Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PEPTIDES RECOGNIZED BY MELANOMA-SPECIFIC Al-, A2- AND
A3-RESTRICTED CYTOTOXIC LYMPHOCYTES, AND USES THEREFOR
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention is directed to peptides that, in
association with Class I MHC molecules, form epitopes
recognized by cytotoxic T-cells specific for human melanoma,
to immunogens comprising said epitopic peptides, and to
related compositions, methods and apparatus.
Description of the Background Art
Melanoma affects 30,000 new patients per year in the
United States. It is a cancer manifested by the unabated
proliferation of melanocytes. Eighty percent of melanoma
patients are diagnosed during their productive years between
the ages of 25 and 65. The incidence of melanoma is rapidly
increasing, in 1935 the lifetime risk of developing melanoma
was 1:1,500 individuals, at present, the risk has risen to
1:105. It is believed that by the year 2000 the risk of
developing melanoma will increase to about 1:70 to 1:90.
Early diagnosis and treatment of this disease is crucial.
Once a primary tumor becomes metastatic the disease is almost
always fatal.
Cytotoxic lymphocyte (CTL) response has been shown to be
an important host defense against malignant cells, Rock et
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al. J. Immunol., (1993), 150:1244.
Lymphocytes isolated from patients having melanoma, when
stimulated in vitro with recombinant interleukin-2 (rIL-2)
and autologous melanoma cells, develop a melanoma specific
cytotoxic response, Vose et al., Nature, (1982), 296:359;
Knuth et al., Proc. Natl. Acad. USA, (1984), 81:3511;
Slingluff et al., Arch. Surg., (1987), 122:1407; Darrow et
al., Cancer, (1988), 62:84; Slingluff et al., J. Natl. Cancer
Inst., (1988), 80:1016; Slingluff et al., Ann. Surg., (1989),
210:194; Muul et al., J. Immunol., (1987), 138:989; Van den
Eynde et al., Int. J. Cancer, (1989), 44:634; Anichini et
al., Int. J. Cancer, (1985), 35:683. The majority of
melanoma-specific effector lymphocytes are CD8+ cytotoxic T
lymphocytes (CTL) that are restricted by class I Major
Histocompatibility Complex (MHC) molecules, Vose et al;
Slingluff et al (1988), supra, Hersey et al., Cancer Immunol.
Immunother., (1986), 22:15. These characteristics are
present whether CTL have been generated from peripheral blood
lymphocytes (PBL), lymph node cells, or tumor infiltrating
lymphocytes.
The evidence that the CTL response to human melanoma is
restricted by class I MHC molecules includes demonstration of
cross-reactivity for allogenic melanoma cells that share a
restricting class I MHC molecule with the autologous tumor.
The HLA-A2 molecule and its variants, of which HLA-A2.1 is by
far the most common, is an effective restricting element for
the melanoma-specific CTL response. Additionally, melanoma-
specific HLA-restricted CTL lyse the majority of A2+
melanomas tested, Darrow et al., J. Immunol., (1989),
142:3329; Wolfel et al., J. Exp. Med., (1989), 170:797; Hom
et al., J. Immunother., (1991), 3:153. By demonstrating
lysis of A2- melanomas transfected with the A2.1 gene, it has
been shown that these transfected melanomas can present the
epitopes recognized by A2-restricted melanoma-specific CTL,
Kawakami et al., J. Immunol., (1992), 148:638. These results
suggest that these CTL recognize A2-restricted epitopes that
are shared by the majority of melanomas, although very little
is known about the number and identity of their epitopes.
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Class I molecules of the Major Histocompatibility
Complex (MHC) bind to peptides derived from intracellular
pathogens or from proteins expressed in tumor cells, and
present them on the cell surface to the host immune system.
The mechanism of peptide presentation involves protein
synthesis and proteolysis in the cytosol, followed by
transport of peptides into the endoplasmic reticulum (ER),
through the action of the TAP transporter molecules.
Peptides then become associated with newly synthesized class
1 molecules, and the resulting complexes move to the cell
surface. Proteins that are membrane associated or secreted
contain signal sequences that cause them to be contransla-
tionally transferred into the ER from membrane-bound
ribosomes. Such proteins would thus be protected from the
action of cytoplasmic proteases. However, since peptide
epitopes do arise from such proteins, although their TAP
dependent expression is unclear, it has been assumed that the
proteolysis to generate these peptide epitopes occurs after
these proteins have been aberrantly translated on cytoplasmic
ribosomes.
Adoptive transfer of tumor stimulated CTL has been as-
sociated with some tumor regressions, Rosenberg et al., N.
Eng. J. Med., (1988), 319:1676.
An alternate approach to augmenting the T-cell response
to melanoma is the use of a vaccine to stimulate CTL in vivo
(active specific immunotherapy). Epitopes for CD8+ CTL are
believed to be short, usually 9- residue peptides that bind
to a cleft on the surface of the class I MHC molecule, Udaka
et al., Cell, (1992), 69:989; VanBleek et al., Nature,
(1990), 348:213; Falk et al., J. Exp. Med., (1991), 174:425.
These peptides, generated from proteolysis of endogenous
proteins in the cytosol, are transported to the endoplasmic
reticulum, where they become associated with newly syn-
thesized class I MHC molecules. They are then transported to
the cell surface, Elliott et al., Nature, (1990), 3348:195.
CTL epitopes have been reconstituted in vitro by allowing
exogenous peptides to bind to MHC molecules on the cell
surface of target cells, Townsend et al., Annu. Rev.
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Immunol., (1989), 7:601. However, because of the complexity
of the peptide mixture associated with class I MHC molecules,
Hunt et al., Science, (1992), 255:1261, the definition of
individual peptides that comprise specific CTL epitopes has
proven extremely difficult.
One method has been to generate genomic or cDNA
libraries from tumor cells followed by transfection of
progressively smaller subsets of these molecular clones into
cells that express the appropriate MHC molecule, but not the
tumor specific epitope. Molecular clones that encode T cell
epitopes are identified by their ability to reconstitute
tumor-specific T cell recognition of the transfected cells.
The exact T cell epitope is then identified by a combination
of molecular subcloning and the use of synthetic peptides
based on the predicted amino acid sequence. See, e.g., P.
van der Brugge, et al., Science 254, 1643 (1991); C.
Traversari, et al., J. Exp. Med. 176, 1453 (1992); B.
Gaugler, et al., ibid. 179, 921 (1994); T. Boon, et al.,
Annu. Rev. Immunol. 12, 337 (1994); A.B.H. Baker, et al., J.
Exp. Med. 179, 1005 (1994); Y. Kawakami, et al., Proc. Natl.
Acad. Sci. USA 91, 6458 (1994); P.G. Coulie, et al., J. Exp.
Med. 180, 35 (1994); Y. Kawakami, et al., ibid. 180, 347
(1994); V. Brichard, et al., ibid. 178, 489 (1993); T.
Wolfei, et al., Eur. J. Immunol. 150, 2955 (1993).
Unfortunately, it is possible to inadvertently identify
clones that encode cross-reacting peptides that are
recognized because of their high level of expression in the
transfectants.
By this genetic method, an HLA-Al restricted T cell
epitope (EADPTGHSY) of a melanoma-associated antigen, MAGE-1,
was identified. Traversari, et al., J. Exp. Med., 176:1453-
57 (1992). MAGE-1 is expressed in about 20-40% of cancers of
several different tissue types, including melanomas, breast
cancers, non-small cell lung cancers, head and neck squamous
cell cancers, and bladder cancer. It is also found in the
normal male testis. The MAGE gene family also includes
another member, MAGE-3, for which a homologous HLA-Al-
restricted CTL epitope (EVDPIGHLY) was determined, although
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only after the first priority date. HLA-A1-restricted CTL
epitopes are of limited utility because only a minority of
melanomas are HLA-Al+. The function of the MAGE gene
products is not known.
5 The genetic approach has also been used to identify HLA-
A2.1-restricted CTL epitopes on tyrosinase. This enzyme is
not tumor-specific; it is expressed by normal melanocytes as
well as melanoma cells. Tyrosinase is involved in melanin
biosynthesis. Autologous CTL recognized tyrosinase-derived
HLA-A2-restricted epitopes (YMNGTMSQV and MLLAVLYCL). See
Wolfel, et al., Eur. J. Immunol., 24:759-64 (1994). However,
these peptides were not recognized by the other CTL lines
tested.
Another tissue-specific protein, gpl00, is the target of
the antibody HMB45, which is specific for melanoma and
melanocytes. Based on the correlation between HMB45 activity
and recognition by a single TIL-derived HLA-A2-restricted
melanoma-specific CTL line, Bakker, et al., J. Exp. Med.,
179:1005-9 (1994) established that transfection of cells with
the gene for gplOO reconstituted the epitope recognized by
this T cell. A subsequent study, using the same T-cell line
to screen transfected cDNA libraries also identified the
peptide LLDGTATLRL as being sufficient to reconstitute
activity. This study was not published prior to Applicants'
first priority date. GplOO is believed to play a role in
melanin biosynthesis.
An HLA-A2.1-restricted epitope (AAGIGILTV) has also been
identified genetically in another melanocytic protein, MART-1
(Melan-A). Kawakami, et al., J. Exp. Med., 180:347-52 (1994)
and Proc. Nat. Acad. Sci. USA, 91:3515-19 (1994), and see
also Coulie, et al., J. Exp. Med., 180:35-42 (1994).
An alternate approach toward characterization of CTL
epitopes is to identify them directly. Naturally occurring
peptides associated with MHC molecules on the tumor cells are
directly extracted, fractionated by HPLC and used to
reconstitute recognition by tumor specific CTL of a non-tumor
cell expressing appropriate MHC molecules. Sequencing can be
performed by Edman degradation. Mandelboim, et al., Nature,
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369:67-71 (1994) (CTL epitope on murine lung carcinoma).
However, Applicants pioneered the use of tandem mass
spectrometry to evaluate HHC-associated peptides. C.L.
Slingluff, et al., J. Immunol. 150, 2955 (1993); D.F. Hunt,
et al., Science 255, 1261 (1992); R.A. Henderson, et al.,
Proc. Natl. Acad. Sci. USA 90, 10275 (1993).
However, when peptides associated with MHC molecules on
tumor cells are extracted, a complex mixture, of up to
10,000-20,000 different peptides of similar size (mostly
nonamers), is obtained. Within this mixture, only a small
number of molecules are likely to correspond to the peptides
of interest. Consequently, their isolation and sequencing
was extremely difficult. Boon, et al., Ann. Rev. Immunol.,
12:337-65 (1994) states, "to our knowledge, the peptide
elution method has not yet ensured the identification of a
peptide recognized by anti-tumor CTL". More colorfully,
Finn, et al., Curr. Op. Immunol., 5:701-8 (1993) likened the
process to "throwing a fish hook into the ocean, hoping to
catch the big one", given, inter alia, the "very low amounts
of peptides".
In the present invention, HLA associated peptides have
been extracted, isolated and identified from different
melanoma lines. These peptides can be used to reconstitute
epitopes for HLA-A2.1- and HLA-A3- restricted melanoma-
specific CTL. These peptides and the stimulated CTL may be
useful for the in vivo immunotherapeutic treatment of
melanoma. Aspects of applicants' invention were described in
Cox, et al., Science, 264:716-719 (1994), which was published
on April 29, 1994.
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SiJMMARY OF THE INVENTION
The present invention relates to immunogens which are
capable of eliciting a melanoma-specific cytotoxic lymphocyte
response in at least some individuals, which response is
directed to peptide epitopes carried by those immunogens, and
to the use of those immunogens in active specific
immunotherapy and immunoprophylaxis against melanoma.
These immunogens may be used as vaccines, in active
specific immunotherapy. The immunogens may be administered
directly or by gene therapy. The epitopic peptides may also
be used to stimulate lymphocytes, the latter then being used
for adoptive immunotherapy.
In one embodiment, a CTL epitope of the present
invention is a sequence which is at least substantially
homologous with a CTL epitope of the melanoma antigens pMel-
17 and gplOO, (these two antigens are essentially identical).
One such epitope is the peptide 946L. Peptide 9461 is
substantially homologous to peptide 946L.
In another embodiment, a CTL epitope of the present
invention is a sequence which is at least substantially
homologous with a CTL epitope of tyrosinase. One such
epitope is the peptide Lys-Cys-Asp-Ile-Cys-Thr-Asp-Glu-Tyr.
Peptides 9461 and 946L, related to a single segment in pMel-
17 (a protein homologous to gpl00), had unexpectedly high
A2.1 CTL stimulatory activity. They also are recognized by
CTL from different individuals.
Another pMel-17-derived peptide (ALLAVGATK) had
acceptable A3 CTL stimulatory activity, and is the first HLA-
A3-associated stimulatory peptide identified in pMel-17 and
one of the few, if any, A3-associated peptides identified in
melanoma antigens generally.
KCDICTDEY is the first Al-restricted epitope to be
identified in tyrosinase and one of the few such epitopes
identified in melanoma antigens generally (Al epitopes have
been identified in MAGE-l (EADPTGHSY) and MAGE-3
(EVDPIGHLY)).
It is advantageous to be able to elicit a melanoma-
specific CTL response from one or more Al-, A2.1- and/or A3-
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restricted CTLs, and preferably all of them. In a similar
manner, a melanoma-specific CTL response may be elicited
which is restricted by other MHC molecules.
According to another aspect of the present
invention, there is provided an isolated HLA-A3 specific
peptide consisting of an amino acid sequence set forth at
SEQ ID NO: 4 or SEQ ID NO: 98.
According to still another aspect of the present
invention, there is provided a composition comprising the
isolated HLA-A3 specific peptide as described herein and an
HLA-A2 specific peptide consisting of the amino acid
sequence of SEQ ID NO: 14, SEQ ID NO: 39, or SEQ ID NO: 9.
According to yet another aspect of the present
invention, there is provided a composition comprising the
isolated, HLA-A3 specific peptide as described herein and an
HLA-Al specific peptide, the amino acid sequence of which is
set forth at SEQ ID NO: 93.
Additional embodiments of the present invention
are described below.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A Melanoma specific recognition of autologous
tumor by VMM18 CTL. VMM18 cells (solid squares) were lysed by
the CTL in a 4 h 51Cr release assay, while minimal lysis of
non-melanoma targets K562 (open squares), VMM12-EBV (open
circles) and the HLA-A3- melanoma DM6 (open triangles) was
observed.
Figure 1B Recognition of VMM18 melanoma by VMM18 CTL was
restricted by the class I MHC molecule HLA-A3. Lysis of
autologous melanoma was inhibited after incubation of target
cells with W6/32 (solid diamonds) and GAP-A3 (solid squares)
MAbs, specific for class I MHC and HLA-A3 respectively.
Incubation with L243 (open circles) had little effect on
recognition of autologous melanoma. Specific lysis of
autologous melanoma was 65a (dotted line), while lysis of
VMM12-EBV was 1.5% (solid line). The effector:target ratio
used was 10:1.
Figure 2 VMM18 CTL recognize a shared antigen expressed
by HLA-A3+ melanomas. Lysis of hot (51chromium labeled)
autologous and HLA-A3+ allogeneic melanoma cells (see legend)
was inhibited by cold (unlabelled) VMM18 melanoma cells (top
fig.), but not by cold (unlabelled) HLA-A3- DM6 melanoma cells
(bottom fig.). 2 x 104 VMM18 CTL were incubated with 1.4 x 104
unlabelled (cold) VMM18 or DM6 melanoma cells for 1 h at 37 C,
prior to the addition of 2 x 10351Cr-labelled targets as
indicated, giving a final E:T ratio of 10:1.
Figure 3 Expression of Pmel-17 reconstitutes recognition
of non-melanoma HLA-A3+ target cells by VMM18 CTL. VMM18 CTL
lysed 51Cr-labeled autologous melanoma cells VMM18 (solid
squares) as well as a non-melanoma HLA-A3+ cell line VMM12-
EBV infected with recombinant vaccinia virus expressing Pmel-
17 (vac-Pmel-17, closed circles). Minimal lysis of uninfected
VMM12-EBV cells (open circles), or cells infected with
control recombinant vaccinia virus expressing influenza
nucleoprotein (vac-NP, open triangles), was observed.
Figure 4 Relative ability of Pmel-17 peptides to
sensitize non-melanoma target cells for recognition by VMM18
CTL. "Cr-labelled T2-A3 cells were incubated with Pmel-17
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peptides ALLAVGATK (solid squares) and LLAVGATK (solid
triangles) and the control HLA-A3 binding peptide QVPLRPMTYK,
from the HIV Nef protein (open circles).
Figures 5A-B. Recognition of autologous and HLA-matched
5 melanomas by melanoma-reactive CTL. In 19A), VMM12 CTL are
evaluated for lysis of a panel of target cells. The VMM12
CTL recognize shared melanoma antigens presented by HLA-Al
(VMM15 melanoma cells share HLA-A1 with VMM12), and by HLA-A3
(VMM10 melanoma cells share HLA-A3 with VMM12). Similarly,
10 in 19B), VMM15 CTL are evaluated in the same manner. VMM15
CTL recognize shared melanoma antigens presented by HLA-Al
(VMM12 melanoma cells) and by either HLA-A1, -A25, or -B8
(VMM14 melanoma cells).
Figures 6A-B. HLA-Al+ CTL lines recognize tyrosinase
peptides on HLA-Al. In 20A), VMM12 CTL are capable of
lysing C1R-Al cells infected with a vaccinia-tyrosinase
construct. In 20B), VMM15 CTL also recognize tyrosinase.
Figures 7A-D. List of peptides synthesized and tested
for recognition by VMM12 and VMM15 CTL. These peptides were
predicted from the defined sequence of tyrosinase, accounting
for some possible alternate sequences and for possible post-
translational modifications. Those listed in the 3rd
synthesis were not tested. Figs. 21A-D refers to syntheses
1-4, respectively.
Figure 8. VMM15 CTL recognize peptides containing
KCDICTDEY in association with HLA-Al. C1R-Al cells were
pulsed with 10 uM, 1 uM and 0.1 uM concentrations of
synthetic peptides prior to addition of VMM15 CTL.
Background lysis of CiR was approximately 10%. Direct
cytotoxicity by the peptides themselves was negligible (open
diamonds), averaging 0-2%. An epitope for VMM15 CTL was
reconstituted by three of the test peptides, numbers 5, 12,
and 15, corresponding to KCDICTDEY, DAEKCDICTDEY, and
EKCDICTDEY as marked.
Figure 9. VMM12 CTL recognize a peptide containing
KCDICTDEY in association with HLA-A1. CiR-A1 cells were
pulsed with peptides at 1 to 0.01 uM concentrations prior to
adding VMM12 CTL. The peptides themselves were not cytolytic
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(open diamonds). The peptide DAEKCDICTDEY reconstituted an
epitope for these VMM12 CTL, although weakly.
Figure 10. Amino acid sequence of tyrosinase, with the
position of KCDICTDEY highlighted and underlined. The high
proportion of cystine residues and acidic residues are noted
relative to the proportion in the intact protein.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
OF THE INVENTION
The present invention relates to certain melanoma-
specific CTL epitopes, and their incorporation into
immunogens for immunoprophylactic and immunotherapeutic
purposes. For the purpose of the present invention, a
melanoma-specific CTL epitope is an epitope which is
recognized by a T-cell receptor of at least some cytotoxic
lymphocytes of at least some individuals in the population of
interest, and which is more frequently or strongly associated
with melanoma cells than with at least some other cancer
and/or normal cells. There may be some cross-reactivity, for
example, with other cells of melanocytic lineage. Absolute
specificity is not required, provided that a useful prophy-
lactic, therapeutic or diagnostic effect is still obtained.
Melanoma-Specific CTL Epitopes
The melanoma-specific CTL epitopes of the present
invention are peptides, typically 9-13 amino acids in length,
which are sufficiently similar to a melanoma-specific epitope
recognized by a melanoma-specific CTL to be useful, under
suitable conditions of use, to protect an individual from
melanoma, or to be useful in the diagnosis of melanoma or of
a patient's ability to fight a melanoma by a CTL response.
Preferably, these epitopes are identical to or otherwise
substantially homologous with melanoma-specific peptide
epitopes recognized by melanoma-specific CTLs.
The family of melanoma epitopes which are recoverable
from an individual is dependent on the nature of the binding
site of the Class I MHC (HLA) molecules expressed by the
individual, and, as a result of the polymorphism of the Class
I MHC (HLA) molecules, can vary considerably from one
individual to another. For the purpose of the present
invention, the melanoma cell line used as a source of
melanoma-specific CTL epitopes may be any melanoma cell line;
similarly, the Class I MHC (HLA) molecule may be any such
molecule borne by a melanoma which is capable of binding to
and presenting a melanoma-specific epitope, including, but
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not limited to, the various allelic forms of Class I MHC
molecules, including but not limited to those enumerated in
Table I. Among the Class I molecules, the principal genetic
loci are denoted as HLA-A, HLA-B, and HLA-C. The preferred
epitopic sequence may vary depending on the restriction
system.
Application of active specific immunotherapy to a
heterogeneous melanoma patient population would be
facilitated by identification of CTL epitopes presented in
association with a wide range of class I MHC molecules.
