Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 95/08572 ~2 ~ 7~ PCT/US94/10728
K~CTION OF T--CEI-L RECEPTOR8 AND ANTIGEN IN AUTO I ~ r~u~:
DT~ R
ACKNOWLEDGEMENTS
This invention was supported in parts by grants from
NIH. The U.S. Government may have rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of
application Serial No. 08/066,325, filed May 21, 1993 which
is a file wrapper continuation of application Serial No.
07/877,444, filed April 30, 1992, which is continuation-in-
part of application Serial No. 07/517,245 filed May 1, 1990,
10 and International Application Serial No. PCT/US91/02991
filed May 1, 1991. This application is a continuation-in-
part of Application Serial. No. 07/379,500 filed July 12,
1989, which is a continuation-in-part of Application Serial.
No. 07/086,694, filed August 17, 1987, which disclosures are
specifically incorporated herein by reference.
INTRODUCTION
Technical Field
The field of the subject invention is diagnosis and
treatment of diseases, particularly autoimmune diseases.
Backqround
Autoimmune diseases are a result of a failure of the
immune system to avoid recognition of self. The attack by
the immune system of host cells can result in a large number
of disorders, including such neural diseases as multiple
Wos5/o8572 ~72~12 PCT~S94/10728 ~
sclerosis and myasthenia gravis diseases of the joints, such
as rheumatoid arthritis, attacks on nucleic acids, as
observed with systemic lupus erythematosus and such other
diseases associated with various organs, as psoriasis,
juvenile onset diabetes, Sjogren's disease, and thyroid
~s~sD. These ~;~eAses can have a variety of symptoms,
which can vary from minor and irritating to life-
threat~in~.
Despite the extensive research efforts that have been
involved with elucidating the basis for these diseases, the
~;seA~e~ for the most part have been recalcitrant to an
understAn~ing of their etiology in the development of
therapeutic modes. Many of the diseases are believed to be
associated with lymphocytic involvement, which can result in
lS attack and degradation of proteins, cytotoxicity, and the
like.
In the case of cancer, tumor infiltrating lymphocytes
(TIL) are believed to be part of the body's defense
mechAnism to destroy the tumor. Efforts have been made to
expand T-cells found in tumor tissue and return the culture
expanded cells to the host.
The complexity of the immune system has been a daunting
barrier to an understanding of the autoimmune diseases and
the immune response to neoproliferative diseases. In
attempting to understand the mechanisms involved with the
immunological response, there is substantial interest in
understAn~ing in what manner the system degenerates to
attack self. By understAn~;ng the relationships between the
components of the immune system, the manner in which the
immune system distinguishes between self and non-self, and
the components the immune system associated with a
particular disease, ways may be developed to diagnose
individuals who may be susceptible to autoimmune diseases
and provide therapies to protect such susceptible
individuals from autoimmune disease during its onset and
~ WO95/08572 PCT~S94110728
2 ~ 7~
-3-
during its progress or to treat individuals with specific
T-cells.
Relevant Literature
Multiple sclerosis (MS) is an inflammatory disease of
the central nervous system characterized by myelin
destruction (McFarlin and McFarland, New Engl. J. Med.
307:1183- 1251 (1982)). At the site of demyelination,
depletion of oligodendroglia cells and proliferation of
astrocytes is usually observed. Raine and Traugott,
Immunoregulatory ProcPssec in Experimental Allergic
Encephalomyelitis and Multiple Sclerosis, Elsevier, New
York, 151-212 (1984); Prineas and Wright, Lab. Invest
38:409-421 (1978). There is an accumulation of
morphologically identifiable macrophages, plasma cells and
T lymphocytes, characteristic of an inflammatory response in
the brain. Prineas, Handbook of Clinical Neurology, 3,
Elsevier, New York, (1985) pp. 213-257. MHC Class II,
positive antigen presenting cells and activated T-cells
secreting various cytokines are present. Woodroofe et al.,
J. Neurol. Sci. 74, 135-152 (1986); Hafler and Weiner, Ann.
Neurol. 22, 89-93 (1987); Hafler and Weiner, Immunol. Rev.
100, 307-332 (1987); Hoffman, J. Exp. Med. 170, 607-612
(1989). Several lines of evidence suggest that
T lymphocytes migrate from the peripheral blood through the
CNS compartment and participate directly in the promotion of
brain lesions. Hoffman et al., J. Immunol. 136, 3239-3245
(1986); Traugott, J. Neuroimmunol. 4, 201-221 (1985). In
studies of MS plaque tissue with monoclonal antibodies, it
has been shown that the majority of T-cells have the helper
inducer CD4 positive phenotype. Sobel et al., J. Exp. Med.
167, 1313-1322 (1988). Also, by restriction fragment length
polymorphism analysis, T-cell receptor Va and V~ genes have
been shown to contribute to the genetic control of
susceptibility to this disease. Beall et al., J,
Neuroimmunol. 21, 59-66 (1989); Seboun et al., Cell 57,
Woss/o8s72 ~ PCT~S94/10728
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1095-1100 (1989); Oksenberg et al., Proc. Natl. Acad. Sci.
USA 86, 988-992 (1989) describe the use of TIL cells in the
treatment of tumors (Barth et al., J. Immunol. 1~4, 1531
(1990) ) .
HLA-DR2Dw2 is associated with increased susceptibility
to MS. Terasaki et al. Science 193:1245- 1247 (1976).
Susceptibility to MS has been associated with certain MHC
Class II genes. Oksenberg and Steinman, Current Opinion in
Immunology 2:619-621 (1990). At the cellular level,
oligoclonality of T-cells has been described in the
cerebrospinal fluid (CSF) of MS patients. Lee et al., Ann.
Neurol. 29:33-40 (1991). Oksenberg et al., Nature
3~5:344-346 (1990) describes the use of PCR to amplify TCR
Va sequences from transcripts derived from MS brain lesions.
Wucherpfennig et al. Science 2~8:1016-1019 (1990) and Ota
et al., Nature 3~6:183 (1990) report studies of T-cell
clones in man that recognize myelin basic protein.
SUMMARY OF THE INVENTION
The relationship of particular sequences of the Va
and/or Vb subunits of the helper T-cell receptor is
esta~lished by identifying invasive T-cells in tissue from
autoimmune or neoplastic lesions. The particular variable
regions may be identified from germline rearrangement, mRNA
or the T-cell receptor subunit sequences. The oligoclonal
regions of the T-cell receptor ("TcR") or the cells having
such regions are then used for therapeutic applications for
the treatment of the diseases.
With disease causing T-cells, peptides with the
sequence of the T cell receptor or peptides with the
sequence of the antigen bound by the T cell receptor may be
used by themselves to block binding, for the formation of
antibodies, or the preparation of cytotoxic molecules
specific for the target T-cell. The amino acid sequence of
the peptides may be modified for improved activity.
Wo9~ PCT~S94/10728
Sequences based on motifs associated with the disease are
provided for diagnosis and therapy.
In conjunction with the restricted repertoire of the
TcR, the MHC phenotype is also relevant to susceptibility to
particular autoimmune and neoplastic diseases. By screening
for the presence of the susceptible phenotype, counseling
and monitoring can be provided to minimize the occurrence
and/or severity of the disease.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for determining
T-cell receptor variable regions related to autoimmune
diseases. Also specific MHC profiles may be identified
associated with specific autoimmune diseases, which will
provide for monitoring of such patients to identify
initiation of symptoms of such disease.
For autoimmune disease, by identifying specific T- cell
receptor (TcR) variable regions associated with the disease,
therapies are employed to inhibit the attack of the T-cells
having such variable regions on the target cells or
proteins. The therapies may involve ablation of T-cells
carrying the particular variable regions, administration of
agents associated with inhibition of the T-cell receptor to
the target cell, or prevention of the degenerative effects
of the binding of the T-cell to the target cell or protein.
These agents include peptides with the sequence, or a
modified sequence, of the T cell receptor or the antigen
bound by the T cell receptor, which agents may be used to
block binding, for the formation of antibodies, or the
preparation of cytotoxic molecules specific for the target
T-cell. For neoproliferative diseases, the T-cells having
the appropriate T-cell receptor may be concentrated and
expanded and returned to the host.
The T-cell receptor has two subunits involved in
binding, either a and b, or g and d. The variable regions
associated with the subunits have a similar organization to
Woss/08572 ~ p~ ~ PCT~S94110728
--6--
those of the immunoglobulins, the b and g subunits having a
variable region which comprises exons associated with the V,
D and J regions, while the a and d subunits comprise exons
associated with the V and J regions. By rearrangement of
germline DNA, the exons are joined to the constant or
conserved region and by subsequent splicing of the
meCc~nger RNA, an open reading frame is achieved which
encodes the subunit. Depending upon the particular genetic
inheritance of the host, the spectrum of the variable region
loci of an individual will be different from other
individuals. In addition, not all variable region exons
present may rearrange to form a functional T-cell receptor
subunit.
For some autoimmune ~iseAces, one may wish to
distinguish between a lesion associated with a chronic
condition or an acute condition. For example, for multiple
sclerosis, the chronic condition is exemplified by the
preC~ce of large numbers of macrophages and a relatively
low number of T-cells in comparison to acute phase which has
lower levels of macrophages and higher levels of T-cells.
The cells may be identified in accordance with conventional
histocytochemistry t~hn;ques, using antibodies to surface
markers, as appropriate.
By determining the loci which are rearranged to form
functional variable regions, which variable regions are
associated with autoimmune lesions, one can diagnose the
nature of an autoimmune disease, establish the existence of
a chronic episode, and treat the disease, prophylactically
or therapeutically, by inhibiting the degenerative effect of
the T-cells. By determining the loci which are rearranged
to form functional variable regions effective against
tumors, these cells may be used to combat the tumor.
T-cell receptors may be divided into two categories:
the CD4 helper-inducer T-cell receptors, which T-cell
receptors bind to Class II MHC; and the CD8 suppressor-
cytotoxic T- cell receptors, which T-cell receptors bind to
~ W09St08s72 PCT~S94/10728
21~
Class I MHC. For the most part, the T-cells associated with
such diseases as multiple sclerosis are the CD4 positive
phenotype, though CD8 csn also play a role.
The autoimmune diseases of significant prevalence
include multiple sclerosis, associated with destruction of
myelin and glial cells, rheumatoid arthritis, associated
with joint lesions, systemic lupus erythematosus (SLE),
associated with the deposition of autoantibodies and immune
complexes, psoriasis, pemphigus vulgaris, juvenile onset
diabetes, associated with destruction of beta cells in
islets of Langerhans, Sjogren's disease, thyroid disease,
Hashimoto's thyroiditis, myasthenia gravis, as well as many
others.
The variable (V) loci by themselves or in conjunction
with the J loci of the T-cell receptors of T- cells found at
the site of the lesion may be identified in a number of
different ways. Particularly, the lesion or plaque is
isolated and total RNA, from which cDNA may be prepared, or
DNA is prepared according to standard procedures. To
provide for more accurate results, the cDNA is amplified by
any convenient t~çhn;que, such as the polymerase chain
reaction (PCR), cloning, or the like. In the case of the
polymerase chain reaction, primers are employed which will
identify the particular variable region which either has
been expressed as identified by cDNA, or has been
rearranged, so as to be associated with a J and C region.
Primers may be selected in accordance with the known
sequences of conserved regions of the T-cell receptor
subunits. It is found, that there will usually be 1 and no
more than about 7, usually not more than about 5, generally
only about 3 common, variable regions of the T- cell
receptor subunits associated with the lesions. Therefore,
with neural disorders, where the tissue is available from a
deceased or from a biopsy having the particular disorder,
one may relate the presence of T-cells in the lesions to the
~ e and, further, relate the particular V regions and J
W095/08572 PCT~S94/10728
-8-
regions associated with the T-cells present in the lesion
with the MHC antigen type of the deceased. In this ~nner~
one may provide for identification of the variable regions
of humans suffering from the disease with the MHC type and
be able to treat the ~;s~Ase accordingly. With genomic DNA,
one would establish the presence of the rearrangement in the
DNA by having primers which relate to substantially
conserved regions of the variable region and the joining of
the constant region, either coding or non-coding regions.
Alternatively, by cloning, one could sequence the DNA and
establish the identity of the variable region. Isolation of
mRNA from the tissue in question, reverse transcription to
cDNA and then amplification and identification of the
rearranged V-C product will also define the disease related
marker. Similarly, with neoproliferative tissue, the tissue
may be isolated and the T-cells effective for combating the
neoproliferative cells identified.
Instead of the nucleic acid as the basis for the
diagnosis, by having a battery of monoclonal antibodies,
various t~chn;ques may be employed for identifying the
binding of the antibody to the T-cells. Thus, flow
cytometry, e.a. a FACS scan, may be employed, where the
antibodies are labeled with a fluorescer and those T-cells
to which the antibodies bind would then identify the
particular variable region.
A 100% correlation is normally not to be expected, nor
will it be necessarily achieved. It will usually be
satisfactory that in at least 60%, preferably 70%, of the
hosts positive for the disease, the shared variable region
locus associated with the disease is present in a population
of host T-cells, particularly in rearranged form.
Similarly, in fewer than about 50%, preferably in fewer than
about 30% of the hosts which do not present the symptoms of
the ~;s~se, the rearranged variable region is absent.
These percentages should be based upon a statistically
significant number of hosts.
W095/~8572 2~ ~ 51 2 PCT~S9J/1~728
T-cells with a shared variable region will express the
variable region gene from a single V region subfamily. Gene
segments which display greater than 75% nucleotide sequence
similarity over the length of the V region gene are
considered members of the same subfamily (Crew, et al (1981)
Cell 25:59-66). The sequence similarity is calculated for
the V region exon itself, and does not include sequences
encoded by the D and J segments and N additions.
Depending upon the particular disease, various tissues
may be employed for identifying the T-cells. For neural
diseases such as multiple sclerosis, brain plaques or
cerebrospinal fluid may be employed as a source of the
T-cells. Similarly, for myasthenia gravis, muscle, thymus
tissue or T-cells responsive to acetylcholine receptor may
be employed. For rheumatoid arthritis, the synovium may be
employed. For other diseases such as thyroiditis, or
Grave's disease, thyroid tissue, or in systemic lupus
erythematosus, kidney tissue may be employed as the source
of T-cells.
