U.S. patent application number 10/136145 was filed with the patent office on 2003-11-20 for melanoma associated antigenic polypeptide, epitopes thereof and vaccines against melanoma.
Invention is credited to Adema, Gosse Jan, Figdor, Carl Gustav.
Application Number | 20030216559 10/136145 |
Document ID | / |
Family ID | 26136040 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030216559 |
Kind Code |
A1 |
Adema, Gosse Jan ; et
al. |
November 20, 2003 |
Melanoma associated antigenic polypeptide, epitopes thereof and
vaccines against melanoma
Abstract
A melanoma associated antigen known as gp100 and peptides
derived from the antigen. Gp100 and its peptide derivatives can be
used in vaccines for the treatment of melanoma. Another aspect of
the invention is host-cells capable of expressing gp100 for the
gp100-derived peptides. Furthermore, tumor-infiltrating lymphocytes
(TILs) specifically recognizing gp100 are described, as are
vaccines with these TILs. Also disclosed are diagnostics for the
detection of melanoma and for the monitoring of vaccination.
Inventors: |
Adema, Gosse Jan; (Nijmegen,
NL) ; Figdor, Carl Gustav; (Nijmegen, NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
26136040 |
Appl. No.: |
10/136145 |
Filed: |
May 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10136145 |
May 1, 2002 |
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08388852 |
Feb 15, 1995 |
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6500919 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
C07K 14/4748 20130101;
A61K 39/00 20130101; A61P 35/00 20180101; Y10S 530/822
20130101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 16, 1994 |
EP |
94200337.7 |
Dec 21, 1994 |
EP |
94203709.4 |
Claims
What is claimed is:
1. A peptide consisting of from about 8 to about 12 contiguous
amino acids of an amino acid sequence, said amino acid sequence
selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:21 and SEQ ID NO:22 in combination with a pharmaceutically
acceptable carrier or diluent.
2. A peptide consisting of from about 8 to about 12 contiguous
amino acids of an amino acid sequence, said amino acid sequence
selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:21 and SEQ ID NO:22.
3. The peptide of claim 2, wherein the amino acid sequence is that
of SEQ ID NO:4.
4. The peptide of claim 2, wherein the amino acid sequence is that
of SEQ ID NO:6.
5. The peptide of claim 2, wherein the amino acid sequence is that
of SEQ ID NO:21.
6. The peptide of claim 2, wherein the amino acid sequence is that
of SEQ ID NO:22.
7. An immunogenic carrier or marker coupled to the peptide of claim
2.
8. A nucleotide sequence, comprising a nucleotide sequence encoding
a melanoma associated antigen, said antigen comprising the amino
acid sequence of SEQ ID NO:2.
9. The nucleotide sequence according to claim 8, comprising the
nucleotide sequence of SEQ ID NO:1.
10. A nucleotide sequence, comprising a nucleotide sequence
encoding an immunogenic peptide, said peptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22
and SEQ ID NO:23.
11. The nucleotide sequence according to claim 10, comprising a
nucleotide sequence selected from the group consisting of the
sequences of SEQ ID NOS: 3, 5, 7 and 9.
12. A test kit for the detection of melanoma cells, comprising at
least one primer and a labeled probe directed to the nucleotide
sequence according to claim 8 or its complementary sequence.
13. A method for the detection of melanoma cells, comprising: a.
obtaining a specimen from a patient; b. subjecting the specimen to
methods for the amplification of a nucleotide sequence according to
claim 8 or a part of said sequence coding for an immunogenic
fragment; c. reacting amplified nucleic acid with a complementary
labeled probe; and d. detecting nucleic acid labeled with the
probe.
14. A cloning vehicle comprising the nucleotide sequence of claim
8.
15. A host cell transfected or transformed with the cloning vehicle
according to claim 14.
16. The host cell according to claim 15 selected from the group
consisting of murine EL4 cells, murine P8.15 cells and human BLM
cells.
17. The host cell according to claim 15, which is an antigen
presenting cell.
18. The host cell according to claim 15, which also produces
co-stimulating molecules.
19. A vaccine comprising an antigen or epitope thereof, said
antigen comprising the amino acid sequence of SEQ ID NO:2, said
vaccine comprising an antigen presenting cell that has been
preloaded with a peptide comprising the antigen or epitope.
20. A vaccine comprising a host cell according to claim 15.
21. A vaccine comprising the nucleotide sequence according to claim
8.
22. A vaccine comprising a T cell receptor against a nucleotide
sequence or cells expressing said T cell receptor, wherein said
nucleotide sequence comprises a nucleotide sequence encoding a
melanoma associated antigen, said antigen comprising the amino acid
sequence of SEQ ID NO:2.
23. A vaccine comprising an antigen or epitope thereof, said
antigen comprising the amino aid sequence of SEQ ID NO:2, said
vaccine additionally comprising at least one compound selected from
the group consisting of an adjuvant, one or more cytokines,
antibodies directed against CD2, CD3, CD27, CD28 or other T cell
surface antigens, and helper epitopes that stimulate CD4+ or CD8+ T
cells.
24. A method for the generation of antigen reactive tumor
infiltrating lymphocytes, comprising: a. taking a sample of a
melanoma from a patient; b. isolating tumor infiltrating
lymphocytes from the sample; c. reacting said lymphocytes with an
antigen comprising the amino acid sequence of SEQ ID NO:2; and d.
isolating the lymphocytes binding to said antigen.
25. Tumor infiltrating lymphocytes capable of binding to a melanoma
associated antigen, said antigen comprising the amino acid sequence
of SEQ ID NO:2.
26. Melanoma cells that express an antigen comprising the amino
acid sequence of SEQ ID NO:2, said cells transfected with a cloning
vehicle having a nucleotide sequence coding for a lymphokine.
27. A vaccine comprising tumor infiltrating lymphocytes according
to claim 25 and a pharmaceutically acceptable carrier or
diluent.
28. An antibody directed to an immunogenic peptide, said peptide
comprising an immunogenic fragment of an antigen comprising the
amino acid sequence of SEQ ID NO:2.
29. A vaccine comprising the antibody according to claim 28.
30. A method for monitoring immunotherapy, comprising detecting the
presence of antibodies directed to an immunogenic peptide, said
peptide comprising an immunogenic fragment of an antigen comprising
the amino acid sequence of SEQ ID NO:2.
31. A kit for the detection of antibodies according to claim 28,
comprising a conjugate of a detectable marker and an immunogenic
peptide comprising an immunogenic fragment of an antigen comprising
the amino acid sequence of SEQ ID NO:2.
32. A cloning vehicle comprising the nucleotide sequence of claim
10.
33. The host cell of claim 15 cotransfected with a cloning vehicle
comprising a nucleotide sequence coding for an MHC class I
allele.
34. A vaccine comprising the nucleotide sequence according to claim
10.
35. A vaccine comprising a T cell receptor against a peptide or
cells expressing said T cell receptor, said peptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22 and SEQ ID NO:23.
36. A vaccine comprising a peptide and a pharmaceutically
acceptable carrier or diluent, said peptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22
and SEQ ID NO:23, said vaccine additionally comprising at least one
compound selected from the group consisting of an adjuvant, one or
more cytokines, antibodies directed against CD2, CD3, CD27, CD28 or
other T cell surface antigens, and helper epitopes that stimulate
CD4+ or CD8+ T cells.
37. The vaccine according to claim 19 comprising, in addition, at
least one compound selected from the group consisting of an
adjuvant, one or more cytokines, antibodies directed against CD2,
CD3, CD27, CD28 or other T cell surface antigens, and helper
epitopes that stimulate CD4+ or CD8+ T cells.
38. The vaccine according to claim 20 comprising, in addition, at
least one compound selected from the group consisting of an
adjuvant, one or more cytokines, antibodies directed against CD2,
CD3, CD27, CD28 or other T cell surface antigens, and helper
epitopes that stimulate CD4+ or CD8+ T cells.
39. The vaccine according to claim 21 comprising, in addition, at
least one compound selected from the group consisting of an
adjuvant, one or more cytokines, antibodies directed against CD2,
CD3, CD27, CD28 or other T cell surface antigens, and helper
epitopes that stimulate CD4+ or CD8+ T cells.
40. The vaccine according to claim 22 comprising, in addition, at
least one compound selected from the group consisting of an
adjuvant, one or more cytokines, antibodies directed against CD2,
CD3, CD27, CD28 or other T cell surface antigens, and helper
epitopes that stimulate CD4+ or CD8+ T cells.
41. The vaccine according to claim 34 comprising, in addition, at
least one compound selected from the group consisting of an
adjuvant, one or more cytokines, antibodies directed against CD2,
CD3, CD27 or other T cell surface antigens, and helper epitopes
that stimulate CD4+ or CD8+ T cells.
42. The vaccine according to claim 35 comprising, in addition, at
least one compound selected from the group consisting of an
adjuvant, one or more cytokines, antibodies directed against CD2,
CD3, CD27, CD28 or other T cell surface antigens, and helper
epitopes that stimulate CD4+ or CD8+ T cells.
43. A vaccine comprising melanoma cells according to claim 26 and a
pharmaceutically acceptable carrier or diluent.
44. An antibody directed to a peptide, said peptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22 and SEQ ID NO:23.
45. An immunogenic peptide having 5-20 contiguous amino acids of
gp100, said peptide being capable of reacting with
T-lymphocytes.
46. The immunogenic peptide of claim 45, wherein said peptide is at
least about 9 to 10 amino acids in length.
47. The immunogenic peptide of claim 46 having the sequence
KTWGQYWQV (SEQ ID NO:22) or KTWGQYWQVL (SEQ ID NO:21).
48. The immunogenic peptide of claim 46 wherein the peptide is
LLDGTATLRL (SEQ ID NO:4).
49. The immunogenic peptide of claim 46, wherein said peptide
contains at least one amino acid modification of said gp100
sequence.
50. The immunogenic peptide of claim 47, wherein said peptide
contains at least one amino acid modification of said gp100
sequence.
51. The immunogenic peptide of claim 48, wherein said peptide
contains at least one amino acid modification of said gp100
sequence.
52. The peptide of claim 5, wherein said modification includes at
least one amino acid substitution in said peptide sequence.
53. An immunogenic peptide having the formula selected from the
group consisting of X.sub.1X.sub.2X.sub.3GQYWQX.sub.4 or
X.sub.1X.sub.2X.sub.3PGPVTX.sub.4 wherein: X.sub.1 is any naturally
occurring amino acid; X.sub.2 is any hydrophobic aliphatic amino
acid; X.sub.3 is any naturally occurring amino acid; and X.sub.4 is
any hydrophobic aliphatic amino acid.
54. The peptide of claim 53, wherein the amino acid for X.sub.1 is
selected from the group consisting of tyrosine and lysine.
55. The peptide of claim 53, wherein X.sub.2 is selected from the
group consisting of leucine and threonine.
56. The peptide of claim 53, wherein X.sub.3 is tyrosine.
57. The peptide of claim 53, wherein X.sub.4 is selected from the
group consisting of alanine and valine.
58. The immunogenic peptide of claim 45 wherein said peptide is
recognized by HLA-A2 restricted tumor infiltrating lymphocyte.
59. The immunogenic peptide of claim 53 wherein said peptide is
recognized by HLA-A2 restricted tumor infiltrating lymphocyte.
60. The immunogenic peptide of claim 45 wherein said peptide is a
native, synthetic or recombinant peptide.
61. The immunogenic peptide of claim 53 wherein said peptide is a
native, synthetic or recombinant peptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of, and claims priority
from, U.S. patent application Ser. No. 08/388,852, filed Feb. 19,
1995, now U.S. Pat. No. ______, which itself claims priority from
EP 94200337.7, filed Feb. 16, 1994, and EP 94203709.4, filed Dec.
21, 1994. U.S. patent application Ser. No. 08/388,852, EP
94203709.4 and 94200337.7 are hereby incorporated by this reference
as if set forth in their entirety herein.
TECHNICAL FIELD
[0002] The present invention is concerned with cancer treatment and
diagnosis, especially with a melanoma associated antigen, epitopes
thereof, vaccines against melanoma, tumor infiltrating T
lymphocytes recognizing the antigen and diagnostics for the
detection of melanoma and for the monitoring of vaccination.
BACKGROUND
[0003] Tumor cells may emancipate themselves from restrictive
growth control by oncogene activation, and/or by the inactivation
of tumor suppression genes. The course of tumor progression
proceeds by a series of gradual, stepwise changes in different
"unit characteristics", i.e., phenotypic traits, many of which are
known to be determined or at least influenced by the altered
expression of defined oncogenes and/or tumor suppressive genes.
Emancipation of the cell from immunological host restriction may
follow multistep pathways similar to the emancipation from growth
control.
[0004] A problem often encountered in cancer immunotherapy is the
lack of immunogenicity of the tumor. This escape of the immune
control system can be understood on the basis of phenotype
differences encountered in neoplastic cells (differences found in
Burkitt's lymphoma cells according to Klein, G. and Boon, T., Curr.
Opinion in Immunol. 5, 687-692, 1993):
[0005] decreased ability to process and present antigens;
[0006] decreased ability to stimulate autologous T cells;
[0007] complete downregulation of immunogenic proteins associated
with transformed cells;
[0008] no or low expression of leukocyte adhesion molecules or
other accessory molecules; and
[0009] selective downregulation of certain MHC class I and class II
alleles.
