Gly His Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 13 372 DNA Homo sapiens 13
caggtgcaac tagtgcagtc cggcgccgaa gtgaagaaac ccggtgcttc cgtgaaagtc
60 agctgtaaaa ctagtagata caccttcact gaatacacca tacactgggt
tagacaggcc 120 cctggccaaa ggctggagtg gataggaggt attaatccta
acaatggtat tcctaactac 180 aaccagaagt tcaagggccg ggtcaccatc
accgtagaca cctctgccag caccgcctac 240 atggaactgt ccagcctgcg
ctccgaggac actgcagtct acttctgcgc cagaagaaga 300 atcgcctatg
gttacgacga gggccatgct atggactact ggggtcaagg aacccttgtc 360
accgtctcct ca 372 14 124 PRT Homo sapiens 14 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr
Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40
45 Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Thr Ser Ala Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Phe Cys 85 90 95 Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp
Glu Gly His Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 15 372 DNA Homo sapiens 15 caggtgcaac
tagtgcagtc cggcgccgaa gtgaagaaac ccggtgcttc cgtgaaagtc 60
agctgtaaaa ctagtggata caccttcact gaatacacca tacactgggt tagacaggcc
120 cctggccaaa ggctggagtg gataggaggt attaatccta acaatggtat
tcctaactac 180 aaccagaagt tcaagggccg ggtcaccatc accgtagaca
cctctgccag caccgcctac 240 atggaactgt ccagcctgcg ctccgaggac
actgcagtct actactgcgc cagaagaaga 300 atcgcctatg gttacgacga
gggccatgct atggactact ggggtcaagg aacccttgtc 360 accgtctcct ca 372
16 124 PRT Homo sapiens 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Ile His Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn
Pro Asn Asn Gly Ile Pro Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly
Arg Val Thr Ile Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 17 220 PRT Homo sapiens 17 Asp Ile Val Met Ser Gln Ser Pro Ser
Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys
Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Arg Asn Gln Lys Asn
Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys
Leu Leu Ile Phe Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro
Asp Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Asn Leu Thr 65 70
75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Asp Cys Gln
Gln 85 90 95 Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195
200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220
18 453 PRT Homo sapiens 18 Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Met Ser Cys Lys Thr Ser
Arg Tyr Thr Phe Thr Glu Tyr Thr 20 25 30 Ile His Trp Val Arg Gln
Ser His Gly Lys Ser Leu Glu Trp Ile Gly 35 40 45 Gly Ile Asn Pro
Asn Asn Gly Ile Pro Asn Tyr Asn Gln Lys Phe Lys 50 55 60 Gly Arg
Ala Thr Leu Thr Val Gly Lys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80
Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 85
90 95 Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp
Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210
215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330
335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu
Ser Pro Gly Lys 450 19 321 DNA Homo sapiens 19 cgtactgtgg
ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag
120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac
agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc
tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc
catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 20
107 PRT Homo sapiens 20 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 21 990 DNA
Homo sapiens 21 gcctccacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc
300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact
cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc
tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc
cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc
gggtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990 22 330 PRT Homo sapiens 22 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 23 427 DNA Homo sapiens 23 aagcttgccg ccaccatgga
ttcacaggcc caggttctta tgttactgcc gctatgggta 60 tctggtacct
gtggggacat tgtgatgtca cagtctccat cctccctagc tgtgtcagtt 120
ggagagaagg ttactatgag ctgcaagtcc agtcagagcc ttttatatag tcgtaatcaa
180 aagaactact tggcctggtt ccagcagaag ccagggcagt ctcctaaact
gctgattttc 240 tgggcatcca ctagggaatc tggggtccct gatcgcttca
caggcagtgg atttgggacg 300 gatttcaatc tcaccatcag cagtgtgcag
gctgaggacc tggcagttta tgactgtcag 360 caatatttta gctatccgct
cacgttcggt gctgggacca agctggagct gaaacgtgag 420 tggatcc 427 24 133
PRT Homo sapiens 24 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Pro
Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln
Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr
Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Arg Asn
Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln 50 55 60 Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp 85 90 95
Phe Asn Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100
105 110 Asp Cys Gln Gln Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Ala Gly
Thr 115 120 125 Lys Leu Glu Leu Lys 130 25 457 DNA Homo sapiens 25
aagcttgccg ccaccatggg atggagctgg gtctttctct ttctcctgtc aggaactgca
60 ggtgtcctct ctgaggtcca gctgcaacag tctggacctg agctggtgaa
gcctggggct 120 tcagtaaaga tgtcctgcaa gacttctaga tacacattca
ctgaatacac catacactgg 180 gtgagacaga gccatggaaa gagccttgag
tggattggag gtattaatcc taacaatggt 240 attcctaact acaaccagaa
gttcaagggc agggccacat tgactgtagg caagtcctcc 300 agcaccgcct
acatggagct ccgcagcctg acatctgagg attctgcggt ctatttctgt 360
gcaagaagaa gaatcgccta tggttacgac gagggccatg ctatggacta ctggggtcaa
420 ggaacctcag tcaccgtctc ctcaggtgag tggatcc 457 26 143 PRT Homo
sapiens 26 Met Gly Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Gly Thr
Ala Gly 1 5 10 15 Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys
Thr Ser Arg Tyr Thr Phe 35 40 45 Thr Glu Tyr Thr Ile His Trp Val
Arg Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Gly Ile
Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys
Gly Arg Ala Thr Leu Thr Val Gly Lys Ser Ser Ser 85 90 95 Thr Ala
Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110
Tyr Phe Cys Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His 115
120 125 Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135 140 27 8068 DNA Homo sapiens 27 gaattccagc acactggcgg
ccgttactag ttattaatag taatcaatta cggggtcatt 60 agttcatagc
ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 120
ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
180 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
ctgcccactt 240 ggcagtacat caagtgtatc atatgccaag tacgccccct
attgacgtca atgacggtaa 300 atggcccgcc tggcattatg cccagtacat
gaccttatgg gactttccta cttggcagta 360 catctacgta ttagtcatcg
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 420 gcgtggatag
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 480
gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
540 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
gagctcgttt 600 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt
tttgacctcc atagaagaca 660 ccgggaccga tccagcctcc gcggccggga
acggtgcatt ggaacgcgga ttccccgtgc 720 caagagtgac gtaagtaccg
cctatagagt ctataggccc acccccttgg cttcttatgc 780 atgctatact
gtttttggct tggggtctat acacccccgc ttcctcatgt tataggtgat 840
ggtatagctt agcctatagg tgtgggttat tgaccattat tgaccactcc cctattggtg
900 acgatacttt ccattactaa tccataacat ggctctttgc cacaactctc
tttattggct 960 atatgccaat acactgtcct tcagagactg acacggactc