Once the rearranged variable region(s) are established,
one may then identify T-cell variable region allele(s) or
T-cell receptors in a host, in association with the HLA or
MHC restriction, as indicative of the propensity for the
disease or the existence of the disease or the
neoproliferative cell responsive T-cells. Where it appears
that the disease is associated with a clonal or oligoclonal
population of T-cells, the presence of one or more of the
T-cell receptors having the rearranged variable regions
associated with the disease will indicate the greater or
lesser likelihood for the occurrence of the disease. A
clonal population will have 100% of the T-cells expressing
one V. or Vb region gene and one VDJ segment. An oligoclonal
population will have from less than 100% of the T-cells
expressing one V~ or Vb region gene and one VDJ segment, to
50% of the T-cells expressing no more than 12 V~ or Vb region
WO95/0857Z 2~ PCT~S94/1072~ ~
--10--
genes, with no more than six VDJ segments per expressed V
region gene.
For diagnosis of autoimmune diseases, either the
nucleic acid or antigen may be detected. For nucleic acid
detection, DNA or RNA in cells may be isolated by any
convenient means and by employing appropriate probes, in
conjunction with t~ch~;ques, such as Southern transfer, dot-
blots, or the like, the presence of the rearranged V region
may be detected. Depending upon the nature of the disease,
there may be an opportunity for prophylactic intervention to
reduce the potential for the disease occurring.
If one wished to determine the number of cells which
are expressing the T-cell receptors associated with the
disease, this can be achieved in a number of ways. The
messenger RNA may be isolated from T-cells and probed with
an appropriate probe for the V gene region. By employing
Northern t~chn;ques, one can detect the presence of the
messenger encoding the T-cell receptor 2nd obtain a
qualitative value for the amount of T-cell receptor being
expressed containing the particular V region gene.
Alternatively, one may prepare cDNA from the messenger and
using the polymerase chain reaction, amplify the amount of
messenger and determine the number of T-cells expressing the
particular variable region in this manner.
More conveniently, one may use antibodies as described
previously which are specific for the V region and/or the J
region alleles or potentially the combination V-J for the a
subunit. In this way, one may detect the V region and the
J region, With the ~ subunit, intervention of the D region
makes it unlikely to find a antibody which would be specific
for the VDJ se~uence, but the V and/or J region may be
detected individually.
Antibodies may be prepared in accordance with
conventional ways, particularly employing the monoclonal
antibody techniques as described, for example in U.S. Patent
~ W095/08572 PCT~S94tlO728
21 7~
--11--
Nos. 4,690,893; 4,713,325; 4,714,681; 4,716,111: and
4,720,459.
Any of a number of techniques may be employed for
identifying the presence of a T-cell receptor binding to the
particular monoclonal antibody or anti-serum. A wide
variety of labels have been used for detection, such as
particles, enzymes, chromophores, fluorophores,
chemiluminescence, and the like. Any particular label or
techn; que which is employed is not critical to this
invention and any convenient technique may be employed. The
t~chn i ques may be either competitive or non-competitive
methodologies, including sandwich methodologies. The cells
will usually be lysed to provide membrane-free proteins in
accordance with conventional t~chn;ques. Cellular debris
may be removed and the protein extracted and harvested.
Alternatively, intact cells may be employed and detected by
fluorescence activated cell sorting or the like.
For therapeutic purposes, there may be an interest in
using human antibodies. Normally, one will not be permitted
to immunize a human host with the T-cell receptor or
fragment thereof to activate T-cells specific for the
sequence of interest. However, there are alternatives, in
that mice or other lower mammals may be immunized, and the
genes encoding the variable regions of the antibodies
specific for the T-cell region of interest isolated and
manipulated by joining to an appropriate human constant
region, and optionally, the complementary determining
regions (CDR) used to replace the CDRs of a human antibody
by genetic engineering. The resulting chimeric construct,
comprising a lower mammal variable region or CDRs and a
human constant region may then be transformed into a
microorganism or mammalian host cell in culture,
particularly a lymphocyte, and the hybrid antibodies
expressed. Of particular interest would be IgG constant
regions. See, for example, EPA 85.305604.2. Also recent
techniques suggest random association of immunoglobulin
Wo95/08s72 PCT~S94110728
-12-
genes from a human host for expression in a non-human cell
host e.a. prokaryotic, and screening for affinity.
In some instances, it may be satisfactory to use mouse
antibodies, where tolerance can be achieved or some degree
of immune suppression may be involved. ~mmune suppression
may be achieved with cyclosporine, irradiation, anti-leu3
(anti-CD4) (U.S. Patent No. 4,681,760), or the like.
The antibodies may be used in a variety of ways, for
example, for inhibiting binding between the T-cell and the
target cell, for killing of T-cells, or for isolating the
T-cells. In the first situation, the entire antibody may be
administered, or Fab fragments, or even only the Fv region.
By removing all or a portion of the constant region, there
may be a reduction in the immune response. For selectively
killing the T-cells carrying the particular V region, one
may use a variety of immunotoxins, which may include the
antibody or specific binding fragment thereof, bonded to all
or a portion of a plant toxin, such as ricin, abrin, etc.,
or diphtheria toxin. By employing an appropriate antibody
isotype, e.a., IgM or IgG3, the complement cascade may be
enlisted. Alternatively, a radioactive substituent may be
used which provides for a lethal dosage upon binding of the
antibody to the host cell. Another choice is to use an
antibody or fragment thereof conjugated with a cytolytic
agent for specific elimination of the undesired T-cells.
Finally, the T-cell can be removed by extracorporeal means,
such as plasmapheresis, where the plasma may be passed
through or over antibodies bound to a support, with the
undesired T-cells being selectively removed.
For therapeutic purposes, the antibody may be
formulated with conventional pharmaceutically or
pharmacologically acceptable vehicles for administration,
conveniently by injection. Vehicles include deionized
water, saline, phosphate-buffered saline, Ringer's
solution, dextrose solution, Hank's solution, etc. Other
additives may include additives to provide isotonicity,
~ W095/08S72 PCT~S94110728
~1 ~2~ ~
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buffers, preservatives, and the like. The antibody or
derivative thereof will usually be formulated in purified
form at concentrations in the range of about 0.05 to
lO ~g/ml. The antibody may be administered parenterally,
typically intravenously or intramuscularly, as a bolus,
intermittently or in a continuous regimen.
Desirably, the dose should deplete or at least bind
about 75% of the undesired T-cells, preferably at least
about 90%. Typical doses for adult humans will be in a
range of about lO to lO0 mg. Doses for children or other
animal species may be extrapolated from the adult human dose
based on relative body weight.
Instead of antibodies, oligopeptides may be employed,
having the same or substantially the same sequence as the
oligopeptide sequence identified as being diagnostic of the
autoimmune disease. These sequences will be oligopeptides
of at least 8, usually at least lO more usually at least 12,
and preferably at least 18 amino acids, and generally not
more than about 60 amino acids, usually not more than about
50 amino acids, of the T-cell receptor subunit chain. While
the entire subunit(s) may be employed, usually not more than
about 50 number % of the amino acids will be employed,
particularly excluding the conserved or constant region.
All or at least a portion of the variable region, capable of
binding to the target protein (the protein recognized by the
T-cell receptor) and/or MHC antigen, will be present. The
MHC antigen may be by itself or bound to a fragment of the
target protein, which fragment will normally include the
particular locus associated with the disease.
Of particular interest is the demonstration that the
variable rearrangements of T-cells associated with specific
sites of autoimmune ~is~ have a restricted repertoire, so
that a relatively small number of T-cell variable regions of
both the a and b subunits will be observed. Furthermore, as
will be discussed below, a limited repertoire of MHC type
will be associated with the disease and as to these
-
W095/08572 ~ ~ 2~ PCT~S94/10728
- repertoires, there will generally be a restricted repertoire
of T-cell receptor variable regions.
In particular, with MS patients it is found in the
brain, particularly in the plaques associated with the
disease, that T-cells may be isolated which have rearranged
germline DNA to provide for expression of the T-cell
receptor. This may be contrasted with brains of normal
healthy individuals, where the cells found in the brain have
unrearranged germline DNA.
By identifying a particular Class II haplotype or
molecular phenotype, one can then identify particular Va and
Vb variable regions assoGiated with an autoimmune disease.
Once the autoimmune associated T-cell receptor or variable
regions are identified, one may than use the various
therapies which are described in the subject application for
prophylaxis or treatment.
As previously indicated, of particular interest are
specific V regions and J regions of both the a and b ~h~ i n.~
of the T-cell receptor. For sequences of human and mouse V
regions, see Concannon, et al., Proc. Natl. Acad. Sci. USA
83:6598-6602 (1986). Of the regions of interest of CD4
T-cell associated with multiple sclerosis, among Va families
are 8-10, 12 and 16, particularly 10. Other regions of
interest include 1, 5 and 7.
Of the J regions, of particular interest are Ja
regions, more particularly the Ja region GGGTACCr.A~.ATGACGAA-
CCCACCTTTGGGACAGGCACTCAGCTAAAAGTGCAACTC.
Of the Vb regions, are the families 5, 6, 7 and 12 more
particularly 5 and 6, and of the Vb 5 family, particularly
30 5.1 and 5.2.
In addition, certain amino acid sequence motifs are
seen in the CDR3 region of the TCR. The sequences appear
more frequently than would be expected and follow the
sequence LCAS(S) (where the parentheses indicate the
optional presence of the amino acid), particularly LCASS.
The next amino acid will be one having a long chain,
~ W095/08572 PCT~S94110728
21 7~
-15-
neutral, preferably hydrocarbon, such as L, I and V, or Q,
particularly L. The next amino acid may be neutral or
charged positively or negatively charged, may be short (2-4
carbon atoms) or long (5 to 6 carbon atoms), and may include
G, A, P, L, I or V, as well as D, E, K, and R, particularly
G, A, V, D and R, more particularly R and G, which are
involved with sequences specific for the MBP sequence 87 to
106. The next amino acid will frequently be G, A or S, so
that the motif will be L-X-(G, A or S), particularly (G).
An alternative motif is P following LCAS(S), where the next
amino acid is PT.
These peptides may be isolated free of flanking amino
acids or may include up to a total of about 20 flanking
amino acids. Alternatively, flanking regions may be
provided which are not naturally occurring sequences. The
peptides may be modified and used as described previously.
For the most part, the sequence will be derived from Jb
l and 2, more particularly l.2, l.6, 2.l, 2.3, 2.5, 2.6 and
2.7.
20This same approach may be used to identify the
rearrangement and expression of T-cell receptor subunits,
both for restriction as to Class I and II MHC, to identify
sequences associated with pathogenesis.
The presence of pathogenic T-cells may be detected with
various probes, such as AGC CTA CGC; AGC TTG CGC; AGC CTG
CGG; TTG CGC; and AGC CTA CGC AGC TTG CGC AGC CTG CGG TTG
CGC (specific for LRGA). The probes may be as few as 6
nucleotides and as many as 30 nucleotides, usually being not
more than about 3l nucleotides.
30The peptides may serve as vaccines, to obtain an immune
response, to ablate the pathogenic T-cells. Immune
responses may be achieved in accordance with conventional
ways. The peptides may be conjugated to an immunoassay,
introduced into a viral vector so as to be fused to the
envelope or capsid protein, fused to proteins using
recombinant technology, and the like.
W09s/08s72 ~ 2 PCT~Ss4/10728
The oligopeptide may be joined to other peptides,
proteins, or polyalkyleneoxy compounds for a variety of
reasons, such as to provide for enhanced stability,
toleration, ease of preparation or purification, or the
like. The subject peptides may be used to inhibit the
binding of the T-cell receptor to the target peptide.
The peptide may be formulated in substantially the same
manner as described for the antibodies. The amount of the
active ingredient administered will vary widely depending on
the particular composition, the particular host, the number
and frequency of administrations, the manner of
administration, etc. Usually there will be from about 0.01
to 10 ~g/kg of host, more usually from about 0.05 to 5 ~g/kg
of host, where the concentration may range from about
10 ~g/ml to about 1 mg/ml.
The manner of administration may vary widely, depending
upon the formulation and nature of the active ingredient.
~; n; ~tration may be parenteral, intravascular,
peritoneally, subcutaneous, oral, etc., may employ
catheters, pumps, constant diffusion membranes, etc.
The oligopeptides may be prepared in a variety of ways,
conveniently, in accordance with conventional synthetic
procedures. Where larger sequences are involved, such as 30
amino acids or more, recombinant DNA techniques may be
employed, where the gene may be synthesized in accordance
with conventional ways, such as commercially available DNA
synthesizers, e~pAn~ed employing the polymerase chain
reaction, and then inserted into an appropriate vector
having the necessAry transcriptional and translational
initiation and termination regions. The resulting vector is
then transformed into a host in which the expression vector
is replicated and functional expression is obtained. The
product may be secreted and harvested from the medium or
when not secreted and retained cytoplasmically, the cells
are harvested, lysed, and the desired protein isolated and
purified in accordance with conventional ways.
W095/08572 PCT~Ss4/10728
Instead of the oligopeptide, anti-idiotype antibodies
may be employed. By preparing a monoclonal antibody to the
idiotype of the antibody to the subject oligopeptide, the
anti-idiotype may mimic the oligopeptide and serve to
compete for the MHC with the T-cell receptor for the MHC
antigen. The anti-idiotype may provide greater stability on
administration, as well as other advantages.
T-cells can be inhibited from reacting with MHC
antigens which may result in pathogenesis by employing
ribozymes specific for one or both subunits of the T-cell
receptor. For the Class I TcR, the ribozyme would be
directed against the a- subunit, while for the Class II TcR,
either the a- and/or the b-subunit could be the target. The
ribozyme would comprise a sequence having complementarity to
the sequence encoding the CDR3. Unnatural nucleotides may be
used to enhance stability, such as the presence of thio
linkages or replacement of oxygen in the phosphate group
with carbon groups or the like. Alternatively, antisense
seguences could be used which were specific for the target
subunits. Administration of the ribonucleotides would be in
accordance with conventional means in relation to the
transport of the ribonucleic acid across the blood-brain
barrier.