[0010] MHC Class I/II antigens are often downregulated in solid
tumors. This may affect all class I/II antigens, or only part of
them. Viral and cellular peptides that can sensitize appropriate
target cells for cytotoxic T lymphocyte mediated lysis may fail to
do so when produced in cells with a low level of expression of MHC
class I antigen. Cytotoxic sensitivity may be induced, at least in
some cases by raising the level of MHC class I/II antigen
expression by interferon .gamma. and tumor necrosis factor
.alpha..
[0011] However, during the stepwise changes from normal to tumor
tissue, tumor-associated antigens appear. These antigens can be
exposed through various mechanisms:
[0012] they can be molecules that are masked in some way during
normal cell development, but where the neoplastic change induces
removal of the masking protection for the immunosystem;
[0013] deletion of some molecules from the plasma membrane may
alter the profile of adjacent molecules in a given membrane patch
and thus, in effect, generate a new profile that might become
immunogenic to the host;
[0014] a membrane alteration accompanying neoplastic transformation
may expose new, previously hidden regions of a molecule, or may
result in addition of new structural features to an existing
molecule; and
[0015] shedding and disintegration of tumor cells may expose the
immune system to nuclear, nucleolar, or cytoplasmic components that
are normally hidden in the cell.
[0016] The characteristics of tumor-associated antigens are very
much dependent on the origin of the tumor carrying them. The
existence of antigens associated with animal tumors was documented
in the last century, and the antigenic character of human cancers
has been well established, primarily through recent studies with
monoclonal antibodies.
[0017] Attempts to isolate and chemically characterize these
antigens have encountered serious difficulties, many having to do
with a lack of reagents suitable for precipitation of the
antigen-bearing molecules from a solution.
[0018] Like many other stimuli, the tumor-associated antigens
activate not one, but a whole set of defense mechanisms--both
specific and unspecific, humoral and cellular. The dominant role in
vivo resistance to tumor growth is played by T lymphocytes. These
cells recognize tumor-associated antigens presented to them by
antigen presenting cells (APCs), and will be activated by this
recognition, and upon activation and differentiation, attack and
kill the tumor cells. A special class of these types of lymphocytes
is formed by the tumor infiltrating lymphocytes (TILs) that can be
found in solid tumors.
[0019] EP 147,689 suggests activating T lymphocytes with an
antigenic substance linked to an insoluble carrier in vitro and
then to administer these activated lymphocytes to a tumor
patient.
[0020] Conventional chemotherapy is relatively ineffective in the
treatment of patients with metastasic melanoma, and approximately
6000 patients die of this disease in the United States each
year.
[0021] Rosenberg et al. (New Eng. J. Med. 319(25), 1676-1681, 1988)
showed the beneficial effect of immunotherapy with autologous TILs
and interleukin-2 (IL-2) in melanoma patients. This therapy
constitutes resecting the tumor deposit, isolating the TILs, in
vitro expansion of the TILs, and infusion into the patient under
concurrent treatment of high and toxicity inducing doses of IL-2.
The TILs used by Rosenberg are directed to and are able to
recognize melanoma-associated antigens. It has been our goal to
isolate such a melanoma-associated antigen in order to be able to
use the antigen and/or its epitopes for the development of an
immunotherapy for melanoma patients.
[0022] Melanoma antigens were described by Old, L. (1981) who
identified six antigenic glycoproteins and three glycolipids
occurring in 120 melanoma cell lines.
[0023] Also vaccines with melanoma antigens have been described: in
U.S. Pat. Nos. 5,030,621 and 5,194,384 a polyvalent vaccine has
been made by culturing melanoma cells and subsequent isolation of
excreted melanoma-specific antigens from the culture medium.
[0024] Some specific antigens have already been proposed for
therapy and diagnosis of melanoma type of cancer: the peptide p97
has been disclosed in U.S. Pat. No. 5,262,177 and U.S. Pat. No.
5,141,742, while a 35 kD protein has been mentioned in EP
529,007.
SUMMARY OF THE INVENTION
[0025] We now have found a melanoma-associated polypeptide,
characterized in that it comprises the amino acid sequence of SEQ
ID NO:2.
[0026] This melanocyte lineage-specific antigenic polypeptide (also
mentioned gp100) is recognized by the monoclonal antibody
NKI-beteb, which antibody has proven suitable for diagnostic
purposes. The antigens recognized by this antibody are
intracellular proteins of approximately 10 kd (gp10) and 100 kd
(gp100). The latter is also detectable in a culture medium of
melanoma cells (Vennegoor, C. et al, Am. J. Pathol. 130, 179-192,
1988). It has also been found that the gp100 antigen reacts with
other melanoma-specific antibodies such as HMB-50 (described by
Vogel, A. M. and Esclamado, R. M., Cancer Res. 48, 1286-1294, 1988)
or HMB-45 (described by Gown, A. M. et al., Am. J. Pathol. 123,
195-203, 1986). Since the proteins reacting with these monoclonal
antibodies have been shown to be glycosylated in melanoma cells,
differences have been found in mobility when analyzed by
SDS-PAGE.
[0027] Although this gp100 antigen is predominantly expressed
intracellularly, it has now been established that it is a suitable
immunogenic antigen, because it has been demonstrated that these
intracellular proteins can be processed and presented as peptides
in the context of MHC molecules to cells of the immune system. In
fact, tumor-infiltrating lymphocytes derived from tumors of
melanoma patients have been found which react with the antigen.
[0028] Therefore, the gp100 polypeptide is a potential target for
cellular responses against carcinoma and thus a suitable subject
for therapy and diagnosis in melanoma patients.
BRIEF DESCRIPTION OF THE FIGURES
[0029] The present invention is further described by way of example
with reference to the accompanying figures, in which:
[0030] FIG. 1 shows the genomic organization of part of the human
gp100/Pmel17 gene. A (SEQ ID NO:33) and A' (SEQ ID NO:34) represent
the introns which are removed in transcripts corresponding to gp100
cDNA and Pmel17 cDNA respectively. Exon sequences are indicated in
capitals and intron sequences as small letters. The best fit to the
branch point sequence (Ruskin, B. et al., 1984) is underlined.
[0031] FIG. 2 shows an alignment of the carboxyterminal part of
members of the gp100 (SEQ ID NO:2)/pMel17 family. Identical amino
acids (-) and gaps (*) are indicated. Conserved cysteine residues
(#) are indicated as well.
[0032] FIG. 3(A) Gp100 deletion mutants encoding parts of the gp100
protein are shown (numbers indicate amino acids in the gp100
protein as indicated in SEQ ID NO:2).
[0033] FIG. 3(B) Recognition by TIL 1200 of cells transfected with
HLA-A2.1 and the gp100 deletion mutants shown in FIG. 3A.
[0034] FIG. 4(A) Five peptides derived from the gp100 148-166
region (SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ
ID NO:8, SEQ ID NO:4 and residues 280-288 of SEQ ID NO:2,
respectively), varying from, an 8-mer to an 11-mer, were tested for
recognition by TIL 1200. Specific lysis was detected at an effector
to target ratio of 30:1.
[0035] FIG. 4(B) Titration of gp100 peptides (SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:8, respectively)
identified in FIG. 4(A) for recognition by TIL 1200 (E/T ratio
30:1).
[0036] FIG. 5. Binding of gp100 and viral epitopes to HLA-A2.1.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Gp100 is a type I transmembrane protein, which has a
threonine-rich domain containing repetitive amino acid sequences
present in the middle of the protein (amino acids 309-427). This
threonine-rich domain, which may be subjected to extensive O-linked
glycosylation, is preceded by a histidine-rich region (amino acids
182-313) and followed by a cysteine-rich domain (amino acids
475-566). Based on hydrophobicity plot analysis (Kyte, J. and
Doolittle, R. F., 1982), a single transmembrane domain bordered by
charged residues is present in the carboxy-terminal part (amino
acids 591-611) of gpl 00. The predicted cytoplasmic domain is 45
amino acids long. Five putative N-linked glycosylation sites are
present, consistent with gp100 being a glycoprotein.
[0038] The term "polypeptide" refers to a molecular chain of amino
acids, does not refer to a specific length of the product and may
be modified in vivo or in vitro, for example, by glycosylation,
amidation, carboxylation or phosphorylation; thus, inter alia,
peptides, oligopeptides and proteins are included within the
definition of polypeptide.
[0039] Of course, functional derivatives, as well as fragments, of
the polypeptide according to the invention are also included within
the scope of the present invention. Functional derivatives include
polypeptides differing in one or more amino acids in the overall
sequence, which have deletions, substitutions, inversions or
additions. Amino acid substitutions that can be expected not to
essentially alter biological and immunological activities have been
described. Amino acid replacements between related amino acids or
replacements that have occurred frequently in evolution are, inter
alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see, Dayhof, M.
D., Atlas of protein sequence and structure, Nat. Biomed, Res.
Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on this
information Lipman and Pearson developed a method for rapid and
sensitive protein comparison (Science, 227, 1435-1441, 1985) and
determining the functional similarity between homologous
polypeptides.
[0040] Functional derivatives which still show immunological
activity towards the monoclonal antibody NKI-beteb or HMB-50 or
HMB-45 are included within the scope of this invention.
[0041] Furthermore, as functional derivatives of these peptides are
included peptides derived from gp100 that are able to induce target
cell lysis by tumor infiltrating lymphocytes.
[0042] In addition, with functional derivatives of these peptides
are also included addition salts of the peptides, amides of the
peptides and specifically the C-terminal amides, esters and
specifically the C-terminal esters and N-acyl derivatives
specifically N-terminal acyl derivatives and N-acetyl
derivatives.
[0043] The polypeptides according to the invention can be produced
either synthetically or by recombinant DNA technology. Methods for
producing synthetic polypeptides are well known in the art.
[0044] The organic chemical methods for peptide synthesis are
considered to include the coupling of the required amino acids by
means of a condensation reaction, either in homogenous phase or
with the aid of a so-called solid phase. The condensation reaction
can be carried out as follows:
[0045] a) condensation of a compound (amino acid, peptide) with a
free carboxyl group and protected other reactive groups with a
compound (amino acid, peptide) with a free amino group and
protected other reactive groups, in the presence of a condensation
agent;
[0046] b) condensation of a compound (amino acid, peptide) with an
activated carboxyl group and free or protected other reaction
groups with a compound (amino acid peptide) with a free amino group
and free or protected other reactive groups.
[0047] Activation of the carboxyl group can take place, inter alia,
by converting the carboxyl group to an acid halide, oxide,
anhydride, imidazolide or an activated ester, such as the
N-hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl
ester.
[0048] The most common methods for these condensation reactions
are: the carbodiimide method, the azide method, the mixed anhydride
method and the method using activated esters, such as described in
The Peptides, Analysis, Synthesis, Biology, Vol. 1-3 (Ed. Gross, E.
and Meienhofer, J.) 1979, 1980, 1981 (Academic Press, Inc.).
[0049] Production of polypeptides by recombinant DNA techniques is
a method which is generally known, but which has a lot of
possibilities all leading to somewhat different results. The
polypeptide to be expressed is coded for by a DNA sequence or, more
accurately, by a nucleic acid sequence.
[0050] It has been found that the amino acid sequence of gp100
closely resembles the amino acid sequence of the already known
melanoma-associated peptide pMel17, disclosed in Kwon, B. S.
(1991).
[0051] The amino acid differences between gp100 and Pmel17 consist
of substitutions at amino acid position 274 (T-C/PRO-LEU) and 597
(C-G/ARG-PRO) and a stretch of seven amino acids absent in gp100 at
position 587. A single nucleotide difference at position 762 (C-T)
does not result in an amino acid substitution. Gp100 is also 80%
homologous to a putative protein deduced from a partial cDA clone
(RPE-1) isolated from a bovine retinal cDNA library (Kim, R. Y. and
Wistow, G. J., 1992) and 42% homologous to a chicken melanosomal
matrix protein, MMP115 (Mochii, M., 1991). See also, FIG. 2.
[0052] Also, the invention includes the nucleic acid sequence
comprising the sequence encoding the gp100 polypeptide.
[0053] Preferably, the sequence encoding gp100 is the sequence of
SEQ ID NO:1.
[0054] As is well known in the art, the degeneracy of the genetic
code permits substitution of bases in a codon resulting in another
codon still coding for the same amino acid, e.g., the codon for the
amino acid glutamic acid is both GAT and GAA. Consequently, it is
clear that for the expression of a polypeptide with an amino acid
sequence shown in SEQ ID NO:2, use can be made of a derivate
nucleic acid sequence with such an alternative codon composition
thereby differing from the nucleic acid sequence shown in SEQ ID
NO:1.
[0055] "Nucleotide sequence", as used herein, refers to a polymeric
form of nucleotides of any length, both to ribonucleic acid (RNA)
sequences and to deoxyribonucleic acid (DNA) sequences. In
principle this term refers to the primary structure of the
molecule. Thus, this term includes double and single stranded DNA,
as well as double and single stranded RNA, and modifications
thereof.
[0056] The nucleotide sequence of gp100 contains 2115 base pairs
(bp) and terminates with a poly(A) tract of 15 nucleotides that is
preceded by the consensus polyadenylation sequence AATAAA (SEQ ID
NO:31). An open reading frame (ORF) extending from nucleotide 22
through 2007 is present in gp100 DNA. This ORF starts with an ATG
codon within the appropriate sequence context for translation
initiation and codes for a protein of 661 amino acids. The
amino-terminal 20 amino acids fit all criteria for signal
sequences, including a potential cleavage site after ALA at
position 20 (-1), which would indicate that mature gp100 contains
641 amino acids (approximately 70 kD).