tgtattttta caggatgggg 1020 tctcatttat tatttacaaa ttcacatata
caacaccacc gtccccagtg cccgcagttt 1080 ttattaaaca taacgtggga
tctccacgcg aatctcgggt acgtgttccg gacatgggct 1140 cttctccggt
agcggcggag cttctacatc cgagccctgc tcccatgcct ccagcgactc 1200
atggtcgctc ggcagctcct tgctcctaac agtggaggcc agacttaggc acagcacgat
1260 gcccaccacc accagtgtgc cgcacaaggc cgtggcggta gggtatgtgt
ctgaaaatga 1320 gctcggggag cgggcttgca ccgctgacgc atttggaaga
cttaaggcag cggcagaaga 1380 agatgcaggc agctgagttg ttgtgttctg
ataagagtca gaggtaactc ccgttgcggt 1440 gctgttaacg gtggagggca
gtgtagtctg agcagtactc gttgctgccg cgcgcgccac 1500 cagacataat
agctgacaga ctaacagact gttcctttcc atgggtcttt tctgcagtca 1560
ccgtccttga cacgcgtctc gggaagcttg ccgccaccat ggattcacag gcccaggttc
1620 ttatgttact gccgctatgg gtatctggta cctgtgggga cattgtgatg
tcacagtctc 1680 catcctccct agctgtgtca gttggagaga aggttactat
gagctgcaag tccagtcaga 1740 gccttttata ttctagaaat caaaagaact
acttggcctg gttccagcag aagccagggc 1800 agtctcctaa actgctgatt
ttctgggcat ccactaggga atctggggtc cctgatcgct 1860 tcacaggcag
tggatttggg acggatttca atctcaccat cagcagtgtg caggctgagg 1920
acctggcagt ttatgactgt cagcaatatt ttagctatcc gctcacgttc ggtgctggga
1980 ccaagctgga gctgaaacgt gagtggatcc atctgggata agcatgctgt
tttctgtctg 2040 tccctaacat gccctgtgat tatgcgcaaa caacacaccc
aagggcagaa ctttgttact 2100 taaacaccat cctgtttgct tctttcctca
ggaactgtgg ctgcaccatc tgtcttcatc 2160 ttcccgccat ctgatgagca
gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 2220 aacttctatc
ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 2280
aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc
2340 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg
cgaagtcacc 2400 catcagggcc tgagctcgcc cgtcacaaag agcttcaaca
ggggagagtg ttagagggag 2460 aagtgccccc acctgctcct cagttccagc
ctgaccccct cccatccttt ggcctctgac 2520 cctttttcca caggggacct
acccctattg cggtcctcca gctcatcttt cacctcaccc 2580 ccctcctcct
ccttggcttt aattatgcta atgttggagg agaatgaata aataaagtga 2640
atctttgcac ctgtggtgga tctaataaaa gatatttatt ttcattagat atgtgtgttg
2700 gttttttgtg tgcagtgcct ctatctggag gccaggtagg gctggccttg
ggggaggggg 2760 aggccagaat gactccaaga gctacaggaa ggcaggtcag
agaccccact ggacaaacag 2820 tggctggact ctgcaccata acacacaatc
aacaggggag tgagctggaa atttgctagc 2880 gaattcttga agacgaaagg
gcctcgtgat acgcctattt ttataggtta atgtcatgat 2940 aataatggtt
tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 3000
ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
3060 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
gtgtcgccct 3120 tattcccttt tttgcggcat tttgccttcc tgtttttgct
cacccagaaa cgctggtgaa 3180 agtaaaagat gctgaagatc agttgggtgc
acgagtgggt tacatcgaac tggatctcaa 3240 cagcggtaag atccttgaga
gttttcgccc cgaagaacgt tttccaatga tgagcacttt 3300 taaagttctg
ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg 3360
tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
3420 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
tgagtgataa 3480 cactgcggcc aacttacttc tgacaacgat cggaggaccg
aaggagctaa ccgctttttt 3540 gcacaacatg ggggatcatg taactcgcct
tgatcgttgg gaaccggagc tgaatgaagc 3600 cataccaaac gacgagcgtg
acaccacgat gcctgcagca atggcaacaa cgttgcgcaa 3660 actattaact
ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 3720
ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
3780 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
tggggccaga 3840 tggtaagccc tcccgtatcg tagttatcta cacgacgggg
agtcaggcaa ctatggatga 3900 acgaaataga cagatcgctg agataggtgc
ctcactgatt aagcattggt aactgtcaga 3960 ccaagtttac tcatatatac
tttagattga tttaaaactt catttttaat ttaaaaggat 4020 ctaggtgaag
atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 4080
ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
4140 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
tttgtttgcc 4200 ggatcaagag ctaccaactc tttttccgaa ggtaactggc
ttcagcagag cgcagatacc 4260 aaatactgtc cttctagtgt agccgtagtt
aggccaccac ttcaagaact ctgtagcacc 4320 gcctacatac ctcgctctgc
taatcctgtt accagtggct gctgccagtg gcgataagtc 4380 gtgtcttacc
gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 4440
aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
4500 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
cggacaggta 4560 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
gagcttccag ggggaaacgc 4620 ctggtatctt tatagtcctg tcgggtttcg
ccacctctga cttgagcgtc gatttttgtg 4680 atgctcgtca ggggggcgga
gcctatggaa aaacgccagc aacgcggcct ttttacggtt 4740 cctggccttt
tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 4800
ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
4860 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
ttctccttac 4920 gcatctgtgc ggtatttcac accgcatatg gtgcactctc
agtacaatct gctctgatgc 4980 cgcatagtta agccagtata cactccgcta
tcgctacgtg actgggtcat ggctgcgccc 5040 cgacacccgc caacacccgc
tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 5100 tacagacaag
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 5160
ccgaaacgcg cgaggcagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
5220 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
caggctcccc 5280 agcaggcaga agtatgcaaa gcatgcatct caattagtca
gcaaccatag tcccgcccct 5340 aactccgccc atcccgcccc taactccgcc
cagttccgcc cattctccgc cccatggctg 5400 actaattttt tttatttatg
cagaggccga ggccgcctcg gcctctgagc tattccagaa 5460 gtagtgagga
ggcttttttg gaggcctagg cttttgcaaa aagctagctt cacgctgccg 5520
caagcactca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca
5580 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa
gggaaaacgc 5640 aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
tggcgatagc tagactgggc 5700 ggttttatgg acagcaagcg aaccggaatt
gccagctggg gcgccctctg gtaaggttgg 5760 gaagccctgc aaagtaaact
ggatggcttt cttgccgcca aggatctgat ggcgcagggg 5820 atcaagatct
gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt 5880
gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
5940 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
gcccggttct 6000 ttttgtcaag accgacctgt ccggtgccct gaatgaactg
caggacgagg cagcgcggct 6060 atcgtggctg gccacgacgg gcgttccttg
cgcagctgtg ctcgacgttg tcactgaagc 6120 gggaagggac tggctgctat
tgggcgaagt gccggggcag gatctcctgt catctcacct 6180 tgctcctgcc
gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 6240
tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
6300 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
ggctcgcgcc 6360 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
ggcgaggatc tcgtcgtgac 6420 ccatggcgat gcctgcttgc cgaatatcat
ggtggaaaat ggccgctttt ctggattcat 6480 cgactgtggc cggctgggtg
tggcggaccg ctatcaggac atagcgttgg ctacccgtga 6540 tattgctgaa
gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 6600
cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
6660 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
agatttcgat 6720 tccaccgccg ccttctatga aaggttgggc ttcggaatcg
ttttccggga cgccggctgg 6780 atgatcctcc agcgcgggga tctcatgctg
gagttcttcg cccaccccgg gctcgatccc 6840 ctcgcgagtt ggttcagctg
ctgcctgagg ctggacgacc tcgcggagtt ctaccggcag 6900 tgcaaatccg
tcggcatcca ggaaaccagc agcggctatc cgcgcatcca tgcccccgaa 6960
ctgcaggagt ggggaggcac gatggccgct ttggtcccgg atctttgtga aggaacctta
7020 cttctgtggt gtgacataat tggacaaact acctacagag atttaaagct
ctaaggtaaa 7080 tataaaattt ttaagtgtat aatgtgttaa actactgatt
ctaattgttt gtgtatttta 7140 gattccaacc tatggaactg atgaatggga
gcagtggtgg aatgccttta atgaggaaaa 7200 cctgttttgc tcagaagaaa
tgccatctag tgatgatgag gctactgctg actctcaaca 7260 ttctactcct