The protective compositions may be used in vitro or n
vivo by adding to groups of cells comprising lymphocytes and
cells associated with the autoimmune disease or target
protein. By adding the protective composition, usually a
protein such as an antibody or peptide having the
appropriate variable region sequence, one can prevent the
destruction of the cells and/or target protein. Tissue
destruction may result in the loss of cells, or in the loss
of function in the cells that are present. Where cells are
involved, the T-cells will be restricted by the major
- histocompatibility antigen of the target cells, with the
target cells usually being syngeneic with the T-cells.
W O 95/08572 PC~rnUS94/10728
-18-
Other oligopeptides are provided which are identified
as inducing an autoimmune response to a self antigen, or a
portion thereof, and are capable of binding to an MHC
antigen of a host susceptible to the autoimmune disease.
The compositions may be employed to enhance protection, by
serving to tolerize the host and prevent immune attack
against the endogenous protein or cell producing the
endogenous protein. For toleration, the subject peptides
may be conjugated to syngeneic spleen cells, or be linked to
an innocuous immunogen to which the host has been previously
immunized, such as tetanus toxin, bovine serum albumin, etc.
Adjuvants are normally avoided.
Sequences which may be employed for toleration will be
sequences from proteins endogenous to the host involved with
autoimmune diseases, which include such proteins as the
neurological proteins found in the peripheral nervous system
(PNS) or the central nervous system (CNS) and the
acetylcholine receptor (AChR). These proteins are
designated as PO which is found in the PNS and CNS, Pl, in
myelin basic protein, the predor;n~nt CNS protein of myelin,
P2, a predominant PNS myelin protein, PLP, a proteolipid
protein, a PNS and CNS myelin constituent, and the
acetylcholine receptor. Pl is involved in post-immunization
encephalomyelitis and may be involved in multiple sclerosis.
P2 is involved in post-;~munization neuritis (Guillain-Barre
syndrome) a major complication, for example, in the swine
flu irmllnization program and the acetylcholine receptor is
involved in myasthenia gravis and may play a role in post-
immunization myositis. Other autoimmune diseases, such as
rheumatoid arthritis, lupus erythematosus, myasthenia
gravis, multiple sclerosis, post-immunization myositis,
post-immunization neuritis, and juvenile diabetes are
treated with the same methods.
The particular protein of interest will be screened for
the presence of a subject motif, and one or more sequences
including the motif selected. Where the histocompatibility
~ W095/08572 PCT~S94/10728
2~ 72~12
--19--
genotype (haplotype) of the intended recipient is known, one
se~uence may be preferred over another. However, where the
haplotype is not known, or the composition may be
administered to a number of different hosts, it will
frequently be desirable to combine a number of the sequences
as oligopeptides in the same composition. The oligopeptides
may be present as the individual peptides, or may be joined
together in a single sequence, with or without intervening
bridges, where any bridges will be other than the naturally
occurring intervening sequences of the immunogen.
Desirably, any such sequence would have fewer than about 100
amino acids, more usually fewer than about 60 amino acids.
If there are a plurality of motifs present in the immunogen,
all or fewer than all of the sequences including the motifs
may be employed in a single composition. Usually, there
will be not more than ten different motif comprising
oligopeptides, more usually not more than about six
different oligopeptides in the composition.
There will usually be more than one partial sequence in
the immunogen comprising the subject motif. The
oligopeptide comprising the subject motif may be from any
site of the immunogen sequence, that is N-terminal or
C-terminal proximal or central, where the oligopeptide
sequence will normally be substantially homologous with from
9-15 amino acids of the immunogen sequence, although longer
sequences may also be employed. Usually, the difference in
homology between the natural sequence and the oligopeptide
which is employed will be not more than two lesions, more
usually not more than 1 lesion, which may be insertions,
deletions, or conservative or non-conservative
substitutions. The composition may comprise one or more
different oligopeptides, with the following sequence:
charged amino acid, two hydrophobic amino acids, and at
least one of the next two amino acids being a polar amino
acid, where the charged or polar amino acid may be
substituted by glycine, usually not more than one being
W O 95/08572 PC~rrUS94/10728 ~
5~
-20-
substituted by glycine. The charged amino acids are
aspartic acid, glutamic acid, lysine, arginine, and
histidine (D, E, K, R, H). The hydrophobic amino acids are
alanine, proline, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, and tyrosine, that is both the
aliphatic and aromatic neutral or substantially neutral
amino acids having not more than one heteroatom, e..,
chalcogen, on the side chain (A, P, V, L, I, M, F, W, and
Y). The polar amino acids will be the charged amino acids,
as well as serine, threonine, asparagine, and glutamine (S,
T, N, and Q).
Usually, the motif sequence present in the oligopeptide
will be at other than the C-terminus of the oligopeptide,
desirably being at the N-terminus and not closer to the
C-terminus than the center of the sequence, where the
second, third, or fourth amino acid of the motif (depending
upon whether there are four or five amino acids in the
motif) is the central amino acid. The N-terminal amino acid
may be the same as the inducing peptide or may have an
internal amino acid of the inducing peptide as the N-
terminal amino acid of the oligopeptide.
Alternatively, the oligopeptide sequence may be derived
by binding assays, where an oligopeptide is selected which
is capable of forming a ternary complex with the disease
inducing T-cell receptor and MHC molecule. The
oligopeptides will usually have at least about nine amino
acids and need not have more than about 30 amino acids,
usually not having more than about 20 amino acids. The
compositions are prepared in a variety of ways in accordance
with conventional synthetic techniques, particularly
automated synthesizers. The subject oligopeptides may be
joined covalently to other organic molecules, either
proteinaceous or non-proteinaceous.
The oligopeptide sequence may be distinguished from the
natural sequence. In some cases sequence analogs will be
prepared with stepwise substitution of the amino acids with
~ W O 95/08572 PC~rnUS94/10728
~1 7,~
-21-
alanine or valine, particularly alanine. Each of the
peptides may then be tested for their binding affinity to a
host Class II MHC associated with restriction of T-cells
involved with the autoimmune disease. Once a substitution
has been identified as reducing or enhancing MHC antigen
binding affinity, the same site may be further substituted
with other amino acids to determine whether further
enhancement may be achieved. Amino acids associated with T-
cell recognition may also be substituted to dim;n;~h T-cell
stimulation. Thus, those amino acids of the oligopeptide
associated with T-cell recognition may be modified to reduce
T-cell stimulation in vivo, while not significantly
affecting MHC antigen binding. In this way a strong
blocking oligopeptide may be achieved, without inducing the
autoimmune action of the T-cells. Generally the total
number of amino acids substituted will not exceed 3, ranging
from 1 to 3, usually 1 to 2. The reduction or enhancement
in binding will usually be at least about 10-fold, more
usually at least about 100 fold, and preferably at least
about 1000-fold.
An alternative oligopeptide analog will have a
functional group at the N-terminus, where the functional
group would generally be from about 1 to 6, usually l to 3
carbon atoms, and may be alkyl or acyl, such as methyl,
ethyl, propyl, isopropyl, hexyl, cyclohexyl, formyl, acetyl,
propionyl or the like.
Depending upon the particular application, the subject
compositions may be administered in a variety of ways, by
themselves or in conjunction with various additives. Various
carriers may be employed which are physiologically
acceptable, such as water, alcohol, saline, phosphate
buffered saline, sugar, mineral oil, etc. Other additives
may also be included, such as stabilizers, detergents,
= flavoring agents, thickeners, etc. The amount of active
ingredient administered will vary widely depending upon the
particular composition, the particular host, the number and
W095/08572 ~ ~ PCT~S94/10728
-22-
frequency of administrations, the manner of a~ini~tration,
etc. Usually, there will be from about 0.0l to l0 ~g/kg of
host more usually from about 0.05 to 5 ~g/kg of host, where
the concentration may range from l0 ~g/ml to l mg/ml.
Transplantation or MHC antigens have polymorphic
regions, where the individual alleles are associated with
specific hosts. For the most part, the host will be
diploid and heterozygous, so that each host will have two
haplotypes, meaning that there will be two different copies
of a particular transplantation antigen type from the same
locus, unless the host is homozygous at that particular
locus. Therefore, as to an individual host or a plurality
of hosts, mixtures of oligopeptides will usually be
employed. The subject oligopeptides may be a~; n; stered
concurrently or consecutively with the oligopeptides of the
T-cell receptor.
For identifying T-cells associated with combating
neoproliferative diseases, by identification of the variable
region associated with such T-cells, tissue may be obtained
by biopsy, surgical intervention or the like, the mRNA or
DNA may be isolated from the tissue sample and in the case
of mRNA, cDNA prepared in accordance with conventional ways.
The DNA sample may then be assayed using a primer specific
for the various Va or Vb regions to identify the predominant
variable region associated with the disease. Once a number
of patients have been screened, the likely variable
region(s) associated with a particular tumor will have been
identified. One may then use the affinity separations, e.q.
panning, affinity chromatography, etc. for isolating the
desired T-cells. The cells may then be expanded in culture
using a conventional growth medium, with or without the
addition of fetal calf serum, interleukins, e.q. IL-2, or
the like. The cells may then be harvested after expansion by
at least l00 and restored to the donor.
W095/08S72 ~ 21 7%~ PCT~S94110728
-23-
Alternatively, one may have a stored supply of T- cells
of the appropriate variable region and either matched or
unmatched as to MHC, particularly Class I. These cells may
then be administered by injection into the neoproliferative
- 5 tissue site, or into the blood stream. From 103 to 109 cells
may be a~i n; ctered to a human host in a physiologically
acceptable medium, the amount varying with the number of
cells available, the manner of administration, the
frequency of administration, and the like.
In addition to identifying T-cell variable regions
associated with specific autoimmune diseases such as
multiple sclerosis, one may in addition identify specific
molecular phenotypes associated with susceptibility to
autoimmune disease. The phrase "molecular phenotype" is
used instead of the designation "haplotype" since in the
absence of segregation analysis in families, it cannot be
certain whether these genes are all in a cis configuration
on a single chromosome. A phenotype associated with
multiple sclerosis is DRBl 1501, DQAl 0102 and DQBl 0602.
This phenotype may be further broken down into la,
associated with DPBl 0401 and lb 0402. This particular
phenotype is associated with V~ family rearrangements,
particularly V~ 5.l, 5.2 and 6.
By identifying MHC molecular phenotypes of individuals
suffering an autoimmune disease, one can establish certain
molecular phenotypes which provide for a susceptibility to
the disease. In the case of multiple sclerosis one can look
to see for rearrangement of T-cells and the prevalence of
the various families and members of the families of the Vb
and Va T-cell receptors. Once these are identified, one can
use this information to ablate the T-cells associated with
the disease. Thus, by isolating diseased tissue, e.a.,
plaques, and identifying T-cells having rearrangements, one
can identify a family of V~ and Va regions which are
associated with the disease, so that once the molecular
phenotype has been identified as providing susceptibility to
wogs~8s72 ~ PCT~Ss4/10728
-24-
autoimmune disease one will also know which T-cells to
ablate or target.
The upregulation of the MHC antigens on neural cells at
lesions affords an opportunity to direct specifically
various agents to the site of the lesion. Thus, one may use
a variety of radionuclides, nmr agents, or other agents
which provide a detectable signal for identifying the site
of the lesion. The agent providing the signal may be joined
to various carriers, such as antibodies for the MHC, or
fragments of antibodies, e.a. Fab, Fv, etc., immunodo~;nAnt
se~uences which are peptides of about l0, usually 12, amino
acids or more, which have a high affinity for the MHC
antigen. The presence of hematopoietic cells at the lesion,
will further augment the presence of the agent at the
lesion. The signal at the lesion should be greatly
enhanced, as compared to other regions of the brain.
Besides diagnosis, one may use the selective presence
of the MHC antigens for therapeutic purposes. Thus, one may
direct various therapeutic agents, by conjugating the agent
to the MHC antigen specific marker, by using the MHC antigen
specific marker for directing therapeutic agent containing
liposomes to the lesion site. Agents may include inhibitors
of TNFa, down regulators of MHC antigen expression, e.g.
~-interferon, TGF-~, and a-fetoprotein, peptides which block
the MHC antigen-TcR interaction, inhibitors of generalized
degradative pathways, such as reducing agents and superoxide
dismutase for singlet oxygen, etc.
Since the disease results in some permeabilization of
the blood-brain barrier, the opportunity to introduce drugs
across the barrier is enhanced. One may still use
injections at specific sites, permeabilizing agents, or
employ naturally occurring transport mechanisms.
The various agents will be administered in accordance
with their individual nature and in accordance with their
purpose. Inert physiologically acceptable carriers may be
employed, such as deionized water, saline, and the like.
Woss/o8s72 ~ PCT~S94/10728
-25-
Concentrations and the use of other additives or components
will be based on experience with like reagents and may be
determined empirically.
The following examples are offered by way of
illustration and not by of limitation.
W09~08572 ~ PCT~S94/1072
-26-
~PERIMENTAL
I. TcR Va ExPression in Brain Plaques of MultiDle Qclerosis
Samples were taken from brain plaques of 3 patients
with chronic progressive MS, and 3 controls (non MS). Total
RNA and cDNA (from 5 ~g RNA) were prepared according to
s~n~Ard procedurec. cDNA was also prepared from 1 ~g RNA
isolated from a pool of peripheral blood lymphocytes from
five different individuals, stimulated with 3 ~g/ml of PHA.
cDNAs were amplified by PCR for 40 cycles in the presence of
lO ~Ci of [32P]dATP (Amersham). Samples were analyzed by gel
electrophoresis with ethidium bromide to identify the
specific fragment band. After separation, bands were
excised and radioactivity was determined. Results are
expressed in median cpm. All TcR 5' primers amplify TCR
sequences from germ line DNA using a specific 3' Va primer
for each family. The following tables indicate the primers
employed and the results.
T~ble l.T-cell Receptor a Primers.