Besides HLA-A2, the most commonly expressed class I MHC
molecules are Al and A3, then B7 and B8. Approximately 90% of
the melanoma patient population should express one or more of
these molecules or HLA-A2. Peptides from MAGE-1 and MAGE-3
have been identified as HLA-A1-restricted CTL epitopes, and a
few peptides have been identified for some of the less common
MHC molecules, including A24, A31, and B44. Little work has
been done toward identification of HLA-A3-restricted
responses, and-except for the peptides from MAGE proteins -
little work has been done toward identification of HLA-Al-
restricted responses.
Preferably, the epitope is one restricted by one of the
more prevalent forms (in the melanoma patient population) of
these loci. The loci HLA-Al, HLA-A2, HLA-A3, HLA-B7 and HLA-
B8 are of greatest interest. Within HLA-A2, HLA-A2.1 is of
particular interest.
Preferably, the CTL epitopes of the present invention,
in the cytotoxicity assay described hereafter, when used in
oligopeptide form to reconstitute epitopes for suitable CTL,
achieve, at the dosage resulting in maximal lysis of target
cells exposed to the stimulated CTL, a percentage lysis of
target cells which is at least 10 percentage points higher
(more preferably, at least 20 points higher) the background
level of lysis of the target cells by the CTLs (i.e., in
absence of the peptide).
Preferably, the peptide concentration at which the
epitope-stimulated CTLs achieve half the maximal increase in
lysis relative to background is no more than about 1 mM,
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preferably no more than 1 M, more preferably no more than
about 1 nM, still more preferably no more than about 100 pM,
most preferably no more than about 10 pM. For the peptides
946L and 9461, half-maximal lysis of T2 cells is observed
with concentrations of peptide in the pM range. In contrast,
the MAGE-1 peptide EADPTGHSY had half-maximal lysis between 1
and 100 nM (prob about 10); while the tyrosinase peptides
YMNGTMSQV and MLLAVLYCL reported by Boon induced half-maximal
lysis (even with pre-treatment with MA2.1 antibody) at over
10 nM.
ALLAVGATK is at present the only pMel-17 derived peptide
known to be immunogenic in the context of HLA-A3, which is
expressed by 200 of the patient population. It achieves
half-maximal lysis of T2 cells expressing HLA-A3 at a
concentration of about 10 nM. While not as potent as our
A2.1 peptides, its potency is acceptable.
Preferably the epitope is recognized by CTLs from at
least two different individuals, more preferably at least
five different individuals.
More preferably, the CTL epitope satisfies two or more
of the above desiderata.
The 946L peptide, although recognized by HLA-A2.1-
restricted melanoma-specific CTL, may not be optimal at
present. It is known that some residues on the nonamer
peptide are particularly important for binding of the peptide
to the MHC molecule (residues 2,9), while others are
particularly important for Tc recognition (residues 4-8).
The other residues may be important for either or both. It
is proposed that amino acid substitutions for the 946 peptide
may be useful at increasing immunogenicity, particularly by
attempting to change residues that may increase binding to
the MHC such as changing residue 9 to a valine or residue 3
to anything other than glutamic acid (E). Using existing
knowledge about which of these residues may be more likely to
affect binding either to the MHC or to the TcR, a rational
approach to this process may be employed. The resulting
peptides, if more effective, could be used for any of the
purposes described herein. (refs: E.L. Huczko et al. J.
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Immunol. 151:2572, 1993; J. Ruppert et al. Cell 754: 929,
1993; Madden Dr et al. Cell 75:693-708, 1994.) It is
possible to predict peptides binding to specific Class I MHC
molecules by identifying amino acid sequences fitting
5 described binding motifs within known protein sequences. In
attempting to identify epitopes for melanoma-specific CTL,
these peptides can be screened for their ability to sensitize
non-melanoma targets for recognition by melanoma specific
CTL.
10 Therefore, in addition to epitopes which are identical
to the naturally occurring melanoma-specific epitopes, the
present invention embraces epitopes which are substantially
homologous with such epitopes, and therefore melanoma-
specific in their own right.
15 The term "substantially homologous", when used in
connection with amino acid sequences, refers to sequences
which are substantially identical to or similar in sequence
with each other, giving rise to a homblogy in conformation
and thus to similar (or improved) biological activity. The
term is not intended to imply a common evolution of the
sequences.
An epitope is considered substantially homologous to a
reference epitope if it has at least 100 of an immunological
activity of the reference epitope and differs from the
reference epitope by no more than one non-conservative
substitution not suggested by a known binding motif of the
pertinent MHC molecule. Any number of highly conservative,
conservative or semi-conservative substitutions, or non-
conservative substitutions suggested by known binding motifs,
subject to the activity limitation, are permitted.
Kast, et al., J. Immunol, 152:3904-12 (1994) sets forth
HLA-A specific peptide binding motifs for the HLA molecules
Al, A2.1, A3, All and A24. Engelhard, et al., in Sette, ed.,
Naturally Processed Peptides, 57:39-62 (1993) explored the
features that determined binding to HLA-A2.1 and HLA-B7. See
also Hobohim et al; Eur. J. Immunol., 23:1271-6 (1993);
Kawakami, et al., J. Immunol., 154:3961-8 (1995). Based on
these and other sources, the preferred and tolerated AAs for
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various HLA molecules include (but are not limited to) the
following:
Table 10
Molecule Position Preferred AA tolerated AA
Al 2 T, S, M
3 D, E A, S
9 Y
A2.1 2 L, M I, V, A, T
9 L, V, I A, M, T
A3 2 L, M, I, V, S C, G, D
A, T, F
9 K, R, Y, H, F A
All 2 M, L, I, V, S C, D, F
A, T, G, N
9 K R, H, Y
A24 2 Y, F, W M
9 F, L, I, W
B7 1 A M, S, R, L
2 P V
3 R A, K, S, M
9 L I, A, V
B8 3 K not known
5 K not known
9 L not known
B27 2 R not known
9 R, K, H not known
B35 2 P not known
9 y not known
B53 2 P not known
If a position is not listed, studies revealed a greater
variability of AAs than for the listed positions. For listed
positions, AAs not listed may be tolerated, especially if
they are conservative or semi-conservative substitutions for
"preferred" or "tolerated" AAs.
An example of a peptide variant which satisfies the
known binding motif is YLEPGPVTV. This differs from 946L at
position 9. However, V is a preferred a.a. at position 9 of
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HLA-A2.1 binding peptides.
Substantially homologous peptide epitopes may be
identified by a variety of techniques. It is known in the
art that one may synthesize all possible single substitution
mutants of a known peptide epitope. For a nonpeptide, there
are (20x9-1=179) such mutants. Geysen, et al., Proc Nat.
Acad. Sci. (USA), 81:3998-4002 (1984). While the effects of
different substitutions are not always additive, it is
reasonable to expect that two favorable or neutral single
substitutions at different residue positions in the epitope
can safely be combined in most cases.
One may also synthesize a family of related single or
multiple substitution mutants, present the mixture to the
HLA-A2.1 positive lymphoblastoid cell line T2 (or other cell
line capable of presenting melanoma-specific CTL epitopes),
and expose the T2 cells to melanoma-specific CTLs. If the T2
cells are lysed, the effective epitopes may be identified
either by direct recovery from the T2 cells or by a
progressive process of testing subsets of the effective
peptide mixtures. Methods for the preparation of degenerate
peptides are described in Rutter, USP 5,010,175, Haughten, et
al., Proc. Nat. Acad. Sci. (USA), 82:5131-35 (1985), Geysen,
et al., Proc. Nat. Acad. Sci. (USA), 81:3998-4002 (1984);
W086/06487; W086/00991.
Multiple mutagenesis may be used to screen a few residue
positions intensely or a larger number of positions more
diffusely. One approach is to explore at least a
representative member of each a.a. type at each position,
e.g., one representative of each of exchange groups I-V as
hereafter defined. Preferably, Gly and Pro are screened in
addition to one other group I residue. Preferably, at least
one screened residue is an H-bonding resiude. If a positive
mutant features a particular representative, like amino acids
can be explored in a subsequent library. If, for example, a
Phe substitution improves binding, Tyr and Trp can be
examined in the next round.
In the case of the peptide 946L (SEQ. ID. No.:14), a
possible multiple mutagenesis strategy would be as follows:
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Parental Tyr Leu Glu Pro Glv Pro Val Thr Ala
Possible Phe Ile Asp Ala Pro Ala Ile Ala Thr
Mutations Trp Val Ser Ala Ser Leu Ser Ser
Met Thr Ser Thr Met Pro Pro
Ala Gly Thr Gly Gly Gly
Thr Leu
Val
Ile
Met
For peptide 1030, a possible strategy would be:
Parental Tvr Met ASp Gly Thr Met Ser Gln Val
Phe Val Glu Pro Ala Val Ala Asn Ile
Trp Ile Ala Ser Ile Thr Leu
Leu Ser Pro Leu Pro Met
Ala Thr Gly Gly Ala
Thr Thr
Other strategies are, of course, possible. For example,
the Asp/Glu and Gln/Asn sets can be merged. It is known from
comparison of peptide 1030 with the homologous tyrosinase
segment that substitution of Asn for Asp in position 3
reduces CTL activity 100-fold. However, a multiple
mutagenesis strategy could identify compensating mutations at
other sites.
For our preferred A3 peptide, a possible multiple
mutagenesis strategy would be
Ala Leu Leu Ala Val G1v Ala Thr Lys
Thr Ile Ile Thr Ile Ala Thr Gly Arg
Ser Val Val Ser Leu Thr Ser Ala His
Pro Met Met Pro Met Ser Pro Ser Tyr
Gly Ser Gly Pro Gly Pro Phe
Cys Ala
Gly
Asp
Ala
Thr
Phe
For our preferred Al peptide, a possible multiple
mutagenesis strategy would be
Lys Cys Asp Ile Cys Thr Asp Glu Tvr
Arg Thr Glu Leu Thr Ala Glu Asp Phe
His Ser Val Ser Ser Trp
Ala Met Ala Pro
Met Gly Gly
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These strategies take into account both conservative
substitutions for the wild type AAs, and the known Al, A2.1
and A3 binding motifs.
The person of ordinary skill in the art, in determining
which residues to vary, may also make comparisons of the
sequences of the naturally processed MHC associated peptides,
and may obtain 3D structures of the MHC: peptide: TCR
complexes, in order to identify residues involved in MHC or
TCR binding. Such residues may either be left alone, or
judiciously mutated in an attempt to enhance MHC or TCR
binding.
It is also possible to predict substantially homologous
epitopes by taking into account studies of sequence
variations in families of naturally occurring homologous
proteins. Certain amino acid substitutions are more often
tolerated than others, and these are often correlatable with
similarities in size, charge, etc. between the original amino
acid and its replacement. Insertions or deletions of amino
acids may also be made. N- and C-terminal truncations or
extensions are more likely to be tolerated than internal
deletions or insertions. With regard to truncation, the
peptide may be truncated by one or more amino acids and still
be substantially homologous, however, it cannot be fewer than
five amino acids. Extensions are permissible, however, note
that larger peptides are digested in vivo prior to
presentation.
Conservative substitutions may be made in the amino acid
sequence of the proteins of interest without compromising the
desired properties of the peptides, i.e., induction of
cytotoxic T-lymphocytes in a patient when administered
thereto.
Conservative substitutions are herein defined as
exchanges within one of the following five groups:
I. Small aliphatic, nonpolar or slightly
polar residues:
Ala, Ser, Thr, Pro, Gly
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II. Polar, negatively charged residues: and
their amides
Asp, Asn, Glu, Gln
III. Polar, positively charged residues:
5 His, Arg, Lys
IV. Large, aliphatic, nonpolar residues:
Met, Leu, Ile, Val, Cys
V. Large, aromatic residues:
Phe, Tyr, Trp
10 Within the foregoing groups, the following substitutions
are considered "highly conservative":
Asp/Glu
His/Arg/Lys
Phe/Tyr/Trp
15 Met/Leu/Ile/Val
Semi-conservative substitutions are defined to be
exchanges between two of groups (I)-(V) above which are
limited to supergroup (A), comprising (I), (II) and (III)
above, or to supergroup (B), comprising (IV) and (V) above.
20 Also, Ala is considered a semi-conservative substitution for
all non group I amino acids.
It will be appreciated that highly conservative
substitutions are less likely to affect activity than other
conservative substitutions, conservative substitutions are
less likely to affect activity than merely semi-conservative
substitutions, and semi-conservative substitutions less so
than non-conservative substitutions.
Although a substitution mutant, either single or
multiple, of the peptides of interest may not have quite the
potency of the original peptide, such a mutant may well be
useful.
Substitutions are not limited to the genetically
encoded, or even the naturally occurring amino acids. When
the epitope is prepared by peptide synthesis, the desired
amino acid may be used directly. Alternatively, a genetical-
ly encoded amino acid may be modified by reacting it with an
organic derivatizing agent that is capable of reacting with
selected side chains or terminal residues. The following
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21
examples of chemical derivatives are provided by way of
illustration and not by way of limitation.
Aromatic amino acids may be replaced with D- or
L-naphylalanine, D- or L-Phenylglycine, D- or L-2-thieney-
lalanine, D- or L-1-, 2-, 3- or 4-pyreneylalanine, D- or
L-3-thieneylalanine, D- or L-(2-pyridinyl)-alanine, D- or
L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or
L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)- phenyl-
glycine, D-(trifluoromethyl)-phenylalanine, D-p-fluoro-
phenylalanine, D- or L-p-biphenylphenylalanine, D- or
L-p-methoxybiphenylphenylalanine, D- or L-2-indole-
(alkyl)alanines, and D- or L-alkylainines where alkyl may be
substituted or unsubstituted methyl, ethyl, propyl, hexyl,
butyl, pentyl, iso-propyl, iso-butyl, sec-isotyl, iso-pentyl,
non-acidic amino acids, of C1-C20.
Acidic amino acids can be substituted with non-
carboxylate amino acids while maintaining a negative charge,
and derivatives or analogs thereof, such as the non-limiting
examples of (phosphono)-alanine, glycine, leucine,
isoleucine, threonine, or serine; or sulfated (e.g., -SO3H)
threonine, serine, tyrosine.
Other substitutions may include unnatural hyroxylated
amino acids made by combining "alkyl" (as defined and
exemplified herein) with any natural amino acid. Basic amino
acids may be substituted with alkyl groups at any position of
the naturally occurring amino acids lysine, arginine,
ornithine, citrulline, or (guanidino)-acetic acid, or other
(guanidino)alkyl-acetic acids, where "alkyl" is define as
above. Nitrile derivatives (e.g., containing the CN-moiety
in place of COOH) may also be substituted for asparagine or
glutamine, and methionine sulfoxide may be substituted for
methionine. Methods of preparation of such peptide
derivatives are well known to one skilled in the art.
In addition, any amide linkage can be replaced by a
ketomethylene moiety, e.g. (-C(=O)-CHz-) for (-(C=O)-NH-).
Such derivatives are expected to have the property of
increased stability to degradation by enzymes, and therefore
possess advantages for the formulation of compounds which may
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22
have increased in vivo half lives, as administered by oral,
intravenous, intramuscular, intraperitoneal, topical, rectal,
intraocular, or other routes.
In addition, any amino acid can be replaced by the same
amino acid but of the opposite chirality. Thus, any amino
acid naturally occurring in the L-configuration (which may
also be referred to as the R or S configuration, depending
upon the structure of the chemical entity) may be replaced
with an amino acid of the same chemical structural type, but
of the opposite chirality, generally referred to as the D-
amino acid but which can additionally be referred to as the
R- or the S-, depending upon its composition and chemical
configuration. Such derivatives have the property of greatly
increased stability to degradation by enzymes, and therefore
are advantageous in the formulation of compounds which may
have longer in vivo half lives, when administered by oral,
intravenous, intramuscular, intraperitoneal, topical, rectal,
intraocular, or other routes.
The thiol group of cysteine reacts very rapidly with
alkyl halides, such as iodoacetate, iodoacetamide, methyl
iodine, and so on, to give the corresponding stable alkyl
(substituted or unsubstituted) derivatives, such as -CH2-S-
CH3. The thiol group can also add across double bonds such as
those of N-ethylmaleimide or of maleic anhydride, and it can
open the ring of ethyleneimine, providing a new site for
tryptic cleavage. Thiols form complexes with various metal
(especially mercury, silver, arsenic, copper, iron, zinc,
cobalt, molybdenum, manganese and cadmium ions) and
organometal ions (e.g., R-Hg+, such as para-mercuribenzoic
acid).
The thiol group may be oxidized to yield a disulfide
bond or a sulfonate. A thiol may be converted to a disulfide
by thiol-disulfide exchange, for example, exchange with an
aromatic disulfide such as dithionitrobenzoic acid (DTNB) or
Ellman's reagnet. Of course, a cysteine residue may be
disulfide bonded to a cysteine residue in the same or a
different peptide, or to a free cysteine. By way of further
examples, some of which are already embraced by the general
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23
discussion above, cysteinyl residues may be reacted with
alpha-haloacetates (and corresponding amines), such as
2-chloroacetic acid or chloroacetamide, to give carboxymethyl
or carboxyamidomethyl derivatives. Cysteinyl residues may
also be derivatized by reaction with compounds such as
bromotrifluoroacetone, alpha-bromo-
beta-(5-imidozoyl)propionic acid, chioroacetyl phosphate,
N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues may be derivatized by reaction with
compounds such as diethylprocarbonate e.g., at pH 5.5-7.0
because this agent is relatively specific for the histidyl
side chain, and para-bromophenacyl bromide may also be used;
e.g., where the reaction is preferably performed in 0.1 M
sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues may be reacted with
compounds such as succinic or other carboxylic acid
anhydrides. Derivatization with these agents is expected to
have the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing alp-
ha-amino-containing residues include compounds such as
imidoesters/e.g., as methyl picolinimidate; pyridoxal
phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; 0-methylisourea; 2,4
pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
Arginyl residues may be modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin
according to known method steps. Derivatization of arginine
residues requires that the reaction be performed in alkaline
conditions because of the high pKa of the guanidine
functional group. Furthermore, these reagents may react with
the groups of lysine as well as the arginine epsilon-amino
group.
The specific modification of tyrosyl residues per se is
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24
well-known, such as for introducing spectral labels into
tyrosyl residues by reaction with aromatic diazonium
compounds or tetranitromethane. N-acetylimidizol and
tetranitromethane may be used to form 0-acetyl tyrosyl
species and 3-nitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) may be
selectively modified by reaction with carbodiimides
(R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl-
(4-ethyl) carbodiimide or 1- ethyl-3-(4-azonia-4,4-
dimethylpentyl) carbodiimide. Furthermore, aspartyl and
glutamyl residues may be converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues may be readily
deamidated to the corresponding glutamyl and aspartyl
residues. Alternatively, these residues may be deamidated
under mildly acidic conditions. Either form of these
residues falls within the scope of the present invention.
Derivatization with bifunctional agents is useful for
cross-linking the peptide to a water-insoluble support matrix
or to other macromolecular carriers, according to known
method steps. Commonly used cross-linking agents include,
e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, in-
cluding disuccinimidyl esters such as 3,3'- dithiobis(suc-
cinimidylpropionate), and bifunctional maleimides such as
bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S.
Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;
4,229,537; and 4,330,440, may be employed for protein
immobilization.
Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or
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threonyl residues, methylation of the alpha-amino groups of
lysine, arginine, and histidine side chains (Creighton, T.E.,
Proteins: Structure and Molecule Properties, W.H. Freeman &
Co., San Francisco, pp. 79-86 (1983)), acetylation of the
5 N-terminal amine, methylation of main chain amide residues
(or substitution with N-methyl amino acids) and, in some
instances, amidation of the C-terminal carboxyl groups,
according to known method steps. Glycosylation is also
possible.
10 Derivatized moieties may impart altered affinity for
their target, altered immunogenicity, or improved solubility,
absorption, biological half life, and the like, or attenuated
undesirable side effects. Moieties capable of mediating such
effects are disclosed, for example, in Remington's
15 Pharmaceutical Sciences, 16th ed., Mack Publishing Co.,
Easton, PA (1980).
Modifications are not limited to the side chains of the
amino acids. One may also modify the peptidyl linkage
itself, e.g., -NRCO- (where R is alkyl or aryl), instead
20 of -NHCO-, as in the so-called "peptoids."
The peptides may also comprise isoteres of two or more
residues in the immunogenic peptide. An isotere as defined
here is a sequence of two or more residues that can be
sustituted for a second sequence because the steric
25 conformation of the first sequence fits a binding site
specific for the second sequence. The term specifically
includes peptide backbone modifications well known to those
skilled in the art. Such modifications include modifications
of the amide nitrogen, the cx-carbon, amide carbonyl, complete
replacement of the amide bond, extensions, deletions or
backbone crosslinks. See, generally, Spatola, Chemistry and
Biochemistry of Amino Acids, peptides and Proteins, Vol. VII
(Weinstein ed., 1983).
It is also possible to construct and use so-called
peptide mimetics whose conformation is similar to that of a
peptide but do not have a peptide-like molecular formula. In
effect, in a mimetic, all of the residues of the peptide are
replaced by one or more isoteres as defined above.