ccaaaaaaga agagaaaggt agaagacccc aaggactttc cttcagaatt 7320
gctaagtttt ttgagtcatg ctgtgtttag taatagaact cttgcttgct ttgctattta
7380 caccacaaag gaaaaagctg cactgctata caagaaaatt atggaaaaat
attctgtaac 7440 ctttataagt aggcataaca gttataatca taacatactg
ttttttctta ctccacacag 7500 gcatagagtg tctgctatta ataactatgc
tcaaaaattg tgtaccttta gctttttaat 7560 ttgtaaaggg gttaataagg
aatatttgat gtatagtgcc ttgactagag atcataatca 7620 gccataccac
atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga 7680
acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
7740 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
tcactgcatt 7800 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
tgtctggatc taataaaaga 7860 tatttatttt cattagatat gtgtgttggt
tttttgtgtg cagtgcctct atctggaggc 7920 caggtagggc tggccttggg
ggagggggag gccagaatga ctccaagagc tacaggaagg 7980 caggtcagag
accccactgg acaaacagtg gctggactct gcaccataac acacaatcaa 8040
caggggagtg agctggaaat ttgctagc 8068 28 240 PRT Homo sapiens 28 Met
Asp Ser Gln Ala Gln Val Leu Met Leu Leu Pro Leu Trp Val Ser 1 5 10
15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala
20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser
Gln Ser 35 40 45 Leu Leu Tyr Ser Arg Asn Gln Lys Asn Tyr Leu Ala
Trp Phe Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
Phe Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe
Thr Gly Ser Gly Phe Gly Thr Asp 85 90 95 Phe Asn Leu Thr Ile Ser
Ser Val Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Asp Cys Gln Gln
Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 115 120 125 Lys Leu
Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145
150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235 240 29 7731 DNA Homo sapiens 29 ttgaagacga aagggcctcg
tgatacgcct atttttatag gttaatgtca tgataataat 60 ggtttcttag
acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 120
atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
180 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
cccttattcc 240 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
gaaacgctgg tgaaagtaaa 300 agatgctgaa gatcagttgg gtgcacgagt
gggttacatc gaactggatc tcaacagcgg 360 taagatcctt gagagttttc
gccccgaaga acgttttcca atgatgagca cttttaaagt 420 tctgctatgt
ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg 480
catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
540 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
ataacactgc 600 ggccaactta cttctgacaa cgatcggagg accgaaggag
ctaaccgctt ttttgcacaa 660 catgggggat catgtaactc gccttgatcg
ttgggaaccg gagctgaatg aagccatacc 720 aaacgacgag cgtgacacca
cgatgcctgc agcaatggca acaacgttgc gcaaactatt 780 aactggcgaa
ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 840
taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
900 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
cagatggtaa 960 gccctcccgt atcgtagtta tctacacgac ggggagtcag
gcaactatgg atgaacgaaa 1020 tagacagatc gctgagatag gtgcctcact
gattaagcat tggtaactgt cagaccaagt 1080 ttactcatat atactttaga
ttgatttaaa acttcatttt taatttaaaa ggatctaggt 1140 gaagatcctt
tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 1200
agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
1260 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
tgccggatca 1320 agagctacca actctttttc cgaaggtaac tggcttcagc
agagcgcaga taccaaatac 1380 tgtccttcta gtgtagccgt agttaggcca
ccacttcaag aactctgtag caccgcctac 1440 atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 1500 taccgggttg
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 1560
gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
1620 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
ggtatccggt 1680 aagcggcagg gtcggaacag gagagcgcac gagggagctt
ccagggggaa acgcctggta 1740 tctttatagt cctgtcgggt ttcgccacct
ctgacttgag cgtcgatttt tgtgatgctc 1800 gtcagggggg cggagcctat
ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 1860 cttttgctgg
ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 1920
ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
1980 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
ttacgcatct 2040 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
atctgctctg atgccgcata 2100 gttaagccag tatacactcc gctatcgcta
cgtgactggg tcatggctgc gccccgacac 2160 ccgccaacac ccgctgacgc
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 2220 caagctgtga
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 2280
cgcgcgaggc agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
2340 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
gactaatttt 2400 ttttatttat gcagaggccg aggccgcctc ggcctctgag
ctattccaga agtagtgagg 2460 aggctttttt ggaggcctag gcttttgcaa
aaagctagct tacagctcag ggctgcgatt 2520 tcgcgccaaa cttgacggca
atcctagcgt gaaggctggt aggattttat ccccgctgcc 2580 atcatggttc
gaccattgaa ctgcatcgtc gccgtgtccc aaaatatggg gattggcaag 2640
aacggagacc taccctggcc tccgctcagg aacgagttca agtacttcca aagaatgacc
2700 acaacctctt cagtggaagg taaacagaat ctggtgatta tgggtaggaa
aacctggttc 2760 tccattcctg agaagaatcg acctttaaag gacagaatta
atatagttct cagtagagaa 2820 ctcaaagaac caccacgagg agctcatttt
cttgccaaaa gtttggatga tgccttaaga 2880 cttattgaac aaccggaatt
ggcaagtaaa gtagacatgg tttggatagt cggaggcagt 2940 tctgtttacc
aggaagccat gaatcaacca ggccacctca gactctttgt gacaaggatc 3000
atgcaggaat ttgaaagtga cacgtttttc ccagaaattg atttggggaa atataaactt
3060 ctcccagaat acccaggcgt cctctctgag gtccaggagg aaaaaggcat
caagtataag 3120 tttgaagtct acgagaagaa agactaacag gaagatgctt
tcaagttctc tgctcccctc 3180 ctaaagctat gcatttttat aagaccatgg
gacttttgct ggctttagat ctttgtgaag 3240 gaaccttact tctgtggtgt
gacataattg gacaaactac ctacagagat ttaaagctct 3300 aaggtaaata
taaaattttt aagtgtataa tgtgttaaac tactgattct aattgtttgt 3360
gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa tgcctttaat
3420 gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc
tactgctgac 3480 tctcaacatt ctactcctcc aaaaaagaag agaaaggtag
aagaccccaa ggactttcct 3540 tcagaattgc taagtttttt gagtcatgct
gtgtttagta atagaactct tgcttgcttt 3600 gctatttaca ccacaaagga
aaaagctgca ctgctataca agaaaattat ggaaaaatat 3660 tctgtaacct
ttataagtag gcataacagt tataatcata acatactgtt ttttcttact 3720
ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg tacctttagc
3780 tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt
gactagagat 3840 cataatcagc cataccacat ttgtagaggt tttacttgct
ttaaaaaacc tcccacacct 3900 ccccctgaac ctgaaacata aaatgaatgc
aattgttgtt gttaacttgt ttattgcagc 3960 ttataatggt tacaaataaa
gcaatagcat cacaaatttc acaaataaag catttttttc 4020 actgcattct
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcta 4080
ataaaagata tttattttca ttagatatgt gtgttggttt tttgtgtgca gtgcctctat
4140 ctggaggcca ggtagggctg gccttggggg agggggaggc cagaatgact
ccaagagcta 4200 caggaaggca ggtcagagac cccactggac aaacagtggc
tggactctgc accataacac 4260 acaatcaaca ggggagtgag ctggaaattt
gctagcgaat tccagcacac tggcggccgt 4320 tactagttat taatagtaat
caattacggg gtcattagtt catagcccat atatggagtt 4380 ccgcgttaca
taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 4440
attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
4500 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
tgtatcatat 4560 gccaagtacg ccccctattg acgtcaatga cggtaaatgg
cccgcctggc attatgccca 4620 gtacatgacc ttatgggact ttcctacttg
gcagtacatc tacgtattag tcatcgctat 4680 taccatggtg atgcggtttt
ggcagtacat caatgggcgt ggatagcggt ttgactcacg 4740 gggatttcca
agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca 4800
acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
4860 tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag 4920 acgccatcca cgctgttttg acctccatag aagacaccgg
gaccgatcca gcctccgcgg 4980 ccgggaacgg tgcattggaa cgcggattcc
ccgtgccaag agtgacgtaa gtaccgccta 5040 tagagtctat aggcccaccc
ccttggcttc ttatgcatgc tatactgttt ttggcttggg 5100 gtctatacac
ccccgcttcc tcatgttata ggtgatggta tagcttagcc tataggtgtg 5160
ggttattgac cattattgac cactccccta ttggtgacga tactttccat tactaatcca
5220 taacatggct ctttgccaca actctcttta ttggctatat gccaatacac
tgtccttcag 5280 agactgacac ggactctgta tttttacagg atggggtctc
atttattatt tacaaattca 5340 catatacaac accaccgtcc ccagtgcccg
cagtttttat taaacataac gtgggatctc 5400 cacgcgaatc tcgggtacgt
gttccggaca tgggctcttc tccggtagcg gcggagcttc 5460 tacatccgag
ccctgctccc atgcctccag cgactcatgg tcgctcggca gctccttgct 5520
cctaacagtg gaggccagac ttaggcacag cacgatgccc accaccacca gtgtgccgca
5580 caaggccgtg gcggtagggt atgtgtctga aaatgagctc ggggagcggg
cttgcaccgc 5640 tgacgcattt ggaagactta aggcagcggc agaagaagat
gcaggcagct gagttgttgt 5700 gttctgataa gagtcagagg taactcccgt
tgcggtgctg ttaacggtgg agggcagtgt 5760 agtctgagca gtactcgttg
ctgccgcgcg cgccaccaga cataatagct gacagactaa 5820 cagactgttc
ctttccatgg gtcttttctg cagtcaccgt ccttgacacg cgtctcggga 5880
agcttgccgc caccatggga tggagctggg tctttctctt tctcctgtca ggaactgcag
5940 gtgtcctctc tgaggtccag ctgcaacagt ctggacctga gctggtgaag
cctggggctt 6000 cagtaaagat gtcctgcaag acttctagat acacattcac
tgaatacacc atacactggg 6060 tgagacagag ccatggaaag agccttgagt
ggattggagg tattaatcct aacaatggta 6120 ttcctaacta caaccagaag
ttcaagggca gggccacatt gactgtaggc aagtcctcca 6180 gcaccgccta
catggagctc cgcagcctga catctgagga ttctgcggtc tatttctgtg 6240
caagaagaag aatcgcctat ggttacgacg agggccatgc tatggactac tggggtcaag
6300 gaacctcagt caccgtctcc tcaggtgagt ggatcctctg cgcctgggcc
cagctctgtc 6360 ccacaccgcg gtcacatggc accacctctc ttgcagcctc
caccaagggc ccatcggtct 6420 tccccctggc accctcctcc aagagcacct
ctgggggcac agcggccctg ggctgcctgg 6480 tcaaggacta cttccccgaa
ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg 6540 gcgtgcacac
cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg 6600
tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc
6660 ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa
actcacacat 6720 gcccaccgtg cccagcacct gaactcctgg ggggaccgtc
agtcttcctc ttccccccaa 6780 aacccaagga caccctcatg atctcccgga
cccctgaggt cacatgcgtg gtggtggacg 6840 tgagccacga agaccctgag
gtcaagttca actggtacgt ggacggcgtg gaggtgcata 6900 atgccaagac
aaagccgcgg gaggagcagt acaacagcac gtaccgggtg gtcagcgtcc 6960
tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca
7020 aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag
ccccgagaac 7080 cacaggtgta caccctgccc ccatcccggg aggagatgac
caagaaccag gtcagcctga 7140 cctgcctggt caaaggcttc tatcccagcg
acatcgccgt ggagtgggag agcaatgggc 7200 agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc tccttcttcc 7260 tctacagcaa
gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct 7320
ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg
7380 gtaaatgagt gcgacggccg gcaagccccg ctccccgggc tctcgcggtc
gcacgaggat 7440 gcttggcacg taccccctgt acatacttcc cgggcgccca
gcatggaaat aaagcaccgg 7500 atctaataaa agatatttat tttcattaga
tatgtgtgtt ggttttttgt gtgcagtgcc 7560 tctatctgga ggccaggtag
ggctggcctt gggggagggg gaggccagaa tgactccaag 7620 agctacagga
aggcaggtca gagaccccac tggacaaaca gtggctggac tctgcaccat 7680
aacacacaat caacagggga gtgagctgga aatttgctag cgaattaatt c 7731 30
472 PRT Homo sapiens 30 Met Gly Trp Ser Trp Val Phe Leu Phe Leu Leu
Ser Gly Thr Ala Gly 1 5 10 15 Val Leu Ser Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met
Ser Cys Lys Thr Ser Arg Tyr Thr Phe 35 40 45 Thr Glu Tyr Thr Ile
His Trp Val Arg Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile
Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn 65 70 75 80 Gln
Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Gly Lys Ser Ser Ser 85 90
95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110 Tyr Phe Cys Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu
Gly His 115 120 125 Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215
220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340
345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys 465 470 31 339 DNA Homo sapiens 31
gacattgtga tgacccaatc tccagactct ttggctgtgt ctctagggga gagggccacc
60 atcaactgca agtccagtca gagcctttta tattctagaa atcaaaagaa
ctacttggcc 120 tggtatcagc agaaaccagg acagccaccc aaactcctca
tcttttgggc tagcactagg 180 gaatctgggg tacctgatag gttcagtggc
agtgggtttg ggacagactt caccctcacc 240 attagcagcc tgcaggctga
agatgtggca gtttattact gtcagcaata ttttagctat 300 ccgctcacgt
tcggacaagg gaccaaggtg gaaataaaa 339 32 113 PRT Homo sapiens 32 Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30 Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg
Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly
Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Phe Ser Tyr Pro Leu
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys 33 113 PRT
Homo sapiens 33 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val
Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln
Ser Leu Leu Tyr Ser 20 25 30 Arg Asn Gln Lys Asn Tyr Leu Ala Trp
Phe Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Phe
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser
Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Asp Cys Gln Gln 85 90 95 Tyr
Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110 Lys 34 113 PRT Homo sapiens 34 Asp Ile Val Met Thr Gln Ser Pro
Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn
Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Arg Asn Gln Lys
Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr 65
70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln 85 90 95 Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110 Lys 35 8068 DNA Homo sapiens 35
gaattccagc acactggcgg ccgttactag ttattaatag taatcaatta cggggtcatt
60 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
gcccgcctgg 120 ctgaccgccc aacgaccccc gcccattgac gtcaataatg
acgtatgttc ccatagtaac 180 gccaataggg actttccatt gacgtcaatg
ggtggagtat ttacggtaaa ctgcccactt 240 ggcagtacat caagtgtatc
atatgccaag tacgccccct attgacgtca atgacggtaa 300 atggcccgcc
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 360
catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
420 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
acgtcaatgg 480 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
tgtcgtaaca actccgcccc 540 attgacgcaa atgggcggta ggcgtgtacg
gtgggaggtc tatataagca gagctcgttt 600 agtgaaccgt cagatcgcct
ggagacgcca tccacgctgt tttgacctcc atagaagaca 660 ccgggaccga
tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc 720
caagagtgac gtaagtaccg cctatagagt ctataggccc acccccttgg cttcttatgc
780 atgctatact gtttttggct tggggtctat acacccccgc ttcctcatgt
tataggtgat 840 ggtatagctt agcctatagg tgtgggttat tgaccattat
tgaccactcc cctattggtg 900 acgatacttt ccattactaa tccataacat
ggctctttgc cacaactctc tttattggct 960 atatgccaat acactgtcct
tcagagactg acacggactc tgtattttta caggatgggg 1020 tctcatttat
tatttacaaa ttcacatata caacaccacc gtccccagtg cccgcagttt 1080
ttattaaaca taacgtggga tctccacgcg aatctcgggt acgtgttccg gacatgggct
1140 cttctccggt agcggcggag cttctacatc cgagccctgc tcccatgcct
ccagcgactc 1200 atggtcgctc ggcagctcct tgctcctaac agtggaggcc