PrimerClone SequenceFomily
Member~
Va 1 HAP 10 5'-TTGCCCT~A~Ar~ATGCCAGAG-3' 1.1,
1.2, 1.3
Va 2 HAP 26 5'-GTGTTCCCAGAGGGAGCCATTGCC-3' 2.1, 2.2
Va 3 HAP 05 5'-GGTGAACAGTCAACAGGGAGA-3' 3.1
Va 4 HAP 08 5'-ACAAGCATTACTGTACTCCTA-3' 4.1
Va 5 HAP 35 5'-GGCCCTGAACATTCAGGA-3' 5.1
Va 6 HAP 01 5'-GTCACTTTCTAGCCTGCTGA-3' 6.1
Va 7 HAP 21 5'-AGGAGCCATTGTCt:A~ATAAA-3~ 7.1, 1. 2
Va 8 HAP 41 5'-GGA(~AC.AATGTGGAGCAGCATC-3' 8.1, 1. 2
WO9S/08572 1 7~ PCTnUS94/10728
-27-
Va 9 HAP 36 5'-ATCTCAGTGCTTGT~ATAATA-3' 9.1
Va 10 HAP 58 5'-ACCCAGCTGGTGGAGCAGAGCCCT-3' 10.1
Va 11 HAP 02 5'-AGAAAGCAAGGACCAAGTGTT-3' 11.1
Va 12 PGA 5 5'-CAGAAGGTAACTCAAGCGCAGACT-3' 12.1
5 ~Ab13)
Va 13 AB 11 5'-GCTTAT~-A~AACACTGCGT-3' 13.1
Va 14 AB 21 5'-GCAGCTTCCCTTCCAGCAAT-3' 14.1
Va 15 AC 24 5'-AGAACCTGACTGCCCAGGAA-3' 15.1
Va 16 AE 212 5'-CATCTCCATGGACTCATATGA-3' 16.1
Va 17 AF 211 5'-GACTATACTAACAGCATGT-3' 17.1
Va 18 AC 9 5'-TGTCAGGCAATGACAAGG-3' 18.1
Ca tAb51) PGA 5 5'-AATAGGTCGACACACTTGTCACTGGA-3' Ca
W095/08572 ~ ;, rCI/US94/10728
28-
e ~ ~ 8 o ~C C ~ 8
~0 ~ ~,~ O ~ ~r ~ ~ 5 ~ ~ . 9 ~ ~
Z .~ 8 ~ b
x o ~ o o ~ O ~` ~ ~ a
$ ~ ~ .e ~ 6
O, O ~ o ~ ~ Za ~ ~ ~ Eb~ ~
,~ ~ ~ _ _ o~ ~ ~c~ ~ 6 ~t ~i
S '`
g ~ ~_ O ~3 g 00 ~ 3
o ~ ~ o ~ 8 ~ " ~
ô 2 ~ o ~ ~ y
O o ~ O,C~ gy ~
~ $ ~0 ~ V~ r ~ ~ V O ~ ?~
._ ~ -- ~ ~ ~o ~ r~ æ v~ o -- -- ' o
~ O ~ ~ ~ ~ E ~ ~
`'' ~ 8 ` ' ~ 6 o E
o . = ~ o ~ 3 ~
o ~- o o 8 ~ 6
.Z C 0 6 6, ~1 E
~ r~ ~ ~ ~ 0 ~ ~ 6 ~ ~
Oo~ ~ æ ~ O ~ ~ e 6 ~ c~ o ~ ~
E- ~ o o~ ~ o ~ ~ c _~E-
~ ~ 3 ~ ~ 6 ~
8 ~ ,o ~o o ~ c.
oO ~ -- ~ ~ ~ ~ ~ ~ Y U~-Z5,0 ~ C~
z 3 ~r e~ 8 ~ i z ;3 ~ 3 tt c ~
~ r ~ ~ ~ g ~ o ~ ~
~ m ~ m ~ m ~ 9
L~ V ~ L E~ V ~ 1 8 ~ ~ C
-
W095/08S72 21 ~ - PCT~S94/10728
-29-
The T-cell receptors present in the brain of MS
patient 1 were amplified and subjected to gel
electrophoresis, where control brain cDNA, MS parietal
region brain cDNA, MS occipital region brain cDNA, PGA5, a
full length TcR a cDNA (Sim, et al., Nature 312, 771-775
(1984)) were comrAred. Two ~1 of cDNA was combined in a
100 ~1 reaction volume, with 1 unit of DNA Taq polymerase
(Perkin Elmer-Cetus), 10 ~1 10x reaction buffer, 50 ~M each
dNTPs, and 1 ~M of each primer. The PCR profile used was:
denaturation 96C for 60 sec., annealing 45C for 60 sec.
and extension 72C for 120 sec., for a total of 35 cycles on
a DNA Thermal Cycler (Perkin Elmer-Cetus). One tenth of
each sample was independently run in a 4% Nusiev gel, and an
appropriate size fraction was cut from the gel. The agarose
piece was frozen and thawed 3 times, and 2 ~1 of the
supernatant were directly reamplified with the same primers
for an additional 25 cycles. Actin sequences were
successfully amplied from brain cDNA, but not from the PGA 5
control using the following primers: (5'-AC~ r-ACGGACCACCGC-
CCTG-3', 5'-CACGTTGTGGGTGACGCCGTC-3'). Va and Ca
transcripts were amplified from both MS brain cDNA and PGA 5
templates, but not from the control MS brain cDNA with
primersAB 13-14(5'-CAGAAGGTAACTGCAGCGCAGACT-3',5'-TTGGGG-
ATCCAGAGCACAGAAGTATACTGC-3'), which include the restriction
sites PstI and BamHI and define a 286 bp fragment of the
Val2.1 region gene and AB 41-42 (5'-CAGAACCCTGACCCTGCCGTG-
TAC-3', 5'-GTGTCCACAGTTTAGGTTCGTATCTGT-3', which include a
SalI site and define a 340 bp fragment of the Ca region
transcript, respectively. Note that rearranged TcRa
sequences could be amplified from cDNA of the MS brain
prepared from the occipital region using the Val2.1 primer
AB 13 and Ca primer AB 42.
Junctional region sequences were derived from the
Val2.1-JC amplification from the occipital region of the MS
brain. 100 ~1 of the PCR reaction were phenol:chloroform
extracted twice with 1/1 volume, chloroform extracted once
W095/08572 ~2~ PCT~Ss4/10728 ~
-30-
with 1/1 volume, and dialyzed through a Centricon 30
(Amicon) with 2 ml of TE buffer for 30 min. at 5000 rpm.
The sample was recovered and the DNA digested for 3 hr. with
BamHI and PstI (or SalI). After digestion, the sample was
phenol:chloroform extracted and then chloroform extracted
once, passed through the same Centricon column as described
above, and brought to a final volume of 20 ~l. 3 ~l of
sample was put into a 10 ~l ligation with 200 ng of
Bam HI/PstI or BamHI/SalI cut M13mpl8 and ligated overnight
at 16~ with T4 DNA ligase (New England Biolabs).
Transformation into E. coli JM101 was done according to
standard procedures and positive plaques were selected by
hybridization to 32P-labelled TcR probes. 30 clones were
sequenced by the dideoxy chain termination method using
35S-dATP and Sequenase (U.S. Biochemicals).
The results in Table 2 show that in the amplification
of the cDNA of one patient and the actin control, that actin
could be amplified from the brain cDNAs but not from PGA5,
a full length cDNA clone which contains the Val2.1 segment.
Also, evident were lesser amounts of a smaller PCR product
corresponding to the Val2.1 gene in the patient but not in
the control sample. To ensure that the one Va family was
amplified, genomic and brain Val2.1 PCR products were
analyzed using restriction endonucleases and compared to the
known restriction map. Only the expected fragments were
observed, consistent with the notion that only the Val2.1
family was amplified. When colonies containing cloned Va
PCR products were screened with a Val2.1 region probe,
approximately 20% were positive. DNA from several of these
colonies was sequenced and found to be identical to the
published Val2.1 sequences (Sim, et al., Nature 312, 771-775
(1984)). Thus, the restriction fragment length
polymorphisms (RFLP) recently associated with MS
susceptibility must be in a sequence flanking to the TcR
Va gene. (Oksenberg, et al., Proc. Natl. Acad. Sci. USA 86,
988-992 (1989)).
W09~0857~ -31- ~1 72 ~ rCT~S94/1~728
The above results demonstrated that PCR could amplify
the receptor transcripts from post-mortem brain samples,
starting from 5 ~g of total RNA without the necessity of n
vitro e~p~ncion of T-cells. Similarly, Ca sequences were
amplified from MS brain cDNAs, but not from the control
sample. A subsequent amplification using primers
complementary to the Va and the Ca TcR regions produced a
major band when the control PGA5 and cDNA from the occipital
region of the MS brain were used as a template, indicating
the presence of rearranged TcR transcripts in the sample.
The Va and Ca amplifications from the parietal region brain
library most probably represent real transcripts from
rearranged chromosomes, as has been found in other cDNA
libraries from T-cell lines (Loh, et al., Science 243, 217-
220 (1989).
No PCR product was observed using primers correspondingto the Vb8 family, even though these primers are able to
amplify the gene from buffy coat extracted genomic DNA.
This TcR V region was recently reported to be associated
with susceptibility to MS (Beall, et al., J. Neuroimmunol
21, 59-66 (1989).
To provide further evidence that the DNA produced
during the PCR amplification was an authentic amplified
product of rearranged TcR genes, the PCR products were
sequenced after double screening of colonies with Va and Ca
probes. Only two different J regions were seen in the
thirty sequences examined, both different from the PGA5 Ja
sequence. Eleven sequences contain the Ja O family found in
clone HAP41 (Yoshikai, et al., J. EXP. Med. 164, 90-103
(1986). Fourteen sequences had a previously undescribed Ja
sequence, GGGTACCGAGATGACGAACCCACCTTTGGGACAGGCACTCAGCTAAAAG-
TGGAACTC.
In order to completely analyze the TcR Va usage in MS
brains, 18 different Va specific oligonucleotides for use as
5' PCR primers, based on published sequences for these gene
families were prepared (Yoshikai, et al., su~ra (1986);
WO9~t08572 ~ 5~ PCT~S94110728
-32-
Xline, et al., Proc. Natl. Acad. Sci. USA 84, 6884-6888
(1987)). Optimal conditions for amplification with each
primer were ascertained with genomic DNA using a specific
3' Va primer for each TcR Va family and with reverse-
transcribed RNA isolated from PHA stimulated peripheralblood lymphocytes. Using 5' Va primers and a common 3' Ca
primer, the results from amplification of brain cDNA show
that in each brain only a few TcR V gene families are
preferentially expressed and rearranged. The Va 10 and 12
were detected in MS brains 1 and 2. MS brain 2 also
expressed the Va 8 and the Va 16. In MS brain 3, the Va 8,
9 and 10 families were efficiently amplified. The Va 10 was
thus common to all three samples.
In order to analyze the usage of Va genes, we analyzed
cDNA reverse transcribed from mRNA isolated from uveal
melanoma specimens. Eighteen different Va specific
oligonucleotides representing the major human TCR Va
families were used for the 5' primers and a Ca sequence was
used for the 3'-primer (Table 1). Total RNA was extracted
from each of eight uveal melanoma samples and was reverse
transcribed. Total RNA from melanoma tissue was prepared
in the presence of guanidinium thiocyanate in the method
using RNAzol~ (Cinna/Biotec, TX) (Choi, et al., Proc. Natl.
~cad. Sci. USA 86, 8941 (1989)), and references cited
therein). 2 ~g of total RNA was used for the synthesis of
single strand cDNA using reverse transcriptase. In a final
volume of 20 ~1 lxPCR buffer (50 mM KCl, 20 mM Tris-Cl,
pH 8.4, 2.5 mM MgCl), 1 mM of NTP's, 20 units of RNAsin,
100 pmoles of random hexamer (Pharmacia) and 200 units of
BRL MnMllTV reverse transcriptase were incubated with RNA
(2 ~g) for 40 minutes at 42~ s~k;, et al., Ibid., 85,
5698 (1988)). The reaction mixture was heated at 95~ for
5 minutes, then quickly chilled on ice. The DNA was then
ready for PCR. The resulting cDNA was amplified using
individual sets of Va- Ca primers with primers for
melanotransferrin, a specifi~ marker for melanoma. Each Va
W095/08572 . ~ PCT~ss4/1072
-33-
primer yielded a band of 300 to 400 bp on ethidium bromide-
fit~ i ~; ng of the electrophoresed PCR product.
Analysis of Va expression in TIL from melanoma
specimens is shown in Table 3.
WO95/08572 2 ~.~ 2~ 12 PCr~US94/~0728
--34--
T~b1Q 3.Usage of
TCR Va Gene in Uveal Melanomas
Case Va Familie8
iber
1 Va2 Va7
2 Va6 Va7 Val
3 Va7 Val Val
` 3 4
4 Va7 Val Val
3 4
Va7
10 6 Va7 Val
o
7 Val
8 Va7 Va8 Va9 Val
TCR Va families expressed in human uveal melanoma. A
single stranded cDNA sample was amplified using Va-specific
primer with a Ca primer at a final concentration of l ~M in
each reaction. The amplification was performed with 2.5
units of Taq polymerase (ampli Taq~: Perkin Elmer) on a
Perkin-Elmer DNA thermal cycler (Cetus). The PCR cycle
profile was 9SC denaturation for 1 min. annealing of
primers at 55~ for 1 min, extension primers at 72~ for 1
min for 35 cycles. PCR products were separated on 1% regular
agarose/3% Nuseive~ agarose gels (FMC Corporation) and
expression of Va families was considered positive when a
rearranged band (300-400 bp) was visualized with ethidium-
WO9SJ'~iJ/~ l ?f 7~ 94~l0728
-35-
bromide staining. Experiments were repeated three times per
sample. Results were identical with a different aliquot of
each sample.
All TCR 5' primers amplify TCR sequences from germline
DNA using a specific 3' Va primer for each family. We have
detected a Va-Ca rearrangement of all TCR gene members in a
variety of activated T-cells including single rearrangements
of specific Va members in T-cell clones reactive to
pertussis toxin, to Borrelia bergdorfei, and alloantigens as
well as rearrangements of all Va members in pooled T- cells
stimulated by PHA.
Among seven of eight cases only one to three Va genes
(Va 7, 8, 9, 12 and 14) were detected. In seven of eight
cases Va7 was expressed and rearranged. Melanotransferrin
was amplified in all cases. Amplification artifacts due to
contArinAting DNA were excluded by performing controls in
which no amplification was observed without cDNA samples or
with genomic DNA. Identical results were obtained after a
further experiment using different aliquots of each
patient's tumor.