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The Melanoma-Specific Immunogen
The melanoma-specific immunogen of the present invention
is a molecule corresponding to or otherwise comprising a
melanoma-specific CTL epitope as previously described. The
immunogen may comprise one or more melanoma-specific CTL
epitopes, which may be the same or different. Preferably,
the immunogen is chosen so that at least one epitope is
effective in each of two or more restriction systems, e.g.,
HLA-A1 and HLA-A3; HLA-A1 and HLA-A2; HLA-A2 and HLA-A3; and
HLA-Al, -A2 and -A3. In some instances, a single epitope may
be effective in more than one restriction system. For
example HLA-A2 and HLA-69, or HLA-A3 and HLA-All, are pairs
of MHC molecules having similar peptide binding motifs.
Otherwise, for the immunogen to be effective in more than one
restriction system, two or more epitopes (at least one for
each MHC molecule of interest) will need to be provided.
These epitopes may be separate or overlapping.
It should be noted that instead of linking epitopes
within a single immunogen, the compositions of the present
invention may include two or more immunogens which present
different epitopes.
If the immunogen comprises a plurality of such epitopes,
they may be linked directly, or through a spacer of some
kind, or by noncovalent means such as an avidin:biotin
complex. The immunogen may take any form that is capable of
eliciting a melanoma-specific cytotoxic immune response. By
way of example and not of limitation, the immunogen may be a
fusion of a plurality of CTL epitopes which is sufficiently
large to be immunogenic, a conjugate of one or more epitopes
to a soluble immunogenic macromolecular carrier, such as
serum albumin, keyhole limpet hemocyanin, or dextran, a
recombinant virus engineered to display the epitope on its
surface, or a conjugate of a plurality of epitopes to a
branched lysine core structure, a so-called "multiple
antigenic peptide" (see Posnett, et al., J. Biol. Chem.,
263:1719-25, 1988).
The immunogenic conjugate may also comprise moieties
intended to enhance the immune response, such as a T helper
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27
peptide, a cytokine or an adjuvant; a targeting agent, such
as an antibody or receptor ligand or ligand analogue; or a
stabilizing agent, such as a lipid.
For instance, the ability of the peptides to induce CTL
activity can be enhanced by linkage to a sequence which
contains at least one epitope that is capable of inducing a T
helper cell response. Particularly preferred immunogenic
peptides/T helper conjugates are linked by a spacer molecule.
The spacer is typically comprised of relatively small,
neutral molecules, such as amino acids or amino acid
mimetics, which are substantially uncharged under
physiological conditions. The spacers are typically selected
from, e.g., Ala, Gly, or other neutral spacers of nonpolar
amino acids or neutral polar amino acids. It will be
understood that the optionally present spacer need not be
comprised of the same residues and thus may be a hetero- or
homo-oligomer. When present, the spacer will usually be at
least one or two residues, more usually three to six
residues. Alternatively, the CTL peptide may be linked to
the T helper peptide without a spacer.
The immunogenic peptide may be linked to the T helper
peptide either directly or via a spacer either at the amino
or carboxy terminus of the CTL peptide. The amino terminus
of either the immunogenic peptide or the T helper peptide may
be acylated.
Besides one or more of the novel melanoma-specific CTL
epitopes described herein, the immunogen may present one or
more such epitopes already known in the art, such as the
following:
Table A. Peptide epitopes for human tumor-specific CTL
Protein MHC Peptide sequence Tumor type
restriction
Tyrosinase A2 MLLAYLYCL Melanoma
Tyrosinase A24 AFLPWHRLF, Melanoma
AFLPWHRLFL
Tyrosinase B44 SEIWRDIDF Melanoma
I II I II I
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gp100 Pmel17 A2 KTWGQYWQV Melanoma
gp100 Pmel17 A2 ITDQVPFSV Melanoma
gp100 Pme117 A2 VLYRYGSFSV Melanoma
gp100 Pmell7 A2 LLDGTATLRL Melanoma
MART-1 MelanA A2 AAGIGILTV Melanoma
MART-1 MelanA A2 ILTVILGVL Melanoma
gp75 TRP-1 A31 ---- Melanoma
MAGE-1 Al EADPTGHSY Melanoma, other
tumors '
MAGE-1 Cw*1601 SAYGEPRKL Melanoma, other
tumors 1
MAGE-3 Al EVDPIGHLY Melanoma,
other tumors 2
MAGE-3 A2 FLWGPRALV Melanoma,
I other tumors 2
BAGE Cw*1601 AARAVFLAL Melanoma,
other tumors 3
GAGE-1,2 Cw6 YRPRPRRY Melanoma,
other tumors 4
HER-2/neu A2 KIFGSLAFL, Ovarian Cancer
VMAGVGSPYV
HER-2/neu A2 IISAVVGIL Ovarian
Cancer, NSCLC
CEA A2 YLSGANLNL Colon Cancer
p15 A24 (E)AYGLDFYIL Melanoma and
normal tissues
43kD protein A2 QDLTMKYQIF Melanoma
MUM-1 gene product B*4402 EEKLIVVLF Melanoma
mutated across
intron/exon junction
mutated beta-catenin A24 SYLDSGIHF Melanoma
1 MAGE-1: expressed in Melanoma (36%), Bladder CA (19%),
Breast CA (180), Head & neck CA (250), Non-small cell lung CA
(NSCLC, 340), Sarcomas (11%), Prostate CA (150) [50]
2 MAGE-3: expressed in Melanoma (650), Bladder CA (34%),
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Breast CA (11%), Head & neck CA (48%), Non-small cell lung CA
(NSCLC, 310), Sarcomas (110), Prostate CA (15%) [50]
3 BAGE: expressed in Melanoma (22%), Bladder CA (15%), Breast
CA (10%), Head and neck CA (<100), NSCLC (<10%) [50]
4 GAGE-1, -2: expressed in Melanoma (24%), Bladder CA (12%),
Breast CA (90), Head & neck CA (190), NSCLC (19%), Sarcomas
(25%), Prostate cancers (10%) [50]
5 Isoleucine (I) at position 5 is the result of mutation.
The wild type sequence si EEKLSVVLF.
6 Phenylalanine (F) at pos. 9 is the result of mutation. The
wild type sequence is SYLDSGIHS.
If it is desirable to present more than one CTL epitope,
rather than presenting all of the epitopes on a single
immunogen, they may be presented on two or more different
immunogens. These may be administered separately, or as part
of a mixture, e.g., a mixture of epitopic peptides.
Mode of Production
The peptide portion of the immunogens of the present
invention may be produced by any conventional technique,
including
(a) nonbiological synthesis by sequential
coupling of component amino acids,
(b) production by recombinant DNA techniques
in a suitable host cell, and
(c) chemical or enzymatic modification of a
sequence made by (a) or (b) above.
Gene Expression. The peptides disclosed herein may be
produced, recombinantly, in a suitable host, such as bacteria
from the genera Bacillus, Escherichia, Salmonella, Erwinia,
and yeasts from the genera Hansenula, Kluyveromyces, Pichia,
Rhinosporidium, Saccharomyces, and Schizosaccharomyces, or
cultured mammalian cells such as COS-1. The more preferred
hosts are microorganisms of the species Pichia pastoris,
Bacillus subtilis, Bacillus brevis, Saccharomyces cerevisiae,
Escherichia coli and Yarrowia lipolytica. Any promoter,
regulatable or constitutive, which is functional in the host
may be used to control gene expression.
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It has been found that peptide fragments from the
protein pMEL17 reconstitute HLA A2.1 and A3 epitopes. The
pMEL17 gene is a single-stranded cDNA reading 5' to 3'. The
gene encoding for pMEL17, is:
5 GGAAGAACAC AATGGATCTG GTGCTAAAAA GATGCCTTCT TCATTTGGCT
GTGATAGGTG CTTTGCTGGC TGTGGGGGCT ACAAAAGTAC CCAGAAACCA
GGACTGGCTT GGTGTCTCAA GGCAACTCAG AACCAAAGCC TGGAACAGGC
AGCTGTATCC AGAGTGGACA GAAGCCCAGA GACTTGACTG CTGGAGAGGT
GGTCAAGTGT CCCTCAAGGT CAGTAATGAT GGGCCTACAC TGATTGGTGC
10 AAATGCCTCC TTCTCTATTG CCTTGAACTT CCCTGGAAGC CAAAAGGTAT
TGCCAGATGG GCAGGTTATC TGGGTCAACA ATACCATCAT CAATGGGAGC
CAGGTGTGGG GAGGACAGCC AGTGTATCCC CAGGAAACTG ACGATGCCTG
CATCTTCCCT GATGGTGGAC CTTGCCCATC TGGCTCTTGG TCTCAGAAGA
GAAGCTTTGT TTATGTCTGG AAGACCTGGG GCCAATACTG GCAAGTTCTA
15 GGGGGCCCAG TGTCTGGGCT GAGCATTGGG ACAGGCAGGG CAATGCTGGG
CACACACACC ATGGAAGTGA CTGTCTACCA TCGCCGGGGA TCCCGGAGCT
ATGTGCCTCT TGCTCATTCC AGCTCAGCCT TCACCATTAC TGACCAGGTG
CCTTTCTCCG TGAGCGTGTC CCAGTTGCGG GCCTTGGATG GAGGGAACAA
GCACTTCCTG AGAAATCAGC CTCTGACCTT TGCCCTCCAG CTCCATGACC
20 CTAGTGGCTA TCTGGCTGAA GCTGACCTCT CCTACACCTG GGACTTTGGA
GACAGTAGTG GAACCCTGAT CTCTCGGGCA CCTGTGGTCA CTCATACTTA
CCTGGAGCCT GGCCCAGTCA CTGCCCAGGT GGTCCTGCAG GCTGCCATTC
CTCTCACCTC CTGTGGCTCC TCCCCAGTTC CAGGCACCAC AGATGGGCAC
AGGCCAACTG CAGAGGCCCC TAACACCACA GCTGGCCAAG TGCCTACTAC
25 AGAAGTTGTG GGTACTACAC CTGGTCAGGC GCCAACTGCA GAGCCCTCTG
GAACCACATC TGTGCAGGTG CCAACCACTG AAGTCATAAG CACTGCACCT
GTGCAGATGC CAACTGCAGA GAGCACAGGT ATGACACCTG AGAAGGTGCC
AGTTTCAGAG GTCATGGGTA CCACACTGGC AGAGATGTCA ACTCCAGAGG
CTACAGGTAT GACACCTGCA GAGGTATCAA TTGTGGTGCT TTCTGGAACC
30 ACAGCTGCAC AGGTAACAAC TACAGAGTGG GTGGAGACCA CAGCTAGAGA
GCTACCTATC CCTGAGCCTG AAGGTCCAGA TGCCAGCTCA ATCATGTCTA
CGGAAAGTAT TACAGGTTCC CTGGGCCCCC TGCTGGATGG TACAGCCACC
TTAAGGCTGG TGAAGAGACA AGTCCCCCTG GATTGTGTTC TGTATCGATA
TGGTTCCTTT TCCGTCACCC TGGACATTGT CCAGGGTATT GAAAGTGCCG
AGATCCTGCA GGCTGTGCCG TCCGGTGAGG GGGATGCATT TGAGCTGACT
GTGTCCTGCC AAGGCGGGCT GCCCAAGGAA GCCTGCATGG AGATCTCATC
GCCAGGGTGC CAGCCCCCTG CCCAGCGGCT GTGCCAGCCT GTGCTACCCA
GCCCAGCCTG CCAGCTGGTT CTGCACCAGA TACTGAAGGG TGGCTCGGGG
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ACATACTGCC TCAATGTGTC TCTGGCTGAT ACCAACAGCC TGGCAGTGGT
CAGCACCCAG CTTATCATGC CTGTGCCTGG GATTCTTCTC ACAGGTCAAG
AAGCAGGCCT TGGGCAGGTT CGGCTGATCG TGGGCATCTT GCTGGTGTTG
ATGGCTGTGG TCCTTGCATC TCTGATATAT AGGCGCAGAC TTATGAAGCA
AGACTTCTCC GTACCCCAGT TGCCACATAG CAGCAGTCAC TGGCTGCGTC
TACCCCGCAT CTTCTGCTCT TGTCCCATTG GTGAGAATAG CCCCCTCCTC
AGTGGGCAGC AGGTCTGAGT ACTCTCATAT GATGCTGTGA TTTTCCTGGA
GTTGACAGAA ACACCTATAT TTCCCCCAGT CTTCCCTGGG AGACTACTAT
TAACTGAAAT AAATACTCAG AGCCTGAAAA A
The peptide 946L YLEPGPVTA reconstitutes an A2.1
epitope. Its native encoding gene sequence is TAC CTG GAG
CCT GGC CAA GTC ACT GCC. Because this peptide has proven
immunologic activity, it is ideal for specific immunization.
Such immunization may be accomplished either directly, or by
use of a vaccine consisting of virus (e.g., Vaccinia)
encoding or HLA-A2 cells expressing a genetic sequence
encoding this peptide. =The peptide ALLAVGATK, which
corresponds to residues 17-25 of pMel-17, reconstitutes an A3
epitope.
Also promising is the gene sequence encoding tyrosinase-
related peptide 1030, YMDGTMSQV, natively encoded by TAT ATG
GAT GGA ACA ATG TCC GAG GTA, which reconstitutes an A2-
epitope, and that encoding KCDICTDEY, which reconstitutes an
Al epitope of tyrosinase.
The Genetic Code can readily be used to design a gene
encoding an arbitrary amino acid sequence, such as that of
the preferred HLA-A1 epitope, KCDICTDEY, or the preferred
HLA-A3 epitope, ALLAVGATK. Preferably, where more than one
codon could be used to encode a particular amino acid,
consideration is given to the codon preferences of the
intended host organism.
These sequences may be constructed in such a manner,
including the appropriate expression systems for use in gene
therapy procedures. Because several different nucleotide
sequences may encode a single amino acid, alternate DNA
sequences may also encode these peptides.
Standard reference works setting forth the general
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32
principles of recombinant DNA technology include Watson,
J.D., et al., Molecular Biology of the Gene, Volumes I and
II, The Benjamin/Cummings Publishing Company, Inc.,
publisher, Menlo Park, CA (1987); Darnell, J.E., et al.,
Molecular Cell Biology, Scientific American Books, Inc.,
publisher, New York, N.Y. (1986); Lewin, B.M., Genes II,
John Wiley & Sons, publishers, New York, N.Y. (1985); Old,
R.W., et al., Principles of Gene Manipulation: An
Introduction to Genetic Engineering, 2d edition, University
of California Press, publisher, Berkeley, CA (1981);
Sambrook, J., et al., Molecular Cloning: A Laboratorv
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1989); and Ausubel, et al., Current Protocols in Molecular
Biology, Wiley Interscience, N.Y., (1987, 1992).
-
Chemical Peptide Synthesis. Chemical peptide synthesis
is a rapidly evolving area in the art, and methods of solid
phase peptide, synthesis are well-described in the followinq
references:
Merrifield, B., J. Amer. Chem. Soc. 85:2149-2154 (1963);
Merrifield, B., Science 232:341-347 (1986); Wade, J.D., et
al., Biopolymers 25:S21-S37 (1986); Fields, G.B., Int. J.
Polypeptide Prot. Res. 35:161 (1990); MilliGen Report Nos. 2
and 2a, Millipore Corporation, Bedford, MA, 1987; Ausubel, et
al, supra, and Sambrook, et al, supra.
In general, as is known in the art, such methods involve
blocking or protecting reactive functional groups, such as
free amino, carboxyl and thio groups. After polypeptide bond
formation, the protective groups are removed (or de-protect-
ed). Thus, the addition of each amino acid residue requires
several reaction steps for protecting and deprotecting.
Current methods utilize solid phase synthesis, wherein the
C-terminal amino acid is covalently linked to an insoluble
resin particle large enough to be separated from the fluid
phase by filtration. Thus, reactants are removed by washing
the resin particles with appropriate solvents using an
automated programmed machine. The completed polypeptide
chain is cleaved from the resin by a reaction which does not
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33
affect polypeptide bonds.
In the more classical method, known as the "tBoc
method," the amino group of the amino acid being added to the
resin-bound C-terminal amino acid is blocked with
tert-butyloxycarbonyl chloride (tBoc). This protected amino
acid is reacted with the bound amino acid in the presence of
the condensing agent dicyclohexylcarbodiimide, allowing its
carboxyl group to form a polypeptide bond the free amino
group of the bound amino acid. The amirio-blocking group is
then removed by acidification with trifluoroacetic acid
(TFA); it subsequently decomposes into gaseous carbon dioxide
and isobutylene. These steps are repeated cyclically for
each additional amino acid residue. A more vigorous
treatment with hydrogen fluoride (HF) or trifluoro-
methanesulfonyl derivatives is common at the end of the
synthesis to cleave the benzyl-derived side chain protecting
groups and the polypeptide-resin bond.
More recently, the preferred "Fmoc" technique has been
introduced as an alternative synthetic approach, offering
milder reaction conditions, simpler activation procedures and
compatibility with continuous flow techniques. This method
was used, e.g., to prepare the peptide sequences disclosed in
the present application. Here, the oc-amino group is
protected by the base labile 9-fluorenylmethoxycarbonyl
(Fmoc) group. The benzyl side chain protecting groups are
replaced by the more acid labile t-butyl derivatives.
Repetitive acid treatments are replaced by deprotection with
mild base solutions, e.g., 20a piperidine in dimethyl-
formamide (DMF), and the final HF cleavage treatment is
eliminated. A TFA solution is used instead to cleave side
chain protecting groups and the peptide resin linkage
simultaneously.
At least three different peptide-resin linkage agents
can be used: substituted benzyl alcohol derivatives that can
be cleaved with 95o TFA to produce a peptide acid, methanolic
ammonia to produce a peptide amide, or 1% TFA to produce a
protected peptide which can then be used in fragment
condensation procedures, as described by Atherton, E., et
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34
al., J. Chem. Soc. Perkin Trans. 1:538-546 (1981) and
Sheppard, R.C., et al., Int. J. Polyeptide Prot. Res.
20:451-454 (1982). Furthermore, highly reactive Fmoc amino
acids are available as pentafluorophenyl esters or dihydro-
oxobenzotriazine esters derivatives, saving the step of
activation used in the tBoc method.
Pharmaceutical Methods and Preparations
The preferred animal subject of the present invention is
a primate mammal. By the term "mammal" is meant an
individual belonging to the class Mammalia, which, of course,
includes humans. The invention is particularly useful in the
treatment of human subjects, although it is intended for
veterinary uses as well. By the term "non-human primate" is
intended any member of the suborder Anthropoidea except for
the family Hominidae. Such non-human primates include the
superfamily Ceboidea, family Cebidae (the New World monkeys
including the capuchins, howlers, spider monkeys and squirrel
monkeys) and family Callithricidae (including the marmosets);
the superfamily Cercopithecoidea, family Cercopithecidae
(including the macaques, mandrills, baboons, proboscis
monkeys, mona monkeys, and the sacred hunaman monkeys of
India); and superfamily Hominoidae, family Pongidae
(including gibbons, orangutans, gorillas, and chimpanzees).
The rhesus monkey is one member of the macaques.
The term "protection", as used herein, is intended to
include "prevention," "suppression" and "treatment."
"Prevention" involves administration of the protein prior to
the induction of the disease. "Suppression" involves
administration of the composition prior to the clinical
apipearance of the disease. "Treatment" involves
administration of the protective composition after the
appearance of the disease.
It will be understood that in human and veterinary
medicine, it is not always possible to distinguish between
"preventing" and "suppressing" since the ultimate inductive
event or events may be unknown, latent, or the patient is not
ascertained until well after the occurrence of the event or
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events. Therefore, it is common to use the term
"prophylaxis" as distinct from "treatment" to encompass both
"preventing" and "suppressing" as defined herein. The term
"protection," as used herein, is meant to include
5 "prophylaxis." It should also be understood that to be
useful, the protection provided need not be absolute,
provided that it is sufficient to carry clinical value. An
agent which provides protection to a lesser degree than do
competitive agents may still be of value if the other agents
10 are ineffective for a particular individual, if it can be
used in combination with other agents to enhance the level of
protection, or if it is safer than competitive agents.
The composition may be administered parentally or
orally, and, if parentally, either systemically or topically.
15 Parenteral routes include subcutaneous, intravenous
intradermal, intramuscular, intraperitoneal, intranasal,
transdermal, or buccal routes. One or more such routes may
be employed. Parenteral administration can be, e.g., by
bolus injection or by gradual perfusion over time.
20 Alternatively, or concurrently, administration may be by the
oral route. The immunization is preferably accomplished
initially by intramuscular injection followed by intradermal
injection, although any combination of intradermal and
intramuscular injections may be used.
25 It is understood that the suitable dosage of a immunogen
of the present invention will be dependent upon the age, sex,
health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of
the effect desired. However, the most preferred dosage can
30 be tailored to the individual subject, as is understood and
determinable by one of skill in the art, without undue
experimentation. This will typically involve adjustment of a
standard dose, e.g., reduction of the dose if the patient has
a low body weight.