[0057] The most striking difference between gp100 and Pmel17 cDNAs
is the inframe deletion of 21 bp in gp100 cDNA (FIG. 2). Comparison
of the nucleotide sequence of genomic DNA with the sequence of
gp100 cDNA revealed the presence of an intron (102 bp) just at the
position of the 21 bp insertion in Pmel17 cDNA. The exon/intron
boundaries nicely fit the consensus 5' donor and 3' acceptor splice
site sequences (Padgett, 1986). In the genomic DNA, the sequence
comprising the additional 21 bp in Pmel17 cDNA is located directly
upstream of the 3' cleavage site used to generate gp100 RNA and is
preceded by an alternative 3' acceptor splice site. Whereas the
gp100-specific 3' acceptor splice site fits the consensus sequence,
the Pmel17-specific 3' acceptor splice site appears to be
sub-optimal in that it lacks a pyrimidine-rich region. Sub-optimal
RNA processing sites are present in many alternatively processed
messenger RNA precursors and have been implicated to function in
regulation of alternative RNA processing (reviewed by Green, M. R.,
1991). Collectively, these data prove that the transcripts
corresponding to gp100 and Pmel17 cDNAs are generated by
alternative splicing of a single primary transcript.
[0058] A further part of the invention are peptides, which are
immunogenic fragments of the gp100 polypeptide.
[0059] Immunogenic fragments are fragments of the gp100 molecule,
which still have the ability to induce an immunogenic response,
ie., that it is either possible to evoke antibodies recognizing the
fragments specifically, or that it is possible to find T
lymphocytes that have been activated by the fragments.
[0060] As previously stated, it has been known that the immunogenic
action of tumor-associated antigens is often elicited through a T
cell activating mechanism (Townsend, A. R. M. and Bodmer, H., Ann.
Rev. Immunol. 7, 601-624, 1989). Cytotoxic T lymphocytes (CTLs)
recognizing melanoma cells in a T cell receptor (TCR)-dependent and
MHC-restricted manner have been isolated from tumor-bearing
patients (reviewed by Knuth, A., 1992). Brichard et al. (1993) have
shown that a peptide derived from tyrosinase, an other
melanocyte-specific antigen, is recognized by a CTL clone.
[0061] It is known that the activation of T cells through the MHC
molecule necessitates processing of the antigen of which short
pieces (for example, 8-12 mers) are presented to the T
lymphocyte.
[0062] The immunogenic oligopeptides located in the gp100 sequence
form also part of the invention.
[0063] We have found immunogenic peptide sequences of the gp100
sequence which are not only able to bind with the MHC I molecule,
but which also have been demonstrated to recognize tumor
infiltrating lymphocytes which have been isolated from a melanoma
patient.
[0064] Several peptides have been found: the peptides having the
amino acid sequences V-L-P-D-G-Q-V-I-W-V (SEQ ID NO:6),
M-L-G-T-H-T-M-E-V (SEQ ID NO: 24), R-L-M-K-Q-D-F-S-V (SEQ ID
NO:25), V-W-K-T-W-G-Q-Y-W-Q-V-L (SEQ ID NO:10) and
L-L-D-G-T-A-T-L-R-L (SEQ ID NO:4) have been found to bind to the
MHC HLA-A2.1 molecule. In addition, the latter two peptides are
recognized by anti-melanoma cytotoxic T lymphocytes in the context
of HLA-A2.1.
[0065] Preferably, these peptides are flanked by non-related
sequences, i.e., sequences with which they are not connected in
nature, because it has been found that such flanking enhances the
immunogenic properties of these peptides, probably through a better
processing and presentation by APCs.
[0066] Another part of the invention is formed by nucleotide
sequences comprising the nucleotide sequences coding for the above
mentioned peptides.
[0067] Next to the use of these sequences for the production of the
peptides with recombinant DNA techniques, which will be exemplified
further, the sequence information disclosed in the sequence
listings for gp100 or its epitopes can be used for diagnostic
purposes.
[0068] From these sequences, primers can be derived as basis for a
diagnostic test to detect gp100 or gp100-like proteins by a nucleic
acid amplification technique for instance the polymerase chain
reaction (PCR) or the nucleic acid sequence based amplification
(NASBA) as described in U.S. Pat. No. 4,683,202 and EP 329,822,
respectively.
[0069] With PCR large amounts of DNA are generated by treating a
target DNA sequence with oligonucleotide primers such that at
primer extension product is synthesized which is separated from the
template using heat denaturation and in turn serves as a template,
resulting in amplification of the target sequence. When RNA is to
be amplified with PCR, the RNA strand is first transcribed into a
DNA strand with the aid of reverse transcriptase.
[0070] With the aid of NASBA, large amounts of single stranded RNA
are generated from either single stranded RNA or DNA or double
stranded DNA. When RNA is to be amplified the ssRNA serves as a
template for the synthesis of a first DNA strand by elongation of a
first primer containing a ssRNA polymerase recognition site. The
formed DNA strand, in turn, serves as the template for the
synthesis of a second, complementary, DNA strand by elongation of a
second primer, resulting in a double stranded active RNA-polymerase
promoter site, and the second DNA serves as a template for
synthesis of large amounts of the first template, the ssRNA, with
the aid of RNA polymerase.
[0071] Detection of the amplified nucleotide sequence is
established by hybridizing a complementary detection probe to the
amplified nucleic acid. This probe can be labeled and/or
immobilized on a solid phase. Detection of the label can be
performed through methods known in the art. Detection of nucleic
acids bound through the probe to the solid phase can be done by
compounds capable of selective detection of nucleic acids.
[0072] As previously mentioned, the nucleotide sequences can be
used for the production of gp100 or one of its epitopes with
recombinant DNA techniques. For this, the nucleotide sequence must
be comprised in a cloning vehicle that can be used to transform or
transfect a suitable host cell.
[0073] A wide variety of host cell and cloning vehicle combinations
may be usefully employed in cloning the nucleic acid sequence. For
example, useful cloning vehicles may include chromosomal,
non-chromosomal and synthetic DNA sequences such as various known
bacterial plasmids, and wider host range plasmids such as pBR 322,
the various pUC, pGEM and pBluescript plasmids, bacteriophages,
e.g., lambda-gt-Wes, Charon 28 and the M13 derived phages and
vectors derived from combinations of plasmids and phage or virus
DNA, such as SV40, adenovirus or polyoma virus DNA (see also,
Rodriquez, R. L. and Denhardt (1988); Lenstra, 1990).
[0074] Useful hosts may include bacterial hosts, yeasts and other
fungi, plant or animal hosts, such as Chinese Hamster Overy (CHO)
cells or monkey cells and other hosts.
[0075] Vehicles for use in expression of the peptides will further
comprise control sequences operably linked to the nucleic acid
sequence coding for the peptide. Such control sequences generally
comprise a promoter sequence and sequences which regulate and/or
enhance expression levels. Furthermore, an origin of replication
and/or a dominant selection marker are often present in such
vehicles. Of course, control and other sequences can vary depending
on the host cell selected.
[0076] Techniques for transforming or transecting host cells are
quite known in the art (see, for instance, Maniatis et al., 1982
and 1989).
[0077] It is extremely practical if, along with the information for
the peptide, the host cell also is co-transformed or co-transfected
with a vector that carries the information for an MHC molecule to
which said peptide is known to bind. Preferably the MHC molecule is
HLA-A2.1 HLA-AL or HLA-A3.1, or any other HLA allele that is known
to be present in melanoma patients. HLA-A2.1 is especially
preferred because it has been established (Anichini A., 1993) that
melanoma cells carry antigens recognized by HLA-A2.1 restricted
cytotoxic T cell clones from melanoma patients.
[0078] Host cells especially suited for the expression of gp100 are
the murine EL4 and P8.15 cells. For expression of gp100 human BLM
cells (described by Katano, M., 1984) are especially suited because
they already are able to express the MHC molecule HLA-A2.1
[0079] Gp100 or any of its peptides or their nucleotide sequences
mentioned above can be used in a vaccine for the treatment of
melanoma.
[0080] In addition to an immunogenically effective amount of the
active peptide, the vaccine may contain a pharmaceutically
acceptable carrier or diluent. Such carriers and diluents are well
known to those of skill in the art.
[0081] The immunogenicity of the peptides of the invention,
especially the oligopeptides, can be enhanced by cross-linking or
by coupling to an immunogenic carrier molecule (i.e., a
macromolecule having the property of independently eliciting an
immunological response in a patient, to which the peptides of the
invention can be covalently linked).
[0082] Covalent coupling to the carrier molecule can be carried out
using methods well known in the art, the exact choice of which will
be dictated by the nature of the carrier molecule used. When the
immunogenic carrier molecule is a protein, the peptides of the
invention can be coupled, e.g., using water-soluble carboimides
such as dicyclohexylcarbodiimide, or glutaraldehyde.
[0083] Coupling agents such as these can also be used to cross-link
the peptides to themselves without the use of a separate carrier
molecule. Such cross-linking into polypeptides or peptide
aggregates can also increase immunogenicity.
[0084] Examples of pharmaceutically acceptable carriers or diluents
useful in the present invention include stabilizers such as SPGA,
carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose,
dextran), proteins such as albumin or casein, protein-containing
agents such as bovine serum or skimmed milk and buffers (e.g.,
phosphate buffer).
[0085] Optionally, one or more compounds having adjuvant activity
may be added to the vaccine. Suitable adjuvants are for example
aluminum hydroxide, phosphate or oxide, oil-emulsions (e.g., Bayol
F.sup.(R) or Marcol 52.sup.(R)), saponins or vitamin-E
solubilisate.
[0086] The vaccine according to the present invention can be given
inter alia intravenously, intraperitoneally, intranasally,
intradermally, subcutaneously, or intramuscularly.
[0087] The useful effective amount to be administered will very
depending on the age and weight of the patient and mode of
administration of the vaccine.
[0088] The vaccine can be employed to specifically obtain a T cell
response, but it is also possible that a B cell response is
elicited after vaccination. If so, the B cell response leads to the
formation of antibodies against the peptide of the vaccine, which
antibodies will be directed to the source of the antigen
production, i.e., the tumor cells. This is an advantageous feature,
because in this way the tumor cells are combated by responses of
both immunological systems.
[0089] Both immunological systems will even be more effectively
triggered when the vaccine comprises the peptides as presented in
an MHC molecule by an antigen presenting cell (APC). Antigen
presentation can be achieved by using monocytes, macrophages,
interdigitating cells, Langerhans cells and especially dendritic
cells, loaded with one of the peptides of the invention. Loading of
the APCs can be accomplished by bringing the peptides of the
invention into or in the neighborhood of the APC, but it is more
preferable to let the APC process the complete gp100 antigen. In
this way, a presentation is achieved which mimics the in vivo
situation most realistically. Furthermore, the MHC used by the cell
is of the type that is suited to present the epitope.
[0090] An overall advantage of using APCs for the presentation of
the epitopes is the choice of APC cell that is used in this
respect. It is known from different types of APCs that there are
stimulating APCs and inhibiting APCs.
[0091] Preferred are the listed cell types, which are so-called
"professional" antigen presenting cells, characterized in that they
have co-stimulating molecules, which have an important function in
the process of antigen presentation. Such co-stimulating molecules
are, for example, B7, CTLA-4, CD70, or heat stable antigen
(Schwartz, 1992).
[0092] Fibroblasts, which have also been shown to be able to act as
an antigen-presenting cell, lack these co-stimulating
molecules.
[0093] It is also possible to use cells already transfected with a
cloning vehicle harboring the information for gp100 and which are
cotransfected with a cloning vehicle that comprises the nucleotide
sequence for an MHC class I molecule, for instance the sequence
coding for HLA A2.1, HLA A1 or HLA A3.1. These cells will act as an
antigen presenting cell and will present gp100-fragments in the MHC
class I molecules which are expressed on their surface. It is
envisaged that this presentation will be enhanced when the cell is
also capable of expressing one of the above-mentioned
co-stimulating molecules or a molecule with a similar function.
This expression can be the result of transformation or transfection
of the cell with a third cloning vehicle having the sequence
information coding for such a co-stimulating molecule, but it can
also be that the cell already was capable of production of
co-stimulating molecules.
[0094] Instead of a vaccine with these cells, which next to the
desired expression products, also harbor many elements which are
also expressed and which can negatively affect the desired
immunogenic reaction of the cell, it is also possible that a
vaccine is composed with liposomes which expose MHC molecules
loaded with peptides, and which, for instance, are filled with
lymphokines. Such liposomes will trigger an immunologic T cell
reaction.
[0095] By presenting the peptide in the same way as it is also
presented in vivo, an enhanced T cell response will be evoked.
Furthermore, by the natural adjuvant working of the relatively
large antigen presenting cells, a B cell response also is
triggered. This B cell response will lead to the formation of
antibodies directed to the peptide-MHC complex. This complex is
especially found in tumor cells where it has been shown that in the
patient epitopes of gp100 are presented naturally, which are thus
able to elicit a T cell response. It is this naturally occurring
phenomenon that is enlarged by the vaccination of APCs already
presenting the peptides of the invention. By enlarging not only an
enlarged T cell response will be evoked, but also a B cell response
that leads to initiation of antibodies directed to the MHC-peptide
complex.