The amplified products obtained with the Va7 primer
were further identified by hybridization with Va7 and Ca
specific oligonucleotide probes. In all cases where the Va7
rearranged product was visualized on agarose gel
electrophoresis with ethidium bromide staining, a positive
hybridization was observed on dot blotting to the Va7
oligonucleotide probe (5'-CTG GAG CTC CTG TAG AAG GAG-3').
Amplified melanotransferrin did not hybridize with this
probe at all. In addition, the Va7-Ca amplified product
hybridized to a Ca oligonucleotide probe (5'-CAG AAC CCT GAC
CCT GCC GTG TAC-3') but not with Val and Va4 specific
oligonucleotide probes.
Additional characterization of the Va7 amplified
products was obtained by restriction mapping with the
endonucleases, DdeI, KpnI and HinfI. The restriction
Woss/o8s72 ~ PC~S94/10728
-36-
pattern was consistent with the known map of Va7. (yochikA;~
et al., J. EXP. Med., 11~, 90 (1968)).
In this manner, the variable region(s) associated with
each of the different neoproliferative tissues may be
determined. A substantially homogeneous composition of
T-cells may then be administered for treatment of the
particular neoproliferative tissue. Instead of isolating
mature T-cells, pre-T-cells may be isolated, activated with
the tumor tissue or appropriate protein and the T-cells
having the appropriate variable region(s) isolated and used
for therapy. The T-cells may also be used for prophylaxis
by administering T-cell compositions after chemotherapy,
irradiation and/or surgical intervention.
TCR Va and Vb rearrangements were studied in 16 MS
brains and in 10 control brains. TCRVa-Ja-Ca and Vb-Db-Jb-
Cb rearrangements were confirmed with Southern blotting and
hybridization of the PCR product obtained by amplification
with 1 of 18 Va or 1 of 21 V~ specific oligonucleotide
primers. The following table indicates the primers.
WO 95/08572 ~ i94/10728
37 21 7~
5 ~ o ~ Y ~ y 5- b 5 ' ~
J c ~ ~ ~ ~ r~ ~J 6 g C) ~ 6~ ~ 6 6 6 J ~)
~ 6 6 ~ ; ~ Jj g6 r ) ~ 6 6,7 ~ 6 ,~
6 _ _) ~ 6 6 ) ,~ 6 ; ~ E6 6 6 r~7 6
g r ~) C ~ C g o ~ C r
~Jr? E- ? 6, ~ - 6, _~ 6 E- - c~ r? ~ 6 r) ~ e~
~ ~ . . O _ ~ ~ ~ u~ x o~ o
E-- ~ ~ ~ ul ~ ~ 1- x ~ -- -- ~ ~ ~ ~ ~ ~ ~~~
r ~ ~ ~ ~ ~ ~ ~ ~
~ ~ 6
a ~ ~ D
r~ V J ~) V ~S r ~ ~ 6 ~ 6 c~ -- 6 ~ V
" _) ~ V ~: ~ 6 ~ V ~ c 6 C ~ ) r_ c ) ~5:
c ~ ~ ~ 6 rJ r ) c- 5 ~ Cj ~ V -- 6 6 _ t.) E- E-
, - - V C~ ~ E- C~ V ~ C V ~: ;~ C ) V 6 6 c~ 6
~~ ? V 6, ;~ V, ~ ~? 6, ~ 6 C~ V V 6 C) V ~ 6
w095~ 2 ~ 1 ~ PCT~S94110728 -
-38-
Coded human brain samples were obtained from the Rocky
Mountain Multiple Sclerosis Center in Englewood, Colorado
and La Trobe University, Victoria, Australia. Samples
included rapidly frozen and cryopreserved autopsy samples
from different regions of 16 MS brains and 10 non-MS
controls. Each sample was homogenized and the total RNA was
extracted using the RNAzol method (Cinna/Biotecx,
Friendswood, TX), (Chonzynski and Sacchi, Anal. Biochem.
162, 156 (1987)). Approximately 0.25 ~g of total RNA was
reverse transcribed into a first cDNA strand in a 10 ~l
reaction contA;n;ng 1 ~l lOxPCR buffer (100 mM Tris-HCl
pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% (w/v) gelatin
(Perkin, Elmer, Norwalk, CT), 1 ~l of 10 mM dioxynucleotide
triphosphates, 0.25 U random h~mers (Pharmacia,
Piscataway, NJ) and 100 U of Superscript MuLV-reverse
transcriptase (BRL, Gaithersburg, MD). The reaction mix was
incubated at room temperature for 10 min, followed by
incubations at 42~ for 45 minutes and 95~ for 5 minutes.
The mix was then quick chilled on ice. cDNA was subjected to
enzymatic amplification by the PCR method. 10 ~l cDNA was
combined in a 50 ~l reaction mix with 4 ~l lOx PCR buffer,
1.25 U Taq polymerase, 0.5 ~M of Ca or Cb primer and 0.5 ~M
of Va or Vb specific oligonucleotide primer (Table 4).
The PCR profile used was: Denaturation 95~ for 60 sec
annealing 55~ for 60 sec and extension 72~ for 60 sec for
35 cycles in a DNA Thermal Cycler.
DNA Isolation and HLA Typing: High molecular weight
DNA was extracted from tissue samples according to standard
procedures. HLA-DRB1, DQA1, DQB1 and DPBl typing was
performed by PCR, dot blotting and hybridization with allele
specific oligonucleotide probes (Helmuth, et al., Am. J.
Hum. Genet. ~7, 515 (1990); and Bugawan, et al.,
Immunoqenetics 32, 231 (1990)).
Specificity was confirmed by identifying single
rearrangements in antigen specific T-cell clones for B.
bergdorfei, pertussis toxin and acetylcholine receptor. No
w095/08572 ~ 1 7~ 2 PCT~S94/10728
-39-
amplification was detected in a colon carcinoma cell line.
All samples were coded with the molecular biologist blinded
to the origin of the specimen other than 3 MS brains and 3
controls.
A limited number of TCR Va gene arrangements were seen
in 15 of 16 of the MS specimens.
WO 95/08572 PCI/US94110728--
40_
~ l` '` ~ '` _
_ _
_
N ~1~1 N ~J ~l
_
L~ O O O O
0 0 0 1~ Q
'.0
~I
N
3 0 ~ uc ~ u 'O u~ Q ~ D u ~o ~ ~
2 _ 2 2
2 N
2 ~ J 5 2 ~ ~ 2 2
Q N
O O O O O OO O O
O Q -- O Q O= 2
n~ o o o o o o o o ~ o
Q Q o O O O O O C _ O
o o No No o ~ ~J No
O O O O O OO O O
~n _ o N -- N _ -- ; N
V~ ~ _ O O O O O O O O
_ _ N _ N oo o o
t O O O O O O O O O O
Q O O O O O O O O O O
V~ _ _ _ _ _o o
--U'~ U~
~ ~ _ U~ -- ~ o 10` o o~
a~ O
R '' 'J ~ ~ ~ ~ ~ ~ o
~d ~ J y C~ ~ N J
~ o u~ o Ir. o Ir~
WO 95/08572 PCT/US94110728
2~ 7~$,1~
o
N
0 _ 0 _ 0
_ . _
`O
U~ _ _ _
~ `S ~ ~r
_
N N N N 1~1 N N N
:~ -- _ =-- _
O O O
-- _ _ _
0 0 0 m 0 0 0 0 0 0
-- -- ~
0 0 D u~o D u ~) ou ~ o ç D u ~O D ~
-- _ 2
N _
2 ' 2
O ~J ~ 1 N
O O O O O O O ~ O
O O -- O O O -- O _
m o O O O O O O O o
~ o o o o o o o o ~ o
o o o o o o o o
N o o o o o o0 o ~O
L~ m ~ o o o o o o o o
g O O O O O O O O O
~ _ N N
J O O O O _ N
O O O O O O O O O
~: O O O O O O O O O O
g O O O O O O O O O O
V~ Vl V~ V~`O U~ o o ~O
m ~O _ u~ o
o _ -- ---- o o o o
O
y o_~ N _ S
In O U7 0 n o u~
~1 ~1 ~ N -
WO95/08572 ~ 42- PCI'IUS94/1072~--
I ~
U~
-
N N l~J N N
_ _ _
t~ O O O
O~ O'
0 ~ 1~
~ `O `O
I.~J N
0:~ -- -- --
~1
0 1~ ~ _r O 1
N
O ~ -- ~ ~ ~ ` --
-- N _ _ N
o o N oN o o~o ~o o~
-- --O _ _ ~,
m ` _ N
C~ O ~ OO O O O O O O
O O OO O O
-- O OO O O O O O O
~n tn o oo o o O O N
~,~ O O OO O O ~ ~ ~ '`J
`O OO O O O _ O O
`~ O O O O O O O O
C ~ ~ OO O O O O O O
Q O O OO O O O O O O
r~) N _ _ _ N
CO N--
o O _ _ _ ~~ _ N
~ ~~ O--O `~ O O Z Z :C
~ ~ ~'n ~ ~ 3
_ -- < ~-- ~: N N `O
t~ S ~ ~ ~ ~ _ N
~ O ~ o In o u~ .
WO 95/08S72 2 ~ 7 2 ~ PCT/US94110728
-43--
N
O'
0 0 0
r~
`O
U~
_
N N N N N
_
_ _
O. C~.
0 0 ~O ~ 0
N 2
~n _
U~
1.5.1 . N N
m
0 ~ 9 D U a~ lo
-
~ -
~ O ~ ~ _ ~ -- -- --
Q r _ ; --
N _ _ N
m o ~o o o o o N
O O O O O O O O O
V~ _ N
o o oNoO oN o O
~O O O O O O O O O
o o o o ~ O O
O o _ _ N -- ~o O --o ôl
O O O O O O O O O O O
0 o 8 ~ ~ ~
m o o o o o o o o o o o
O O O-- O O -- O O-- O O Z 2 2
_ _ ~ ~ _ -- C O ~ -- ~
= Y ~: O ~ -- _ --
1~ 0 ~-) o Ir~ O U')
W O 95/08572 ~ PC~rrUS94/10728 -
~ ~ -44-
W
~o _
ô _ _ . _
_ ~ _
o o o o
m N o o
O O O O
U~ o 1.~ U-l N
J O O O O
O ~) o O O
O O O O
m O~O N
o: O o o _ O
~ U ~
~ J W g
I~7 o In
~ wogs~ 172~1~ PCI/US94/10728
-45--
N
C~.
`O
~r
-
>
L~ _
~
N
rJ~
V~ 1'7
D _ _ ~ _
O `~ ~ N r~
O O O O
r o O ~
O O O O
c m O O N
O O O O O
O O O O
O O O O O
O
~2: o
D Z O O _ O
~ _ S g S
U~ O Ul O
W 0 95/08572 ~ ~ PC~rrUS94/10728
-46-
In one specimen, no Va genes of the 18 families
surveyed were rearranged, although this patient had two V~
rearrangements. The number of TCR Va genes transcribed
ranged from 0 to 9 per brain, with a mean of 4.4 + 2.8
(+ 1 SD). TCR V~ rearrangements were more diverse, with a
range of 2 to 13 per brain, with a mean of 7.0 + 3.4
(+ 1 SD). TCR Va or Vb transcripts were not detected in any
of the 10 brains of individuals who died of non-neurologic
rl ~ F~5l-~es .
All of the 16 MS patients were typed for the Class II
HLA loci HLA-DRB1, DQA1, DQB1 and DPB1 using PCR and
sequence-specificoligonucleotideprobehybridization.Eight
of 16 patients were DRB1*1501, DQAl*0102, DQBl*0602 and
either DPBl*0401 or 0402. This molecular HLA-DR:DQ
haplotype, which corresponds to the cellular type
HLA-DR2Dw2, is associated with increased susceptibility to
MS in certain caucasoid populations. Patients who were of
the above indicated molecular phenotype showed an increased
frequency of certain Va and Vb rearrangements.
W09S~ 21 ~S12 PCTIUS94/10728
-47-
-
~, o o o
U~
o
+ ~ ~ r~
-
a
~j t, O ~--- O o
.-- ,~,
~ ~ _ X
a *O - - ~ ~ b e
E- a *- ,- 8 1
,Q C
E- o u, æ j 3 0 0
E ~~ 1' D ~ E v ~
~ y ~ ~ ~ o y .~ ~ I O, ~ I c
WO95/08572 ~ PCT~S94/1072~ -
-48-
As evidenced by the above table, of 8 patients with the
indicated phenotype, 7 had rearrangements of Vb 5.2 and all
8 rearranged either Vb 5.1 or 5.2 or both. Vb 6 was
transcribed in 6 of 8 MS brains with the above phenotype
compared to 4 of 8 MS brains of patients who were not of the
indicated phenotype and 6 control brains which were not of
the indicated phenotype. Other frequent rearrangements seen
in patients with the indicated phenotype were Vb 7, 6/8,
Vb 12, 4/8, Va 16, 6/8, Va 5, Va 7, Va 12 and Va 1 in 4/8.
Cloninq and Seauencinq of PCR-Amplified cDNA: PCR amplified
cDNA samples were cloned into M13 for sequence analysis.
Samples were digested with Pst I and SacI or with Pst I and
Bam HI to cut restriction sites in the oligonucleotide
primers. Centricon centrifugal microconcentrators (Amicon,
Danvers MA) were used to concentrate and desalt the digested
PCT products. After transformation in JM101 competent
cells, clones containing TCR b inserts were identified by
hybridization with a TCR b C-region HRP- labelled probe.
Single stranded DNA from positive clones was prepared, and
V WCb sequences determined by the dideoxy chain termination
method with an AmpliTaq sequencing kit (Perkin-Elmer).
WO 95/08572 2 1 7 2 ~ 1 2 PCT/US94tlO728
e
~49~
Tab~e 7. CDR3 Sequences of TCR Rearrangements Amplified
from MS Brains and Controls.