agacttaggc acagcacgat 1260 gcccaccacc accagtgtgc cgcacaaggc
cgtggcggta gggtatgtgt ctgaaaatga 1320 gctcggggag cgggcttgca
ccgctgacgc atttggaaga cttaaggcag cggcagaaga 1380 agatgcaggc
agctgagttg ttgtgttctg ataagagtca gaggtaactc ccgttgcggt 1440
gctgttaacg gtggagggca gtgtagtctg agcagtactc gttgctgccg cgcgcgccac
1500 cagacataat agctgacaga ctaacagact gttcctttcc atgggtcttt
tctgcagtca 1560 ccgtccttga cacgcgtctc gggaagcttg ccgccaccat
ggagacagac acactcctgc 1620 tatgggtgct gctgctctgg gttccaggtt
cctccggaga cattgtgatg acccaatctc 1680 cagactcttt ggctgtgtct
ctaggggaga gggccaccat caactgcaag tccagtcaga 1740 gccttttata
ttctagaaat
caaaagaact acttggcctg gtatcagcag aaaccaggac 1800 agccacccaa
actcctcatc ttttgggcta gcactaggga atctggggta cctgataggt 1860
tcagtggcag tgggtttggg acagacttca ccctcaccat tagcagcctg caggctgaag
1920 atgtggcagt ttattactgt cagcaatatt ttagctatcc gctcacgttc
ggacaaggga 1980 ccaaggtgga aataaaacgt gagtggatcc atctgggata
agcatgctgt tttctgtctg 2040 tccctaacat gccctgtgat tatgcgcaaa
caacacaccc aagggcagaa ctttgttact 2100 taaacaccat cctgtttgct
tctttcctca ggaactgtgg ctgcaccatc tgtcttcatc 2160 ttcccgccat
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 2220
aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt
2280 aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag
cctcagcagc 2340 accctgacgc tgagcaaagc agactacgag aaacacaaag
tctacgcctg cgaagtcacc 2400 catcagggcc tgagctcgcc cgtcacaaag
agcttcaaca ggggagagtg ttagagggag 2460 aagtgccccc acctgctcct
cagttccagc ctgaccccct cccatccttt ggcctctgac 2520 cctttttcca
caggggacct acccctattg cggtcctcca gctcatcttt cacctcaccc 2580
ccctcctcct ccttggcttt aattatgcta atgttggagg agaatgaata aataaagtga
2640 atctttgcac ctgtggtgga tctaataaaa gatatttatt ttcattagat
atgtgtgttg 2700 gttttttgtg tgcagtgcct ctatctggag gccaggtagg
gctggccttg ggggaggggg 2760 aggccagaat gactccaaga gctacaggaa
ggcaggtcag agaccccact ggacaaacag 2820 tggctggact ctgcaccata
acacacaatc aacaggggag tgagctggaa atttgctagc 2880 gaattcttga
agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 2940
aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
3000 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
aaccctgata 3060 aatgcttcaa taatattgaa aaaggaagag tatgagtatt
caacatttcc gtgtcgccct 3120 tattcccttt tttgcggcat tttgccttcc
tgtttttgct cacccagaaa cgctggtgaa 3180 agtaaaagat gctgaagatc
agttgggtgc acgagtgggt tacatcgaac tggatctcaa 3240 cagcggtaag
atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 3300
taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
3360 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
cagaaaagca 3420 tcttacggat ggcatgacag taagagaatt atgcagtgct
gccataacca tgagtgataa 3480 cactgcggcc aacttacttc tgacaacgat
cggaggaccg aaggagctaa ccgctttttt 3540 gcacaacatg ggggatcatg
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3600 cataccaaac
gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa 3660
actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
3720 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
ggtttattgc 3780 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
attgcagcac tggggccaga 3840 tggtaagccc tcccgtatcg tagttatcta
cacgacgggg agtcaggcaa ctatggatga 3900 acgaaataga cagatcgctg
agataggtgc ctcactgatt aagcattggt aactgtcaga 3960 ccaagtttac
tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 4020
ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
4080 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
ctttttttct 4140 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
ccagcggtgg tttgtttgcc 4200 ggatcaagag ctaccaactc tttttccgaa
ggtaactggc ttcagcagag cgcagatacc 4260 aaatactgtc cttctagtgt
agccgtagtt aggccaccac ttcaagaact ctgtagcacc 4320 gcctacatac
ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 4380
gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
4440 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
aactgagata 4500 cctacagcgt gagctatgag aaagcgccac gcttcccgaa
gggagaaagg cggacaggta 4560 tccggtaagc ggcagggtcg gaacaggaga
gcgcacgagg gagcttccag ggggaaacgc 4620 ctggtatctt tatagtcctg
tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 4680 atgctcgtca
ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 4740
cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
4800 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
gaacgaccga 4860 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
atgcggtatt ttctccttac 4920 gcatctgtgc ggtatttcac accgcatatg
gtgcactctc agtacaatct gctctgatgc 4980 cgcatagtta agccagtata
cactccgcta tcgctacgtg actgggtcat ggctgcgccc 5040 cgacacccgc
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 5100
tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
5160 ccgaaacgcg cgaggcagct gtggaatgtg tgtcagttag ggtgtggaaa
gtccccaggc 5220 tccccagcag gcagaagtat gcaaagcatg catctcaatt
agtcagcaac caggctcccc 5280 agcaggcaga agtatgcaaa gcatgcatct
caattagtca gcaaccatag tcccgcccct 5340 aactccgccc atcccgcccc
taactccgcc cagttccgcc cattctccgc cccatggctg 5400 actaattttt
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 5460
gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctagctt cacgctgccg
5520 caagcactca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag
ccagtccgca 5580 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
atctggacaa gggaaaacgc 5640 aagcgcaaag agaaagcagg tagcttgcag
tgggcttaca tggcgatagc tagactgggc 5700 ggttttatgg acagcaagcg
aaccggaatt gccagctggg gcgccctctg gtaaggttgg 5760 gaagccctgc
aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg 5820
atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
5880 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
gggcacaaca 5940 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
gcgcaggggc gcccggttct 6000 ttttgtcaag accgacctgt ccggtgccct
gaatgaactg caggacgagg cagcgcggct 6060 atcgtggctg gccacgacgg
gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 6120 gggaagggac
tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 6180
tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
6240 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
cacgtactcg 6300 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
gagcatcagg ggctcgcgcc 6360 agccgaactg ttcgccaggc tcaaggcgcg
catgcccgac ggcgaggatc tcgtcgtgac 6420 ccatggcgat gcctgcttgc
cgaatatcat ggtggaaaat ggccgctttt ctggattcat 6480 cgactgtggc
cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 6540
tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
6600 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
tctgagcggg 6660 actctggggt tcgaaatgac cgaccaagcg acgcccaacc
tgccatcacg agatttcgat 6720 tccaccgccg ccttctatga aaggttgggc
ttcggaatcg ttttccggga cgccggctgg 6780 atgatcctcc agcgcgggga
tctcatgctg gagttcttcg cccaccccgg gctcgatccc 6840 ctcgcgagtt
ggttcagctg ctgcctgagg ctggacgacc tcgcggagtt ctaccggcag 6900
tgcaaatccg tcggcatcca ggaaaccagc agcggctatc cgcgcatcca tgcccccgaa
6960 ctgcaggagt ggggaggcac gatggccgct ttggtcccgg atctttgtga
aggaacctta 7020 cttctgtggt gtgacataat tggacaaact acctacagag
atttaaagct ctaaggtaaa 7080 tataaaattt ttaagtgtat aatgtgttaa
actactgatt ctaattgttt gtgtatttta 7140 gattccaacc tatggaactg
atgaatggga gcagtggtgg aatgccttta atgaggaaaa 7200 cctgttttgc
tcagaagaaa tgccatctag tgatgatgag gctactgctg actctcaaca 7260
ttctactcct ccaaaaaaga agagaaaggt agaagacccc aaggactttc cttcagaatt
7320 gctaagtttt ttgagtcatg ctgtgtttag taatagaact cttgcttgct
ttgctattta 7380 caccacaaag gaaaaagctg cactgctata caagaaaatt