35 Prior to use in humans, a drug will first be evaluated
for safety and efficacy in laboratory animals. In human
clinical studies, one would begin with a dose expected to be
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safe in humans, based on the preclinical data for the drug in
question, and on customary doses for analogous drugs (if
any). If this dose is effective, the dosage may be
decreased, to determine the minimum effective dose, if
desired. If this dose is ineffective, it will be cautiously
increased, with the patients monitored for signs of side
effects. See, e.g., Berkow, et al.,.eds., The Merck Manual,
15th edition, Merck and Co., Rahway, N.J., 1987; Goodman, et
al., eds., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford,
N.Y., (1990); Avery's Drua Treatment: Principles and Practice
of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS
Press, LTD., Williams and Wilkins, Baltimore, MD. (1987),
Ebadi, Pharmacoloay, Little, Brown and Co., Boston, (1985).
The total dose required for each treatment may be
administered in multiple doses (which may be the same or
different) or in a single dose, according to an immunization
schedule, which may be predetermi-ned or ad hoc. The schedule
is selected so as to be immunologically effective, i.e., so
as to be sufficient to elicit an effective CTL response to
the antigen and thereby, possibly in conjunction with other
agents, to provide protection. The doses adequate to
accomplish this are defined as "therapeutically effective
doses." (Note that a schedule may be immunologically
effective even though an individual dose, if administered by
itself, would not be effective, and the meaning of
"therapeutically effective dose" is best interpreted in the
context of the immunization schedule.) Amounts effective for
this use will depend on, e.g., the peptide composition, the
manner of administration, the stage and severity of the
disease being treated, the weight and general state of health
of the patient, and the judgment of the prescribing
physician, but generally range for the initial immunization
(that is for therapeutic or prophylactic administration) from
about 1.0 g to about 5000 g of peptide for a 70 kg patient,
followed by boosting dosages of from about 1.0 g to about
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37
1000 g of peptide pursuant to a boosting regimen over weeks
to months depending upon the patient's response and condition
by measuring specific CTL activity in the patient's blood.
It must be kept in mind that the peptides and compositions of
the present invention may generally be employed in serious
disease states, that is, life-threatening or potentially life
threatening situations. In such cases, in view of the
minimization of extraneous substances and the relative
nontoxic nature of the peptides, it is possible and may be
felt desirable by the treating physician to administer
substantial excesses of these peptide compositions.
The doses may be given at any intervals which are
effective. If the interval is too short, immunoparalysis or
other adverse effects can occur. If the interval is too
long, immunity may suffer. The optimum interval may be
longer if the individual doses are larger. Typical intervals
are 1 week, 2 weeks, 4 weeks (or one month), 6 weeks, 8 weeks
(or two months) and one year. The appropriateness of
administering additional doses, and of increasing or
decreasing the interval, may be reevaluated on a continuing
basis, in view of the patient's immunocompetence (e.g., the
level of antibodies to melanoma-associated antigens).
The concentration of CTL stimulatory peptides of the
invention in the pharmaceutical formulations can vary widely,
i.e., from less than about 0.10, usually at or at least about
2o to as much as 20o to 50% or more by weight, and will be
selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular mode of administration
selected.
In one embodiment, the immunogen is dissolved or
suspended in an aqueous carrier. A variety of aqueous
carriers may be used, e.g., water, buffered water, 0.9%
saline, 0.3% glycine, hyaluronic acid and the like. These
compositions may be sterilized by conventional, well known
sterilization techniques, or may be sterile filtered. The
resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with
a sterile solution prior to administration. The compositions
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38
may contain pharmaceutically acceptable auxiliary substances
as required to approximate physiological conditions, such as
pH adjusting and buffering agents, tonicity adjusting agents,
wetting agents and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, calcium
chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The peptides of the invention may also be administered
via liposomes, which serve to target the peptides to a
particular tissue, as well as increase the half-life of the
peptide composition. Liposomes include emulsions, foams,
micelles, insoluble monolayers, liquid crystals, phospholipid
dispersions, lamellar layers and the like. In these
preparations the peptide to be delivered is incorporated as
part of a liposome, alone or in conjunction with a molecule
which binds to, e.g., a receptor prevalent among melanocytes
or melanomas, or with other therapeutic or immunogenic
compositions. Thus, liposomes filled with a desired peptide
of the invention can be directed to the site of target cells,
where the liposomes then deliver the selected
therapeutic/immunogenic peptide compositions. Liposomes for
use in the invention are formed from standard vesicle-forming
lipids, which generally include neutral and negatively
charged phospholipids and a sterol, such as cholesterol. The
selection of lipids is generally guided by consideration of,
e.g., liposome size, acid lability and stability of the
liposomes in the blood stream. A variety of methods are
available for preparing liposomes, as described in, e.g.,
Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.
Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019369.
For targeting to the melanoma cells, a ligand to be
incorporated into the liposome can include, e.g., antibodies
or fragments thereof specific for cell surface determinants
of the desired melanoma cells. A liposome suspension
containing a peptide may be administered intravenously,
locally, topically, etc. in a dose which varies according to,
inter alia, the manner of administration, the peptide being
delivered, and the stage of the disease being treated.
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39
For solid compositions, conventional nontoxic solid
carriers may be used which include, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, and the like. For oral
administration, a pharmaceutically acceptable nontoxic
composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously
listed, and generally 10-950 of active ingredient, that is,
one or more peptides of the invention, and more preferably at
a concentration of 25 s-75%.
For aerosol administration, the immunogenic peptides are
preferably supplied in finely divided form along with a
surfactant and propellant. Typical percentages of peptides
are 0.01%-20o by weight, preferably 1%-10%. The surfactant
must, of course, be nontoxic, and preferably soluble in the
propellant. Representative of such agents are the esters or
partial esters of fatty acids containing from 6 to 22 carbon
atoms, such as caproic, octanoic, lauric, palmitic, stearic,
linoleic, linolenic, olesteric and oleic acids with an
aliphatic polyhydric alcohol or its cyclic anhydride. Mixed
esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.10-20o by weight of the
composition, preferably 0.25-50. the balance of the
composition is ordinarily propellant. A carrier can also be
included, as desired, as with, e.g., lecithin for intranasal
delivery.
In addition to the peptides or analogues of the
invention, a pharmaceutical composition may contain suitable
pharmaceutically acceptable carriers, such as excipients,
carriers and/or auxiliaries which facilitate processing of
the active compounds into preparations which can be used
pharmaceutically.
The appropriate dosage form will depend on the disease,
the immunogen, and the mode of administration; possibilities
include tablets, capsules, lozenges, dental pastes,
suppositories, inhalants, solutions, ointments and parenteral
depots. See, e.g., Berker, su ra, Goodman, supra, Avery,
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supra and Ebadi, supra=
However, it is expected that each vaccine preparation will
include 1-100 g of the peptide epitope.
5 The composition may also include an adjuvant. Typical
adjuvants include proteins, peptides, carbohydrates, lipids
and liposaccharides. An example of a currently popular
adjuvant is DETOX (Ribi Immunochemicals)(muramyl dipeptide
and cell wall fragments from Mycobacterium phlei). Other
10 adjuvants include QS-21, MontanideMISA-21, incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide,
alum, DEAE-dextran, saponin, and mineral oil. Montanide ISA-
51 is manufactured by Seppic, Inc. (75 Quai D'Orsay, 75321,
Paris, France). Its composition is manide oleate in mineral
15 oil solution.
QS-21 is manufactured by Cambridge Biotech (365
Plantation Street, Worcester, MA 01605-2376). It is a
triterpene glycoside isolated from the bark of a South
American tree (Quillaja saponaria). The tradename for QS-21
20 is Stimulon". Its molecular formula is C92O46H148, and its
molecular weight is 1,990. Its complete chemical name is
3-0-/3-D-galactopyranosyl- (1->2) - [O-D-xyl_opyranosyl- (1-
>3)]-(3-D-glucuronpyranosyl-quillaic acid 28-0-0-D-
apiofuranosyl- (1->3) -o-D-xylopyranosyl- (1->4) -a-L-
25 rhamnopyranosyl-(1->2)-3-[5-O-a-L-arabinofuranosyl 3,5-
dihydroxy-6-methyloctanoyl]-3,5-dihydroxy-6-
methyloctanoyl]-(.i-D-fucopyranoside.
If desired, the adjuvant may be conjugated to the
epitope and not simply a part of a mixture. See Deres, et
30 al, Nature, 342:561-4 (1989).
The composition may also include an immunomodulator,
especially cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7,
IL-12, Interferon-alpha, Interferon-gamma, Granulocyte
Macrophage Colony Stimulating Factor (GMCSF), Tumor Necrosis
35 Factor (TNF)-alpha, and TNF- beta.
The composition may also include antigen-presenting
cells, such as dendritic cells or macrophages. Preferably,
the APCs are harvested, e.g., from peripheral blood or bone
*Trade-mark
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marrow, conjugated, covalently or noncovalently (e.g., by
pulsing) to the immunogen, e.g., a peptide, and administered
to the patient.
The composition may also include a molecule which
activates or helps in activating CTLs, such as a CD-28
stimulatory molecule (e.g., B7.1, B7.2, or anti-CD28). If
the molecule may be administered in place of the molecule
itself.
CD80 (B7 BB1) is expressed on activated B cells and
dendritic cells. It is a ligand for CD28 and CTLA-4. It has
been found to represent two (partially homologous) proteins,
B7-1 and B7-2. See Ramarathinam, et al. T cell costimulation
by B7/BB1 induces CD8 T-cell-dependent tumor rejection: an
important role of B7/BB1 in the induction, recruitment, and
effector function of antitumor T cells. J.Exp. Med. 1994:
1790: 1205-1214; Freeman et al. Cloning of B7-2: a CTLA-4
counter-receptor that costimulates human T cell
proliferation. Science 1993, 262: 909-911; Li et al.
Costimulation of tumor-reactive CD4+ and CD8+ T lymphocytes
by B7, a natural ligand for CD28, can be used to treat
established mouse melanoma. J. Immunol. 1994, 153: 421-428;
Hodge et al. Admixture of a recombinant vaccinia virus
containing the gene for the costimulator molecule B7 and a
recombinant vaccinia virus containing a tumor-associated
antigen gene results in enhanced specific T-cell responses
antitumor immunity. Cancer Res. 1995, 55: 3598-3603.
A pharmaceutical composition according to the present
invention may further comprise at least one cancer chemo-
therapeutic compound, such as one selected from the group
consisting of an anti-metabolite, a bleomycin peptide
antibiotic, a podophyllin alkaloid, a Vinca alkaloid, an
alkylating agent, an antibiotic, cisplatin, or a nitrosourea.
A pharmaceutical composition according to the present
invention may further or additionally comprise at least one
viral chemotherapeutic compound selected from gamma globulin,
amantadine, guanidine, hydroxybenzimidazole, interferon-ca,
interferon-0, interferon-ry, thiosemicarbarzones, methisazone,
rifampin, ribvirin, a pyrimidine analog, a purine analog,
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42
foscarnet, phosphonoacetic acid, acyclovir, dideoxy-
nucleosides, or ganciclovir. See, e.g., Katzung, supra, and
the references cited therein on pages 798-800 and 680-681,
respectively-
As an alternative to a pharmaceutical composition
comprising the immunogen of the present invention, per se,
the pharmaceutical composition may instead comprise a vector
comprising an expressible gene encoding such an immunogen.
The pharmaceutical composition and method would then be
chosen so that the vector was delivered to suitable cells of
the subject, so that the gene would be expressed and the
immunogen produced in such a manner as to elicit an immune
response. A preferred vector would be a Vaccinia virus, such
as a construct containing a minigene encoding the peptide
946L (YLEPGPVTA), 9461 ((YIEPGPVTA), 1030 (SEQ. iD. N0.:78)
or ALLAVGATK. A gene encoding the protein pMel-17 is also of
some interest. In the case of genes encoding naturally
occurring proteins, or peptide fragments thereof, one may,
but need not, use the DNA sequence which encodes the proteins
or peptides in nature. A preferred route for immunization
would be scarification. A preferred immunization protocol
would be 10E6 to 10E8 pfu/dose in the initial injection,
followed up with boosters at 1,3 and 12 months. The boosters
could be the previously described immunogen-containing
composition.
In the case of genes encoding naturally occurring
proteins, or peptide fragments thereof, one may, but need
not, use the DNA sequence which encodes the proteins or
peptides in nature.
Recombinant vaccinia virus constructs have been used for
immunization against hepatitis B (Moss, et al., Nature, 311,
67, 1984), herpes simplex virus (Wacchsman, et al., Biosci.
Rep. 8, 323; 334, 1988) , parainfluenza type 3 (Spriggs, et
al., J. Virol., 62, 1293, 1988), and Lassa fever virus
(Fisher-Hoch, et al., Proc. Natl. Acad. Sci. USA, 86, 317,
1989). Vaccinia virus constructs comprising gene for cancer-
associated antigens have also been prepared (Lathe, et al.,
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43
Nature, 326, 878, 1987; Bernards, et al., Proc. Natl. Acad.
Sci. USA, 84, 6854, 1987; Estin, et al., Proc. Natl. Acad.
Sci. USA, 85, 1052, 1988).
Alternatively or additionally, the composition may
comprise melanoma-specific CTL. Antigenic peptides may be
used to elicit CTL ex vivo. Ex vivo CTL responses to a
melanoma antigen are induced by incubating in tissue culture
the patient's CTL precursor cells (CTLp) together with a
source of antigen-presenting cells (APC) and the appropriate
immunogenic peptide. After an appropriate incubation time
(typically 1-8 weeks), in which the CTLp are activated and
mature and expand into effector CTL, the cells are infused
back into the patient, where they will destroy their specific
target cell. In order to optimize the in vitro conditions
for the generation of specific cytotoxic T cells, the culture
of stimulator cells may be maintained in an appropriate
serum-free medium.
Prior to incubation of the stimulator cells with the
cells to be activated, e.g., precursor CD8+ cells, an amount
of antigenic peptide is added to the stimulator cell culture,
of sufficient quantity to become loaded onto the human Class
I molecules to be expressed on the surface of the stimulator
cells. In the present invention, a sufficient amount of
peptide is an amount that will allow about 200, and
preferably 200 or more, human Class I MHC molecules loaded
with peptide to be expressed on the surface of each
stimulator cell. Preferably, the stimulator cells are
incubated with at least 1 mg/ml, more preferably >20 g/ml
peptide.
Resting or precursor CD8+ cells are then incubated in
culture with the appropriate stimulator cells for a time
period sufficient to activate the CD8+ cells. Preferably,
the CD8+ cells are activated in an antigen-specific manner.
The ratio of resting or precursor CD8+ (effector) cells to
stimulator cells may vary from individual to individual and
may further depend upon variables such as the amenability of
an individual's lymphocytes to culturing conditions and the
nature and severity of the disease condition or other
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44
condition for which the within-described treatment modality
is used. Preferably, however, the lymphocyte:stimulator cell
ratio is in the range of about 1:5 to 20:1, more preferably
3:1 to 5:1. The effector/stimulator culture may be
maintained for as long a time as is necessary to stimulate a
therapeutically useful or effective number of CD8+ cells.
The induction of CTL in vitro requires the specific
recognition of peptides that are bound to allele specific MHC
class I molecules on APC. The number of specific MHC/peptide
complexes per APC is crucial for the stimulation of CTL,
particularly in primary immune responses. While small
amounts of peptide/MHC complexes per cell are sufficient to
render a cell susceptible to lysis by CTL, or to stimulate a
secondary CTL response, the successful activation of a CTL
precursor (pCTL) during primary response requires a
significantly higher number of MHC/peptide complexes.
Peptide loading of empty major histocompatability complex
molecules on cells allows the induction of primary cytotoxic
T lymphocyte responses.
Often it is useful, in the generation of peptide-
specific CTL, to stimulate with mutant cell lines that have
some empty MHC molecules. An exmample is the human lymphoid
cell line, T2. However, mutant cell lines expressing every
MHC molecule are not yet available. Thus, in some cases, it
may be useful to strip endogenous MHC-associated peptides
from the surface of APC, followed by loading the resulting
empty MHC molecules with the immunogenic peptides of
interest. The use of non-transformed (non-tumorigenic), non-
infected cells, and preferably, autologous cells of patients
as APC is desirable for the design of CTL induction protocols
directed towards development of ex vivo CTL therapies. This
application discloses methods for stripping the endogenous
MHC-associated peptides from the surface of APC followed by
the loading of desired peptides.
A stable MHC class I molecule is a trimeric complex
formed of the following elements: 1) a peptide usually of
8 - 10 residues, 2) a transmembrane heavy polymorphic protein
chain which bears the peptide-binding site in its al and u2
CA 02249390 1998-09-18
WO 97/34613 PCT/US97/04958
domains, and 3) a non-covalently associated non-polymorphic
light chain, (3Zmicroglobulin. Removing the bound peptides
and/or dissociating the 02microglobulin from the complex
renders the MHC class I molecules nonfunctional and unstable,
5 resulting in rapid degradation. All MHC class I molecules
isolated from PBMCs have endogenous peptides bound to them.
Therefore, the first step is to remove all endogenous
peptides bound to MHC class I molecules on the APC without
causing their degradation before exogenous peptides can be
10 added to them.
Two possible ways to free up MHC class I molecules of
bound peptides include the culture temperature from 37 C to
26 C overnight to destabilize ,(3Zmicroglobulin and stripping
the endogenous peptides from the cell using a mild acid
15 treatment. The methods release previously bound peptides
into the extracellular environment allowing new exogenous
peptides to bind to the empty class I molecules. The cold-
temperature incubation method enables exogenous peptides to
bind efficiently to the MHC complex, but requires an
20 overnight incubation at 26 C which may slow the cell's
metabolic rate. It is also likely that cells not actively
synthesizing MHC molecules (e.g., resting PBMC) would not
produce high amounts of empty surface MHC molecules by the
cold temperature procedure.
25 Harsh acid stripping involves extraction of the peptides
with trifluoroacetic acid, pH 2, or acid denaturation of the
immunoaffinity purified class I-peptide complexes. These
methods are not feasible for CTL induction, since it is
important to remove the endogenous peptides while preserving
30 APC viability and an optimal metabolic state which is
critical for antigen presentation. Mild acid solutions of pH
3 such as glycine or citrate-phosphate buffers have been used
to identify endogenous peptides and to identify tumor
associated T cell epitopes. The treatment is especially
35 effective, in that only the MHC class I molecules are
destabilized (and associated peptides released), while other
surface antigens remain intact, including MHC class II
molecules. Most importantly, treatment of cells with the
CA 02249390 1998-09-18
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46
mild acid solutions do not affect the cell's viability or
metabolic state. The mild acid treatment is rapid since the
stripping of the endogenous peptides occurs in two minutes at
4 C and the APC is ready to perform its function after the
appropriate peptides are loaded. The technique is utilized
herein to make peptide-specific APCs for the generation of
primary antigen-specific CTL. The resulting APC are
efficient in inducing peptide-specific CD8+ CTL.
Activated CD8+ cells may be effectively separated from
the stimulator cells using one of a variety of known methods.
For example, monoclonal antibodies specific for the
stimulator cells, for the peptides loaded onto the stimulator
cells, or for the CD8+ cells (or a segment thereof) may be
utilized to bind their appropriate complementary ligand.
Antibody-tagged molecules may then be extracted from the
stimulator-effector cell admixture via appropriate means,
e.g., via well-known immunoprecipitation or immunoassay
methods.
Effective, cytotoxic amounts of the activated CDB+ cells
can vary between in vitro and in vivo uses, as well as with
the amount and type of cells that are the ultimate target of
these killer cells. The amount will also vary depending on
the condition of the patient and should be determined via
consideration of all appropriate factors by the practitioner.
Preferably, however, about 1 X 106 to about 1 X 1012, more
preferably about 1 X 108 to about 1 X 1011, and even more
preferably, about 1 X 109 to about 1 X 1010 activated CD8+
cells are utilized for adult humans, compared to about 5 X 106
- 5 X 10' cells are used in mice.
Preferably, as discussed above, the activated CD8+ ce?,-
are harvested from the cell culture prior to administratior.
of the CD8+ cells to the individual being treated. It is
important to note, however, that unlike other present and
proposed treatment modalities, the present method preferably
uses a cell culture system that is not tumorigenic.
Therefore, if complete separation of stimulator cells and
activated CD8+ cells is not achieved, there is no inherent
danger known to be associated with the administration of a
CA 02249390 1998-09-18
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47
small number of stimulator cells, whereas administration of
mammalian tumor-promoting cells may be extremely hazardous.
Methods of re-introducing cellular components are known
in the art and include procedures such as those exemplified
in U.S. Patent No. 4,844,893 to Honsik, et al. and U.S.
Patent No. 4,690,915 to Rosenberg. For example,
administration of activated CD8+ cells via intravenous
infusion is appropriate.
Adoptive transfer of melanoma-specific CTL has been
accompanied by tumor shrinkage in a large minority of
patients with advanced melanoma and by disappearance of all
detectable tumor in a smaller proportion of patients.