Vb5.2/3 N-D--N- J C13
XL--1
LCASS ; LPGTPYGYFGSGTRLTW ~Jb 1.2) EDLKN
~, LCASS . LPGTP~jY~-GSGTRLTW tJb 1.2) EDLNK
r- LCASS LRLANSPLHFGNGTRLTVT ~Jb 1.6) EDLNK
LCASS ~ LDRLYNSPLHFGNGTRLTVT (Jb 1.6) EDLNK
LCAS QLRLANSPLHFGNGTRLTVT (Jb 1.6) EDLNK
LCASS QLRLANSPLHFGNGTRLTVT ~Jb 1.~6) EDLNK
LCASS F LGYNSPLHFGNGTRLTVT ~Jb 1.6) EDLNK
LCASS ~ QPTVYNNEQ~-~GQRIRLLVL ~Jb 2.1) EDLKN
LCASS SDGRMSTQYFGPGTRLLVL (Jb 2.3) EDLKN
LCASS LVAGSIYEQYFGPGTRLTVT (Jb 2.7) EDLKN
LCASS SEREGRAQYFGQGTRLTVL (Jb ?) EDLKN
. LCASS GGEGRAQYFGQGTRLTVL (Jb ?) EDLKN
LCASS LDGVPYGYTFGSGTGLTW (Jb 1.2) DLNK
LCASS LDGVPYGYTFGSGTRLTW (Jb 1.2) EDLNK
.LCASS LDGVNYGYTFGSGTRLTW (Jb 1.2) EDLNK
.LCASS LVGRGPYGYTFGSGTRLTW (Jb 1.2~ EDLNK
~ LCASS LGGVPYGYTFGSGTGLTW (Jb 1.2) EDLNK
LCASS LRGTPYGYTFGSGTRLTW (Jb 1.2) EDLNK
LCASS QPAVYNEQFFGPGTRLTVL (Jb 2.1) EDLKN
~LCASS LELAGYNEQFFGPGTRLTVL (Jb 2.1) EDLKN
. LCASS LGGSEE DTQYFGPGTRLTVL (Jb 2.3) EDLKN
LCASS LGGSEETQYFGPGTRLLVL (Jb 2.5) EDLKN
LCASS LGGSVETQYFGPGTRLLVL (Jb 2.5) EDLKN
~'LCASS LGSGTL QETQYFGPGTRLLVL (Jb 2.5) EDLKN
LCASS LASGTL QETQYFGPGTRLLVL (Jb 2.5) EDLKN
LCASS LASGTL QETQYFGPGTRLLVL (Jb 2.5) EDLKN
LCASS -~ PTGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
LCASS ~ PTGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
LCASS QGSTFGAGSRLTVL (Jb 2.6) EDLKN
'iLCASS SGANVLTFGAGSRLTVL (Jb 2.6) EDL~CN
~`LCASS ~ LGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
LCASS LRGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
LCASS LVAGSIYEQYFGPGTRLTVT (Jb 2.7) EDLKN
LCASS LVAGSIYEQYFGPGTRLTVT (Jb 2.7) EDLKN
LCASS LVAGSIYEQYFGPGTRLTVT (Jb 2.7) EDLKN
W095/08572 ~ PCTrUS94/10728 -
~50-
Vb5.2/3 ~-D-N-J C~
-; LCAS TLRLGNSPLHFGNGTRLTVT (Jb l.6) EDLNK
LCASS DSSETQYFGPGTRLLVL (Jb 2.5) EDLKN
_ LCASS LRGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
LCASS LRGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
. LCASS PTGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
~ LCASS LVAGIYFQYFGPGTRL~VT ~Jb 2.7) EDLKN
- LCASS LVAGSIYEQYFGPSTRLTVT (Jb 2~7) EDLKN
;LCASS LVAGSIYEQYFGPSTRLTVT (Jb 2.7) EDLKN
Mu~le ;nf~ltr~tln~ lym~ho~yt~
LCASS t LGSPGYR TNEKLFFGSGTQLSVL (Jb l.4) EDLNK
LCASS ~ FTGAY YNEQFFGPGTRLTVL (Jb 2.l) EDLKN
.-LCASS ' RRTSGFVH DTQYFGPGTRLTVL (Jb 2.3) EDLKN
;LCAS: ARRTSGFV TDTQYFGPGTRLTVL (Jb 2.3) EDLKN
~LCAS TARRTSGFV TDTQYFGPGTRLTVL (Jb 2.3) EDLKN
~LCA TARRTSGFV TDTQYFGPGTRLTVL (Jb 2.3) EDLKN
LCA' TARRTSGFV TDTQYFGPGTRLTVL (Jb 2.3) EDLKN
'LCA TARRTSGFV TDTQYFGPGTRLTVL (Jb 2.3) EDLKN
, LCA-~ TARRTSGFV TDTQYFGPGTRLTVL (Jb 2.3) EDLKN
. LCAS ' RQGARTGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
- JO tPBT.~
'LCASS ' VALQDR. Y~y~GsGTGLT W (Jb l.2) EDLNK
LCASS ~ TVRGSQPQHFGDGTRLSIL (Jb l.S) EDLN~
~'LCASS ~ PGMKNIQYFGAGTRLSVL (Jb 2.4) EDLKN
LCASS~ DSPSGQETQYFGPGTRLTVL (Jb 2.5) EDLKN
.'LCASSj RPGNIRETQYFGPGTRLSVL (Jb 2.5) EDLNK
,LCASS ~ RSQGARTGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
B~ (PBT.~)
:LCASS~ DAGYNSPLHFGNGTRLTVT (Jb l.6) EDLNK
, LCASS' YRTQLNSPLHFGNGTRLTVT (Jb l.6) EDLNK
LCASS3 LEHRPTAKNIQYFGAGTRLSVL (J~ 2.4~ EKLKN
LCASS~ PERGANVLTFGAGSRLTVL (Jb 2.6) EDLKN
LCASSS QEASYEQYFGPGTRLTVT (J~ 2.7) EKLKN
LCAS ~ RLVRDLSH EQYFGPSTRLTVT (Jb 2.7) EDL~N
WO9~ PCT~S94/1072
-51-
Tsblo 8. Nucleotide sequence Homology in the Use of
~uAr~GlY and LeuGlyGlyGlu.
Sample N-D-N-J
KL3 AGCAGC CTA CGC GGG GCC AAC S S LRGAN (Vb5.2/Jb2.6)
' AGCAGC TTA CGC GGG ACA CCC S LRGTP ~Vb5.2/Jbl.2)
KLl AGCAGC TTG CGC TTG GCT AAT SS LRLAN (Vb5.2/Jbl.6)
AGC CAG TTG CGC TTG GCT AAT S Q LRLA (Vb5.2/Jbl.6)
AGCAGC CAG TTG CGC TTG GCT A~T SS Q LRLA (Vb5.2/Jbl.6)
AGCAGC TTG GAT CGC TTG TAT AAT SS LDRLA ~Vb5.2/Jbl.6)
LJl AGC ACG TTG CGC TTG GGT S T LRLG (Vb5.2/Jbl.6)
AGCAGC CTA CGG GGG GCC AAC SS LRGAN (Vb5.2/Jb2.6)
AGCAGC CTA CGG GGG GCC AAC SS LRGAN (Vb5.2~Jb2.6)
MS18^ ACGACG TTG AGG GGG GCG CTA SS LRGAL (Vb5.2/Jb2.4)
BFl* AGCAGC CTC AGG GGG SS LRG (Vb6/Jbl.6)
E* AGCAGC ATA AGG GGA AGC SS IRGS (Vb6/Jb2.7)
BD3~ AGCAGC ATC GTC AGG GGA TCG SS IVRGS (Vb6/Jb2.7)
phll# AGCAGT TTA AGG GCG GGA SS LRAG (Vb8/Jbl.l)
12H6+ AGCAGC CTC CGG GAC TTT SS LRDF (Vb13/Jb2.1)
KL3 AGCAGC TTG GGA GGG GTA CCC TAT SS LGGVPY (Vb5.2/Jbl.2)
AGCAGC TTG GGA GGG TCC GAA GAG SS LGGSEE (Vb5.2/Jb2.3)
AGCAGC TTG GGA GGG TCC GAA GAG SS LGGSEE (Vb5.2/Jb2.5)
AGCAGC TTG GGA GGG TCC GTT GAG SS LGGSVE (Vb5.2/Jb2.5)
4~ AGCAGC CTG GGG GGC GAA SS LGG~ (Vb8.2/Jb2.5)
CDR3 usage in human MBP 88-99 specific T cell line
(Martin, et al., 1991 J.E.M. 173:19-24)
* CDR3 usage in rat spinal cord derived T cell clones
specific for BP 85-99 (Gold et al., 1992 J.I. 148:1712-
1717)
@ CDR3 usage in rat lymph node derived T cell clone
specific for BP 85-99 (Gold et al., 1992)
# Cone derived from a human tonsil cDNA library
(Tillinghast et al., 1986 Science 233:879-883)
+ Noncytolytic mouse T cell clone specific for the
influenza virus strain A/PR8/34 (Morahan et al., 1989)
W095l08s72 c~ PCT/US94/10728--
--52--
Table 9.
n y ~ ~ ~o y
~ O O _ O ~ O -- O. _ _ O _ O
> ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ O ~ ~
c~O ~ _ O _ _ O _ _ ~ O _ O
~ ~ y y ~ O -- C~ _ _ _ O
,~, ~ O ~ _ O O O O -- ~ ~ O o O O O O
y ~ ~ _ O ~ ~ O,
-- o ` O`
y~ O. y ~ y ~ ~O y ~ y 0~ 00
` oo ~ ~ y ~ - - o. ~ o
O.c4 ~r ~ cr.~ ~ ~r y -- o,
~ - o - o ~ ~ o - ~ - - - ~ o o
o~y y `o ~ `o y ~ -- co o. o ~o
c, o g ~ ~ æ g ~o ~0 ~0 ~ 0~ 0 ~ " ~
3 ZS o 8 8 o
3 t. ~ æ
O O O O O ~ O O O -- g O ~ ~ ~
C~ O O O O O O O O O O O O O O ~ O
O O O O O O O O O O O ~ O O
m ~ ~ - .~i _; _ _ _ _ _ _ - ,~ - ~
g O O O O ~o ~., ,~~ O O O o
es <' , ~
O o o o o o o o o o o o o o o
V~ _ _ ~, ~
~ o ~ o o ~, _ o O o _ _ , O
_~ Z ' o o o o o o o o o o -- o o ~ o
:: C~ ~ o o o g o O O o o O o O ~ _ _ O O
~ ~ m e~ ~ < ~ ~ ~ u
~ Woss/o8s72 ~ ~ 72 51 2 PCT~S94110728
, ~ .
Flow Cytometry: Peripheral blood mononuclear cells
(PBMC) were prepared by Ficoll-Hypaque density gradient
centrifugation as described (Jackson and Warner, 1985).
Briefly, 20 ml of blood at room temperature were diluted
with an equal volume of saline, underlayered with
Histopaque-1077-1 (Sigma, St. Louis, M0) and centrifuged 30
min. at 400 g. PBMC were washed twice by centrifugation at
250 g for 10 min with st~ining solution (saline cont~in;~g
1% fetal calf serum). Three-color staining of 20 ~1 of
2Xl07 PBMC suspensions was performed in 96 well plates
(Costar, Cambridge, MA) at 4~ by incubating 20 ~1 of FITC-
conjugated Diversi-T ab TCR Screening Panel (T Cell
Sciences, Cambridge, MA) monoclonal antibodies, 20 ~1 of
phycoerythrin conjugated anti-Leu-3a (CD4) (Becton
Dickinson, San Jose, CA) and 20 ~1 of PerCP anti-Leu-2a
(CD8) (Becton Dickinson) for 30 min. The PBMC were washed
three times in st~; n; ng solution and fixed with 1%
formaldehyde. Fluorescence analysis was carried out on a
Becton Dickinson FACScan.
II. Prevention of EAF with Competitive Peptides
Materials and Methods
The determinant of myelin basic protein (MBP) P5-17
contains a pattern P-S-Q-R-H-G-S-K-Y-L-A-T-A. Using an
algorithm for the subject motif for predicting T-cell
clones, the epitope of the clone Fl-28, an MBP-specific T-
cell was isolated (Zamvil, et al., J. EXP. Ned. (1985)
162:2107), which clone recognizes the autoantigen myelin
basic protein. A peptide corresponding to amino acids P35-
47 containing two patterns GILDB and RFFS was synthesized
and shown to be stimulatory. The peptide determinant tested
was GILDSIGRFFSGDRGAP. The stimulatory epitope was shown
with overlapping peptides to actually consist of LDSIGRFFSG-
DRGAP (Zamvil, et al., Nature (1986) 324:258).
W095/08572 ~ ~ 2~ PCT~S94110728
-54-
P35-47 of the human myelin basic protein (MBP)
comprising the RFFS motif was found to be stimulatory with
mouse T-cells restricted by MHC I-E~UEbU. P5-17 of the human
myelin basic protein comprising the motif KYLAT was found to
be stimulatory with mouse T-cells restricted by I-A,UAbu or
I-A,~ A~U; and P89-101 of the human myelin basic protein
comprising the motif HFFK was found to be stimulatory with
mouse T-cells restricted by I-A.sAbs.
The algorithm can be used to define what immunogenic
part of an autoantigen shares sequence homology with
pathogens. For example, in the case of MBP P35-47 shared
with paramyxoviruses and influenza and MBP 89-101 shared
with picornavirus, the critical sequence triggering the T-
cell is shared with a pathogen.
In another disease, myasthenia gravis, a T-cell epitope
was discovered by using the subject algorithm to construct
synthetic peptides of the acetylcholine receptor. The
peptide AChR P215-232, DTPYLDITYHFVMQRLPL was particularly
stimulatory in a number of myasthenics. Other stimulatory
peptides included 277-291 and 330-347 which followed the
subject algorithm. Antigen-specific T-cell clones are
isolated from peripheral blood lymphocytes (PBL), cultured
in vivo with antigen and syngeneic irradiated PBL as antigen
presenting cells (APL) (Cunningham, et al., J. Gen. Virol.
(1985) 66:249); Eckles, et al., Nature (1981) 301:716).
SYnthetic MBP PePtides: Peptides corresponding to the
amino acid sequences of rat (R) and bovine (B) MBP
(Martenson, 1984, In Experimental Allergic
Enc~ph~lomyelitis. A Useful Model for Multiple Sclerosis.
Alvard, ed. Alan Liss, N.Y.), were synthesized as described
previously using solid phase techniques (Erickson and
Merrifield, 1976, in The Proteins, Vol. 2, Neurath, ed.