atggaaaaat attctgtaac 7440 ctttataagt aggcataaca gttataatca
taacatactg ttttttctta ctccacacag 7500 gcatagagtg tctgctatta
ataactatgc tcaaaaattg tgtaccttta gctttttaat 7560 ttgtaaaggg
gttaataagg aatatttgat gtatagtgcc ttgactagag atcataatca 7620
gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
7680 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
gcttataatg 7740 gttacaaata aagcaatagc atcacaaatt tcacaaataa
agcatttttt tcactgcatt 7800 ctagttgtgg tttgtccaaa ctcatcaatg
tatcttatca tgtctggatc taataaaaga 7860 tatttatttt cattagatat
gtgtgttggt tttttgtgtg cagtgcctct atctggaggc 7920 caggtagggc
tggccttggg ggagggggag gccagaatga ctccaagagc tacaggaagg 7980
caggtcagag accccactgg acaaacagtg gctggactct gcaccataac acacaatcaa
8040 caggggagtg agctggaaat ttgctagc 8068 36 240 PRT Homo sapiens 36
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5
10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser
Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Arg Asn Gln Lys Asn Tyr Leu
Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile Phe Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Phe Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile
Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln
Gln Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr 115 120 125 Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135
140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 37 372
DNA Homo sapiens 37 caggtgcaac tagtgcagtc cggcgccgaa gtgaagaaac
ccggtgcttc cgtgaaagtc 60 agctgtaaaa ctagtagata caccttcact
gaatacacca tacactgggt tagacaggcc 120 cctggccaaa ggctggagtg
gataggaggt attaatccta acaatggtat tcctaactac 180 aaccagaagt
tcaagggccg ggccaccttg accgtaggca agtctgccag caccgcctac 240
atggaactgt ccagcctgcg ctccgaggac actgcagtct actactgcgc cagaagaaga
300 atcgcctatg gttacgacga gggccatgct atggactact ggggtcaagg
aacccttgtc 360 accgtctcct ca 372 38 124 PRT Homo sapiens 38 Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr 20
25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Gly Lys Ser
Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Ile Ala Tyr
Gly Tyr Asp Glu Gly His Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 39 124 PRT Homo sapiens 39 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr
20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu
Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn Tyr
Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Gly Lys
Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Arg Ile Ala
Tyr Gly Tyr Asp Glu Gly His Ala Met Asp 100 105 110 Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 40 124 PRT Homo sapiens 40
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5
10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu
Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn
Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp
Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Ile
Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp 100 105 110 Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 41 124 PRT Homo sapiens
41 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr
Glu Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro
Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val
Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg
Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp 100 105 110 Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 42 7731 DNA Homo
sapiens 42 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
tgataataat 60 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
cgcggaaccc ctatttgttt 120 atttttctaa atacattcaa atatgtatcc
gctcatgaga caataaccct gataaatgct 180 tcaataatat tgaaaaagga
agagtatgag tattcaacat ttccgtgtcg cccttattcc 240 cttttttgcg
gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 300
agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
360 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
cttttaaagt 420 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
caagagcaac tcggtcgccg 480 catacactat tctcagaatg acttggttga
gtactcacca gtcacagaaa agcatcttac 540 ggatggcatg acagtaagag
aattatgcag tgctgccata accatgagtg ataacactgc 600 ggccaactta
cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 660
catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
720 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
gcaaactatt 780 aactggcgaa ctacttactc tagcttcccg gcaacaatta
atagactgga tggaggcgga 840 taaagttgca ggaccacttc tgcgctcggc
ccttccggct ggctggttta ttgctgataa 900 atctggagcc ggtgagcgtg
ggtctcgcgg tatcattgca gcactggggc cagatggtaa 960 gccctcccgt
atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 1020
tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
1080 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
ggatctaggt 1140 gaagatcctt tttgataatc tcatgaccaa aatcccttaa
cgtgagtttt cgttccactg 1200 agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt ttctgcgcgt 1260 aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 1320 agagctacca
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 1380
tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
1440 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
agtcgtgtct 1500 taccgggttg gactcaagac gatagttacc ggataaggcg
cagcggtcgg gctgaacggg 1560 gggttcgtgc acacagccca gcttggagcg
aacgacctac accgaactga gatacctaca 1620 gcgtgagcta tgagaaagcg
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 1680 aagcggcagg
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 1740
tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
1800 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc 1860 cttttgctgg ccttttgctc acatgttctt tcctgcgtta
tcccctgatt ctgtggataa 1920 ccgtattacc gcctttgagt gagctgatac
cgctcgccgc agccgaacga ccgagcgcag 1980 cgagtcagtg agcgaggaag
cggaagagcg cctgatgcgg tattttctcc ttacgcatct 2040 gtgcggtatt
tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata 2100
gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
2160 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
cgcttacaga 2220 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
tttcaccgtc atcaccgaaa 2280 cgcgcgaggc agcatgcatc tcaattagtc
agcaaccata gtcccgcccc taactccgcc 2340 catcccgccc ctaactccgc
ccagttccgc ccattctccg ccccatggct gactaatttt 2400 ttttatttat
gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg 2460
aggctttttt ggaggcctag gcttttgcaa aaagctagct tacagctcag ggctgcgatt
2520 tcgcgccaaa cttgacggca atcctagcgt gaaggctggt aggattttat
ccccgctgcc 2580 atcatggttc gaccattgaa ctgcatcgtc gccgtgtccc
aaaatatggg gattggcaag 2640 aacggagacc taccctggcc tccgctcagg
aacgagttca agtacttcca aagaatgacc 2700 acaacctctt cagtggaagg
taaacagaat ctggtgatta tgggtaggaa aacctggttc 2760 tccattcctg
agaagaatcg acctttaaag gacagaatta atatagttct cagtagagaa 2820
ctcaaagaac caccacgagg agctcatttt cttgccaaaa gtttggatga tgccttaaga
2880 cttattgaac aaccggaatt ggcaagtaaa gtagacatgg tttggatagt
cggaggcagt 2940 tctgtttacc aggaagccat gaatcaacca ggccacctca
gactctttgt gacaaggatc 3000 atgcaggaat ttgaaagtga cacgtttttc
ccagaaattg atttggggaa atataaactt 3060 ctcccagaat acccaggcgt
cctctctgag gtccaggagg aaaaaggcat caagtataag 3120 tttgaagtct
acgagaagaa agactaacag gaagatgctt tcaagttctc tgctcccctc 3180
ctaaagctat gcatttttat aagaccatgg gacttttgct ggctttagat ctttgtgaag
3240 gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat
ttaaagctct 3300 aaggtaaata taaaattttt aagtgtataa tgtgttaaac