(Rosenberg et al, NEM 319: 1676-1680, 1988) and in animal
studies appears to be particularly promising for the treat-
ment of solid tumors (Rosenberg SA et al. Science 233:1318-
1321). One of the problems with existing methods for CTL
generation is that they require the resection of large
metastic tumor deposits to initiate the process. If the
requirement for available autologous tumor could be
circumvented, then patients with no measurable disease but a
high risk of recurrence (eg, melanoma patients with primary
tumors greater than 4 mm thick or with microscopic tumor
metastatic to regional nodes) could be treated with adoptive
therapy even if their tumor were removed and fixed in
formalin and no other gross tumor was evident. These
patients have a very high likelihood of harboring
micrometastic disease for which no other effective therapy is
now available; so most will die of the melanoma. It is
possible that the presence of bulky tumor suppresses the
autologous immune response; so treatment of patients without
bulky disease would be an attractive goal. Especially in
murine systems, CTL have been generated and maintained by
stimulation with cells to which the peptide epitope has been
bound. We propose that, e.g., HLA-A2.1+ or HLA-A3+ cells
(autologous B cells, macrophages, or dendritic cells,
ideally), would be pulsed in vitro with peptide (e.g.,
peptide 946, YXEPGPVTA) and used as in vitro simulators for
autologous lymph node cells or peripheral blood lymphocytes.
CA 02249390 1998-09-18
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48
The patients could be pre-stimulated with a peptide vaccine
prior lymphocyte harvest if the existing response was
inadequate. Lymphocytes stimulated with peptide in vitro
could then be expanded to 1010 or 1011 cells, then re-infused
into the patients in a manner analogous to that used for LAK
cell therapy. It is expected that the adoptively transferred
CTL would survive best with IL-2 infusion at low to
intermediate doses, and that putative inhibitors of Tc
suppression (eg: cyclophosphamide) may be employed also,
prior to the infusions of CTL.
Clinical studies with adoptive immunotherapy using A2-
restricted tumor infiltrating lymphocytes (TIL) have shown a
strong correlation between Pmel-17/gplOO reactivity and
positive clinical responses of patients treated with those
TIL. Kawakami, et al., J. Immunol., 154:3961-8 (1995).
Melanoma-Specific Diagnostic Agents
A melanoma-specific diagnostic agent is (1) a molecule
which is or which comprises a melanoma-specific epitope as
previously defined, and which is labeled, immobilized, or
otherwise rendered suitable for diagnostic use, or (2) an
antibody which specifically binds such a melanoma-specific
epitope, and which is labeled, immobilized, or otherwise
rendered suitable for diagnostic use, or (3) a T-cell line
(e.g., murine or human), which specifically recognizes a
melanoma-specific epitope.
Diagnostic Uses and Compositions
The relationship between the host's immune response and
his or her tumor is poorly understood. Better understanding
of that response depends on evaluation of the specific
responses against individual epitopes, such as the 946
peptide. If patients do have an immune response to 946
naturally, then evaluation and quantitation of that by
precursor frequency analysis of the CTL in the patient's
blood pool may permit some assessment of the protection that
person's immune system is providing. As new therapies become
available for melanoma, it may be useful to screen patients
CA 02249390 1998-09-18
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49
for the presence of the 946 peptide on their tumor and the
presence of CTL in their blood pool with specificity for the
946 peptide on HLA-A2. In like manner one may screen for
ALLAVGATK peptides on the tumor and for anti-ALLAVGATK CTLs
in the blood of A3+ patients. These findings may determine
whether further augmentation of the immune response is
indicated or whether other, non-immunologic, therapy should
be employed. A parallel to this is the determination on
breast cancers of the presence of estrogen and progesterone
receptors before considering hormonal therapy or
chemotherapy.
Thus, the peptides of the present invention may be used
to screen a sample for the presence of an antigen with the
same epitope, or with a different but cross-reactive epitope,
or for the presence of CTLs which specifically recognize the
corresponding epitopes. The sample will normally be a
biological fluid, such as blood, urine, lymphatic fluid,
amniotic fluid, semen, saliva, tears, milk, or cerebrospinal
fluid, or a fraction or derivative thereof, or a=biological
tissue, in the form of, e.g., a tissue section or homogenate.
The preferred sample is blood, or a fraction or derivative
thereof.
Assays may be divided into two basic types, hetero-
geneous and homogeneous. In heterogeneous assays, the
interaction between the affinity molecule and the analyte
does not affect the label, hence, to determine the amount or
presence of analyte, bound label must be separated from free
label. In homogeneous assays, the interaction does affect
the activity of the label, and therefore analyte levels can
be deduced without the need for a separation step.
Assays may also be divided into competitive and non-
competitive formats. In the competitive format, the analyte
competes with a labeled analyte analogue for binding to a
binding partner. In a common noncompetitive format called a
sandwich assay, the analyte is first bound by a capture
reagent, and then by a tag reagent.
In order to detect the presence, or measure the amount,
of an analyte, the assay must provide for a signal producing
CA 02249390 1998-09-18
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system (SPS) in which there is a detectable difference in the
signal produced, depending on whether the analyte is present
or absent (or, in a quantitative assay, on the amount of the
analyte). The detectable signal may be one which is visually
5 detectable, or one detectable only with instruments.
Possible signals include production of colored or luminescent
products, alteration of the characteristics (including
amplitude or polarization) of absorption or emission of
radiation by an assay component or product, and precipitation
10 or agglutination of a component or product. The term
"signal" is intended to include the discontinuance of an
existing signal, or a change in the rate of change of an
observable parameter, rather than a change in its absolute
value. The signal may be monitored manually or automatically.
15 The component of the signal producing system which is
most intimately associated with the diagnostic reagent is
called the "label". A label may be, e.g., a radioisotope, a
fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an
electron-dense compound, an agglutinable particle.
20 The radioactive isotope can be detected by such means as
the use of a gamma counter or a scintillation counter or by
autoradiography. Isotopes which are particularly useful for
the purpose of the present invention are 3H, 1251, 1311, 35S , 14C,
and, preferably, 1251 .
25 It is also possible to label a compound with a
fluorescent compound. When the fluorescently labeled anti-
body is exposed to light of the proper wave length, its
presence can then be detected due to fluorescence. Among the
most commonly used fluorescent labelling compounds are
30 fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
Alternatively, fluorescence-emitting metals such as 125Eu,
or others of the lanthanide series, may be attached to the
35 binding protein using such metal chelating groups as diethyl-
enetriaminepentaacetic acid (DTPA) and ethylenediamine-
tetraacetic acid (EDTA).
The peptides also can be detectably labeled by coupling
CA 02249390 1998-09-18
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51
to a chemiluminescent compound. The presence of the chem-
iluminescently labeled antibody is then determined by
detecting the presence of luminescence that arises during the
course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds are luminol,
isolumino, theromatic acridinium ester, imidazole, acridinium
salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label
the peptides. Bioluminescence is a type of chemiluminescence
found in biological systems in which a catalytic protein
increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by
detecting the presence of luminescence. Important bio-
luminescent compounds for purposes of labeling are luciferin,
luciferase and aequorin.
Enzyme labels, such as horseradish peroxidase, alkaline
phosphatase, malate dehydrogenase, staphylococcal nuclease,
6-V-steroid isomerase, yeast alcohol dehydrogenase, cx-glycero
phosphate dehydrogenase, triose phosphate isomerase,
asparaginase, glucose oxidase, 0-galactosidase, ribonuclease,
glucose-6-phosphate dehydrogenase, glucoamylase and acetyl-
choline esterase, are preferred. When an enzyme label is
used, the signal producing system must also include a
substrate for the enzyme. If the enzymatic reaction product
is not itself detectable, the SPS will include one or more
additional reactants so that a detectable product appears.
A label may be conjugated, directly or indirectly (e.g.,
through a labeled antibody), covalently (e.g., with SPDP) or
noncovalently, to the peptide, to produce a diagnostic
reagent. Similarly, the peptide may be conjugated to a solid
phase support to form a solid phase ("capture") diagnostic
reagent. Suitable supports include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases,
natural and modified celluloses, polyacrylamides, agaroses,
and magnetite. The nature of the carrier can be either
soluble to some extent or insoluble for the purposes of the
present invention. The support material may have virtually
any possible structural configuration so long as the coupled
CA 02249390 1998-09-18
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52
molecule is capable of binding to its target. Thus the
support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be
flat such as a sheet, test strip, etc.
Additionally, the peptides may be used as a diagnostic
tool to evaluate whether other immunotherapeutic treatments
(tumor vaccines of any kind, adoptive transfer of CTL, etc)
are having a beneficial effect.
Also the peptides 946L (YLEPGPVTA) and 9461 (YIEPGPVTA)
have low to intermediate affinity for the HLA-A2.1 molecule.
This is illustrated in Figure 11. For this reason, they will
be useful as control peptides for the evaluation of candidate
peptide/MHC binding affinity. Because they represent a low
affinity range, they can be used in laboratory studies on
binding affinity of other peptides. This methodology, in a
preferred embodiment, would likely include: binding the
peptide to T2 cells, then evaluating lysis of the T2 cells by
any of various standard methods, such as a proliferative
response of the CTL, or cytokine release by the CTL exposed
to the T2 cells+ peptide.
Fibroblasts GM126 were obtained from the National
Institute of General Medical Sciences Human Genetic Mutant
Cell Repository, Bethesa, MD. Melanoma lines DM6, DM13,
DM14, and DM93 were the gift of Drs. Hilliard F. Siegler and
Timothy L. Darrow. VMM1 and VMM5 are melanoma cell lines
established from metastatic melanoma resected from patients
at the University of Virginia (Charlottesville, VA). VBT2
(squamous cell lung carcinoma), VAO1 (adenocarcinoma of the
ovary), and VAB5 (adenocarcinoma of the breast) are cell
lines also established at this institution. JY, MICH, MWF,
23.1, RPMI 1788, and Herluff are EBV-transformed B lympho-
blastoid lines. K562 is a NK-sensitive human erythroleukemia
line. The cell line T2 is derived from the fusion of a T
cell line, CEM, and a human B cell mutant, LCL 721.174. This
cell line expresses HLA-A2.1 molecules but has an Ag-
processing defect that is associated with enhanced presenta-
tion of exogenous peptides.
CA 02249390 1998-09-18
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53
HLA Tvpes of Cell Lines
The HLA types of several cell lines are listed in Table
1.
CA 02249390 1998-09-18
WO 97/34613 PCT/US97/04958
54
~a
U +
õ~.~ N + + I + + r r r r r r r r r r r r
q~ + r r + r r r i r r r r r r r r r
~
a
a
r r r r r r r r r r r r r r r r r r r
a r ~ r N r r r r r r r r r r r r r r r r
U ~ 1 I 1 I 1 1 N 1 1 I I 1 1 1 I I I r 1
Ln
?D
m Q rn
~+ a
0 Ln n
o = i .M r r r r r r r , . , ,
y ~ r CV r ~ V' sT r (~ r11 r r r i r r r r t!1 r V' 1 67
~
J.1
-~1 U
A ~ N ~ r r C r r r r r
C' q . q , 0 n. r r r
m r-I 'I. N 2 r Z C r +=1 N r t r ~ r r r r
V M r~1 ~ r r r i r i
m
.'S
m
N
(. M
~ ro a $1, tD 'D . .
La 3 3 .,
n=
m 0
a
a M f0 . ' rn
co r=+ H CN rLni r ~ rn c~
aD =V~ N ~ r M N
~
N M f='1 ri Ol U9 r O I~ . r i r U1 N [:
-7 H rl tfl m H rl rl t!1 M M 1 l0 N O7 r I r H ll: I- H N
~
y a ~'
N
a r-I rn
=~ M N f'1 rl rl W 0 M M N N
,[ (V C t0 to l11 M
11 ; N N N N N . . .
H ' rl ri .i r e-I . rl '=t rl N
~ = = ri . . . . . . V' . ~ M . . . , . . . . .
N N ri N '=1 ri r-i M N M N N N N N N r N N N N N
m
~ E N x
~ r, 'ro ro m ro ro ~c co ro rt v u u Q ~ ~ ~
E E E E E E E E E FC G U at ~ 0
y O 0 0 0 0 0 0 0 0
U n5 tj u Li 0 },
m U ro rf rt3 c~p ~ ~ ~ r: r~ ==i rn vI C 0 0 .C to to to ~a to
m m ro rt ro ti s~ rs ro 0 v ~, õ
ri ri ri r--1 .-i ri ,~ rl ,--r C r3 Cl 0) r-I Lr
~ a1 aJ N N A1 N a1 Cl W 7 > }=, sa 0 L7 ~ ~.j CQ m m
b ~~~~~ E E~~ a o m m U o c w w w w w w
V-N
C1 ~p MO N N I~
=~ N ... 1 ~ _ N
lD N .-. 0
a ~-. r-i ei ri v ~ m
ri r1 rl ~=-I N Q CD v ON N N W
(1~ N G~ =J
ri r-1 O~ cN 'V= r-1 lf~ N .-1 ln N .Q N N
F O Gq ~ .~ M .1 LO
A A A A ux7 x x ~ ~ > > > ~ U ~ z ~ H S~ =
x cMV
SUBSTITUTE SHEET (RULE 26)
CA 02249390 2006-11-14
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REFERENCE EXAMPLE
The identification of melanoma-specific HLA-A2 epitopes
of pMel-17 and tyrosinase is described in W095/22561,
incorporated by reference herein.
5 EXAMPLE 1
In the present example, we demonstrate that HLA-A3-
restricted CTL recognize shared antigens on autologous and
allogeneic melanoma cells, including an HLA-A3-restricted
peptide derived from Pmel-17/gplOO and one or more peptides
10 not yet identified, but apparently not derived from Pmel-
17/gplOO. These results support the use of Pmel-17/gplOO-
directed immunotherapy for patients who are HLA-A3+, and
suggest that HLA-A3, like HLA-A2, presents multiple shared
melanoma antigens to HLA-A3 restricted CTL.
15 Materials and Methods
Cell lines and HLA typing: The human melanoma cell lines
VMM1, VMM12, VMM18 and VMM34 were derived from patients at
the University of Virginia (Charlottesville, VA). DM6 was
provided by Drs. H.F. Seigler and T.L. Darrow at Duke
20 University (Durham, NC). SkMel-2 was obtained from the
American Type Culture Collection (ATCC, Bethesda, MD).
Immunohistochemical staining of these cell lines with S-100,
HMB-45 and vimentin antibodies was characteristic of
melanoma, while staining for epithelial membrane antigen and
25 cytokeratin was negative (data not shown). The CV-1 and 143B
TK" lines used in the propagation of vaccinia virus were also
obtained from the ATCC. VMM12-EBV is an Epstein-Barr virus
transformed B cell line derived from peripheral blood
mononuclear cells (PBMC) of melanoma patient VMM12. Briefly,
30 the PBMC were incubated with filtered supernatant from the
EBV producing cell line B-958 for 1 h at 37;C, followed by
culture in RPMI 1640 media with 10o fetal calf serum (FCS)
and antibiotics, plus a 1:100 dilution of PHA. K562 is an NK-
sensitive human erythroleukemia line. T2-A3 (an HLA-A3
35 transfectant of the antigen-processing-defective mutant human
lymphoid cell line, T2) was provided by P. Cresswell. HLA
typing was performed by microcytotoxicity assay on autologous
CA 02249390 2006-11-14
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56
lymphocytes (Gentrak). Expression of HLA-A3 by tumor cells
was confirmed by staining with the monoclonal antibody (MAb)
GAP-A3 provided by P. Cresswell.
Production of recombinant vaccinia virus expressing human
Pmel-17
The full-length Pmel-17 cDNA was sub-cloned from pcDNAI/neo
(Invitrogen) into a modified pSC11 vector adjacent to the
vaccinia P7.5 early/late promoter using standard recombinant
DNA methods. Standard dideoxy sequencing was used to confirm
insertion and orientation. A recombinant vacc'inia virus
expressing the protein encoded by this gene (vac-Pmel-17) was
generated using published methods. Briefly, CV-1 cells were
infected with the parental WR strain of vaccinia virus and
transfected (Lipofectin*, Gibco-BRL) with the pSC11.3-Pmel-17
plasmid. Thymidine-kinase negative recombinants were
amplified in 143B TK" cells in the presence of
bromodeoxyuridine (Sigma, St Louis, MO). Recombinants with
beta-galactosidase activity were isolated and cloned through
several rounds of plaque purification. Large-scale stocks
were produced, sucrose purified, and titered in CV-1 cells.
Generation of melanoma-specific cytotoxic T cells: CTL were
generated following the detailed protocols previously
reported. Malignant melanoma was resected from lymph nodes of
patient VMM18. Nodes were mechanically dissociated and
enzymatically digested in Eagle's MEM (GIBCO, Grand Island,
NY) containing 2.501 FCS, 0.1o collagenase B (Boehringer
Mannheim), 0.002o DNAase (Sigma), penicillin 100 U/ml,
streptomycin 100 ug/ml (Pen-Strept, GIBCO) at room
temperature. T cell lines were established from the mixture
of lymphocytes and tumor obtained from the digests, using a
ratio of tumor cells to lymphocytes of 1:1. Cells were
cultured in 24-well tissue culture plates (Linbro, Hamden,
CT) in RPMI 1640 (Sigma) containing 10o FCS, Pen-Strept, and
20 U/ml rIL-2 (Cetus, Emeryville, CA) and were maintained by
repeated stimulation with irradiated (10 Gy) fresh
cryopreserved autologous tumor cells or with the autologous
tumor cell line at a tumor to lymphocyte ratio of 1:10 every
10-12 days. T cell specificity for autologous melanoma was
*Trade-mark
CA 02249390 1998-09-18
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57
confirmed after 28 days of culture. Melanoma specific T
lymphocytes were then expanded by a modification of methods
by E. Goulmy (personal communication), by mixing 1 x 106
specific T-cells with 5 x 106 irradiated (10 Gy) autologous
melanoma stimulators and 10 x 106 irradiated (10 Gy) allogenic
PBL feeders (pooled from at least three donors). The cells
were cultured at 37 C in 80mis RPMI 1640 containing 10% FCS,
Pen-Strept, and 20 U/ml rIL-2 in the edge of an upright T-75
flask (Falcon) , set at a 45 angle. After five days 40ml fresh
culture medium was added to the flask which was then placed
upright for a further two days. T lymphocytes were harvested
and cryopreserved in 2 x 106 aliquots in 90o FCS/10% DMSO for
use in cytotoxic T cell assays. This method was found to
permit significant expansion of T-cell numbers without
changing the specificity of the CTL line (data not shown). T
cells were evaluated by flow cytometry after staining with
fluorescein- or phycoerythrein-conjugated antibodies to CD3,
CD4, CD8 and CD16 (GenTrak Inc., Plymouth Meeting, PA. and
Olympus Corp, Lake Success, NY). Multiple CD8+ VMM18 CTL
lines were generated following this protocol with consistent
results from each. Similar methods were used for generation
of CTL lines from other patients, such as VMM12.
Cytotoxicity assays: Cell mediated lysis of target cells was
determined using a standard 4 h 51Cr-release assay. Briefly,
51Cr-labeled target cells were plated at 2x103 cells/well in
triplicate on 96-well V-bottom plates (Costar, Cambridge, MA)
with indicated ratio of effector cells in a final volume of
200 microliters. Wells containing either culture medium or 1M
HC1 in place of the effector cells served as spontaneous and
maximum 5kCr-release controls, respectively. Plates were
centrifuged at 100 x g for 3 min and incubated at 37 C for 4
h, after which 150 microliters of supernatant from each well
was counted on a gamma counter (ICN). The percent specific
lysis was calculated using the equation: [(experimental
release - spontaneous release) / (maximum release -
spontaneous release)] x 100. Vaccinia infected targets were
generated by infecting cells with 50 pfu/cell of appropriate
recombinant vaccinia virus at 37 C for 5 h, prior to 51Cr-
CA 02249390 2006-11-14
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58
labeling. Antibody blocking assays were performed by
incubating 51Cr-labeled target cells with affinity purified
monoclonal antibodies (MAb) for 1 h at 37 C, prior to
incubation with effector CTL. The MAbs used included W6/32,
specific for a monomorphic determinant on all human class I
MHC molecules; L243, specific for a determinant on human DR
molecules; and GAP-A3, specific for HLA-A3. For cold target
inhibition assays, CTLwere incubated with unlabeled (cold)
target cells for 1 h at 37 C, prior to addition of 51Cr-labeled
(hot) targets.
Reconstitution with synthetic peptides: Peptide sequences
were selected from the reported human sequence of Pmel-
17/gplOO based on predicted HLA-A3 binding motifs. These
peptides were synthesized by standard Fmoc chemistry using a
Gilson model AMS422 peptide synthesizer. Peptides were
reconstituted in CTL assay medium (RPMI 1640, 1001 FCS,
antibiotics) and pre-incubated for 2 h with 2x103 s1Cr labeled
target cells in 100 microliters/well in 96-well plates.
Effector cells were added in 100 microliters assay medium for
a final effector to target (E:T) ratio of 20:1 and the
remainder of the assay was performed as in standard chromium
release assays described above. Wells containing peptide and
target cells but no CTL were used as controls to rule out
toxicity of the peptides themselves. Initial experiments were
performed with unpurified synthetic peptides. Biologically
active peptides identified at initial screening were then
purified to >98% by reversed-phase HPLC on a Vydac*C-4 column
with 0.05o trifluoroacetic acid:water and an acetonitrile
gradient, then re-evaluated in CTL assays.