[0096] The vaccines according to the invention can be enriched by
numerous compounds having an enhancing effect on the initiation and
the maintenance of both the T cell and the B cell response after
vaccination.
[0097] In this way, addition of cytokines to the vaccine will
enhance the T cell response. Suitable cytokines are for instance
interleukines, such as IL-2, IL-4, IL-7, or IL-12, GM-CSF, RANTES,
tumor necrosis factor and interferons, such as IFN.
[0098] In a similar way, antibodies against T cell surface
antigens, such as CD2, CD3, CD27 and CD28 will enhance the
immunogenic reaction.
[0099] Also, the addition of helper epitopes to stimulate CD4.sup.+
helper cells or CD8.sup.+ killer cells augments the immunogenic
reaction. Alternatively, helper epitopes from other antigens can be
used, for instance, from heat shock derived proteins or cholera
toxin.
[0100] Another part of the invention is formed by usage of gp100
reactive tumor infiltrating lymphocytes (TILs). In this method, the
first step is taking a sample from a subject. This is usually done
by resection of a tumor deposit under local anaesthesia. The TILs
present in this specimen are then expanded in culture for four to
eight weeks, according to known methods (Topalian, S. L. et al.,
1987). During this culture the TILs are then checked for reactivity
with gp100 or one of the epitopes derived from gp100. The TILs that
recognize the antigen are isolated and cultured further.
[0101] The tumor infiltrating lymphocytes, reactive with gp100,
which are obtained through this method, form also part of the
invention. One such TIL cell line, designated TIL 1200, has been
found which specifically reacts with gp100 and its epitopes. This
TIL 1200 cell line also expresses the MHC molecule HLA-A2.1.
Furthermore expression of TCR .alpha./.beta., CD3 and CD8 by this
cell line has been demonstrated. Furthermore TIL 1200 recognizes
transfectants expressing both HLA-A2.1 and gp100.
[0102] This TIL 1200 and other TILs recognizing gp100 are suited
for treatment of melanoma patients. For such treatment TILs are
cultured as stated above, and they are given back to the patients
by an intravenous infusion. The success of treatment can be
enhanced by pre-treatment of the tumor bearing host with either
total body radiation or treatment with cyclophosphamide and by the
simultaneous administration of interleukin-2 (Rosenberg, S. A. et
al., 1986).
[0103] The TILs infused back to the patient are preferably
autologous TILs (i.e., those derived from the patient's own tumor)
but also infusion with allogenic TILs can be imagined.
[0104] A further use of the TILs obtained by the method as
described above is for in vivo diagnosis. Labeling of the TILs, for
instance, with .sup.111In (Fisher, 1989) or any other suitable
diagnostic marker, renders them suited for identification of tumor
deposits in melanoma patients.
[0105] Another part of the invention is formed by the T cell
receptor (TCR) expressed by gp100 reactive CTLs. As is well known
in the art, the TCR determines the specificity of a CTL. Therefore,
the cDNA encoding the TCR, especially its variable region, can be
isolated and introduced into T cells, hereby transferring
anti-tumor activity to any T cell. Especially introduction of such
a TCR into autologous T cells and subsequent expansion of these T
cells, will result in large numbers of CTL suitable for adoptive
transfer into the autologous patient.
[0106] Also, cells harboring this T cell receptor can be used for
vaccination purposes.
[0107] A vaccine can also be composed of melanoma cells capable of
expressing gp100. It is possible to isolate these cells from a
patient, using anti-gp100 antibodies, such as NKI-beteb, but is
also possible to produce such melanoma cells from cultured melanoma
cell lines, which either are natural gp100-producers or have been
manipulated genetically to produce gp100. These cells can be
irradiated to be non-tumorogenic and infused (back) into the
patient. To enhance the immunologic effect of these melanoma cells
it is preferred to alter them genetically to produce a lymphokine,
preferably interleukine-2 (IL-2) or granulocyte-macrophage colony
stimulation factor (GM-CSF). Gp110.sup.+ melanoma cells can be
transfected with a cloning vehicle having the sequence coding for
the production of IL-2 or GM-CSF.
[0108] Infusion of such a vaccine into a patient will stimulate the
formation of CTLs.
[0109] Another type of vaccination having a similar effect is
vaccinating with pure DNA, for instance, the DNA of a vector or a
vector virus having the DNA sequence encoding the gp100 antigen or
peptides derived therefrom. Once injected, the virus will infect or
the DNA will be transformed to cells that express the antigen or
the peptide(s).
[0110] Antibodies to any gp100 peptide, including antibodies to
V-W-K-T-W-G-Q-Y-W-Q-V-L (SEQ ID NO:10), and L-L-D-G-T-A-T-L-R-L
(SEQ ID NO:4) are also part of the invention.
[0111] Monospecific antibodies to these peptides can be obtained by
affinity purification from polyspecific antisera by a modification
of the method of Hall, R. et al. (1984). Polyspecific antisera can
be obtained by immunizing rabbits according to standard
immunization schemes.
[0112] "Monospecific antibody", as used herein, means a single
antibody species or multiple antibody species with homogeneous
binding characteristics for the relevant antigen. Homogeneous
binding as used herein refers to the ability of the antibody
species to bind to ligand binding domains of the invention. The
antibody is preferably a monoclonal antibody, more preferably, a
humanized or human monoclonal antibody.
[0113] Monoclonal antibodies can be prepared by any techniques,
such as, for example, immunizing inbred mice, preferably Balb/c
with the appropriate protein by techniques known in the art (see,
e.g., Kohler, G. and Milstein C., 1975). Hybridoma cells are
subsequently selected by growth in hypoxanthine, thymidine and
aminopterin in an appropriate cell culture medium such as
Dulbecco's modified Eagle's medium (DMEM). Antibody producing
hybridomas are cloned, preferably using the soft agar technique of
MacPherson (1973). Discrete colonies are transferred into
individual wells of culture plates for cultivation in an
appropriate culture medium. Antibody producing cells are identified
by screening with the appropriate immunogen. Immunogen positive
hybridoma cells are maintained by techniques known in the art.
Specific anti-monoclonal antibodies are produced by cultivating the
hybridomas in vitro or preparing ascites fluid in mice following
hybridoma injection by procedures known in the art.
[0114] It is preferred to use humanized antibodies. Methods for
humanizing antibodies, such as CDR-grafting, are known (Jones, P.
T. et al., 1986). Another possibility to avoid antigenic response
to antibodies reactive with polypeptides according to the invention
is the use of human antibodies or fragments or derivatives
thereof.
[0115] Human antibodies can be produced by in vitro stimulation of
isolated B-lymphocytes, or they can be isolated from (immortalized)
B-lymphocytes that have been harvested from a human being immunized
with at least one ligand-binding domain according to the
invention.
[0116] Antibodies, as described above, can be used for the passive
vaccination of melanoma patients. A preferred type of antibodies
for this kind of vaccine are antibodies directed against the
above-mentioned peptides presented in connection with the MHC
molecule. To produce these kinds of antibodies, immunization of
peptides presented by APCs is required. Such an immunization can be
performed as described above. Alternatively, human antibodies to
peptide-MHC complexes can be isolated from patients treated with a
vaccine consisting of APCs loaded with one of said peptides.
[0117] The antibodies, which are formed after treatment with one of
the vaccines according to the invention can also be used for the
monitoring of the vaccination. For such a method, serum of the
patients is obtained and the antibodies directed to the peptide for
which they have been vaccinated are detected. Knowing the antibody
titer from this detection, it can be determined if t a need exists
for a booster vaccination.
[0118] Specific detection of the antibodies in the serum can be
achieved by labeled peptides. The label can be any diagnostic
marker known in the field of in vitro diagnosis, but most preferred
(and widely used) are enzymes, dyes, metals and radionuclides, such
as .sup.67Ga, .sup.99mTc, .sup.111In, .sup.113mIn, .sup.123I,
.sup.125I or .sup.131I.
[0119] The radiodiagnostic markers can be coupled directly to the
peptides of the invention or through chelating moieties which have
been coupled to the peptide directly or through linker or spacer
molecules. The technique of coupling of radionuclides to peptides
or peptide-like structures is already known in the field of (tumor)
diagnostics from the numerous applications of labeled antibodies
used both in in vivo and in in vitro tests.
[0120] Direct labeling of peptides can for instance be performed as
described in the one-vial method (Haisma, 1986). A general method
for labeling of peptides through chelators, with or without linker
or spacer molecules, has for instance been described in U.S. Pat.
No. 4,472,509 and U.S. Pat. No. 4,485,086. Chelators using a
bicyclic anhydride of DTPA have been disclosed in Hnatowich, D. J.
et al. (1983). Coupling through diamide dimercaptide compounds has
been disclosed in EP 188,256.
[0121] The invention is further explained by use of the following
illustrative Examples.
EXAMPLES
Example 1
Molecular Characterization of Gp100
[0122] Materials and Methods
[0123] Cells and Monoclonal Antibodies
[0124] The melanoma cell lines Mel-2a, M14, MEWO, BLM (Vennegoor et
al., 1988; van Muijen et al., 1991; Bean et al., 1975; Katano et
al., 1984) and the uveal melanoma cell line Mel 202 (Ksander et
al., 1991) have been previously described. Isolation of normal
human melanocytes from breast or foreskin was performed by the
method of Eisinger and Marko (1982) with modifications by (Smit et
al., 1993).
[0125] Mabs NKI-beteb and HMB-50 have been described previously
(Vennegoor et al., 1988; Vogel and Esclamado, 1988). Mab HMB-45 was
purchased from Enzo Biochem.
[0126] DNA Constructs and Transfections
[0127] The 2.2 kb Eco RI fragment containing gp100 cDNA was
blunt-ended by filling in the ends with Klenow DNA Polymerase and
then closed in both orientations (pSVLgp100+ and pSVLgp100-) in the
Sma I site of the eukaryotic expression vector pSVL (Pharmacia).
pSVL contains the SV40 late promoter and polyadenylation site as
well as the SV40 origin of replication, allowing a very high copy
number during transient expression in COS-7 cells.
[0128] For the construction of the 3' truncated gp100 transcription
unit pSVLgp100+ (@BS) we deleted the sequence between the Bgl II
site in the 3' part of gp100 cDNA and the Sac I site in the
multiple cloning site of the vector. The resulting construct
encodes a truncated gp100 protein in which the carboxy-terminal 133
amino acids of gp100 are replaced by 4 amino acids
(Arg-Ile-Gln-Thr) (SEQ ID NO:32) encoded by vector sequences.
Transient expression of the constructs in COS-7 cells was performed
by using 40 .mu.g/ml lipofection reagent from BRL (Felgner et al.,
1987) and 7.5 .mu.g DNA as described previously (Loenen et al.,
1991).
[0129] Immunofluorescence
[0130] Transfected COS-7 cells were prepared for immunofluorescence
48 hours after the addition of the lipofection/DNA mixture as
described previously (Vennegoor et al., 1988). After incubation
with the primary antibody for 45 minutes, cells were washed and
incubated with fluorescein isothiocyanate (FITC)-labeled goat
F(ab)'.sub.2 anti-mouse IgG (Nordic) for 30 minutes. Preparations
were examined using a confocal laser-scanning microscope at 488 nm
(Biorad MRC 600).
[0131] Metabolic Labeling, Immunoprecipitations and V8 Protease
Mapping
[0132] Immunoprecipitation experiments were performed on
metabolically labeled (L-[.sup.35S]-methionine/cysteine; Amersham)
cells as described by Vennegoor et al. (1988) using either mAb
NKI-betab or HMB-50 covalently linked to protein A-CL 4B sepharose
beads (Pharmacia). In some experiments tunicamycin (75 .mu.g/ml,
Calbiochem) was added during the pre-labeling period and remained
present during the metabolic labeling reaction (12.5 minutes).
Immunoprecipitates were analyzed under reducing conditions (5%
.beta.-mercaptoethanol in SDS-sample buffer) by SDS-PAGE using
5-17.5% polyacrylamide gradient gels. The relative molecular weight
of the proteins was determined using co-electrophorised,
pre-stained molecular weight markers (BRL). Gels were treated with
IM sodium salicylate (pH 5.4) prior to autoradiography (Kodak
XAR).
[0133] V8 protease mapping was performed using the digestion for
proteins in gel slices procedure described by Cleveland et al.
(1977). Briefly, gel slices containing the 100 kD proteins were
placed in the wells of a second SDS-gel (10%) and overlayed with
Staphylococcus aureus V8 protease (2,5 .mu.g/sample, Miles
laboratories). After electrophoresis gels were treated as described
above.
[0134] Molecular Cloning of Part of the Gp100/Pmel17 Gene
[0135] Part of the gp100/Pmel17 gene was amplified by PCR (Taq DNA
Polymerase was from Gibco) on human genomic DNA isolated from
peripheral blood lymphocytes (PBLs) using the following primers:
1497/1516: 5'-TATTGAAAGTGCCGAGATCC-3' (SEQ ID NO:26) and 1839/1857:
5'-TGCAAGGACCACAGCCATC-3' (SEQ ID NO:27) as described previously
(Adema and Baas, 1991). The PCR products were subsequently
amplified using a nested set of primers containing an additional
Eco RI site (5'-TATCTAGAATTCTGCACCAGATACTGAAG-3' (SEQ ID NO: 11)
and 5'-TATCTAGAATTCTGCAAGATGCCCACGATCAG-3' (SEQ ID NO: 12)). The
underlined Eco RI sites in these primers were used to clone the PCR
product in the Eco RI site of pUC 18.