tactgattct aattgtttgt 3360 gtattttaga ttccaaccta tggaactgat
gaatgggagc agtggtggaa tgcctttaat 3420 gaggaaaacc tgttttgctc
agaagaaatg ccatctagtg atgatgaggc tactgctgac 3480 tctcaacatt
ctactcctcc aaaaaagaag agaaaggtag aagaccccaa ggactttcct 3540
tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct tgcttgcttt
3600 gctatttaca ccacaaagga aaaagctgca
ctgctataca agaaaattat ggaaaaatat 3660 tctgtaacct ttataagtag
gcataacagt tataatcata acatactgtt ttttcttact 3720 ccacacaggc
atagagtgtc tgctattaat aactatgctc aaaaattgtg tacctttagc 3780
tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt gactagagat
3840 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
tcccacacct 3900 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
gttaacttgt ttattgcagc 3960 ttataatggt tacaaataaa gcaatagcat
cacaaatttc acaaataaag catttttttc 4020 actgcattct agttgtggtt
tgtccaaact catcaatgta tcttatcatg tctggatcta 4080 ataaaagata
tttattttca ttagatatgt gtgttggttt tttgtgtgca gtgcctctat 4140
ctggaggcca ggtagggctg gccttggggg agggggaggc cagaatgact ccaagagcta
4200 caggaaggca ggtcagagac cccactggac aaacagtggc tggactctgc
accataacac 4260 acaatcaaca ggggagtgag ctggaaattt gctagcgaat
tccagcacac tggcggccgt 4320 tactagttat taatagtaat caattacggg
gtcattagtt catagcccat atatggagtt 4380 ccgcgttaca taacttacgg
taaatggccc gcctggctga ccgcccaacg acccccgccc 4440 attgacgtca
ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 4500
tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
4560 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
attatgccca 4620 gtacatgacc ttatgggact ttcctacttg gcagtacatc
tacgtattag tcatcgctat 4680 taccatggtg atgcggtttt ggcagtacat
caatgggcgt ggatagcggt ttgactcacg 4740 gggatttcca agtctccacc
ccattgacgt caatgggagt ttgttttggc accaaaatca 4800 acgggacttt
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg 4860
tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
4920 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
gcctccgcgg 4980 ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag
agtgacgtaa gtaccgccta 5040 tagagtctat aggcccaccc ccttggcttc
ttatgcatgc tatactgttt ttggcttggg 5100 gtctatacac ccccgcttcc
tcatgttata ggtgatggta tagcttagcc tataggtgtg 5160 ggttattgac
cattattgac cactccccta ttggtgacga tactttccat tactaatcca 5220
taacatggct ctttgccaca actctcttta ttggctatat gccaatacac tgtccttcag
5280 agactgacac ggactctgta tttttacagg atggggtctc atttattatt
tacaaattca 5340 catatacaac accaccgtcc ccagtgcccg cagtttttat
taaacataac gtgggatctc 5400 cacgcgaatc tcgggtacgt gttccggaca
tgggctcttc tccggtagcg gcggagcttc 5460 tacatccgag ccctgctccc
atgcctccag cgactcatgg tcgctcggca gctccttgct 5520 cctaacagtg
gaggccagac ttaggcacag cacgatgccc accaccacca gtgtgccgca 5580
caaggccgtg gcggtagggt atgtgtctga aaatgagctc ggggagcggg cttgcaccgc
5640 tgacgcattt ggaagactta aggcagcggc agaagaagat gcaggcagct
gagttgttgt 5700 gttctgataa gagtcagagg taactcccgt tgcggtgctg
ttaacggtgg agggcagtgt 5760 agtctgagca gtactcgttg ctgccgcgcg
cgccaccaga cataatagct gacagactaa 5820 cagactgttc ctttccatgg
gtcttttctg cagtcaccgt ccttgacacg cgtctcggga 5880 agcttgccgc
caccatggac tggacctggc gcgtgttttg cctgctcgcc gtggctcctg 5940
gggcccacag ccaggtgcaa ctggtgcagt ccggcgccga agtgaagaaa cccggtgctt
6000 ccgtgaaagt cagctgtaaa actagtagat acaccttcac tgaatacacc
atacactggg 6060 ttagacaggc ccctggccaa aggctggagt ggataggagg
tattaatcct aacaatggta 6120 ttcctaacta caaccagaag ttcaagggcc
gggccacctt gaccgtaggc aagtctgcca 6180 gcaccgccta catggaactg
tccagcctgc gctccgagga cactgcagtc tactactgcg 6240 ccagaagaag
aatcgcctat ggttacgacg agggccatgc tatggactac tggggtcaag 6300
gaacccttgt caccgtctcc tcaggtgagt ggatcctctg cgcctgggcc cagctctgtc
6360 ccacaccgcg gtcacatggc accacctctc ttgcagcctc caccaagggc
ccatcggtct 6420 tccccctggc accctcctcc aagagcacct ctgggggcac
agcggccctg ggctgcctgg 6480 tcaaggacta cttccccgaa ccggtgacgg
tgtcgtggaa ctcaggcgcc ctgaccagcg 6540 gcgtgcacac cttcccggct
gtcctacagt cctcaggact ctactccctc agcagcgtgg 6600 tgaccgtgcc
ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc 6660
ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa actcacacat
6720 gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc
ttccccccaa 6780 aacccaagga caccctcatg atctcccgga cccctgaggt
cacatgcgtg gtggtggacg 6840 tgagccacga agaccctgag gtcaagttca
actggtacgt ggacggcgtg gaggtgcata 6900 atgccaagac aaagccgcgg
gaggagcagt acaacagcac gtaccgggtg gtcagcgtcc 6960 tcaccgtcct
gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca 7020
aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac
7080 cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
gtcagcctga 7140 cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag agcaatgggc 7200 agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc tccttcttcc 7260 tctacagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc ttctcatgct 7320 ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg 7380
gtaaatgagt gcgacggccg gcaagccccg ctccccgggc tctcgcggtc gcacgaggat
7440 gcttggcacg taccccctgt acatacttcc cgggcgccca gcatggaaat
aaagcaccgg 7500 atctaataaa agatatttat tttcattaga tatgtgtgtt
ggttttttgt gtgcagtgcc 7560 tctatctgga ggccaggtag ggctggcctt
gggggagggg gaggccagaa tgactccaag 7620 agctacagga aggcaggtca
gagaccccac tggacaaaca gtggctggac tctgcaccat 7680 aacacacaat
caacagggga gtgagctgga aatttgctag cgaattaatt c 7731 43 472 PRT Homo
sapiens 43 Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala
Pro Gly 1 5 10 15 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Thr Ser Arg Tyr Thr Phe 35 40 45 Thr Glu Tyr Thr Ile His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60 Glu Trp Ile Gly Gly Ile
Asn Pro Asn Asn Gly Ile Pro Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys
Gly Arg Ala Thr Leu Thr Val Gly Lys Ser Ala Ser 85 90 95 Thr Ala
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110
Tyr Tyr Cys Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His 115
120 125 Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235
240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360
365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser
Leu Ser Pro Gly Lys 465 470 44 25 DNA Homo sapiens 44 accgtctcct
caggtgagtg gatcc 25 45 26 DNA Homo sapiens 45 cctctcttgc agcctccacc
aagggc 26 46 14 DNA Homo sapiens 46 cctctcttgc agcc 14 47 4 PRT
Homo sapiens 47 Thr Val Ser Ser 1 48 4 PRT Homo sapiens 48 Ser Thr
Lys Gly 1 49 27 DNA Homo sapiens 49 accgtctcct cagcctccac caagggc
27 50 8 PRT Homo sapiens 50 Thr Val Ser Ser Ser Thr Lys Gly 1 5 51
27 DNA Homo sapiens 51 accgtctcct cagcctccac caagggc 27 52 9 PRT
Homo sapiens 52 Thr Val Ser Ser Ala Ser Thr Lys Gly 1 5 53 22 DNA
Homo sapiens 53 gaaataaaac gtgagtggat cc 22 54 27 DNA Homo sapiens
54 cttctttcct caggaactgt ggctgca 27 55 4 PRT Homo sapiens 55 Thr
Val Ala Ala 1 56 24 DNA Homo sapiens 56 gaaataaaac gaactgtggc tgca
24 57 7 PRT Homo sapiens 57 Glu Ile Lys Thr Val Ala Ala 1 5 58 24
DNA Homo sapiens 58 gaaataaaac gaactgtggc tgca 24 59 8 PRT Homo
sapiens 59 Glu Ile Lys Arg Thr Val Ala Ala 1 5 60 20 PRT Homo
sapiens 60 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp
Val Ser 1 5 10 15 Gly Thr Cys Gly 20 61 19 PRT Homo sapiens 61 Met
Gly Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10
15 Val Leu Ser 62 9 DNA Homo sapiens 62 gccgccacc 9 63 37 DNA Homo
sapiens Description of Artificial Sequence DNA Primer 63 cagaaagctt
gccgccacca tggattcaca ggcccag 37 64 6 PRT Homo sapiens 64 Met Asp
Ser Gln Ala Gln 1 5 65 35 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 65 ccgaggatcc actcacgttt cagctccagc