Isolation of naturally processed HLA-A3 associated peptides.
HLA-A3-associated peptides were acid eluted from HLA-A3
molecules affinity-purified from melanoma cells, as
previously described for A2-associated peptides=. Briefly,
VMM18 melanoma cells cultured in cell factories (Nunc,
Naperville, IL), were washed three times in cold PBS,
pelleted, then snap-frozen. Cell pellets were detergent
solubilized in 1% CHAPS, 174 mg/ml PMSF, 5 mg/ml aprotinin,
10 mg/ml leupeptin, 16 mg/mi pepstatin A, 33 mg/ml
*Trade-mark
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iodoacetamide, 0.2a sodium azide and 0.03 mg/ml EDTA for 1 h
at 4 C. After centrifugation at 100,000 x g for 1 h at 4 C,
the pellet of insoluble proteins was discarded, and the
supernatant was filtered (0.2 um), then passed over a protein
A-Sepharose*column precoated with MAb GAP-A3. HLA-A3'
molecules and associated peptides, bound to GAP-A3, were then
eluted with 0.2 N acetic acid, pH 2.7, then peptides were
dissociated at pH 2.1 by bringing the solution to 10o acetic
acid followed by boiling for 5 min. Finally, peptides were
centrifuged through Ultrafree-CL 5000 -KDa filters
(Millipore, Bedford, MA) at 2500 x g for 5 h. Filtrates
containing purified peptides were concentrated using vacuum
centrifugation and stored at -80 C.
HPLC fractionation and co-elution of naturally processed and
synthetic peptides: Extracted HLA-A3 associated peptides were
fractionated by reversed-phase HPLC on a Brownlee narrowbore
C-18 Aquapore column (2.1 mm x 3cm, A, 7mm) and eluted with a
40-minute gradient of 0 to 600 (v/v) acetonitrile/0.085o TFA
in 0.1o TFA. Fractions were collected at 1 minute intervals.
A synthetic peptide, ALLAVGATK, was eluted under identical
conditions to identify its elution point.
Peptide identification and sequencing by mass spectrometry:
Isolated peptides were loaded onto a C18 microcapillary
column (75m i.d. x 12 cm) and gradient-eluted using
acetonitrile and 0.1M acetic acid, with the concentration of
acetonitrile increasing at 25k/min, into a Finnigan-MAT TSQ-
7000 (San Jose, California) triple quadrupole mass
spectrometer equipped with an electrospray ion source. For
mass spectrometric peptide sequencing, collision activated
dissociation (CAD) mass spectra were recorded for m/z 423.
Resul ts
HLA-A3 restricted melanoma specific human CTL recognize one
or more commonly expressed antigens
Cytotoxic T lymphocyte (CTL) lines were generated by
repeated co-culture of lymphocytes, originally harvested from
a tumor involved lymph node, with fresh or cultured
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autologous melanoma cells from patient VMM18 in the presence
of rIL-2 as described. Several CD3+, CD8+, CD4" CTL lines were
derived, which lysed autologous tumor, whereas there was
minimal lysis of the NK target K562, an allogeneic HLA-A3+
5 EBV-transformed B cell line (VMM12-EBV) or the HLA-A3-
melanoma DM6 (Fig.lA). Lysis of autologous tumor was MHC-
class I restricted, based on inhibition with W6/32, a MAb
specific for human class I molecules, but not L243, a MAb
specific for a determinant on human DR molecules (Fig.1B).
10 Furthermore, inhibition observed with GAP-A3, a MAb
recognizing HLA-A3, demonstrates that the VMM18 CTL recognize
one or more peptides presented by HLA-A3 on the surface of
the autologous melanoma cells.
VMM18 CTL lysed several other HLA-A3 matched allogeneic
15 melanomas: VMM1, VMM12, DM122, and SkMel-2, indicating that
one or more shared epitope(s) are presented on the surface of
multiple HLA-A3+ melanomas (Table 101). In cold target
inhibition assays, lysis of allogeneic HLA-A3 matched
melanoma cells by VMM18 CTL was inhibited by unlabeled (cold)
20 autologous melanoma cells (VMM18), but not by HLA-A3- melanoma
cells (DM6) (Fig. 2). This confirms the existence of shared
epitopes restricted by HLA-A3. Lysis of HLA-A3+ non-melanoma
cells such as the squamous lung cancer cell line SkMes-1 and
the lymphoblastoid cell line VMM12-EBV was not observed
25 (Table 101), indicating that these epitopes may be derived
from one or more melanoma-specific proteins.
Identification of an HLA-A3 restricted peptide from the
melanocyte differentiation antigen Pmel-17/gp100
It has been observed that expression by melanoma cells
30 of the melanocyte differentiation antigen Pmel-17 correlates
with recognition by HLA-A2 restricted melanoma specific CTL.
All of the HLA-A3+ melanoma lines recognized by VMM18 CTL
express Pmel-17, as determined by immunohistochemical
staining with antibodies HMB-45 and NKI-beteb. Significantly,
35 VMM34 melanoma cells which are also HLA-A3+ but negative for
Pmel-17 expression, were not recognized by VMM18 CTL.
To determine whether Pmel-17 encodes an epitope
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recognized by HLA-A3 restricted CTL, a recombinant vaccinia
virus (vac-Pmel-17) expressing the full-length protein
encoded by the Pmel-17 cDNA was constructed. Expression of
Pmel-17 by the recombinant vaccinia was confirmed by
infecting C1R-A2, an HLA-A2+ non-melanoma cell line, with
vac-Pmel-17 or an irrelevant recombinant vaccinia encoding
the influenza nucleoprotein, NP (vac-NP). Only the vac-Pmel-
17 infected cells were lysed by VMMS CTL, previously
demonstrated to recognize an HLA-A2 restricted peptide
derived from this antigen (data not shown). When HLA-A3+
VMM12-EBV cells were infected with vac-Pmel-17, they were
lysed by VMM18 CTL. Whereas uninfected VMM12-EBV cells, and
cells infected with a control recombinant vaccinia virus
(vac-NP), were not recognized (Fig.3). Therefore, expression
of Pmel-17/gp100 by VMM12-EBV cells made these cells targets
for lysis by VMM18 CTL, suggesting that the CTL recognized a
peptide derived from Pmel-17/gpl00 and presented by HLA-A3.
Thirty-four peptides from Pmel-17/gpl00 were synthesized
on the basis of peptide binding motifs for HLA-A3. These
peptides were screened for their ability to sensitize
allogeneic HLA-A3+ non-melanoma cells for lysis by VMM18 CTL.
Two of these peptides, the nonamer ALLAVGATK and its amino
terminal truncated octamer LLAVGATK, sensitized VMM12-EBV for
lysis by VMM18 CTL (Table 102). The relative ability of these
peptides to sensitize targets for lysis was determined in a
titration assay using T2-A3, the non-melanoma HLA-A3
transfectant of the antigen processing defective mutant cell
line T2. Half maximal lysis was induced with 1-10 nM and > 1
uM of peptides ALLAVGATK and LLAVGATK respectively, while
recognition of the HLA-A3 binding peptide QVPLRPMTYK, derived
from the HIV Nef protein was not observed (Fig. 4). The
nonamer peptide was able to sensitize targets for VMM18 CTL
recognition at a significantly lower concentration than the
octamer, suggesting that it is more likely to be the
naturally processed peptide to which the CTL were primed.
The nonamer peptide ALLAVGATK is naturally processed and
presented by melanoma cells in association with HLA-A3
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62
To confirm that the HLA-A3 restricted peptide ALLAVGATK
from Pmel-17/gp100 was naturally processed, HLA-A3 associated
peptides were isolated from VMM18 melanoma cells and
fractionated by reversed-phase HPLC, as described. The
synthetic peptide ALLAVGATK (mass of 846 and m/z of 423) was
eluted under identical conditions and found in fraction 14.
Collision activated dissociation (CAD) sequencing of the
peptide(s) m/z 423 was subsequently performed on the HLA-A3
associated peptides eluted in fraction number 14 from VMM18
melanoma cells, confirming its amino acid sequence as
ALLAVGATK, identical to the predicted synthetic peptide. This
confirms that peptide ALLAVGATK from Pmel-17/gplOO is a
naturally processed antigenic peptide, presented by HLA-A3 on
melanoma cells.
Discussion
Evidence of HLA-A3 restricted recognition of melanoma
cells by melanoma specific CTL has been previously observed
however, melanoma antigens presented by HLA-A3 were not
previously identified. In the present report, we have
corroborated the previous finding by demonstrating the
existence of shared melanoma antigens restricted by HLA-A3.
We have also identified a specific naturally-processed
peptide, ALLAVGATK, derived from Pmel-17, as an epitope
recognized by HLA-A3 restricted melanoma specific CTL from
patient VMM18. Since this protein, Pmel-17, is expressed by
the majority of melanoma cells and is a tissue
differentiation antigen of melanocytic origin, this peptide
represents a shared epitope for A3-restricted melanoma-
specific CTL.
Analysis of HLA-A2 associated peptides eluted from the
surface of melanoma cells has demonstrated that the amino
acid sequences of naturally processed MHC-associated peptides
may differ from their respective gene-encoded amino acid
sequences because of post-translational modifications and
that the gene-encoded sequence may not be presented at all.
To confirm that the predicted peptide, ALLAVGATK, is
naturally processed, HLA-A3 associated peptides from VMM18
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63
tumor cells were evaluated directly and sequenced by tandem
mass spectrometry. By this method, it has been confirmed that
this peptide is naturally processed and presented by HLA-A3.
HLA-A2 and -A3 are two of the most commonly expressed
haplotypes in Caucasian populations, representing 46% and 240
respectively. The identification of an HLA-A3 restricted
epitope expands the number of patients (to 600) who might be
targeted for immunization against Pmel-17 antigens. It also
suggests that Pmel-17 directed immunotherapy may be an
important part of immune therapy for melanoma patients of
many different haplotypes.
Although the Pmel-17 derived peptide ALLAVGATK is
recognized by VMM18 CTL, it is not recognized by CTL from
another patient, VMM12. However, VMM12 CTL do recognize and
lyse VMM18 melanoma cells. Because the only Class I MHC
molecule shared by VMM12 and VMM18 is HLA-A3, it is evident
that at least one additional shared CTL epitope is expressed
by both of these tumors.
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WO 97/34613 PCT/US97/04958
64
~J=~
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a
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CA 02249390 1998-09-18
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ct
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CA 02249390 2006-11-14
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66
References for Example 1
1. TRAVERSARI, et al., 1992. Anonapeptide encoded by
human gene MAGE-1 is recognized on HLA-A1 by cytolytic T
lymphocytes directed against tumor antigen MZ2-E. J. Exp.
Med. 176(5):1453.
2. VAN DEN EYNDE, et al., 1995. Anew family of genes
coding for an antigen recognized by autologous cytolytic T
lymphocytes on a human melanoma. J. Exp. Med. 182(3):689-98.
3. BRICHARD, et al., 1993. The tyrosinase gene codes
for an antigen recognized by autologous cytolytic T
lymphocytes on HLA-A2 melanomas. J. Exp. Med. 178(2):489.
4. WOLFEL, et al., 1994. two tyrosinase nonapeptides
recognized on HLA-A2 melanomas by autologous cytolytic T
lymphocytes. European Journal of Immunology 24:759.
5. ROBBINS, et al., 1994. Recognition of tyrosinase by
tumor-infiltrating lymphocytes from a patient responding to
immunotherapy [published erratum appears in Cancer Res 1994
Jul 15;54(14):3952]. Cancer Res. 54(12):3124.
6. SKIPPER, et al., 1995. An HLA-A2 restricted
tyrosinase antigen on melanoma cells results from post-
translational modification. J. Exp. Med., 183:527-34 (1996).
7. KANG, et al., 1995. Identification of a tyrosinase
epitope recognized by HLA-A24-restricted, tumor-infiltrating
lymphocytes. J. Immunol. 155(3):1343.
8. COX, et al., 1994. Identification of a peptide
recognized by five melanoma-specific human cytotoxic t cell
lines. Science 264:716.
9. BAKKER, et al., 1994. Melanoma lineage-specific
antigen gplOO is recognized by melanoma-derived tumor-
infiltrating lymphocytes. J. Exp. Med. 179:1005.
10. KAWAKAMI, et al., 1994. Identification of a human
melanoma antigen recognized by tumor-infiltrating lymphocytes
associated with in vivo tumor rejection. Proc. Natl. Acad.
Sci. USA 91(14):6458.
11. KAWAKAMI, et al., 1994. Cloning of the gene coding
for a shared human melanoma antigen recognized by autologous
T cells infiltrating into tumor. Proc. Natl. Acad. Sci. USA
CA 02249390 1998-09-18
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67
91:3515.
12. COULIE, et al., 1994. A new gene encoding for a
differentiation antigen recognized by autologous cytolytic T
lymphocytes on HLA-A2 melanomas. J. Exp. Med. 180:35.
13. WANG, et al., 1995. Identification of a gene
encoding a melanoma tumor antigen recognized by HLA-A31-
restricted tumor-infiltrating lymphocytes [published erratum
appears in J Exp Med 1995 mar 1; 181(3):1261]. J. Exp. Med.
181 (2) :799.
14. WOLFEL, et al., 1989. Lysis of human melanoma cells
by autologous cytolytic T cell clones. Identification of
human histocompatibility leukocyte antigen A2 as a
restriction element for three differet antigens. J. Exp. Med.
170(3):797.
15. Slingluff, C. L., Jr., A. L. Cox, R. A. Henderson,
D. F. Hunt, and V. H. Engelhard. 1993. Recognition of human
melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes
is mediated by at least six shared peptide epitopes. J.
Immunol. 150 (7) :2955.
16. COX, et al., 1994. Identification of a peptide
recognized by five melanoma-specific human cytotoxic t cell
lines. Science 264:716.
17. ibid.
18. BAKKER, et al., 1995. Identification of a novel
peptide derived from the melanocyte-specific gplOO antigen as
the dominant epitope recognized by an HLA-A2.1-restricted
anti-melanoma CTL line. International Journal of Cancer
62 (1) :97-102.
19. KAWAKAMI, et al., 1995. Recognition of multiple
epitopes in the human melanoma antigen gplOO by tumor-
infiltrating T lymphocytes associated with in vivo tumor
regression. Journal of Immunology 154(8):3961-8.
20. ibid.
21. TRAVERSARI, et al., 1992. Anonapeptide encoded by
human gene MAGE-1 is recognized on HLA-Al by cytolytic T
lymphocytes directed against tumor antigen MZ2-E. J. Exp.
Med. 176(5):1453.
CA 02249390 1998-09-18
WO 97/34613 PCT/US97/04958
68
22. GAUGLER, et al., 1994. Human gene MAGE-3 codes for
an antigen recognized on a melanoma by autologous cytolytic T
lymphocytes. J. Exp. Med. 179:921.
23. HAHN, et al., 1991. Presentation of viral antigen
to class I major histocompatibility complex-restricted
cytotoxic T lymphocyte. Recognition of an immunodominant
influenza hemagglutinin site by cytotoxic T lymphocyte is
independent of the position of the site in the hemagglutinin
translation product. Journal of Experimental Medicine 174
(3) : 733-6.
24. ibid.
25. SLINGLUFF, et al., 1993. Recognition of human
melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes
is mediated by at least six shared peptide epitopes. J.
Immunol. 150 (7) : 2955 .
26. BARNSTABLE, et al., 1978. Production of monoclonal
antibodies to group A erythrocytes, HLA and other human cell
surface antigens--new tools for genetic analysis. Cell 14:9.
27. Lampson, L. A., and R. Levy. 1980. Two populations
of Ia-like molecules on a human B cell line. J. Immunol.
125:293.
28. Berger, A. E., J. E. Davis, and P. Cresswell. 1982.
Monoclonal antibody to HLA-A3. Hybridoma 1:87.
29. KWON, et al., 1991. A melanocyte-specific gene,
Pmel 17, maps near the silver coat color locus on mouse
chromosome 10 and is in a syntenic region on human chromosome
12. Proc. Natl. Acad. Sci. USA 88:9228.
30. Ruppert, J., R. T. Kubo, J. Sidney, H. M. Grey, and
A. Sette. 1994. Class I MHC-peptide interaction: structural
and functional aspects. [Review]. Behring Institute
Mitteilungen::48.
31. DIBRINO, et al., 1993. Endogenous peptides bound to
HLA-A3 possess a specific combination of anchor residues that
permit identification of potential antigenic peptides. Proc.
Natl. Acad. Sci. USA 90 (4) : 1508.
32. SLINGLUFF, et al., 1993. Recognition of human
melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes
is mediated by at least six shared peptide epitopes. J.
CA 02249390 1998-09-18
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69
Immunol. 150 (7) :2955.
33. ENGELHARD, et al., 1993. Mass spectrometric
analysis of peptides associated with the human class I MHC
molecules HLA-A2.1 and HLA-B7 and identification of
structural features that determine binding. [Review].
Chemical Immunology 57 (39): 39-62.
34. SLINGLUFF, et al., 1993. Recognition of human
melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes
is mediated by at least six shared peptide epitopes. J.
Immunol. 150 (7) : 2955 .
35. COX, et al., 1994. Identification of a peptide
recognized by five melanoma-specific human cytotoxic t cell
lines. Science 264:716.
36. KAWAKAMI, et al., 1993. T-cell recognition of human
melanoma antigens. J. Immunother. 14:88.
37. BAKKER, et al., 1994. Melanocyte lineage-specific
antigen gplOO is recognized by melanoma-derived tumor-
infiltrating lymphocytes. J. Exp. Med. 179(3):1005.
38. ADEMA, et al., 1993. Melanocyte lineage-specific
antigens recognized by monoclonal antibodies NKI-beteb, HMB-
50, and HMB-45 are encoded by a single cDNA. American Journal
of Pathology 143 (6): 1579-85.
39. KWON, et al., 1991. A melanocyte-specific gene,
Pmel 17, maps near the silver coat color locus on mouse
chromosome 10 and is in a syntenic region on human chromosome
12. Proc. Natl. Acad. Sci. USA 88:9228.
40. COX, et al., 1994. Identification of a peptide
recognized by five melanoma-specific human cytotoxic t cell
lines. Science 264:716.
41. KUBO, et al., 1994. Definition of specific peptide
motifs for four major HLA-A alleles. Journal of Immunology
152 (8) : 3913 -24 .
42. DIBRINO, et al., 1993. Endogenous peptides bound to
HLA-A3 possess a specific combination of anchor residues that
permit identification of potential antigenic peptides. Proc.
Natl. Acad. Sci. USA 90 (4) :1508.
43. Salter, R. D., D. N. Howell, and P. Cresswell.
1985. Genes regulating HLA class I antigen expression in T-B
CA 02249390 1998-09-18
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lymphoblast hybrids. Immunogenetics 21:235.
44. SLINGLUFF, et al., 1993. Recognition of human
melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes
is mediated by at least six shared peptide epitopes. J.
5 Imrr[unol. 150 (7) : 2955.
45. ENGELHARD, et al., 1993. Mass spectrometric
analysis of peptides associated with the human class I MHC
molecules HLA-A2.1 and HLA-B7 and identification of
structural features that determine binding. [Review].
10 Chemical Immunology 57 (39) : 39-62.
46. Q. Chen and P. Hersey. 1992. MHC-restricted
responses of CD8+ and CD4+ T-cell clones from regional lymph
nodes of melanoma patients. International Journal of Cancer
51 (2): 218-24.
15 47. I. O. Ben-Izhak, P. Stark, R. Levy, R. Bergman and
C. Lichtig. 1994. Epithelial markers in malignant melanoma. A
study of primary lesions and their metastases. American
Journal of Derma topa thol ogy 16 (3): 241-6.
48. H. M. Grey, J. Ruppert, A. Vitiello, J. Sidney, W.
20 M. Kast, R. T. Kubo and A. Sette. 1995. Class I MHC-peptide
interactions: structural requirements and functional
implications. [Review]. Cancer Surveys 22 (37) : 37-49.
49. SKIPPER, et al., 1995. An HLA-A2 restricted
tyrosinase antigen on melanoma cells results from post-
25 translational modification. J. Exp. Med. in press.
50. HUNT, et al., 1992. Characterization of peptides
bound to the class I MHC molecule HLA-A2.1 by mass
spectrometry [see comments]. Science 255 (5049): 1261-3.
51. COX, et al., 1994. Identification of a peptide
30 recognized by five melanoma-specific human cytotoxic t cell
lines. Science 264:716.
52. KAWAKAMI, et al., 1994. Identification of a human
melanoma antigen recognized by tumor-infiltrating lymphocytes
associated with in vivo tumor rejection. PNAS USA
35 91(14):6458-62.
53. KAWAKAMI, et al., 1995. Recognition of multiple
epitopes in the human melanoma antigen gplOO by tumor-
infiltrating T lymphocytes associated with in vivo tumor
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71
regression. Journal of Immunology 154(8):3961-8.
54. GAUGLER, et al., 1994. Human gene MAGE-3 codes for
an antigen recognized on a melanoma by autologous cytolytic T
lymphocytes. J. Exp. Med. 179:921.