[0136] RNA Isolation and Analysis
[0137] Total RNA was isolated using the guanidine thiocyanate
procedure and centrifugation through a cushion of Cesium chloride
(Chirgwin et al., 1979). cDNA was prepared using the Geneamp RNA
PCR kit (Perkin Elmer Cetus) as indicated by the manufacturer. PCR
analysis of the cDNAs was performed for 35 cycles in the presence
of 3 mM MgCl.sub.2 using primers 1497/1516 and 1839/1857 (see
above) as described previously (Adema and Baas, 1991). The reaction
products were size-fractioned on an agarose gel, blotted onto a
nylon membrane (Hybond-N, Amersham) and hybridized to
[.sup.32P]-labeled oligonucleotide probes as described previously
(Adema and Baas, 1991). As probes we used either a gp100-specific
exon/exon junction oligonucleotide (5'-CTTCTTGACCAGGCATGATA-3' (SEQ
ID NO:13)) or a Pmel17-specific oligonucleotide
(5'-TGTGAGAAGAATCCCAGGCA-3' (SEQ ID NO:14)) that corresponds to 20
of the additional 21 nucleotides present in Pmel17 cDNA. In every
hybridization experiment a spot blot containing an oligonucleotide
comprising the Pmel17 exon/exon junction
(5'-GCTTATCATGCCTGTGCCTGGATTCTTCTCACAGGT-3' (SEQ ID NO:15)) was
included as a control.
[0138] Nucleotide Sequence Analysis
[0139] Gp100 cDNA and genomic DNA clones were sequenced by the
dideoxy-nucleotide sequencing method (Sanger et al., 1977) using T7
DNA polymerase (Pharmacia). The sequence of both strands was
determined in each case. Since the genomic DNA clones were obtained
after PCR, the sequence of four independent clones was determined.
Analysis of the DNA sequence was performed using the University of
Wisconsin Genetics Computing Group sequence analysis programs
(Devereux et al., 1984).
[0140] Results
[0141] Expression of Gp100 cDNA in Non-Pigmented COS-7 Cells
Results in Immunoreactivity with mAbs NKI-beteb, HMB-50 and
HMB-45
[0142] Expression of gp100 cDNA in gp100-negative BLM melanoma
cells results in immunoreactivity with the melanocyte
lineage-specific mAbs, NKI-beteb, HMB-50 and HMB-45. To determine
whether expression of gp-100 cl cDNA in non-melanocytic cells also
results in immunoreactivity with these mAbs, we performed transient
expression experiments in COS-7 cells (monkey kidney fibroblasts)
with constructs containing gp100 cDNA in the coding or non-coding
orientation. Only COS-7 cells transfected with the construct
containing the cDNA in the coding orientation (COS-7/pSVLgp100+)
react with all three mAbs. These data demonstrate that
immunoreactivity with mAbs NKI-beteb, HMB-50 and HMB-45 after
expression of gp100 cDNA is not restricted to melanocytic cells. In
addition, these data show that the COS expression system can be
used for further biochemical characterization of the proteins
encoded by gp100 cDNA.
[0143] Analysis of the Proteins Encoded by Gp100 cDNA.
[0144] To characterize the proteins encoded by gp100 cDNA,
COS-7/pSVLgp100+ cells were metabolically labeled and subjected to
immunoprecipitation with mAb NKI-beteb or HMB-50. MoAbs NKI-beteb
and HMB-50 specifically immunoprecipitate proteins of approximately
100 kD (95-110 kd) from extracts of COS-7/pSVLgp100+ cells. The
molecular weight of these proteins is similar (see also below) to
those immunoprecipitated from extracts of metabolically labeled
MEWO cells which express the antigens endogenously (Vennegoor et
al., 1988). Consistent with previous reports (Vennegoor et al.,
1988; Vogel and Esclamado, 1988), both mAbs also recognize a
protein of 10 kD in extracts of MEWO melanoma cells. A protein of
the same size reacts with mAb NKI-beteb in COS-7/pSVLgp100+ cells
and can be discerned with mAb HMB-50 after prolonged exposure (not
shown). We note that the amount of the 10 kd protein varied
considerably between experiments. No specific proteins are
immunoprecipitated by either of the mAbs from extracts prepared
from COS-7 cells transfected with the construct containing the cDNA
in the non-coding orientation.
[0145] Glycoproteins of approximately 100 kD reacting with mAbs
NKI-beteb and HMB-50 have also been found in culture medium of
melanoma cells (Vennegoor et al., 1988; Vogel and Esclamado, 1988).
Comparison of the culture medium of metabolically labeled
COS-7/pSVLgp100+ cells and MEWO cells reveals that both mAbs also
recognize proteins of about 100 kD (see also below) in the culture
medium of these cells. No proteins of 10 kD are immunoprecipitated
by the mAbs from the culture medium of COS-7/pSVLgp100+ cells, as
has been shown for melanoma cells. These data demonstrate that, as
in melanoma cells, the proteins of about 100 kd recognized by mAbs
NKI-beteb and HMB-50 in COS-7/pSVLgp100+ cells are secreted.
[0146] To exclude the possibility that the proteins detected by the
mAbs are derived from endogenous genes induced after transfection
with gp100 cDNA, we performed immunoprecipitation experiments with
COS-7 cells expressing a 3' truncated gp100 transcription unit (see
Materials & Methods for details). Proteins of approximately 85
kd are immunoprecipitated by both mAbs from COS-7 cells expressing
this construct, consistent with a deletion of 129 amino acids. This
finding provides direct evidence that the 100 kd protein recognized
by mAbs NKI-beteb and HMB-50 in COS-7/pSVLgp100+ cells is encoded
by gp100 cDNA.
[0147] The 100 kd Protein Encoded by Gp100 cDNA is Identical to
Gp100
[0148] The proteins of about 100 kD identified by mAbs NKI-beteb
and HMB-50 in COS-7/pSVLgp100+ cells versus MEWO cells have a
slightly different mobility when analyzed by SDS-PAGE. Since the
proteins reacting with these mAbs have been shown to be
glycosylated in melanoma cells (Vennegoor et al., 1988; Vogel and
Esclamado, 1988), these differences could be due to altered
glycosylation, an event frequently observed in the COS expression
system. To confirm this, mAb NKI-beteb was used to
immunoprecipitate proteins from MEWO cells and COS-7/pSVLgp100+
cells cultured in the presence of the glycosylation inhibitor
tunicamycin. In both COS-7/pSVLgp100+ cells and MEWO cells the size
of the proteins of about 100 kd is reduced to two protein bands of
90 kd and 85 kD, confirming that the observed difference in
mobility is due to altered glycosylation.
[0149] To provide further evidence that the proteins recognized by
mAb NKI-beteb in COS-7/pSVLgp100+ cells and MEWO cells are
identical, we performed a V8 protease mapping experiment. The same
protein fragments are obtained after V8 protease digestion of the
major 100 kD protein isolated from COS-7/pSVLgp100+ cells or MEWO
cells. We conclude from these data that gp100 CDNA encodes the
melanocyte lineage-specific glycoprotein gp100 recognized by mAbs
NKI-beteb and HMB-50 in melanoma cells.
[0150] Gp100 is a Type I Transmembrane Protein Highly Homologous to
Pmel17
[0151] The nucleotide sequence of gp100 cDNA was determined. It
contains 2115 base pairs (bp) and terminates with a poly(A) tract
of 15 nucleotides that is preceded by the consensus polyadenylation
sequence AATAAA, (SEQ ID NO:31) (Proudfoot and Brownlee, 1976). An
open reading frame (ORF) extending from nucleotide 22 through 2007
is present in gp100 cDNA. This ORF starts with an ATG codon within
the appropriate sequence context for translation initiation (Kozak,
1987) and codes for a protein of 661 amino acids (SEQ ID NO:1). The
amino-terminal 20 amino acids fit all criteria for signal
sequences, including a potential cleavage site after ALA at
position 20 (von Heyne, 1986), which would indicate that mature
gp100 contains 641 amino acids (approximately 70 kD). Based on
hydrophobicity plot analysis (Kyte and Doolittle, 1982), a single
transmembrane domain bordered by charged residues is present in the
carboxy-terminal part (amino acids 591-611) of gp100. The predicted
cytoplasmic domain is 45 amino acids long. Five putative N-linked
glycosylation sites are present, consistent with gp100 being a
glycoprotein. Furthermore, a histidine-rich domain (amino acids
182-313), a threonine-rich domain (amino acids 309-427) containing
repetitive amino acid sequences, and a cysteine-rich domain
(475-566 amino acids) are present.
[0152] A data base search (Pearson and Lipman, 1988; Altschul et
al., 1990) revealed that gp100 is almost identical to Pmel17,
another melanocyte-specific protein (Kwon et al., 1991). The amino
acid differences between gp100 and Pmel17 consist of substitutions
at position 274 (T-C/PRO-LEU) and 597 (C-G/ARG-PRO) and a stretch
of 7 amino acid absent in gp100 at position 587 (see also, FIG. 2).
A single nucleotide difference at position 782 (C-T) does not
result in an amino acid substitution. Gp100 is also 80% homologous
to a putative protein deduced from a partial cDNA clone (RPE-1)
isolated from a bovine retinal cDNA library (Kim and Wistow, 1992)
and 42% homologous to a chicken melanosomal matrix protein, MMP115
(Mochii et al., 1991).
[0153] Gp100 and Pmel17 are Encoded by a Single Gene
[0154] The most striking difference between gp100 and Pmel17 cDNAs
is the inframe deletion of 21 bp in gp100 cDNA. One possible
explanation for this difference is the existence of two closely
related genes. However, since both cDNAs have identical nucleotide
sequences in their 3' untranslated regions this explanation is not
likely. Another possibility is that both cDNAs correspond to
transcripts generated by alternative splicing of a single primary
transcript. To test this hypothesis, we used PCR to analyze the
genomic DNA corresponding to the part of the gp100 gene surrounding
the putative alternative splice site. Comparison of the nucleotide
sequence of this genomic DNA with the sequence of gp100-cl cDNA
revealed the presence of an intron (102 bp) just at the position of
the 21 bp insertion in Pmel17 cDNA (FIG. 1). The exon/intron
boundaries nicely fit the consensus 5' donor and 3' acceptor splice
site sequences (Padgett et al., 1986). In the genomic DNA, the
sequence comprising the additional 21 bp in Pmel17 cDNA is located
directly upstream of the 3' cleavage site used to generate gp100
RNA and is preceded by an alternative 3' acceptor splice site (FIG.
1). Whereas the gp100-specific 3' acceptor splice site fits the
consensus sequence, the Pmel17-specific 3' acceptor splice site
appears to be sub-optimal in that it lacks a pyrimidine-rich region
(FIG. 1) Sub-optimal RNA processing sites are present in many
alternatively processed messenger RNA precursors and have been
implicated to function in regulation of alternative RNA processing
(reviewed by Green, 1991). Collectively, these data prove that the
transcripts corresponding to gp100 and Pmel17 cDNAs are generated
by alternative splicing of a single primary transcript and thus
originate from a single gene.
[0155] Expression of Gp100 and Pmel17 RNAs in Cells of the
Melanocytic Lineage
[0156] The finding that gp100 and Pmel17 RNAs arise by alternative
splicing of a single primary transcript, raises the question
whether this occurs in a developmentally regulated manner. An RNA
species of 2.5 kb is the major RNA product detected by gp100 cDNA
on Northern blots containing RNA isolated from melanocytic cells.
The same results were obtained by Kwon et al. (1987) using Pmel17-1
cDNA as a probe. However, neither of the probes discriminate
between gp100 and Pmel17 RNAs. To investigate the expression of
gp100 and Pmel17 RNAs in cells of the melanocytic lineage, we
performed a reverse transcriptase/polymerase chain reaction
(RT/PCR) assay followed by Southern blotting and hybridization to
either a gp100 specific exon/exon junction- or a Pmel17-specific
oligonucleotide probe (see, Materials & Methods). Gp100 and
Pmel17 spliced products are both detected in 3 out of 4 cutaneous
melanoma cells, in uveal melanoma cells as well as in neonatal and
adult melanocytes. No products are detected with either probe in
gp100-negative BLM melanoma cells. These results demonstrate that
in all melanocytic cells examined, gp100 and Pmel17 RNAs are
expressed simultaneously.
Example 2
Recognition of Gp100 by Tils
[0157] Material and Methods
[0158] Cell Culture
[0159] TILs were generated by growth of single cell suspensions of
metastatic melanomas with 1,000 U/ml IL-2 (Cetus Corp., Emeryville,
Calif.) and were grown as described previously (Kawakami, 1992).