ttggt 35 66 37 DNA Homo sapiens Description of Artificial Sequence
DNA Primer 66 cagaaagctt gccgccacca tgggatggag ctgggtc 37 67 6 PRT
Homo sapiens 67 Met Gly Trp Ser Trp Val 1 5 68 35 DNA Homo sapiens
Description of Artificial Sequence DNA Primer 68 ccgaggatcc
actcacctga ggagacggtg actga 35 69 36 DNA Homo sapiens Description
of Artificial Sequence DNA Primer 69 gtcatcacaa tgtctccgga
ggaacctgga acccag 36 70 29 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 70 ctccggagac attgtgatga cccaatctc
29 71 45 DNA Homo sapiens Description of Artificial Sequence DNA
Primer 71 gaatataaaa ggctctgact ggacttgcag ttgatggtgg ccctc 45 72
72 DNA Homo sapiens Description of Artificial Sequence DNA Primer
72 cagtcagagc cttttatatt ctagaaatca aaagaactac ttggcctggt
atcagcagaa 60 accaggacag cc 72 73 44 DNA Homo sapiens Description
of Artificial Sequence DNA Primer 73 accccagatt ccctagtgct
agcccaaaag atgaggagtt tggg 44 74 67 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 74 tagcactagg gaatctgggg tacctgatag
gttcagtggc agtgggtttg ggacagactt 60 caccctc 67 75 53 DNA Homo
sapiens Description of Artificial Sequence DNA Primer 75 gtcccttgtc
cgaacgtgag cggatagcta aaatattgct gacagtaata aac 53 76 33 DNA Homo
sapiens Description of Artificial Sequence DNA Primer 76 gctcacgttc
ggacaaggga ccaaggtgga aat 33 77 72 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 77 cagtcagagc cttttatatt ctagaaatca
aaagaactac ttggcctggt tccagcagaa 60 accaggacag cc 72 78 56 DNA Homo
sapiens Description of Artificial Sequence DNA Primer 78 tcccttgtcc
gaacgtgagc ggatagctaa aatattgctg acagtcataa actgcc 56 79 34 DNA
Homo sapiens Description of Artificial Sequence DNA Primer 79
cccaaactcc tcatctattg ggctagcact aggg 34 80 34 DNA Homo sapiens
Description of Artificial Sequence DNA Primer 80 ccctagtgct
agcccaatag atgaggagtt tggg 34 81 17 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 81 tacgcaaacc gcctctc 17 82 18 DNA
Homo sapiens Description of Artificial Sequence DNA Primer 82
gagtgcacca tatgcggt 18 83 16 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 83 aacagctatg accatg 16 84 17 DNA
Homo sapiens Description of Artificial Sequence DNA Primer 84 ccag
tcacgac 17 85 47 DNA Homo sapiens Description of Artificial
Sequence DNA Primer 85 tcag tgaaggtgta tctactagtt ttacagctga
ctttcac 47 86 53 DNA Homo sapiens Description of Artificial
Sequence DNA Primer 86 tagtagatac accttcactg aatacaccat acactgggtt
agacaggccc ctg 53 87 71 DNA Homo sapiens Description of Artificial
Sequence DNA Primer 87 cccttgaact tctggttgta gttaggaata ccattgttag
gattaatacc tcctatccac 60 tccagccttt g 71 88 71 DNA Homo sapiens
Description of Artificial Sequence DNA Primer 88 taactacaac
cagaagttca agggccgggc caccttgacc gtaggcaagt ctgccagcac 60
cgcctacatg g 71 89 63 DNA Homo sapiens Description of Artificial
Sequence DNA Primer 89 gcatggccct cgtcgtaacc ataggcgatt cttcttctgg
cgcagtagta gactgcagtg 60 tcc 63 90 48 DNA Homo sapiens Description
of Artificial Sequence DNA Primer 90 ctatggttac gacgagggcc
atgctatgga ctactggggt caaggaac 48 91 71 DNA Homo sapiens
Description of Artificial Sequence DNA Primer 91 taactacaac
cagaagttca agggccgggt caccatcacc gtagacacct ctgccagcac 60
cgcctacatg g 71 92 27 DNA Homo sapiens Description of Artificial
Sequence DNA Primer 92 ggacactgca gtctacttct gcgccag 27 93 17 DNA
Homo sapiens Description of Artificial Sequence DNA Primer 93
tacgcaaacc gcctctc 17 94 18 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 94 gagtgcacca tatgcggt 18 95 75 DNA
Homo sapiens Description of Artificial Sequence DNA Primer 95
cctttggcca ggggcctgtc taacccagtg tatggtgtat tcagtgaagg tgtatccact
60 agtttccact agttt 75 96 28 DNA Homo sapiens Description of
Artificial Sequence DNA Primer 96 gtcaccgtcc ttgacacgcg tctcggga 28
97 17 DNA Homo sapiens Description of Artificial Sequence DNA
Primer 97 ttggaggagg gtgccag 17 98 22 DNA Homo sapiens Description
of Artificial Sequence DNA Primer 98 gagacattgt gacccaatct cc 22 99
25 DNA Homo sapiens Description of Artificial Sequence DNA Primer
99 gacagtcata aactgccaca tcttc 25 100 23 DNA Homo sapiens
Description of Artificial Sequence DNA Primer 100 ttgacacgcg
tctcgggaag ctt 23 101 22 DNA Homo sapiens Description of Artificial
Sequence DNA Primer 101 ggcgcagagg atccactcac ct 22 102 339 DNA
Homo sapiens 102 gacattgtga tgacccaatc tccagactct ttggctgtgt
ctctagggga gagggccacc 60 atcaactgca agtccagtca gagcctttta
tattctagaa atcaaaagaa ctacttggcc 120 tggttccagc agaaaccagg
acagccaccc aaactcctca tcttttgggc tagcactagg 180 gaatctgggg
tacctgatag gttcagtggc agtgggtttg ggacagactt caccctcacc 240
attagcagcc tgcaggctga agatgtggca gtttatgact gtcaacaata ttttagctat
300 ccgctcacgt tcggacaagg gaccaaggtg gaaataaaa 339 103 339 DNA Homo
sapiens 103 gacattgtga tgacccaatc tccagactct ttggctgtgt ctctagggga
gagggccacc 60 atcaactgca agtccagtca gagcctttta tattctagaa
atcaaaagaa ctacttggcc 120 tggtatcagc agaaaccagg acagccaccc
aaactcctca tctattgggc tagcactagg 180 gaatctgggg tacctgatag
gttcagtggc agtgggtttg ggacagactt caccctcacc 240 attagcagcc
tgcaggctga agatgtggca gtttattact gtcagcaata ttttagctat 300
ccgctcacgt tcggacaagg gaccaaggtg gaaataaaa 339 104 372 DNA Homo
sapiens 104 caggtgcaac tagtgcagtc cggcgccgaa gtgaagaaac ccggtgcttc
cgtgaaagtc 60 agctgtaaaa ctagtagata caccttcact gaatacacca
tacactgggt tagacaggcc 120 cctggccaaa ggctggagtg gataggaggt
attaatccta acaatggtat tcctaactac 180 aaccagaagt tcaagggccg
ggccaccttg accgtaggca agtctgccag caccgcctac 240 atggaactgt
ccagcctgcg ctccgaggac actgcagtct acttctgcgc cagaagaaga 300
atcgcctatg gttacgacga gggccatgct atggactact ggggtcaagg aacccttgtc
360 accgtctcct ca 372 105 372 DNA Homo sapiens 105 caggtgcaac
tagtgcagtc cggcgccgaa gtgaagaaac ccggtgcttc cgtgaaagtc 60
agctgtaaaa ctagtagata caccttcact gaatacacca tacactgggt tagacaggcc
120 cctggccaaa ggctggagtg gataggaggt attaatccta acaatggtat
tcctaactac 180 aaccagaagt tcaagggccg ggtcaccatc accgtagaca
cctctgccag caccgcctac 240 atggaactgt
ccagcctgcg ctccgaggac actgcagtct actactgcgc cagaagaaga 300
atcgcctatg gttacgacga gggccatgct atggactact ggggtcaagg aacccttgtc
360 accgtctcct ca 372 106 372 DNA Homo sapiens 106 caggtgcaac
tagtgcagtc cggcgccgaa gtgaagaaac ccggtgcttc cgtgaaagtc 60
agctgtaaaa ctagtagata caccttcact gaatacacca tacactgggt tagacaggcc
120 cctggccaaa ggctggagtg gataggaggt attaatccta acaatggtat
tcctaactac 180 aaccagaagt tcaagggccg ggtcaccatc accgtagaca
cctctgccag caccgcctac 240 atggaactgt ccagcctgcg ctccgaggac
actgcagtct acttctgcgc cagaagaaga 300 atcgcctatg gttacgacga
gggccatgct atggactact ggggtcaagg aacccttgtc 360 accgtctcct ca 372
107 372 DNA Homo sapiens 107 caggtgcaac tagtgcagtc cggcgccgaa
gtgaagaaac ccggtgcttc cgtgaaagtc 60 agctgtaaaa ctagtggata
caccttcact gaatacacca tacactgggt tagacaggcc 120 cctggccaaa
ggctggagtg gataggaggt attaatccta acaatggtat tcctaactac 180
aaccagaagt tcaagggccg ggtcaccatc accgtagaca cctctgccag caccgcctac
240 atggaactgt ccagcctgcg ctccgaggac actgcagtct actactgcgc
cagaagaaga 300 atcgcctatg gttacgacga gggccatgct atggactact
ggggtcaagg aacccttgtc 360 accgtctcct ca 372 108 124 PRT Homo
sapiens 108 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr
Phe Thr Glu Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly
Ile Pro Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg
Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp 100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
* * * * *