55. CELIS, et al., 1994. Induction of anti-tumor
cytotoxic T lymphocytes in normal humans using primary
cultures and synthetic peptide epitopes. PNAS USA 91(6):2105.
EX.AMPLE 2
A recombinant vaccinia virus has been constructed that
was designed to express the full-length tyrosinase protein.
Appropriate expression of the t<yrosinase protein was
confirmed by infecting tyrosinase-negative non-melanoma cells
with this newly constructed virus and demonstrating their
subsequent recognition by murine tyrosinase specific T cells.
Human HLA-A2-positive lymphoblastoid cells (JY) were infected
with a recombinant vaccinia virus expressing the full-length
tyrosinase protein, labeled, and combined with murine
cytolytic T cells specific for the HLA-A2-restricted
tyrosinase "D" peptide (YMDGTMSQV), which were generated in
our laboratories. Recognition of the vaccinia encoded
tyrosinase was ascertained by measuring target cell lysis in
a standard chromium release assay. As expected, uninfected
JY, and JY infected with a recombinant vaccinia encoding an
irrelevant protein (NP), were not recognized. JY cells
pulsed with the "D" peptide and DM6 melanoma cells served as
positive controls, demonstrating the lytic potential and
specificity of the T cells in this particular assay, as well
as the efficacy of the vaccinia construct as a means of'
inducing expression of tyrosinase in a cell.
A panel of human cytolytic T lymphocytes (CTL) was then
screened for recognition of the tyrosinase protein by the
same method. One human CTL line, VMM12, was found to
specifically recognize tyrosinase. This experiment was
performed as above, except that the CTL were derived from
patient VMM12, and the tyrosinase-negative non-melanoma human
B lymphoblastoid cell line VMM12EBV served as the target
cell. Recognition of VMM12 melanoma tumor cells verifies the
CA 02249390 2006-11-14
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lytic potential of these CTL. VMM12EBV infected with
recombinant vaccinia encoding tyrosinase were recognized and
lysed, whereas VMM12EBV infected with a recombinant vaccinia
construct encoding an irrelevant protein (NP) were not
recognized, demonstrating that the recognition of VMM12EBV
infected with vaccinia-tyrosinase was absolutely dependent on
expression of the tyrosinase protein.
The specific Major Histocompatability Complex (MHC)
molecule recognized by VMM12 CTL in association with the
tyrosinase epitope was determined by repeating the previously
described experiment using target cells (C1R) expressing
individual MHC molecules. Only those targets which shared
expression of HLA-Al with VMM12 were recognized,
demonstrating HLA-Al as the "restriction element". This
experiment was also performed as above, except that
additional target cells, expressing individual HLA molecules
shared with VMM12EBV (Al, A3 & B7), were included. As
observed with VMM12EBV infected with irrelevant vaccinia
viruses (above), uninfected VMM12EBV and uninfected C1R (non-
melanoma) cells were not recognized, as expected. VMM12EBV
(which express the HLA-Al,-A3,-B7, and -B14 MHC molecules)
infected with the tyrosinase-expressing recombinant vaccinia
virus, and VMM12 melanoma tumor cells, were recognized. The
only C1R (lymphoid) target cells that were recognized were
those that expressed both HLA-Al and tyrosinase.
Example 3
Identification of a Tyrosinase Epitope Recognized by Human
Melanoma-Reactive, HLA-Al Restricted CTLs.
Introduction
We have identified the peptide KCDICTDEY (K is N-
terminal), from the tyrosinase protein, as an epitope for
HLA-Al-restricted melanoma-specific cytotoxic T-lymphocytes
(CTL). This work has been done by generating HLA-AI-
restricted melanoma-reactive CTL, creating a vaccinia
construct encoding the intact human tyrosinase gene, then
infecting HLA-A1+ non-melanoma target cells with the vac-
tyrosinase construct. In doing so, VMM12 CTL and VMM15 CTL
both recognize an HLA-Al-associated peptide derived from
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73
tyrosinase. We have since screened a large panel of peptides
that we predicted to bind to HLA-A1, from the defined
sequence of tyrosinase. The peptide KCDICTDEY, when pulsed
onto HLA-Al+ non-melanoma cells (C1R-Al), reconstitutes an
epitope for VMM15 CTL. To a lesser extent, two other
peptides that are longer than 9-residues, but which contain
the entire KCDICTDEY sequence, also reconstitute an epitope
for these CTL. None of 116 other peptides tested worked.
Thus, we believe this is an epitope which can be used as an
immunogen in treating or preventing melanoma in the 20-250 of
patients who express HLA-Al.
Cell lines and HLA typinc.f: The human melanoma cell lines
VMM1, VMM12, VMM15, VMM18, VMM30 and VMM34 were derived from
patients at the University of Virginia (Charlottesville, VA).
Other fresh (uncultured) tumors VMM14 and VMM21 were also
prepared from surgical specimens from patients at the
University of Virginia. DM6 was provided by Drs. H.F.
Seigler and T.L. Darrow at Duke University (Durham, NC).
Immunohistochemical staining of these cell lines with S-100,
HMB-45 and vimentin antibodies was characteristic of
melanoma, while staining for epithelial membrane antigen and
cytokeratin was negative. The CV-1 and 143B TK- lines used in
the propagation of vacciriia virus were also obtained from the
American Type Culture Collection (ATCC, Bethesda, MD). VMM12-
EBV is an Epstein-Barr virus transformed B cell line derived
from peripheral blood mononuclear cells (PBMC) of melanoma
patient VMM12. Briefly, the PBMC were incubated with filtered
supernatant from the EBV producing cell line B-958 for 1 h at
37 C, followed by culture in RPMI 1640 media with 10o fetal
calf serum (FCS) and antibiotics, plus a 1:100 dilution of
PHA. K562 is an NK-sensitive human erythroleukemia line. T2-
A3 (an HLA-A3 transfectant of the antigen-processing-
defective mutant human lymphoid cell line, T2) was provided
by P. Cresswell. HLA typing was performed by
microcytotoxicity assay on autologous lymphocytes (Gentrak).
Expression of HLA-Al by tumor cells was confirmed by staining
with a monoclonal antibody (MAb) from One Lambda.
CTL lines: We have generated human melanoma-specific CTL
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74
lines by in vitro stimulation with autologous tumor, from
patients whose tumors express melanocytic tissue
differentiation antigens and express one or more of the MHC
molecules Al, A3, B7, and B8. Methods for CTL generation
have been described. (Table ill and Figure 5).
Production of recombinant vaccinia virus exDressing the human
genes encodina melanocytic tissue differentiation antigens:
We have examined class I MHC-associated epitopes for the
melanocytic tissue differentiation antigens by using vaccinia
constructs for each of the genes Pmell7/gpl00, tyrosinase,
and MART-1/MelanA. A cDNA clone of the Pmel17 gene
(HUMPMEL17 - Genbank) was generously provided by S.N. Wagner,
Essen, Germany. The tyrosinase gene was provided by Thierry
Boon, Brussels. We have PCR cloned out a cDNA clone of the
MART-1/Melan-A gene from DM6 melanoma cells. The entire
open-reading frame for each of these cDNA's was sub-cloned
into a modified pSCil vector (Ref Hahn JEM 1991) adjacent to
the vaccinia P7.5 early/late promoter using standard
recombinant DNA methods. Standard dideoxy sequencing was used
to confirm insertion and orientation. A recombinant vaccinia
virus expressing the protein encoded by this gene (vac-Pmel-
17) was generated using published methods (Ref Macket J.Virol
1984). Briefly, CV-1 cells were infected with the parental WR
strain of vaccinia virus and transfected (Lipofectin, Gibco-
BRL) with the pSC11.3-Pmel-17 plasmid. Thymidine-kinase
negative recombinants were amplified in 143B TK- cells in the
presence of bromodeoxyuridine (Sigma). Recombinants with
beta-galactosidase activity were isolated and cloned through
several rounds of plaque purification. Large-scale stocks
were produced, sucrose purified, and titered in CV-1 cells.
The resulting recombinant vaccinia viruses were used to
infect the lymphoblastoid cell lines C1R-Al, C1R-A2, C1R-A3,
C1R-B7, and ClR-B8, where C1R is a human lymphoblastoid line
devoid of native expression of HLA-A or HLA-B region
molecules, but expressing low levels of HLA-C and MHC Class
II molecules. In some cases EBV-transformed B cells with
defined MHC expression were used for the infections. This
resulted in transient expression of the antigens of interest.
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These cells were assayed for recognition by CTL in Cr51-
release assays. As a negative control, target cells were
also infected with a recombinant vaccinia virus with an
irrelevant DNA insert (influenza nucleoprotein, NP). Thus,
5 the cell lines listed above permit isolated evaluation of the
expression of antigenic peptides in association with the
common Class I MHC molecules HLA-Al, A2, A3, B7, and B8.
Evaluating recognition of tarcret cells by CTL.
Reactivity was assessed by cytotoxicity in a 4-hour chromium
10 release assay. Positive controls were the autologous tumor
and known cross-reactive tumor lines. A negative control was
uninfected C1R-MHC line and a C1R-MHC line transfected with a
vaccinia construct expressing influenza nucleoprotein, vac-NP
only. Briefly, 51Cr-labeled target cells were plated at 1 -
15 2x103 cells/well in triplicate on 96-well V-bottom plates
(Costar, Cambridge, MA) with indicated ratio of effector
cells in a final volume of 200 microliters. Wells containing
either culture medium or 1M HC1 in place of the effector
cells served as spontaneous and maximum 51Cr-release controls,
20 respectively. Plates were centrifuged at 100 x g for 3 min
and incubated at 37 C for 4 h, after which 150 microliters of
supernatant from each well was counted on a gamma counter
(ICN). The percent specific lysis was calculated using the
equation: [(experimental release - spontaneous release) /
25 (maximum release - spontaneous release)] x 100. Vaccinia
infected targets were generated by infecting cells with 50
pfu/cell of appropriate recombinant vaccinia virus at 37 C for
5 h, prior to 51Cr-labeling.
Peptide synthesis and Reconstitution with synthetic pe,ntides:
30 Peptide sequences were selected from the reported human
sequence of tyrosinase, based on predicted HLA-Al binding
motifs (see table 10). These peptides were synthesized by
standard Fmoc chemistry using a Gilson model AMS422 peptide
synthesizer. Peptides were reconstituted in CTL assay medium
35 (RPMI 1640, 10% FCS, antibiotics) and pre-incubated for 2 h
with 2x103 s'Cr labeled target cells in 100 microliters/well in
96-well plates. Effector cells were added in 100 microliters
assay medium for a final effector to target (E:T) ratio of
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76
20:1 and the remainder of the assay was performed as in
standard chromium release assays described above. Wells
containing peptide and target cells but no CTL were used as
controls to rule out toxicity of the peptides themselves.
Initial experiments were performed with unpurified synthetic
peptides. Biologically active peptides identified at initial
screening were then purified to >98o by reversed-phase HPLC
on a Vydac C-4 column with 0.05% trifluoroacetic acid:water
and an acetonitrile gradient, then re-evaluated in CTL
assays.
Results
Melanoma-reactive CTL lines recognize MHC-associated peptides
from several melanocytic differentiation antigens
The CTL lines listed in Table ill were evaluated for
recognition of peptides derived from the 3 melanocytic tissue
differentiation antigens listed above, in chromium-release
assays, by transient infection with vaccinia constructs
encoding those genes. Examples of their reactivity against
HLA-matched allogeneic melanomas are shown in Figure 5. A
summary of these results with vaccinia constructs are listed
in Table 112 and are shown in Figure 6. Responses to
tyrosinase peptides were observed in half of cases. In
addition, responses to MART-1 and gplOO peptides were
observed in a smaller set of CTL lines.
At least two of the HLA-A1+ CTL lines recognized tyrosinase
peptides in an HLA-Al-restricted manner.
VMM12 CTL and VMM15 CTL were assayed initially on
autologous EBV-B cells as targets. Reactivity against
tyrosinase was observed, so additional studies were performed
to confirm the reactivity and to determine the MHC
restriction (Figure 6). C1R cells that express selected
Class I MHC molecule: only were used as target cells. As
seen in Figure 6, C1R-Al cells infected with vac-tyrosinase
are recognized by VMM12 and VMM15 CTL, confirming that one or
more tyrosinase-derived peptides are recognized by VMM12 and
VMM15 CTL in association with HLA-A1.
The peptide representing residues 243-251 of tyrosinase
reconstitutes an epitope for VMM15 CTL.
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77
A set of peptides were synthesized from the defined amino
acid sequence of tyrosinase, including 9-mers and longe.r
peptides, with tyrosine (Y) at the C-terminal position and T,
S, or M at position 2 and/or D, E, A, or S at position 3
(Figure 7). These were assayed for their ability to "
reconstitute epitopes for melanoma-reactive CTL VMM12 and
VMM15. C1R-A1 cells were pulsed with the peptide at
concentrations ranging from 0.1 to 10 uM in normal assay
medium (RPMI + 10% FCS), then evaluated for recognition in a
chromium-release assay. As shown in Figure 8, three peptides
were recognized by VMM15 CTL, all containing the sequence
KCDICTDEY (tyrosinase residues 243-251). The most effective,
even at the lowest concentration tested, was the 9-mer
peptide KCDICTDEY, but also recognized were a ten-mer,
EKCDICTDEY, and a 13-mer, DAEKCDICTDEY (Figure 8).
Similar reactivity was seen with VMM12 CTL as well,
suggesting that KCDICTDEY is a shared antigen on human
melanoma cells expressing HLA-Al, against which multiple
patients' CTL may be expected to react (Figure 9). The
location of this peptide in the intact protein tyrosinase is
shown in Figure 10.
Discussion
The peptide KCDICTDEY appears to be recognized by CTL
from at least two different patients, in association with
HLA-Al. Although longer peptides also are reactive, the
dominant response seems to be to KCDICTDEY. This peptide is
unusual in its large number of polar amino acid residues,
including two aspartic acid residues, one glutamic acid
residue, and two cystine residues. The tyrosine residue at
position 9 and the aspartic acid at position 3 are important
for binding to the MHC. By a computerized system for
predicting the binding affinity of individual peptides to
HLA-Al (and other HLA haplotypes),(The algorithm for this
software is discussed in Parker, et al., J. Immunol., 152:163
(1994)), this peptide is predicted to the be the tyrosinase
CA 02249390 1998-09-18
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78
peptide with highest affinity for HLA-Al, which may make it
useful for immunization after pulsing on antigen-presenting
cells.
One concern with this peptide is the presence of two
cystine residues, which may be susceptible to interaction
with other sulfhydryl groups on biologic molecules in vitro
and in vivo. Studies on the possibility of this interaction
and its effect on CTL recognition are underway. KCDICTDEY is
associated with half-maximal lysis at approximately 1 ug/ml
(1 uM). Evaluating the possibility of increasing the potency
of this activity is underway, by assessing various amino acid
substitutions and their effects on CTL recognition.
There have been two peptides described as epitopes for
melanoma-reactive HLA-Al-restricted CTL. They are the MAGE-1
and MAGE-3 peptides EADPTGHSY and EVDPIGHLY-While these have
substantial potential value as immunogens, only a subset of
melanoma patients express them. Most other MHC-associated
peptide epitopes are HLA-A2 associated. However, HLA-Al is
expressed in approximately 29% of patients in this country.
We have previously described an HLA-A3-associated epitope
from gplOO, ALLAVGATK. Now, with defined peptide epitopes
known, it is possible to consider the use of a multivalent
peptide vaccine, where all patients expressing either HLA-Al,
HLA-A2, or HLA-A3, which is approximately 700 of the patients
at risk, may be treated with specific vaccine therapy.
CA 02249390 1998-09-18
WO 97/34613 PCT/US97/04958
~ 79
~
U
~
~
0
CJ
~
.,~
-r-I
U
y
> I =,I a) I o 0
,+ +
E=
4 U
41 a ro
3 N
a)
U W
4)
d
U) ul co (D (j) i i
'+ y+ O o ~ ~+ + O + O o
0
E-1 ~I
U
4-1 (D
Q) ,7
r-I 1-) o o o 0 0
rt FC r==I U i i i i i
44 r+ a c~
o v
;:, x b
o ~ 14 _
a4Ji M H CN c u
rn x ~
o U4-) 34 a, u,
U ~4 =.'i (d r-I I- ri l1 M l~ r-1 0O H 00 rl N m r-I
a) o 4-) cn K~
~4 a~
o
44 ~
U ~ 4J W W ~ m [- H ao W W W W
v Q N N tl1 Q ~C W I fl Q (Y1 ao t11 N \O
a1 r1 r 1 r I ~ ~ ~ rl ~ ~ M '-1 r4 1=4
U 1 r=1 Eg R; a' R.' E E 2:
H U U U U ~ U U >
U2 0 ~+
(L) O 0
~ = ri
GQ LI1 aD Lf1 H
m N cYl FC cxl ~ U
[~
. . . . N
U~ ro U 04
H [, rl w r~l lfl lw
E U N ~C !sl < W aC a0 U
= a
~ ro
O r
-~
Gl N l.(1 O
~
O
E
Ln
CA 02249390 1998-09-18
WO 97/34613 PCTIUS97/04958
ri) ra 1 ~ t 1
0 o v a) t a) o 0 1 0
>+ 1 ~ t +
Ul rn i t
a) N o 0 0 ~ 1 0 0 0 0
?+ y+ 1 1
0
-r-I
~
crs
0 N
0 o a) N 0 O C) 0 -H
+ ~
a)
--I
lll N ~
LC) Lf) . [- ~ M M ~ l0 ~
N CQ N N t'7 M U N Cq [-
FC ~C W M ~C (Yl I
\ ("
L~ r-I O m d' Ln Ln 44
r I N M H 00 r-I r- (n M l0' f'M cM M N ill rl r-I M N
di (=Q U F:4 F:~ 4 4 CQ (~i W F:~
~
v
w w w w w w w w w w b
I I t I 1 M I I I 1 I
O Lf1 r{ Op N tQ', l~ Ol N N O ~
0
>
.,~
~ tfl N
LI1 L11 00 l- M M l0
~C ~ U~C al FC CA M U ry cn Ft (s~ ZS 1
\ 41 f~
~ = = r1 O L(1 i.f) 11
r 1 N N rl 00 rl [~ (M M l0 N Ln rl ri N C~ ~ ED
4-1
r
J -1
J7 fJ2
~-I
O ~ ~ m r-i m II II
(Y) > > F t
+
Lfl
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81
Table 112. Summary of CTL reactivities observed
Patient Source of Restricting Class I
ID CTL epitope MHC molecule
VMM10 -n/a
VMM12 Al
VMM14 Tyrosinase unknown
VMM15 Tyrosinase Al
MART-1 unknown
VMM18 Pmel17 A3
MART-1 A3
VMM19 -n/a
VMM21 unknown
VMM30 Tyrosinase unknown
VMM39 -n/a
DM 331 n a
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82
REMARKS
Reference to known method steps, conventional methods
s teps , known methods or conven ti onal methods is not in any
way an admission that any aspect, description or embodiment
of the present invention is disclosed, taught or suggested in
the relevant art.
The foregoing description of the specific embodiments
will so fully reveal the general nature of the invention that
others can, by applying knowledge within the skill of the art
(including the contents of the references cited herein),
readily modify and/or adapt for various applications such
specific embodiments, without undue experimentation, without
departing from the generic concept of the present invention.
Therefore, such adaptations and modifications are intended to
be comprehended within the meaning and range of equivalents
of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology
or phraseology of the present specification is to be
interpreted by the skilled artisan in light of the teachings
and guidance presented herein.
For immunological techniques generally, see Coligan, et
al, Current Protocols in Immunology (NIH: 994); Harlow and
Lane, Antibodies:A laboratory Manual (Cold Spring Harbor
Lab.: 1988).