Melanoma cell lines Mel 397 and Mel 624 were obtained and grown as
reported previously (Kawakami, 1992). HLA-A2.1.sup.+ melanoma cell
lines MeWo (Bean, 1975) and BLM (Katano, 1984) and murine P815
transfectants were grown in DMEM (Gibco, Paisley, Scotland, UK)
plus 7.5% heatinactivated FCS (Gibco). JY, K562, and murine EL4
transfectants were cultured in Iscoves medium (Gibco) plus 7.5%
FCS. Murine cells were grown in the presence of 5.multidot.10.sup.5
M .beta.-ME, and all media contained antibiotics. Isolation of
normal melanocytes from foreskin was performed by the method of
Eisinger and Marko (1982) with modifications as described
previously (Smit, 1989). Melanocytes from passages two to three
were used in chromium release assays.
[0160] DNA Constructs and Transfection.
[0161] Plasmid pBJ1gp100neo was obtained by cloning the EcoRI
fragment of a lambda gp100 cDNA clone in the coding orientation in
the polylinker pBJ1-neo (Lin, 1990). Plasmid pBA2 containing a
genomic fragment encoding HLA-A2.1 and human .beta.-2 microglobulin
was kindly provided by E. J. Baas (The Netherlands Cancer
Institute, Division of Biochemistry, Amsterdam, The Netherlands).
Plasmid pGK-hyg contains the hygromycin phosphotransferase gene (Te
Riele, 1990). For the introduction of the HLA-A2.1 and human
.beta.-2 microglobulin genes, EL4 cells were transfected with 18
.mu.g of pBA2 and 2 .mu.g of pGK-hyg DNA according to the calcium
phosphate coprecipitation procedure (Graham, 1973) using
Calciumphosphate Transfection Systems (Gibco BRL, Gaithersburg,
Md.), 24 hours after transfection, 500 .mu.g/ml hygromycin B
(Calbiochem-Novabiochem Corp., La Jolla, Calif.) was added to the
medium for the selection of stable transfectants. HLA-A2.1.sup.+
gp100.sup.+ EL cells were obtained by transfection of stable
HLA-A2.1.sup.+ E14 clones with 20 .mu.g of pBJ1-gp100neo DNA by
calcium phosphate coprecipitation and were selected with 1 mg/ml
G418. P815 A2.1 and P815 A2.1/gp100 cells were kindly provided by
P. Coulie (Ludwig Ins., Brussels, Belgium).
[0162] mAb and Flow Cytometry
[0163] Phenotypic analysis of melanomas, transfectants, and normal
melanocytes was performed by indirect immunofluorescence followed
by flow cytometry using a FACScan.sup.R (Becton Dickinson &
Co., Mountain View, Calif.). Purified anti-gp100 mAb NKI-beteb
(Vennegoor, 1988) and anti-HLA-A2 mAbs BB7.2 (culture supernatant;
Parham, 1981) and MA2.1 (ascites 1:500 dilution; Parham, 1978) were
used as primary reagents. FITC conjugated GAM-IgG-F(ab').sub.2
(Zymed Laboratories, Inc. S. San Francisco, Calif.) was used for
the second incubation. For the detection of the intracellular gp100
antigen cells were permeabilized in 0.01% digitonin and were
subsequently fixed in 1% paraformaldehyde.
[0164] Chromium Release Assay
[0165] Chromium Release assays were performed as described
previously (Kawakami, 1992). Briefly, 10.sup.6 target cells were
incubated with 100 .mu.Ci Na.sup.51CrO.sub.4 (Amersham Int., Bucks,
UK) for 1 hour. Various amounts of effector cells were then added
to 2.times.10.sup.3 target cells in triplicate wells of U-bottomed
microtiter plates (Costar, Badhoevedorp, The Netherlands) in a
final volume of 150 .mu.l. After 5 hours of incubation, part of the
supernatant was harvested and its radioactive content measured.
Target cells were incubated for 48 hours with 50 U/ml human
(Boehringer, Ingelheim, Germany) or mouse recombinant IFN- (TNO,
Rijswijk, The Netherlands) before use in chromium release
assays.
[0166] TIL 1200
[0167] In search of gp-100 specific cytotoxic T lymphocytes (CTLs)
we focused on HLA-A2.1 as a restriction element because of its
widespread occurrence in Caucasians and its presumptive dominant
role in CTL reactivity against melanoma. A HLA-A2.1.sup.+ TIL line,
TIL 1200 (Shilyansky, J. et al, 1994), was used for this study.
This TIL line expresses TCR .alpha./.beta., CD3 and CD8.
[0168] Results
[0169] HLA-A2.1-Restricted Killing of Melanoma Tumor Cells by TIL
1200 Corresponds to Gp100 Expression
[0170] Cytolytic activity of TIL 1200 was analyzed using a panel of
human melanoma cell lines. TIL 1200 efficiently lysed
HLA-A2.1.sup.+ Mel 624 and MeWo melanoma tumor cells, which both
express gp100, whereas no reactivity towards HLA-A2.1.sup.-
gp100.sup.+ Mel 397 cells was seen. It is interesting to note that
we observed that HLA-A2.1.sup.+ BLM melanoma cells are also
resistant to lysis by TIL 1200. Furthermore, HLA-A2.1.sup.+
EBV-transformed B cells (JY), which also lack gp100 expression, and
K562 cells, were not lysed by TIL 1200. Together, these data
demonstrate that TIL 1200 displays HLA-A2.1-restricted killing
which correlates with gp100 expression.
[0171] TIL 1200 Recognizes HLA-A2.1.sup.+ Gp100.sup.+
Transfectants
[0172] EL4 cells co-transfected with a genomic fragment encoding
HLA-A2.1 together with a plasmid conferring hygromycine resistance
were selected and analyzed by flow cytometry. HLA-A2.1 expressing
cells were subsequently transfected with pBJ1-gp100neo, which
encodes gp100 and confers resistance to G418. Stable transfectants
were selected and were screened for gp100 expression using mAb
NKI/beteb. In collaboration with P. Coulie a similar panel of
transfectants was generated in murine P815 cells (P815 A2.1 and
p815 A2.1/gp100). Using these murine transfectants as target cells
in chromium release assay, we clearly observed gp100 specific lysis
by TIL 1200. The percent specific lysis (25-35%, E/T 30:1) of
murine EL4 A2.1/gp100 and P815 A2.1/gp100 transfectants by TIL 1200
was somewhat lower compared with that observed with HLA-A2.1+
gp100+ human melanoma cells (45-60%, E/T 30:1). This difference may
be explained by nonmatched accessory molecules between human TIL's
and murine transfectants. To overcome this we introduced the gp100
antigen into human HLA-A2.1+ gp100.sup.- BLM melanoma cells by
transfection of pBJ1-gp100neo. Stable BLM gp100 clones were tested
in chromium release assays using TIL 1200. BLM gp100 clones proved
to be as sensitive to lysis by TIL as Mel 624 and MeWo cells which
express the gp100 antigen endogenously. The gp100 specificity of
TIL 1200 was further demonstrated by the absence of lysis of
G418-resistant BLM cells not expressing gpl 00, excluding the
possibility that neomycin-derived peptides are recognized.
[0173] Example 3
[0174] Mapping of Gp100 Epitopes Recognized by TIL 1200 Using Gp100
Deletion mutants.
[0175] Basically, two methods are commonly used in the art to map
epitopes recognized by anti-tumor CTL.
[0176] 1. According to the HLA binding motifs peptides can be
synthesized that reside in the target protein. These peptides can
then be loaded onto cells bearing the appropriate restriction
element, and used as targets for CTL.
[0177] 2. Generation of deletion mutants and expression of these
deletion mutants in for example COS-7 cells together with the
appropriate restriction element. These transfected cells are then
co-cultured with CTL and target cell lysis or
TNF-.alpha./IFN.gamma. production by the CTL are measured.
Transfectants not recognized by the CTL do not express the
peptide.
[0178] Both methods have been done in search for the epitopes of
the invention.
[0179] TIL 1200 Mediated Lysis of Peptide Loaded T2 Cells
[0180] We have chemically synthesized gp100 peptides potentially
recognized by TIL1200. Peptides were synthesized by a solid phase
strategy on an automated multiple peptide synthesizer (Abimed AMS
422) using Fmoc-chemistry (Nijman, 1993). Actual binding of the
peptides to HLA.A2.1 was established with a recently described
peptide binding assay making use of processing defective T2 cells
(Nijman, 1993). This analysis resulted in the identification of gpl
00 derived peptides that strongly bind to HLA-A2.1. Subsequently,
T2 cells loaded with the peptides that strongly bind to HLA-A2.1
were subjected to lysis by TIL1200 using a standard chromium
release assay. In this way the peptide L-L-D-G-T-A-T-L-R-L (SEQ ID
NO:4) has been identified according to this procedure.
Example 4
[0181] Gp100 Epitope Identified by Deletion Mapping
[0182] Gp100 cDNA was inserted into expression vectors pBJ1neo,
pCMVneo (Baker et al, 1990) and pSVL. For the generation of a gp100
cDNA lacking the coding sequences for the peptide 457-466, PCR
reactions were performed with the following combinations of
oligonucleotides:
1 (SEQ ID NO:16) 5'-CATGGAAGTGACTGTCTACC-3'; (SEQ ID NO:17)
5'-CTGAGCGAATTCGGAACCTGTAATACTTTCCG-3'; (SEQ ID NO:18)
5'-CTGAGCGAATTCGTGAAGAGACAAGTCCCCC- -3'; and (SEQ ID NO:19)
5'-TCACAGCATCATATGAGAGTAC-3'
[0183] using the full length gp100 cDNA as a template. PCR products
were digested with Eco RI, ligated and served as a template for a
nested PCR using the following primers: 5'-GCACAGGCCAACTGCAGA-3'
(SEQ ID NO:28) and 5'-TTCAGTATCTGGTGCAGAAC-3' (SEQ ID NO:29). The
Kpn I-Cla I fragment from this PCR product was then exchanged with
the corresponding fragment in pCMVgp100neo to generate
pCMVgp100DEL454-481neo. Gp100 CDNA mutants DEL149-654 and
DEL454-654 were obtained by deletion of the 1.7 kb Hind III and the
0.8 kb Eco RI fragments from pBJ1gp100DEL454-481neo, respectively.
Gp100 cDNA mutants DEL100-654, DEL194-528 and DEL167-508 were
obtained by deletion of the Bgl I-Sac I, Bamh HI-Bgl II and Apa
I-Nsi I fragments from pSVLgp100 respectively.
[0184] BLM cells were transfected with 20 .mu.g of
pCMVgp100DEL454-481neo DNA according to the calcium phosphate
co-precipitation procedure (Graham and van der Eb, 1973) using
Calcium Phosphate Transfection Systems (BRL, Gaithersburg, Md.) and
were selected with 1 mg/ml G418 (Gibco, Paisley, Scotland UK).
[0185] COS-7 cells were cotransfected with 5 .mu.g of
pBJ1HLA-A2.1neo and 5 .mu.g of pBJ1 or pSVL plasmids containing
either full length or deleted gp100 cDNAs using the
DEAE-dextran/chloroquine method (Seed and Aruffo, 1987). After 48
hours of transfection COS-7 cells were used as stimulator cells in
IFN-y release experiments.
[0186] Release Assays
[0187] Chromium release assays were performed as in Example 2.
[0188] For IFN-release assay 10.sup.5 TIL 1200 responder cells were
incubated together with 5.times.10.sup.4 transiently transfected
COS-7 stimulator cells in 300 .mu.l medium in the presence of 100
U/ml IL-2 in a flat bottom 96 well microtiter plate. After 24 hours
of incubation, 100 .mu.l of supernatant was harvested and was
screened for the presence of IFN-y using a IFN-.gamma.-IRMA
immunoradiometric assay kit (Megenix Diagnostics SA, Fleurus,
Belgium).
[0189] Results
[0190] FIG. 3A shows the gp100 cDNA deletion mutants that were
generated. As shown in FIG. 3B, TIL 1200 specifically secreted
IFN-y when stimulated with COS-7 cells transfected with HLA-A2.1
and the full-length gp100 cDNA. Again, TIL 1200 reactivity was
observed against the gp100DEL454-481 mutant. From the other gp100
deletion mutants, only the DEKL100-661 and DEL149-661 constructs
were not recognized, thereby excluding the possibility that TIL1200
was reactive with a peptide located N-terminal from amino acid
position 148 in the gp100 protein. Also, the C-terminal region of
the gp100 protein could be excluded, because TIL 1200 reactivity
could be observed using a mutant construct, DEL454-661, encoding
the first 453 amino acids of gp100. From the observation that a
construct coding within this N-terminal region up to amino acid 166
was able to stimulate TIL 1200 (DEL167-508), it was concluded that
the epitope recognized was located between amino acids 148-166 or
the gp 100 protein.
[0191] HLA-A2.1 Binding
[0192] Several motifs have been described for 9-mer or 10-mer
peptides binding to HLA-A2.1 (Falk et al., 1991; Hunt et al., 1992;
Ruppert et al., 1993) that were deduced from naturally processed
and synthetic HLA-A2.1 binding peptides. The 148-166 region of the
gp100 protein was screened against these motifs and a number of
peptides were synthesized that fitted into a somewhat broader
motif, including threonine residues at position two. These peptides
were loaded onto HLA-A2.1+ T2 cells and tested for their ability to
induce TIL 1200 mediated target cell lysis (FIG. 4(A)). The five
tested peptides were all able to sensitize T2 cells for lysis by
TIL 1200 when used at a concentration of 10 .mu.g/ml. All these
peptides contain the 8-mer peptide TWGQYWQV (SEQ ID NO:8),
corresponding to gp100 amino acids 155-162. All peptides were
titrated to evaluate their relative ability to sensitize T2 target
cells for lysis by TIL 1200. FIG. 4(B) shows that the 9-mer peptide
KTWGQYWQV (SEQ ID NO:22) can be recognized by TIL 1200 when applied
at a concentration of 3 ng/ml, whereas the other peptides had to be
applied at higher concentrations.