A~emic Press, NY, p. 255). Peptides were separated from
the various organic side products and the purity was
determined by high pressure liquid phase column (Merck,
Darmstadt, Germany) and by amino acid analysis. These
~ W095/08572 21 72~12 PCT~S94110728
peptides were not further purified since they all contained
greater than 90% of the desired product. The subject
peptides described above were employed in the following test
procedure. To render autoimmunogenic peptides tolerigenic,
these peptides may be conjugated to lymphocytes (Sriram, et
al., 1983, supra) or by coupling the peptide to a carrier
such as tetanus toxoid or bovine serum albumin, employing
conventional linking groups (Herzenberg, et al., Ann. Rev.
Imm. (1983) 1: 609-632) .
Proliferation Assay: Proliferative responses were
determined as described previously (Zamvil, et al., Nature
(1985) 317:355). lx104 T-cells were cultured with 5x105 X-
irradiated (3,000 rad) PL/J splenic APC in 0.2 ml of culture
media in 96 well flat-bottomed microtiter plates (Falcon,
15 3072) . Peptides were added to culture giving the final
concentrations indicated. At 48 hours incubation, each well
was pulsed with l ~Ci 3H-thymidine and harvested 16 hours
later. The mean c.p.m. thymidine incorporation was
calculated for triplicate cultures. Standard deviations
20 from replicate cultures were within 10% mean value.
DeveloPment of MBP-sPecific T cell clones in the rat:
T cell lines are selected from LN or SC (spinal cords) of
rats ;m~tln;zed with guinea pig myelin basic protein or with
MBP peptide 87-99 (200~g) in CFA. Supernatants from ConA-
25 stimulated Lewis rat splenocytes are used as the source ofIL-2 to e~r~n~ Ag-stimulated T cells. T line cells were
cloned by limiting dilution or FACS sorting. After cloning
subsequent restimulation with antigen was accomplished in
96-well flat bottom plates using 106 irradiated syngeneic
thymocytes/well. After 72 h of stimulation, clones were
refed with growth rnedium and expanded subsequently in 24-
well flat-bottom plates. Restimulation in 24-well plates
was accomplished by using approximately 4XlOs cloned cells
in the presence of 106 irradiated thymocytes and 25 ~g
peptide.
WO95/08572 PCT~S94/10728
~ 56-
Proliferation assays: were performed in 96-well
microtiter plates. Briefly, 2x104 T cells and 106 irradiated
thymocytes/well were incubated with stiumulation medium
only, Con A, or antigen. The cultures were harvested onto
glass fiber filters and TdR uptake was assessed by liquid
scintillation. Mean cpm were calculated from triplicate
wells. In some experiments, competitor peptides, or anti I-A
(OX-6) or anti-I-E antibodies (OX-17) were used to evaluate
which MHC molecules were used to restrict the T cell
response, or to determine whether peptides could antagonize
stimulation by the native peptide.
IIa. Prevention of EAE usinq SYnthetic Peptides N1-20 o~ ~BP
A number of peptides were prepared as shown in the
following table.
TABLE 10
AcN1-20 AcNHASQKRPSQRHGSKYLATAST
AcN1-11
N2-11
Nl-11
N1-20
AcN5-20
AcN9-20
N35-47
N90-101
Antigen presenting cells (APC) were preincubated with
various peptides (67 ~M) as the competitor, for 30 min
Wos~/08572 21 7~ Sl ~ PCT~S94110728
-57-
before the addition of AcN1-11 (from 0.067 to 67 ~M)
together with an I-AU restricted and AcNl-11 specific T-cell
clone, PJR25. In the assay using live APC, the
proliferation of PJR25 to AcN1-11 was inhibited in the
presence of an excess amount of N1-20 or AcN5-20. Inhibition
was evident at the ratio (the competitor:the stimulator) 6:1
or 10:1, and the percentage of inhibition was 79% at 10:1
(p>0.001 compared to medium control, students test) and 52%
at 6:1 (p>0.001) for N1-20 and 50% at 10:1 (p~o.01) for
AcN5-20. The inhibitory effect can be reversed by
increasing the concentration of AcN1-11 in culture.
Peptides N1-11 and AcN2-11 fail to inhibit the response.
Also, inhibition is not evident with peptides N35-47 and
N90-101, which are restricted exclusively to I-EU and I-As
molecules. Peptide AcN9-20, which includes a I-AU
restricted epitope did not inhibit the response in the
presence of live APC. When glutaraldehyde-fixed APC were
used for the inhibitory proliferative response, AcN9-20
inhibited the response (59~) at 10:1 (p>0.01 compared to
medium control), as AcN5-20 (92%) (p>0.01) and N1-20 (91%)
(p>o.oOl) did, where inhibition was not seen with N1-11,
AcN2-11, N35-47 or N90-101.
The inhibitory effect of N1-20 on the proliferation of
PJR25 is time dependent. When N1-20 was added in culture 24
h after the addition of AcN1-11, its inhibitory effect was
reduced. The APC, which was pre-incubated for 18h with 67
~M of N1-20, then extensively washed and fixed with
glutaraldehyde, elicited lowered proliferative responses
(p<0.05 relative to medium control) in the clone than the
fixed APC pre-incubated with control peptide AcN2-11 on
medium alone. These data support the fact that inhibition
occurred at the level of antigen presentation by APC and
exclude the possibility that inhibition in vitro might be
due to non-specific cytotoxicity. Inhibition was observed
not only in the response of PJR25, it was also observed in
the response for another AcN1-11 specific clone, R2.2, whose
W O 9S/08572 ~ S94/10728
-58-
T-cell rec~ptor genes are rearranged differently from that
of PJR25, and in the response of primary lymphocytes
sensitized with AcNl-ll. In contrast, Nl-20 did not inhibit
the I-A8 restricted response of a clone, 3r to N90-101.
Similar competition in vitro was observed not only in clonal
populations, but also in AcNl-11 primed heterogeneous
populations.
~WO 95/08572 PCT/US94110728
_59_ ~17~
~ , ~ o b~ 3 o~
+~ +~ , +~ o +1 o -- o -- -- V~ --
.~ -- o o~ oo ~ O ~ ~ O ~ -C ~ D~
~a~5 o
_
~) E--
~ ~ æ ~ ~ 't E ---
+~ +~ +~ O. +l 0. +~ O, +~ +~ +~ ~ z æ~
~ _ ~ _ O ~ oo ~ K ~
--~ O .C~ C
~ o fi ~ ~ ~ o x
~ ~ g ~ ~ ~ V, E
~ ~ a ~C~-~c~o
C E ~ ' 9 E
~ ~ E ~ _ E _ E 2 4 2 fi ~ E _ j
Z _ _ _ _ _ _ _ _ _ ~ +- ' ~' E
~ - y~
Y O ~ ~ >~
K ~--
o~ o o~ o ~ 8 o ~ o g Oe ~ oO :. 3 S~ Y
~ _ _ _ -- -- -- -- , -- O O O O ~ O t~ _ C
~ 6 ~ ~ Z Z~ z Z` y Y i! o 1~ , E o
Woss/o8s72 ~ PCT~S94/10728
-60-
As shown in the above table, AcN2-11 could not prevent
disease even if the ratio is 6:1 (the competitor AcN1-11),
while injection of Nl-20 significantly (p<0.01 Fisher's
exact test) prevented the clinical development of EAE at the
ratio of 3:1 (p<0.001). In addition, AcN9-20 had a
preventative effect on EAE at the ratio of 3:1 (p<0.001) and
6:1 (p<0.001). Injection of Nl-20 could not prevent EAE
;n~ e~ with the I-As restricted peptide N89-101 SJL/J mice
at the ratio 3:1 or 5:1. In reviewing representative
sections of 20 mice treated with competitors (Nl-20 and
AcN9-20), which did not show any clinical signs of EAE, no
perivascular cuffs of submeningeal cell infiltrates were
evident.
IIb. Bindinq SPecificity of SYnthetic Peptides 89-99 of MBP
We made a set of substituted peptides on the se~uence
V~'~'KNlV'l'~K'l'~, which is identical in rats, mice and human
myelin basic protein (MBP), and corresponds to the I-E
restricted epitope MBP 89-99 in the rat. The peptides are
shown in Table 12. We measured MHC binding in a FACS assay
described in Smilek et al, 1991, Gautam et al 1992a&b.
~ W095/08~72 PCT~S94/10728
~1 72~1~
-61-
Table 12
Set of Alanine Substituted Peptides for Myelin Basic
Protein Epitope in Rat and Man
89 90 91 92 93 94 95 96 97 98 99 100 101
V H F F K N I V T P R T P
A . laIa
A ala
A ala
A 4la
A 5ala
A . 6ala
A ala
A ala
A ala
A ala
A ala
A ala
A ala
The numbering was chosen so that our results can be
~ directly compared to the studies of Martin et al, 1992 on
the human MBP sequence, which is identical, but numbered
differently due to a deletion near the N-terminus of the
myelin basic protein molecule in rodents.
Wos~/08~72 ~ PCT~S94/10728
-62-
We tested these peptides for their ability to cause
EAE when mixed in CFA. As can be seen in Table 13 column 2,
below, the peptides Ala4, Ala5, Ala6, Ala8, and AlalO were
incapable of causing EAE, while Ala9 induced EAE in only 1/6
rats. It was seen that Ala4, Ala6, Ala7, and Ala8 are poor
binders to I-E, implying that these residues 4F,6N,7I, and
8V are critical in MHC binding. The peptides Ala6 and Ala8
weakly ~timulate an encephalitogenic T cell line raised
against native peptide (89-101). When tested on MS18 the
human cytotoxic T cell line which is V~5.2LRGA, and which
recognizes 87-106, the Ala6 and Ala8 peptides cannot serve
as targets for cytotoxicity with HLADRBl*1501 targets. Ala8
can compete with 87-106 to block cytotoxicity of MS18. In
the Lewis rat both Ala6 and Ala8 can block proliferation of
an 8g-101 T cell line when given competitively with native
peptide in vitro. Despite their inability to bind well to
I-E, peptides ala6 and ala8 block the development of EAE
when mixed with native peptide in a 5:1 molar ratio with CFA
(0/6 sick with native plus ala6, 2/6 with native plus ala8,
compared to 11/12 with native peptide alone). These
competitor peptides Ala6 and Ala8, though poor I-E binders
can apparently compete with native peptide and antagonize
the T cell receptor.
WO 95/08572 ~ 1 72 ~ 1 2 Pcr/uss4/l0728
--63--
Table 13:
Summa~ of Assays With Sub~litL~Led Peptides In Rat# and in MS Patient*
~i~ ~ I~Q CTL Prolif. ~rL Comp
~j~ing ~Lf
naLive 21/2114 llM CytUlu~.iC 4+ ne~aLiveMINUS 11/12
Ala 16/6 31 ND 4+ ND ND ND
Ala 26/6 14 ND 3+ ND ND ND
Ala 36/6 11 CYtotoXiC 3+ ND ND ND
Ala 40/6 ~200~ t~LuAic ne~aLive _ ND ND
Ala 50/6 21 ne~a~ive ne~a~ive ND ND ND
Ala 60/21~200ne~aLive l+ ND plus 0/6
Ala 76/6 ~200ne~aLive 2+ plus ND ND
Ala 81/1~~200ne~aLive l+ plus plus 2/6
Ala 91/6 14 Cytoloxic ne~a~ive ND ND ND
Ala 10 0/6 14 cyloloxic 1+ ND ND ND
Ala 11 4/6 20 cyto~oxicne~ative ND ND ND
Ala 12 6/6 14 cyloloxic 1+ ND ND ND
Ala 13 3/6 10 ND 2+ ND ND ND
*Data from Martin et al, 1992 with MS l 8 a CD4+cytotoxic T cell clone specific for
MBP 87-106 restricted by HLA DRB1*1501 DQB1*0602 DPBl*0401, utilizing
V~5.2 with LRGA in the (N)D(N), CDR3 region.
Data shown above demonstrate formulations which are weak MHC binders
which nevertheless antagonize TCR recognition of MBP peptide 87-99, and which
prevent EAE when mixed with MBP in complete Freund's adjuvant in a ~:1 molar
ratio. In addition Ala9 and AlalO are good MHC binders yet are nonencephalitogenic
and are weak stim~ tQrs of an rnr-eph~litogenic T cell line induced with native 87-99
The compound Ala4 is a weaker binder tO I-E (ICso>200 mM), and does not
stimlll~te an 87-99 T cell line, and does not cause EAE. These compounds may
antagonize TCR recognition of native 87-99 as well.
W095/08572 PCT~S94110728
~ 64-
Data shown above demonstrate formulations which are
weak MHC binders which nevertheless antagonize TCR
recognition of MBP peptide 87-99, and which prevent EAE
when mixed with MBP in complete Freund's adjuvant in a 5:l
molar ratio. In addition Ala9 and AlalO are good MHC
binders yet are nonencephalitogenic and are weak
stimulators of an encPph~litogenic T cell line induced
with native 87-99
The compound Ala4 is a weaker binder to I-E (IC50>200
mM), and does not stimulate an 87-99 T cell line, and does
not cause EAE. These compounds may antagonize TCR
recognition of native 87-99 as well.
IIc. Prevention of EAE with SYnthetic PePtides 87-99 of
In the following experiment we demonstrate the
possibility of preventing EAE, by co-;~11n;zing with MBP
and a poor MHC binder, like Ala6 or Ala8, with the
capacity to block an 87-99 response.
Table 14
Competition with Ala6 and Ala5 For Prevention of EAE With
MBP87-99
MBP 87-99ala 6 ala 8 MBP Ac1-20 Incidence
of
0.2 mg ll/12
0.2 mg 0/6
0.2 mg 0/6
0.2 mg 0/6
~W095/08S72 23 72~l2 PCT~S94110728
-65-
l mg 0/6
0.2 mg l mg 6/6
0.2 mg l mg 0/6
0.2 mg l mg 2/6
Incidence of EAE was expressed s number of mice with
clinical EAE/number of mice immunized. For the induction of
EAE, MBP peptide was dissolved in phosphate buffered saline
(PBS) and emulsified with complete Freund's adjuvant (CFA)
in a l:l mixture of PBS and CFA. Mice were injected with
0.2 ml emulsion at the base of the tail. On the same day
and 48 h later, pertussis toxin (List Chemicals, Campbell,
CA) was injected intravenously. Mice were ~ ;ned daily
for signs of EAE. For prevention of EAE, animals were
immunized with MBP 87-99 (0.2 mg), or in a mixture with the
competitor peptide (l mg).