An immunogen is deemed not to occur in nature, even
though its component epitopes do occur in nature, if the
immunogen itself, as a single molecule, does not occur in
nature. For example, a conjugate of 946L to albumin does not
occur in nature even though 946L is a fragment of pMel-17
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83
which is generated by the immune system processing of pMel-17
and complexes with MHC.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: UNIVERSITY OF VIRGINIA PATENT FOUNDATION
(ii) TITLE OF INVENTION: PEPTIDES RECOGNIZED BY MELANOMA-SPECIFIC
Al-,A2-, AND A3-RESTRICTED CYTOTOXIC
LYMPHOCYTES, AND USES THEREFOR
(iii) NUMBER OF SEQUENCES: 226
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,249,390
(B) FILING DATE: 17-MAR-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/013,972
(B) FILING DATE: 19-MAR-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/027,627
(B) FILING DATE: 04-OCT-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
70484-65
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(C) REFERENCE/DOCKET NUMBER: 70484-65
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Tyr Met Asn Gly Thr Met Ser Gln Val
1 5
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Leu Ala Val Leu Tyr Cys Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ala Ala Gly Ile Gly Ile Leu Thr Val
1 5
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Ala Leu Leu Ala Val Gly Ala Thr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Glu Ala Asp Pro Thr Gly His Ser Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Glu Val Asp Pro Ile Gly His Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
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(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Leu Leu Ala Val Gly Ala Thr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Gln Val Pro Leu Arg Pro Met Thr Tyr Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Tyr Leu Glu Pro Gly Val Thr Val
1 5
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Leu Leu Ala Tyr Leu Tyr Cys Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Ala Phe Leu Pro Trp His Arg Leu Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Ala Phe Leu Pro Trp His Arg Leu Phe Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Ser Glu Ile Trp Arg Asp Ile Asp Phe
1 5
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(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Lys Thr Trp Gly Gln Tyr Trp Gln Val
1 5
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Ile Thr Asp Gln Val Pro Phe Ser Val
1 5
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val
1 5 10
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(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Leu Leu Asp Gly Thr Ala Thr Leu Arg 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Ala Ala Gly Ile Gly Ile Leu Thr 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Ile Leu Thr Val Ile Leu Gly Val Leu
1 5
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(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Glu Ala Asp Pro Thr Gly His Ser Tyr
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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Ser Ala Tyr Gly Glu Pro Arg Lys Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Glu Val Asp Pro Ile Gly His Leu Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Phe Leu Trp Gly Pro Arg Ala Leu Val
1 5
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Ala Ala Arg Ala Val Phe Leu Ala Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Tyr Arg Pro Arg Pro Arg Arg Tyr
1 5
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(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Lys Ile Phe Gly Ser Leu Ala Phe Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Val Met Ala Gly Val Gly Ser Pro Tyr Val
1 5 10
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Ile Ile Ser Ala Val Val Gly Ile Leu
1 5
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(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Tyr Leu Ser Gly Ala Asn Leu Asn Leu
1 5
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Glu Ala Tyr Gly Leu Asp Phe Tyr Ile Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Gln Asp Leu Thr Met Lys Tyr Gln Ile Phe
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Glu Glu Lys Leu Ile Val Val Leu Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Ser Tyr Leu Asp Ser Gly Ile His Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2131 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
GGAAGAACAC AATGGATCTG GTGCTAAAAA GATGCCTTCT TCATTTGGCT GTGATAGGTG 60
CTTTGCTGGC TGTGGGGGCT ACAAAAGTAC CCAGAAACCA GGACTGGCTT GGTGTCTCAA 120
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GGCAACTCAG AACCAAAGCC TGGAACAGGC AGCTGTATCC AGAGTGGACA GAAGCCCAGA 180
GACTTGACTG CTGGAGAGGT GGTCAAGTGT CCCTCAAGGT CAGTAATGAT GGGCCTACAC 240
TGATTGGTGC AAATGCCTCC TTCTCTATTG CCTTGAACTT CCCTGGAAGC CAAAAGGTAT 300
TGCCAGATGG GCAGGTTATC TGGGTCAACA ATACCATCAT CAATGGGAGC CAGGTGTGGG 360
GAGGACAGCC AGTGTATCCC CAGGAAACTG ACGATGCCTG CATCTTCCCT GATGGTGGAC 420
CTTGCCCATC TGGCTCTTGG TCTCAGAAGA GAAGCTTTGT TTATGTCTGG AAGACCTGGG 480
GCCAATACTG GCAAGTTCTA GGGGGCCCAG TGTCTGGGCT GAGCATTGGG ACAGGCAGGG 540
CAATGCTGGG CACACACACC ATGGAAGTGA CTGTCTACCA TCGCCGGGGA TCCCGGAGCT 600
ATGTGCCTCT TGCTCATTCC AGCTCAGCCT TCACCATTAC TGACCAGGTG CCTTTCTCCG 660
TGAGCGTGTC CCAGTTGCGG GCCTTGGATG GAGGGAACAA GCACTTCCTG AGAAATCAGC 720
CTCTGACCTT TGCCCTCCAG CTCCATGACC CTAGTGGCTA TCTGGCTGAA GCTGACCTCT 780
CCTACACCTG GGACTTTGGA GACAGTAGTG GAACCCTGAT CTCTCGGGCA CCTGTGGTCA 840
CTCATACTTA CCTGGAGCCT GGCCCAGTCA CTGCCCAGGT GGTCCTGCAG GCTGCCATTC 900
CTCTCACCTC CTGTGGCTCC TCCCCAGTTC CAGGCACCAC AGATGGGCAC AGGCCAACTG 960
CAGAGGCCCC TAACACCACA GCTGGCCAAG TGCCTACTAC AGAAGTTGTG GGTACTACAC 1020
CTGGTCAGGC GCCAACTGCA GAGCCCTCTG GAACCACATC TGTGCAGGTG CCAACCACTG 1080
AAGTCATAAG CACTGCACCT GTGCAGATGC CAACTGCAGA GAGCACAGGT ATGACACCTG 1140
AGAAGGTGCC AGTTTCAGAG GTCATGGGTA CCACACTGGC AGAGATGTCA ACTCCAGAGG 1200
CTACAGGTAT GACACCTGCA GAGGTATCAA TTGTGGTGCT TTCTGGAACC ACAGCTGCAC 1260
AGGTAACAAC TACAGAGTGG GTGGAGACCA CAGCTAGAGA GCTACCTATC CCTGAGCCTG 1320
AAGGTCCAGA TGCCAGCTCA ATCATGTCTA CGGAAAGTAT TACAGGTTCC CTGGGCCCCC 1380
TGCTGGATGG TACAGCCACC TTAAGGCTGG TGAAGAGACA AGTCCCCCTG GATTGTGTTC 1440
TGTATCGATA TGGTTCCTTT TCCGTCACCC TGGACATTGT CCAGGGTATT GAAAGTGCCG 1500
AGATCCTGCA GGCTGTGCCG TCCGGTGAGG GGGATGCATT TGAGCTGACT GTGTCCTGCC 1560
AAGGCGGGCT GCCCAAGGAA GCCTGCATGG AGATCTCATC GCCAGGGTGC CAGCCCCCTG 1620
CCCAGCGGCT GTGCCAGCCT GTGCTACCCA GCCCAGCCTG CCAGCTGGTT CTGCACCAGA 1680
TACTGAAGGG TGGCTCGGGG ACATACTGCC TCAATGTGTC TCTGGCTGAT ACCAACAGCC 1740
TGGCAGTGGT CAGCACCCAG CTTATCATGC CTGTGCCTGG GATTCTTCTC ACAGGTCAAG 1800
AAGCAGGCCT TGGGCAGGTT CGGCTGATCG TGGGCATCTT GCTGGTGTTG ATGGCTGTGG 1860
TCCTTGCATC TCTGATATAT AGGCGCAGAC TTATGAAGCA AGACTTCTCC GTACCCCAGT 1920
TGCCACATAG CAGCAGTCAC TGGCTGCGTC TACCCCGCAT CTTCTGCTCT TGTCCCATTG 1980
GTGAGAATAG CCCCCTCCTC AGTGGGCAGC AGGTCTGAGT ACTCTCATAT GATGCTGTGA 2040
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TTTTCCTGGA GTTGACAGAA ACACCTATAT TTCCCCCAGT CTTCCCTGGG AGACTACTAT 2100
TAACTGAAAT AAATACTCAG AGCCTGAAAA A 2131
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Tyr Leu Glu Pro Gly Pro Val Thr Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
TACCTGGAGC CTGGCCAAGT CACTGCC 27
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
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TATATGGATG GAACAATGTC CGAGGTA 27
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Tyr Ile Glu Pro Gly Pro Val Thr Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Tyr Xaa Glu Pro Gly Pro Val Thr Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
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Gln Leu Arg Ala Leu Asp Gly Gly Asn Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Ala Leu Gln Leu His Asp Pro Ser Gly Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Ala Val Pro Ser Gly Glu Gly Asp Ala Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Thr Val Ser Cys Gln Gly Gly Leu Pro Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Gln Ile Leu Lys Gly Gly Ser Gly Thr Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Pro Leu Ala His Ser Ser Ser Ala Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Ala Leu Asp Gly Gly Asn Lys His Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Phe Leu Arg Asn Pro Pro Leu Thr Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Tyr Leu Ala Glu Ala Asp Leu Ser Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Gln Val Pro Leu Asp Cys Val Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Pro Leu Asp Cys Val Leu Tyr Arg Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Cys Val Leu Tyr Arg Tyr Gly Ser Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Gln Leu Val Leu His Gln Ile Leu Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Ile Leu Lys Gly Gly Ser Gly Thr Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
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(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Ala Val Val Leu Ala Ser Leu Ile Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Leu Ile Tyr Arg Arg Arg Leu Met Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Ala Leu Leu Ala Val Gly Ala Thr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
Gly Val Ser Arg Gln Leu Arg Thr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Thr Leu Ile Gly Ala Asn Ala Ser Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Ala Leu Asn Phe Pro Gly Ser Gln Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 65:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Gln Val Trp Gly Gly Gln Pro Val Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Tyr Val Trp Lys Thr Trp Gly Gln Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Ala Ser Phe Ser Ile Ala Leu Asn Phe
1 5
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(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Leu Leu Ala Val Gly Ala Thr Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
Ala Leu Val Val Thr His Thr Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
Leu Asn Phe Pro Gly Ser Gln Lys
1 5
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(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
Thr Ile Thr Asp Gln Val Pro Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Gln Leu His Asp Pro Ser Gly Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
Asp Leu Ser Tyr Thr Trp Asp Phe
1 5
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(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Val Leu Tyr Arg Tyr Gly Ser Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Leu Val Leu His Gln Ile Leu Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Val Val Leu Ala Ser Leu Ile Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Trp Leu Arg Leu Pro Arg Ile Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Tyr Met Asp Gly Thr Met Ser Gln Val
1 5
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
Ala Lys His Thr Ile Ser Ser Asp Tyr
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(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Ala Pro Val Val Thr His Thr Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
Asp Leu Phe Val Trp Ile His Tyr Tyr
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(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Asp Leu Phe Val Trp Met His Ile Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
Asp Arg Glu Ser Trp Pro Ser Val Phe Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHAR.ACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Asp Tyr Val Ile Pro Ile Gly Thr Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
Glu Phe Cys Ser Leu Thr Gln Tyr
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(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Glu Lys Glu Asp Tyr His Ser Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
Phe Ile Ser Ser Lys Asp Leu Gly Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Phe Gln Asp Tyr Ile Lys Ser Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Gly Asp Glu Asn Phe Thr Ile Pro Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
Ile Val Cys Ser Arg Leu Glu Glu Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Ile Tyr Asp Leu Phe Val Trp Met His Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
Lys Glu Asp Tyr His Ser Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Pro Glu Lys Asp Lys Phe Phe Ala Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
Pro Ile Gly His Asn Arg Glu Ser Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
Pro Leu Leu Met Glu Lys Glu Asp Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Pro Met Phe Asn Asp Ile Asn Ile Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
Arg Glu Ser Trp Pro Ser Val Phe Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
Arg His Arg Pro Leu Gln Glu Val Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
Ser Asp Pro Asp Ser Phe Gln Asp Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
Ser Phe Gln Asp Tyr Ile Lys Ser Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
Ser Lys Asp Leu Asp Tyr Gly Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
Ser Met Asp Ala Leu Leu Gly Gly Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
Ser Met His Asn Ala Leu His Ile Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: ill:
Ser Ser Lys Asp Leu Gly Tyr Asp Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
Ser Ser Met His Asn Ala Leu His Ile Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
Tyr Met Val Pro Phe Ile Pro Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
Ala Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:
Ala Pro Ile Gly His Asn Arg Glu Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:
Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
Asp Leu Phe Val Trp Met His Tyr Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
Asp Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:
Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
Glu Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123:
Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:
Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr
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(2) INFORMATION FOR SEQ ID NO: 125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:
Gly Asp Glu Asp Phe Thr Ile Pro Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:
Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Thr Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:
Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr
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(2) INFORMATION FOR SEQ ID NO: 128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128:
Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129:
Ile Tyr Asp Leu Phe Val Trp Met His Tyr Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:
Ile Tyr Asp Leu Phe Val Trp Ile His Tyr Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:
Ile Tyr Asp Leu Phe Val Trp Met His Tyr Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132:
Leu Ala Lys His Thr Ile Ser Ser Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:
Leu Met Glu Lys Glu Asp Tyr His Ser Leu Tyr
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(2) INFORMATION FOR SEQ ID NO: 134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:
Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:
Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:
Leu Thr Gly Asp Glu Asp Phe Thr Ile Pro Tyr
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(2) INFORMATION FOR SEQ ID NO: 137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:
Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:
Leu Thr Leu Ala Lys His Thr Ile Ser Ser Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:
Met Glu Lys Glu Asp Tyr His Ser Leu Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140:
Pro Asp Ser Phe Gin Asp Tyr Ile Lys Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:
Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142:
Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:
Gln Pro Leu Leu Met Glu Lys Glu Asp Tyr
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(2) INFORMATION FOR SEQ ID NO: 144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144:
Gln Arg His Arg Pro Leu Gln Glu Val Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145:
Gln Ser Ser Met His Asn Ala Leu His Ile Tyr
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146:
Arg Glu Ser Tyr Met Val Pro Phe Thr Pro Leu Tyr
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(2) INFORMATION FOR SEQ ID NO: 147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147:
Arg Arg His Arg Pro Leu Gln Glu Val Tyr
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(2) INFORMATION FOR SEQ ID NO: 148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148:
Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149:
Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr
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(2) INFORMATION FOR SEQ ID NO: 150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:
Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151:
Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152:
Ser Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153:
Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154:
Thr Gly Asp Glu Asp Phe Thr Ile Pro Tyr
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155:
Thr Leu Ala Lys His Thr Ile Ser Ser Asp Tyr
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(2) INFORMATION FOR SEQ ID NO: 156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156:
Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157:
Val Asp Asp Arg Glu Ser Trp Pro Ser Val Phe Tyr
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(2) INFORMATION FOR SEQ ID NO: 158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158:
Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr
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(2) INFORMATION FOR SEQ ID NO: 159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159:
Val Ser Met Asp Ala Leu Leu Gly Gly Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160:
Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Tyr
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(2) INFORMATION FOR SEQ ID NO: 161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:
Ala Met Glu Arg Pro Arg Asp Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162:
Glu Val Ser Thr Pro Gln Ile Leu Thr Tyr
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(2) INFORMATION FOR SEQ ID NO: 163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163:
Ile Thr Thr Ala Cys Ile Arg Ala Ile Tyr
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(2) INFORMATION FOR SEQ ID NO: 164:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164:
Ile Trp Ala Met Thr Ile Ala Ile Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:
Arg Ser Thr Thr Ala Ile Ser Leu Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166:
Thr Thr Ala Cys Ile Arg Ala Ile Tyr
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(2) INFORMATION FOR SEQ ID NO: 167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167:
Val Ser Thr Pro Gln Ile Leu Thr Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168:
Trp Arg Ser Thr Thr Ala Ile Ser Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169:
Tyr Asp Leu Phe Val Trp Ile His Tyr
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(2) INFORMATION FOR SEQ ID NO: 170:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170:
Tyr Asp Leu Phe Val Trp Ile His Tyr Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171:
Tyr Asp Leu Phe Val Trp Met His Tyr
1 5
(2) INFORMATION FOR SEQ ID NO: 172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172:
Tyr Asp Leu Phe Val Trp Met His Tyr Tyr
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(2) INFORMATION FOR SEQ ID NO: 173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173:
Ala Asn Asp Pro Ile Phe Leu Leu His
1 5
(2) INFORMATION FOR SEQ ID NO: 174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174:
Ala Asn Asp Pro Ile Phe Leu Leu His His
1 5 10
(2) INFORMATION FOR SEQ ID NO: 175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175:
Cys Cys Pro Pro Trp Ser Gly Asp Arg
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(2) INFORMATION FOR SEQ ID NO: 176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 176:
Cys Thr Asp Glu Tyr Met Gly Gly Gln
1 5
(2) INFORMATION FOR SEQ ID NO: 177:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177:
Cys Thr Asp Glu Tyr Met Gly Gly Gln His
1 5 10
(2) INFORMATION FOR SEQ ID NO: 178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 178:
Cys Thr Glu Arg Arg Leu Leu Val Arg
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(2) INFORMATION FOR SEQ ID NO: 179:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 179:
Cys Thr Glu Arg Arg Leu Leu Val Arg Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 180:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 180:
Cys Val Ser Ser Lys Asn Leu Met Glu Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 181:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 181:
Asp Gly Thr Pro Glu Gly Pro Leu Arg Arg
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(2) INFORMATION FOR SEQ ID NO: 182:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182:
Asp Ile Asp Phe Ala His Glu Ala Pro Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO: 183:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 183:
Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 184:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 184:
Asp Ser Asp Pro Asp Ser Phe Gln Asp
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(2) INFORMATION FOR SEQ ID NO: 185:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 185:
Asp Val Glu Phe Cys Leu Ser Leu Thr Gln
1 5 10
(2) INFORMATION FOR SEQ ID NO: 186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 186:
Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 187:
Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe
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(2) INFORMATION FOR SEQ ID NO: 188:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 188:
Phe Thr Ile Pro Tyr Trp Asp Trp Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 189:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 189:
Gly Ser Glu Ile Trp Arg Asp Ile Asp Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 190:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 190:
Gly Thr Pro Glu Gly Pro Leu Arg Arg
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(2) INFORMATION FOR SEQ ID NO: 191:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191:
Gly Tyr Glu Ile Trp Arg Asp Ile Asp Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 192:
Ile Phe Asp Leu Ser Ala Pro Glu Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 193:
Leu Pro Glu Glu Lys Gln Pro Leu Leu Met
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(2) INFORMATION FOR SEQ ID NO: 194:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 194:
Leu Ser Ala Pro Glu Lys Asp Lys Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 195:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 195:
Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 196:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 196:
Asn Gly Asp Phe Phe Ile Ser Ser Lys
1 5
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(2) INFORMATION FOR SEQ ID NO: 197:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 197:
Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 198:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 198:
Gln Thr Ser Ala Gly His Phe Pro Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 199:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 199:
Gln Tyr Glu Ser Gly Ser Met Asp Lys
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(2) INFORMATION FOR SEQ ID NO: 200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 200:
Ser Ala Asp Val Glu Phe Cys Leu Ser Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 201:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 201:
Ser Met Asp Lys Ala Ala Asp Phe Ser Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 202:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 202:
Ser Met Asp Lys Ala Ala Asn Phe Ser Phe
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(2) INFORMATION FOR SEQ ID NO: 203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 203:
Ser Ser Asp Tyr Val Ile Pro Ile Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 204:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 204:
Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 205:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 205:
Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr
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(2) INFORMATION FOR SEQ ID NO: 206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 206:
Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 207:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 207:
Tyr Met Val Pro Phe Ile Pro Leu Tyr Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 208:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 208:
Tyr Pro Glu Ala Asn Ala Pro Ile Gly His
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 209:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 209:
Tyr Trp Asp Trp Arg Asp Ala Glu Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 210:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 210:
Ala Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 211:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 211:
Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 212:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 212:
Asp Val Glu Phe Cys Leu Ser Leu Thr Gln Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 213:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 213:
Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 214:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 214:
Ser Thr Pro Met Phe Asn Asp Ile Asn Thr Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 215:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 215:
Arg Arg Met Arg Pro Leu Gln Glu Val Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 216:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 216:
Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 217:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 217:
Val Ser Met Asp Ala Leu Leu Gly Gly Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 218:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 218:
Asp Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 219:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 219:
Leu Ser Ala Pro Glu Lys Asp Lys Phe Phe Ala Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 220:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 220:
Glu Ser Tyr Met Val Pro Phe Ile Pro Leu Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 221:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 221:
Gln Ser Ser Met His Asn Ala Leu His Ile Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 222:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 222:
Thr Leu Ala Lys Met Met Ser Ser Asp Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 223:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 223:
Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr
1 5 10
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(2) INFORMATION FOR SEQ ID NO: 224:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 224:
Gln Arg His Arg Pro Leu Gln Glu Val Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 225:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 225:
Gln Arg His Arg Pro Leu Gin Glu Val Tyr
1 5 10
(2) INFORMATION FOR SEQ ID NO: 226:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 529 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 226:
Met Leu Leu Ala Val Leu Tyr Cys Leu Leu Trp Ser Phe Gln Thr Ser
1 5 10 15
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Ala Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu
20 25 30
Lys Glu Cys Cys Pro Pro Trp Ser Gly Asp Arg Ser Pro Cys Gly Gln
35 40 45
Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala Pro
50 55 60
Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Asp Arg Glu Ser Trp
65 70 75 80
Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser Gly Asn Phe Met
85 90 95
Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys
100 105 110
Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala
115 120 125
Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr
130 135 140
Ile Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys
145 150 155 160
Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu Phe
165 170 175
Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser
180 185 190
Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu
195 200 205
Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln Glu Ile Gln Lys
210 215 220
Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp
225 230 235 240
Ala Glu Lys Cys Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Gln His
245 250 255
Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp
260 265 270
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Gln Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu
275 280 285
Cys Asn Gly Thr Pro Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His
290 295 300
Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe
305 310 315 320
Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala
325 330 335
Asn Phe Asp Phe Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr
340 345 350
Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile
355 360 365
Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro
370 375 380
Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp
385 390 395 400
Leu Gln Arg His Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala
405 410 415
Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu
420 425 430
Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp
435 440 445
Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile
450 455 460
Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser Trp Leu Leu Gly
465 470 475 480
Ala Ala Met Val Gly Ala Val Leu Thr Ala Leu Leu Ala Gly Leu Val
485 490 495
Ser Leu Leu Cys Arg His Lys Arg Lys Gln Leu Pro Glu Glu Lys Gln
500 505 510
Pro Leu Leu Met Glu Lys Glu Asp Tyr His Ser Leu Tyr Gln Ser His
515 520 525
Leu
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