[0193] A comparison was made of the peptides KTWGQYWQV (SEQ ID
NO:22) (gp100 amino acids 155-162), LLDGTATLRL (SEQ ID NO:4) (gp100
amino acids 457-466) and YLEPGPVTA (SEQ ID NO:30) (gp100 amino
acids 280-288, identified by Cox et al., 1994) with three known
viral epitopes presented in HLA-A2.1: the influenza matrix 58-66
peptide (Gotch et al., 1987), the HIV polymerase 510-518 peptide
(Tsomides et al., 1991) and the HIV gp120 197-205 peptide (Dadaglio
et al., 1991). The HLA-A2.1 binding capacity of the above mentioned
epitopes was analyzed by means of an indirect binding assay using
the processing defective cell line T2 (Nijman et al., 1993).
Shortly thereafter, T2 cells were incubated with 12.5 .mu.g of the
epitopes. HLA-A2.1 stabilization at the cell surface was determined
by flow cytometry using mAb BB7.2. The Fluorescence Index is
expressed as the experimental mean fluorescence divided by the mean
fluorescence that is obtained when T2 cells are incubated with a
HLA-A2.1 non-binding peptide at a similar concentration.
[0194] Using this assay, a similar HLA-A2.1 stabilization with the
gp100 280-288 epitope and the tested viral epitopes. Both epitopes
of the invention (KTWGQYWQV (SEQ ID NO:22) and LLDGTATLRL (SEQ ID
NO:4)) bind with a somewhat lower affinity to HLA-A2.1 (FIG. 5).
From this it is concluded that the gp100 epitopes bind to HLA-A2.1
with distinct affinities.
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2 # SEQUENCE LIS #TING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 38 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 2115 base #pairs (B) TYPE: nucleic
acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (F) TISSUE TYPE: Melano #ma (G) CELL TYPE:
Melanocy #te (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 22..
#.2005 (ix) FEATURE: (A) NAME/KEY: misc_ #signal (B) LOCATION: 1...
#81 (ix) FEATURE: (A) NAME/KEY: misc_ #feature (B) LOCATION: 1792
#...1870 (D) OTHER INFORMATION: #/function = "transmembrane region"
(ix) FEATURE: (A) NAME/KEY: misc_ #binding (B) LOCATION: 262.
#..264 (D) OTHER INFORMATION: #/bound moiety = "carbohydrate" (ix)
FEATURE: (A) NAME/KEY: misc_ #binding (B) LOCATION: 337. #..339 (D)
OTHER INFORMATION: #/bound moiety = "carbohydrate" (ix) FEATURE:
(A) NAME/KEY: misc_ #binding (B) LOCATION: 352. #..354 (D) OTHER
INFORMATION: #/bound moiety = "carbohydrate" (ix) FEATURE: (A)
NAME/KEY: misc_ #binding (B) LOCATION: 982. #..984 (D) OTHER
INFORMATION: #/bound moiety = "carbohydrate" (ix) FEATURE: (A)
NAME/KEY: misc_binding (B) LOCATION: 1723...1725 (D) OTHER
INFORMATION: /bound moiety = # "carbohydrate" (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #1: CGCGGAATCC GGAAGAACAC AATGGATCTG
GTGCTAAAAA GATGCCTTCT TC #ATTTGGCT 60 GTGATAGGTG CTTTGCTGGC
TGTGGGGGCT ACAAAAGTAC CCAGAAACCA GG #ACTGGCTT 120 GGTGTCTCAA
GGCAACTCAG AACCAAAGCC TGGAACAGGC AGCTGTATCC AG #AGTGGACA 180
GAAGCCCAGA GACTTGACTG CTGGAGAGGT GGTCAAGTGT CCCTCAAGGT CA #GTAATGAT
240 GGGCCTACAC TGATTGGTGC AAATGCCTCC TTCTCTATTG CCTTGAACTT CC
#CTGGAAGC 300 CAAAAGGTAT TGCCAGATGG GCAGGTTATC TGGGTCAACA
ATACCATCAT CA #ATGGGAGC 360 CAGGTGTGGG GAGGACAGCC AGTGTATCCC
CAGGAAACTG ACGATGCCTG CA #TCTTCCCT 420 GATGGTGGAC CTTGCCCATC
TGGCTCTTGG TCTCAGAAGA GAAGCTTTGT TT #ATGTCTGG 480 AAGACCTGGG
GCCAATACTG GCAAGTTCTA GGGGGCCCAG TGTCTGGGCT GA #GCATTGGG 540
ACAGGCAGGG CAATGCTGGG CACACACACC ATGGAAGTGA CTGTCTACCA TC #GCCGGGGA
600 TCCCGGAGCT ATGTGCCTCT TGCTCATTCC AGCTCAGCCT TCACCATTAC TG
#ACCAGGTG 660 CCTTTCTCCG TGAGCGTGTC CCAGTTGCGG GCCTTGGATG
GAGGGAACAA GC #ACTTCCTG 720 AGAAATCAGC CTCTGACCTT TGCCCTCCAG
CTCCATGACC CCAGTGGCTA TC #TGGCTGAA 780 GCTGACCTCT CCTACACCTG
GGACTTTGGA GACAGTAGTG GAACCCTGAT CT #CTCGGGCA 840 CTTGTGGTCA
CTCATACTTA CCTGGAGCCT GGCCCAGTCA CTGCCCAGGT GG #TCCTGCAG 900
GCTGCCATTC CTCTCACCTC CTGTGGCTCC TCCCCAGTTC CAGGCACCAC AG #ATGGGCAC
960 AGGCCAACTG CAGAGGCCCC TAACACCACA GCTGGCCAAG TGCCTACTAC AG
#AAGTTGTG 1020 GGTACTACAC CTGGTCAGGC GCCAACTGCA GAGCCCTCTG
GAACCACATC TG #TGCAGGTG 1080 CCAACCACTG AAGTCATAAG CACTGCACCT
GTGCAGATGC CAACTGCAGA GA #GCACAGGT 1140 ATGACACCTG AGAAGGTGCC
AGTTTCAGAG GTCATGGGTA CCACACTGGC AG #AGATGTCA 1200 ACTCCAGAGG
CTACAGGTAT GACACCTGCA GAGGTATCAA TTGTGGTGCT TT #CTGGAACC 1260
ACAGCTGCAC AGGTAACAAC TACAGAGTGG GTGGAGACCA CAGCTAGAGA GC #TACCTATC
1320 CCTGAGCCTG AAGGTCCAGA TGCCAGCTCA ATCATGTCTA CGGAAAGTAT TA
#CAGGTTCC 1380 CTGGGCCCCC TGCTGGATGG TACAGCCACC TTAAGGCTGG
TGAAGAGACA AG #TCCCCCTG 1440 GATTGTGTTC TGTATCGATA TGGTTCCTTT
TCCGTCACCC TGGACATTGT CC #AGGGTATT 1500 GAAAGTGCCG AGATCCTGCA
GGCTGTGCCG TCCGGTGAGG GGGATGCATT TG #AGCTGACT 1560 GTGTCCTGCC
AAGGCGGGCT GCCCAAGGAA GCCTGCATGG AGATCTCATC GC #CAGGGTGC 1620
CAGCCCCCTG CCCAGCGGCT GTGCCAGCCT GTGCTACCCA GCCCAGCCTG CC #AGCTGGTT
1680 CTGCACCAGA TACTGAAGGG TGGCTCGGGG ACATACTGCC TCAATGTGTC TC
#TGGCTGAT 1740 ACCAACAGCC TGGCAGTGGT CAGCACCCAG CTTATCATGC
CTGGTCAAGA AG #CAGGCCTT 1800 GGGCAGGTTC CGCTGATCGT GGGCATCTTG
CTGGTGTTGA TGGCTGTGGT CC #TTGCATCT 1860 CTGATATATA GGCGCAGACT
TATGAAGCAA GACTTCTCCG TACCCCAGTT GC #CACATAGC 1920 AGCAGTCACT
GGCTGCGTCT ACCCCGCATC TTCTGCTCTT GTCCCATTGG TG #AGAATAGC 1980
CCCCTCCTCA GTGGGCAGCA GGTCTGAGTA CTCTCATATG ATGCTGTGAT TT #TCCTGGAG
2040 TTGACAGAAA CACCTATATT TCCCCCAGTC TTCCCTGGGA GACTACTATT AA
#CTGAAATA 2100 AATACTCAGA GCCTG # # # 2115 (2) INFORMATION FOR SEQ
ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 661 amino
#acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #2: Met Asp
Leu Val Leu Lys Arg Cys Leu Leu Hi #s Leu Ala Val Ile Gly 1 5 # 10
# 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pr #o Arg Asn Gln Asp
Trp 20 # 25 # 30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Al #a Trp
Asn Arg Gln Leu 35 # 40 # 45 Tyr Pro Glu Trp Thr Glu Ala Gln Arg
Leu As #p Cys Trp Arg Gly Gly 50 # 55 # 60 Gln Val Ser Leu Lys Val
Ser Asn Asp Gly Pr #o Thr Leu Ile Gly Ala 65 #70 #75 #80 Asn Ala
Ser Phe Ser Ile Ala Leu Asn Phe Pr #o Gly Ser Gln Lys Val 85 # 90 #
95 Leu Pro Asp Gly Gln Val Ile Trp Val Asn As #n Thr Ile Ile Asn
Gly 100 # 105 # 110 Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pr #o
Gln Glu Thr Asp Asp 115 # 120 # 125 Ala Cys Ile Phe Pro Asp Gly Gly
Pro Cys Pr #o Ser Gly Ser Trp Ser 130 # 135 # 140 Gln Lys Arg Ser
Phe Val Tyr Val Trp Lys Th #r Trp Gly Gln Tyr Trp 145 1 #50 1 #55 1
#60 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Se #r Ile Gly Thr Gly
Arg 165 # 170 # 175 Ala Met Leu Gly Thr His Thr Met Glu Val Th #r
Val Tyr His Arg Arg 180 # 185 # 190 Gly Ser Arg Ser Tyr Val Pro Leu
Ala His Se #r Ser Ser Ala Phe Thr 195 # 200 # 205 Ile Thr Asp Gln
Val Pro Phe Ser Val Ser Va #l Ser Gln Leu Arg Ala 210 # 215 # 220
Leu Asp Gly Gly Asn Lys His Phe Leu Arg As #n Gln Pro Leu Thr Phe
225 2 #30 2 #35 2 #40 Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Le #u
Ala Glu Ala Asp Leu 245 # 250 # 255 Ser Tyr Thr Trp Asp Phe Gly Asp
Ser Ser Gl #y Thr Leu Ile Ser Arg 260 # 265 # 270 Ala Leu Val Val
Thr His Thr Tyr Leu Glu Pr #o Gly Pro Val Thr Ala 275 # 280 # 285
Gln Val Val Leu Gln Ala Ala Ile Pro Leu Th #r Ser Cys Gly Ser Ser
290 # 295 # 300 Pro Val Pro Gly Thr Thr Asp Gly His Arg Pr #o Thr
Ala Glu Ala Pro 305 3 #10 3 #15 3 #20 Asn Thr Thr Ala Gly Gln Val
Pro Thr Thr Gl #u Val Val Gly Thr Thr 325 # 330 # 335 Pro Gly Gln
Ala Pro Thr Ala Glu Pro Ser Gl #y Thr Thr Ser Val Gln 340 # 345 #
350 Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pr #o Val Gln Met Pro
Thr 355 # 360 # 365 Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Va #l
Pro Val Ser Glu Val 370 # 375 # 380 Met Gly Thr Thr Leu Ala Glu Met
Ser Thr Pr #o Glu Ala Thr Gly Met 385 3 #90 3 #95 4 #00 Thr Pro Ala
Glu Val Ser Ile Val Val Leu Se #r Gly Thr Thr Ala Ala 405 # 410 #
415 Gln Val Thr Thr Thr Glu Trp Val Glu Thr Th #r Ala Arg Glu Leu
Pro 420 # 425 # 430 Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Se #r
Ile Met Ser Thr Glu 435 # 440 # 445 Ser Ile Thr Gly Ser Leu Gly Pro
Leu Leu As #p Gly Thr Ala Thr Leu 450 # 455 # 460 Arg Leu Val Lys
Arg Gln Val Pro Leu Asp Cy #s Val Leu Tyr Arg Tyr 465 4 #70 4 #75 4
#80 Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gl #n Gly Ile Glu Ser
Ala 485 # 490 # 495 Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gl #y
Asp Ala Phe Glu Leu 500 # 505 # 510 Thr Val Ser Cys Gln Gly Gly Leu
Pro Lys Gl #u Ala Cys Met Glu Ile 515 # 520 # 525 Ser Ser Pro Gly
Cys Gln Pro Pro Ala Gln Ar #g Leu Cys Gln Pro Val 530 # 535 # 540
Leu Pro Ser Pro Ala Cys Gln Leu Val Leu Hi #s Gln Ile Leu Lys Gly
545 5 #50 5 #55 5 #60 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Le #u
Ala Asp Thr Asn Ser 565 # 570 # 575 Leu Ala Val Val Ser Thr Gln Leu
Ile Met Pr #o Gly Gln Glu Ala Gly 580 # 585 # 590 Leu Gly Gln Val
Pro Leu Ile Val Gly Ile Le #u Leu Val Leu Met Ala 595 # 600 # 605
Val Val Leu Ala Ser Leu Ile Tyr Arg Arg Ar #g Leu Met Lys Gln Asp
610 # 615 # 620 Phe Ser Val Pro Gln Leu Pro His Ser Ser Se #r His
Trp Leu Arg Leu 625 6 #30 6 #35 6 #40 Pro Arg Ile Phe Cys Ser Cys
Pro Ile Gly Gl #u Asn Ser Pro Leu Leu 645 # 650 # 655 Ser Gly Gln
Gln Val 660 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 30 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1... #30 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #3: CTGCTGGATG GTACAGCCAC CTTAAGGCTG # # 30
(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 10 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #4: Leu Leu Asp Gly Thr
Ala Thr Leu Arg Leu 1 5 # 10 (2) INFORMATION FOR SEQ ID NO: 5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1... #30 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #5: GTATTGCCAG ATGGGCAGGT TATCTGGGTC # # 30
(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 10 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #6: Val Leu Pro Asp Gly
Gln Val Ile Trp Val 1 5 # 10 (2) INFORMATION FOR SEQ ID NO: 7: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (vi) ORIGINAL SOURCE: (F) TISSUE TYPE: Melano #ma (G) CELL TYPE:
Melanocy #te (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1... #24
(xi) SEQUENCE DESCRIPTION: SEQ #ID NO: 7: ACCTGGGGCC AATACTGGCA
AGTT # # 24 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 8 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #8: Thr Trp Gly Gln Tyr Trp Gln Val 1 5 (2) INFORMATION FOR
SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base
#pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (vi) ORIGINAL SOURCE: (F) TISSUE TYPE: Melano #ma (G) CELL TYPE:
Melanocy #te (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1... #36
(ix) FEATURE: (A) NAME/KEY: protein # bind (B) LOCATION: 1... #33
(ix) FEATURE: (A) NAME/KEY: protein # bind (B) LOCATION: 1... #36
(ix) FEATURE: (A) NAME/KEY: protein # bind (B) LOCATION: 7... #33
(ix) FEATURE: (A) NAME/KEY: protein # bind (B) LOCATION: 10.. #.36
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: #9: GTCTGGAAGA CCTGGGGCCA
ATACTGGCAA GTTCTA # # 36 (2) INFORMATION FOR SEQ ID NO: 10: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #10: Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Va #l Leu 1 5 #
10 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY:
misc # feature (B) LOCATION: # 7...12 (D) OTHER INFORMATION:
#/label = EcoRI-site (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #11:
TATCTAGAAT TCTGCACCAG ATACTGAAG # # 29 (2) INFORMATION FOR SEQ ID
NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base #pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: misc #feature (B)
LOCATION: 7...12 (D) OTHER INFORMATION: #/label = EcoRI-site (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #12: TATCTAGAAT TCTGCAAGAT
GCCCACGATC AG # # 32 (2) INFORMATION FOR SEQ ID NO: 13: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #13: CTTCTTGACC AGGCATGATA # # #
20 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #14: TGTGAGAAGA ATCCCAGGCA # # # 20 (2) INFORMATION FOR SEQ
ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base #pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #15:
GCTTATCATG CCTGTGCCTG GATTCTTCTC ACAGGT # # 36 (2) INFORMATION FOR
SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #16:
CATGGAAGTG ACTGTCTACC # # # 20 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #17: CTGAGCGAAT TCGGAACCTG
TAATACTTTC CG # # 32 (2) INFORMATION FOR SEQ ID NO: 18: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #18: CTGAGCGAAT TCGTGAAGAG
ACAAGTCCCC C # # 31 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #19: TCACAGCATC ATATGAGAGT AC # # 22 (2)
INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 11 amino #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #20: Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Va #l 1 5 # 10
(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #21: Lys Thr Trp Gly Gln Tyr Trp Gln Val Leu 1 5 # 10 (2)
INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 9 amino #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #22: Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 (2) INFORMATION
FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9
amino #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #23: Thr
Trp Gly Gln Tyr Trp Gln Val Leu 1 5 (2) INFORMATION FOR SEQ ID NO:
24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino #acids (B)
TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: #24: Met Leu Gly Thr His Thr
Met Glu Val 1 5 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 9 amino #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #25: Arg Leu Met Lys Gln Asp Phe Ser Val 1
5 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #26: TATTGAAAGT GCCGAGATCC # # # 20 (2) INFORMATION FOR SEQ
ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base #pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #27:
TGCAAGGACC ACAGCCATC # # # 19 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: #28: GCACAGGCCA ACTGCAGA # # # 18
(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #29: TTCAGTATCT GGTGCAGAAC # # # 20 (2) INFORMATION FOR SEQ
ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino #acids
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #30: Tyr Leu Glu Pro Gly
Pro Val Thr Ala 1 5 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 6 base p #airs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #31: AATAAA # # # 6 (2) INFORMATION FOR SEQ
ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino #acids
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #32: Arg Ile Gln Thr 1 (2)
INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 61 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY:
exon (B) LOCATION: 1... #8 (ix) FEATURE: (A) NAME/KEY: intron (B)
LOCATION: 9... #55 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION:
56.. #.61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #33: CATGCCTGGT
AGGTCCAGAC ACTGAGTGAA GCAGTGCCTG GGATTCTTCT CA #CAGGTCAA 60 G # # #
61 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY:
exon (B) LOCATION: 1... #8 (ix) FEATURE: (A) NAME/KEY: intron (B)
LOCATION: 9... #52 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION:
53.. #.59 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #34: CATGCCTGGT
AGGTCCGGGC AGCTGGCAAG CAGCAGACAC TGAGTGAAGC AG #TGCCTGG 59 (2)
INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 190 amino #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: #35: Pro Leu Asp Cys Val Leu Tyr Arg Tyr Gly Se #r Phe Ser
Val Thr Leu 1 5 # 10 # 15 Asp Ile Val Gln Gly Ile Glu Ser Ala Glu
Il #e Leu Gln Ala Val Pro
20 # 25 # 30 Ser Gly Glu Gly Asp Ala Phe Glu Leu Thr Va #l Ser Cys
Gln Gly Gly 35 # 40 # 45 Leu Pro Lys Glu Ala Cys Met Glu Ile Ser Se
#r Pro Gly Cys Gln Pro 50 # 55 # 60 Pro Ala Gln Arg Leu Cys Gln Pro
Val Leu Pr #o Ser Pro Ala Cys Gln 65 #70 #75 #80 Leu Val Leu His
Gln Ile Leu Lys Gly Gly Se #r Gly Thr Tyr Cys Leu 85 # 90 # 95 Asn
Val Ser Leu Ala Asp Thr Asn Ser Leu Al #a Val Val Ser Thr Gln 100 #
105 # 110 Leu Ile Met Pro Gly Gln Glu Ala Gly Leu Gl #y Gln Val Pro
Leu Ile 115 # 120 # 125 Val Gly Ile Leu Leu Val Leu Met Ala Val Va
#l Leu Ala Ser Leu Ile 130 # 135 # 140 Tyr Arg Arg Arg Leu Met Lys
Gln Asp Phe Se #r Val Pro Gln Leu Pro 145 1 #50 1 #55 1 #60 His Ser
Ser Ser His Trp Leu Arg Leu Pro Ar #g Ile Phe Cys Ser Cys 165 # 170
# 175 Pro Ile Gly Glu Asn Ser Pro Leu Leu Ser Gl #y Gln Gln Val 180
# 185 # 190 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 197 amino #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #36: Pro Leu Asp Cys Val Leu Tyr Arg Tyr
Gly Se #r Phe Ser Val Thr Leu 1 5 # 10 # 15 Asp Ile Val Gln Gly Ile
Glu Ser Ala Glu Il #e Leu Gln Ala Val Pro 20 # 25 # 30 Ser Gly Glu
Gly Asp Ala Phe Glu Leu Thr Va #l Ser Cys Gln Gly Gly 35 # 40 # 45
Leu Pro Lys Glu Ala Cys Met Glu Ile Ser Se #r Pro Gly Cys Gln Pro
50 # 55 # 60 Pro Ala Gln Arg Leu Cys Gln Pro Val Leu Pr #o Ser Pro
Ala Cys Gln 65 #70 #75 #80 Leu Val Leu His Gln Ile Leu Lys Gly Gly
Se #r Gly Thr Tyr Cys Leu 85 # 90 # 95 Asn Val Ser Leu Ala Asp Thr
Asn Ser Leu Al #a Val Val Ser Thr Gln 100 # 105 # 110 Leu Ile Met
Pro Val Pro Gly Ile Leu Leu Th #r Gly Gln Glu Ala Gly 115 # 120 #
125 Leu Gly Gln Val Arg Leu Ile Val Gly Ile Le #u Leu Val Leu Met
Ala 130 # 135 # 140 Val Val Leu Ala Ser Leu Ile Tyr Arg Arg Ar #g
Leu Met Lys Gln Asp 145 1 #50 1 #55 1 #60 Phe Ser Val Pro Gln Leu
Pro His Ser Ser Se #r His Trp Leu Arg Leu 165 # 170 # 175 Pro Arg
Ile Phe Cys Ser Cys Pro Ile Gly Gl #u Asn Ser Pro Leu Leu 180 # 185
# 190 Ser Gly Gln Gln Val 195 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 192 amino #acids (B)
TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: #37: Pro Leu Asp Cys Val Leu
Tyr Arg Tyr Gly Se #r Phe Ser Leu Thr Leu 1 5 # 10 # 15 Asp Ile Val
Gln Ser Ile Glu Ser Ala Glu Il #e Leu Gln Ala Val Ser 20 # 25 # 30
Ser Ser Glu Gly Asp Ala Phe Glu Leu Thr Va #l Ser Cys Gln Gly Gly
35 # 40 # 45 Leu Pro Lys Glu Ala Cys Met Asp Ile Ser Se #r Pro Gly
Cys Gln Leu 50 # 55 # 60 Pro Ala Gln Arg Leu Cys Gln Pro Val Pro Pr
#o Ser Pro Ala Cys Gln 65 #70 #75 #80 Leu Val Leu His Gln Val Leu
Lys Gly Gly Se #r Gly Thr Tyr Cys Leu 85 # 90 # 95 Asn Val Ser Leu
Ala Asp Ala Asn Ser Leu Al #a Met Val Ser Thr Gln 100 # 105 # 110
Leu Val Met Pro Gly Gln Glu Ala Gly Leu Ar #g Gln Ala Pro Leu Phe
115 # 120 # 125 Val Gly Ile Leu Leu Val Leu Thr Ala Leu Le #u Leu
Ala Ser Leu Ile 130 # 135 # 140 Tyr Arg Arg Arg Leu Met Lys Gln Gly
Ser Gl #u Val Pro Leu Pro Gln 145 1 #50 1 #55 1 #60 Leu Pro His Gly
Arg Thr Gln Trp Leu Arg Le #u Trp Val Ile Phe Arg 165 # 170 # 175
Ser Cys Pro Ile Gly Glu Ser Lys Pro Leu Le #u Ser Gly Gln Gln Val
180 # 185 # 190 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 202 amino #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: #38: Pro Thr Gly Cys Val Leu Tyr Arg Tyr
Gly Th #r Phe Ser Thr Glu Leu 1 5 # 10 # 15 Asn Ile Val Gln Gly Ile
Glu Ser Val Ala Il #e Val Gln Val Val Pro 20 # 25 # 30 Ala Ala Pro
Glu Gly Ser Gly Asn Ser Val Gl #u Leu Thr Val Thr Cys 35 # 40 # 45
Glu Gly Ser Leu Pro Glu Glu Val Cys Thr Va #l Val Ala Asp Ala Glu
50 # 55 # 60 Cys Arg Thr Ala Gln Met Gln Thr Cys Ser Al #a Val Ala
Pro Ala Pro 65 #70 #75 #80 Gly Cys Gln Leu Val Leu Arg Gln Asp Phe
As #n Gln Ser Gly Leu Tyr 85 # 90 # 95 Cys Leu Asn Val Ser Leu Ala
Asn Gly Asn Gl #y Leu Ala Val Ala Ser 100 # 105 # 110 Thr His Val
Ala Val Gly Ser Ile Pro Ser Ar #g Gln Trp His His Ala 115 # 120 #
125 His Arg Gly Ala Ala Leu Gly Thr Ala His Gl #y Arg Cys Ser Gly
His 130 # 135 # 140 Arg Cys Leu His Leu Pro Pro Cys Glu Val Gl #n
Pro Ala Ala Ala His 145 1 #50 1 #55 1 #60 Ser Pro His Gly Pro Pro
Ala Pro Gln Leu Al #a Ala Pro Arg Cys Tyr 165 # 170 # 175 Pro Ala
Phe Ala Ala Ala Pro Gly Phe Trp Gl #y Gly Ser Gln Trp Arg 180 # 185
# 190 Lys Gln Pro Pro Ala Arg Ala Asn Ala Val 195 # 200
* * * * *