As shown in the above table, AcNl-20 could not prevent
disease caused by N87-99. Co-injection of Ala 6 completely
prevented the clinical development of EAE. In addition, Ala
8 had a preventative effect on EAE. This is the first
example of a TCR antagonist which can block the CDR3 region
LRGAN involved in multiple sclerosis.
It is evident from the above results, by modification
of a peptide, particularly an internal peptide of a larger
peptide that combines to an MHC and is associated with an
autoimmune disease or other immune attack on ~ ~lian
- 25 cells, particularly syngeneic cells, the host may be
protected from the immune attack. Thus, as T-cell
immunodominant sequences are identified, these sequences may
be modified by modifying the amino acid sequence to produce
antagonists to the autoimmune disease.
W095~ PCT~S94/10728
-66-
It is evident from the above results that the subject
method can be used for identifying T-cell receptors
associated with degenerative disorders. Thus, by a simple
screening tec~n;que, one may identify those T-cell receptors
which cause or combat disease and by various procedures
inhibit or enhance their activity. The subject invention
provides the capability to diagnose individuals susceptible
to degenerative diseases associated with T- cell receptor
variable regions. By screening degenerative tissue for
T-cell receptors and identifying the specific T-cell
receptors associated with that tissue and the HLA of the
particular host, the relationship between the T-cell
receptor, HLA and the disease may be established.
Contrastingly, when the T- cells are associated with
combatting a neoproliferative disorder, the particular
T-cells may be employed for prophylaxis or therapy.
All publications and patent applications cited in this
specification are herein incorporated by reference as if
each individual publication or patent application were
specifically and individually indicated to be incorporated
by reference.
Although the foregoing invention has been described
in some detail by way of illustration and example for
purposes of clarity of understanding, it will be readily
apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and
modifications may be made thereto without departing from the
spirit or scope of the appended claims.
~ W095/08572 2 1 7 ~ S 1 ~ -67- ` PCTrUS94110728
SEQUENCE LISTING
( 1 ) ~T~N~T~T- INFORMATION:
(i) APPLICANT: Steinman, Lawrence
okQenh~rg, Jorge
Bernard, Claude
ZamvLl, Scott
Mitchell, Denni~ J
Karin, Nathan
(ii) TITLE OF I~v~~ oN: INTERACTION OF T CELL R~u~.O~S AND
ANTIGEN IN AUTOIMMUNE DISEASE
(iii) NUMBER OF ~ N~S: 177
(Lv) CO~k~`PuN~ENCE ~nDR~SS:
'A'l ADDRESSEE: FLEHR, ~n~RAr~, TEST, ALBRITTON & ~T.~RR~RT
,BI STREET: 4 Embarcadero Center, Suite 3400
,C, CITY: San Franci~co
~D STATE: C~lifornia
El CO~h I~: USA
~F ZIP: 94111-4187
(v) COMPUTER RT.~AnpRTT.~ FORM:
'A'I MEDIUM TYPE: Floppy di~k
B COMPUTER: IBM PC compatible
,CI OPERATING SYSTEM: PC-DOS/MS-DOS
DJ SOFTWARE: PatentIn Relea~e ~1.0, VerLion #1.25
(vi) ~uKRE~. APPLICATION DATA: -
(A) APPLICATION NUMBER: PCT/US94/
(B) FILING DATE: 22-SEP-1994
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/125,407
(B) FILING DATE: 22-SEP-1993
(viLi) ATTORNEY/AGENT INFORMATION:
(A) NAME: Rowland, Bertram I
(B) REGISTRATION NUMBER: 20,015
(C) REFERENCE/DOCKET NUMBER: FP55875-4BIR
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 781-1989
(B) TELEFAX: (415) 398-3249
(C) TELEX: 910 277299
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
~A~I LENGTH: 57 base pairs
B TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
DI TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GGGT~CCG~G ATGACGAACC CACCTTTGGG ACAGGCACTC AGCTAAAAGT GCAACTC 57
W 095/08572 ~ ; PCTrUS94/10728
-68-
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 5 amino acids
,B TYPE: amino acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) Mn~-~cun~ TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Cys Ala Ser Ser
l 5
(2) INFORMATION FOR SEQ ID NO:3:
( i ) ~EQU ~:N~ CHARACTERISTICS:
~A'~ LENGTH: 33 ba~e pairs
B TYPE: nucleic acid
,C STRANDEDNESS: single
D, TOPOLOGY: linear
(ii) Mnr~CUr.~ TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AGCCTACGCA GCTTGCG QG CCTGCGGTTG CGC 33
(2) INFORMATION FOR SEQ ID NO:4:
(L) SEQ~N~ CHARACTERISTICS:
~A' LENGTH: 2l base pairs
B TYPE: nucleic acid
C~ STRANDEDNESS: single
~D~ TOPOLOGY: linear
(i$) MOLECULE TYPE: cDNA
(xi) S~Q~I.CE DESCRIPTION: SEQ ID NO:4:
TTGCCCTGAG AGATGCCAGA G 2l
(2) INFORMATION FOR SEQ ID NO:5:
(L) ~yu~N~ CHARACTERISTICS:
~A', LENGTH: 24 base paira
B TYPE: nucleic acid
C STRANDEDNESS: single
l,D, TOPOLOGY: linear
(i~) MOLECULE TYPE: cDNA
(Xi) S :Q~N~' DESCRIPTION: SEQ ID NO:5:
CCCAG AGGGAGCCAT TGCC 24
(2) INFORMATION FOR SEQ ID NO:6:
W O9Sl08572 ~ ~ 7 2 ~ I ~ PCTtUS94tlO728
-69-
( i) ~U~:N~ CHARACTERISTICS:
~A'l LENGTH: 21 ba~e pairs
,B TYPE: nucleic acid
C, STR~NDEDNESS: ~ingle
.Dt TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~:~U~N~: DESCRIPTION: SEQ ID NO:6:
GGTGAACAGT ~-~ACAGGGAG A 21
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 21 base pair~
,BI TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~QD~N~ DESCRIPTION: SEQ ID NO:7:
ACAAGCATTA CTGTACTCCT A 21
2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 18 base pairs
B TYPE: nucleic acid
C, STRANDEDNESS: ningle
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGCCCTGAAC ATTCAGGA 18
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 20 ba~e pairs
B TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~ U~-N~ DESCRIPTION: SEQ ID NO:9:
~ GTCACTTTCT AGCCTGCTGA 20
(2) lN~ukMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
W095/08572 ~ 5 PCT/US94/10728
--70--
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(iL) MrJT~T~CUT~ TYPE: cDNA
(xi) ~r;yur.Ncr; DESCRIPTION: SEQ ID NO:lO:
~r,G~r,cr2.TT GTcr~r-~T~ A 21
~2) lNrOR~qATION FOR SEQ ID NO:ll:
(i) ~;r;yuL~ ; CHARACTERISTICS:
~A' LENGTH: 22 ba~e pairs
B TYPE: nucleic acid
C STR~Nn~nNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Gr-~r-~r-~TG TGGAGCAGCA TC 22
(2) lNrOk~ATION FOR SEQ ID NO:12:
(i) ~riQlJr;N~:r, CHARACTERISTICS:
~A'l LENGTH: 21 ba~e pair~
, B TYPE: nucleic acid
,C, STRANDEDNESS: ~ingle
,D,, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ATCTCAGTGC TTGTGATAAT A 21
(2) INFORMATION FOR SEQ ID NO:13:
(i) ~r.~lJr;Ncr; CHARACTERISTICS:
I~A' LENGTH: 24 ba~e pairs
,BI TYPE: nucleic acid
, C, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ACCCAGCTGG TGGAGCAGAG CCCT 24
(2) INFORMATION FOR SEQ ID NO:14:
(i) ~riyur;NcL CHARACTERISTICS:
IA' LENGTH: 21 ba~e pair~a
, Bl TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
t TOPOLOGY: linear
~ wog~ 7~5I2 -71- - PCTrUS94/10728
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AGAAAGCAAG ~-~r~A~TGT T 21
(2) INFORMATION FOR SEQ ID NO:15:
(i) ~u ~ CHARACTERISTICS:
'A'I LENGTH: 24 base pair~
B, TYPE: nucleic acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~u~ DESCRIPTION: SEQ ID NO:15:
~r~GTAA CTCAAGCGCA GACT 24
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
IA~I LENGTH: 19 ba~e pair~
'B TYPE: nucleic acid
C STRANDEDNESS: single
lD, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GCTTATGAGA ACACTGCGT 19
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 20 base pairs
Bl TYPE: nucleic acid
,C, STRANDEDNESS: single
,,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GCAGCTTCCC TTCCAGCAAT 20
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQu~ CHARACTERISTICS:
'A' LENGTH: 20 base pairs
BI TYPE: nucleic acid
,C STRANDEDNESS: single
l,DJ TOPOLOGY: linear
( ii ) MOnFCUn~ TYPE: cDNA
W 0 95t08572 ~ PC~rtUS94/10728
-72-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AGAACCTGAC TGCc~Ar,r~A 20
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 21 base pairs
B, TYPE: nucleic acid
C, STRANDEDNESS: single
~D) TOPOLOGY: linear
( ii ) MnT~T~`CUT~T` TYPE: cDNA
(xL) ~u~w~: DESCRIPTION: SEQ ID NO:l9:
CATCTCCATG GACTCATATG A 21
(2) INFORMATION FOR SEQ ID NO:20:
(L) SEQUENCE CHARACTERISTICS:
'A'I LENGTH: 19 base pairs
Bl TYPE: nucleic acid
C STRANDEDNESS: ~ingle
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GACTATACTA ACAGCATGT 19
(2) INFORUATION FOR SEQ ID NO:21:
(i) ~yu~w~ CHA~ACTERISTICS:
,'A', LENGTH: 18 base pairs
~B TYPE: nucleic acid
C, STRANDEDNESS: single
D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TGTCAGGCAA TGACAAGG 18
(2) INFORMATION FOR SEQ ID NO:22:
ti) SEQUENCE CHARACTERISTIcs:
'A'l LENGTH: 26 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
~D TOPOLOGY: linear
(iL) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
AATAGGTCGA CACACTTGTC ACTGGA 26
W O95/08572 ~ 1 7 ~ 3- ~ 4/10728
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 22 base pairs
8I TYPE: nucleic acid
,C, STRANDEDNESS: cingle
~Dl TOPOLOGY: linear
( ii ) Mnr-T~CUT T'` TYPE: cDNA
(Xi) ~:Q~:N~ DESCRIPTION: SEQ ID NO:23:
AcrAA~ACGG AC~A~CGCCC TG 22
(2) lN~ORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 21 base pair~
~BI TYPE: nucleic acid
C STR~NDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~yu~:N~: DESCRIPTION: SEQ ID NO:24:
CAC~.~.GG GTGACGCCGT C 21
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
~AI LENGTH: 24 base pair~
B TYPE: nucleic acid
,C STRANDEDNESS: single
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CAGAAGGTAA CTGCAGCGCA GACT 24
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 30 base pair~
B) TYPE: nucleic acid
C) STRANDEDNESS: ~ingle
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
TTGGGGATCC AGAGCACAGA AGTATACTGC 30
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
W O95/08572 ~ ~ ~ 2~ PCTrUS94/10728
-74-
~'A~ LENGTH: 24 base pair~
IB TYPE: nucleic acid
,C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
r~rAArCCTG ACCCTGCCGT GTAC 24
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 27 ba~e pairs
Bl TYPE: nucleic acid
C, STRANDEDNESS: uingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
C~ACAG TTTAGGTTCG TATCTGT 27
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
'A'I LENGTH: 21 base pairs
Bl TYPE: nucleic acid
C STRANDEDNESS: single
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~:yU~N~: DESCRIPTION: SEQ ID NO:29:
CTGGAGCTCC TGTAGAAGGA G 2l
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A' LENGTH: 24 base pairs
B TYPE: nucleic acid
C, STRANDEDNESS: single
~,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
rAr,AA,~CCTG ACCCTGCCGT GTAC 24
(2) INFORMATION FOR SEQ ID NO:3l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
W 0 95108572 ~ 1 7~i 2 PC~rrUS94/10728
-75-
(D) TOPOLOGY: linear
($i) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Gr~r~Ar-~GT TCCCTGACTT GCAC 24
(2) INFORMATION FOR SEQ ID NO:32:
(i) ~yu~:N~ CHARACTERISTICS:
~A' LENGTH: 24 ba~e pairs
'B TYPE: nucleic acid
C, STRPNn~nNESS: single
~D, TOPOLOGY: linear
( ii ) ~nT-~CuT~ TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TCATCAACCA TGCAAGCCTG ACCT 24
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
,~Aj LENGTH: 24 base pairs
B' TYPE: nucleic acid
~C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Gl~.~.AGAG AGAAGAAGGA GCGC 24
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 20 base pairs
'Bl TYPE: nucleic acid
Cl STRANDEDNESS: single
;D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
ACGATCCAGT GTCAAGTCGT 20
(2) INFORMATION FOR SEQ ID NO:3 5:
~ (i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 22 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
l,DI TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
W O95/~~l> ~ 76- PCTrUS94/10728
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
ATACTTCAGT r-Ar-ArACArA GA 22
(2) INFORMATION FOR SEQ ID No:36:
(i) s~wCE CHARACTERISTICS:
/A' LENGTH: 23 ba~e pair~
B, TYPE: nucleic acid
C, STRANn~nNEss: single
~D, TOPOLOGY: linear
(iL) MOLECULE TYPE: cDNA
(Xi) ~ U~N~: D~SrRTPTION: SEQ ID NO:36:
~CC~IAACT ATAGCTCTGG CTG 23
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
/A', LENGTH: 20 base pairs
BI TYPE: nucleic acid
'C, STRANDEDNESS: single
~D, TOPOLOGY: linear
( ii ) MOr.~CUT.~ TYPE: cDNA
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:37:
AGGCCTGAGG GA.CC~.~C 20
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 22 ba~e pairs
~B TYPE: nucleic acid
C, STRANDEDNESS: single
l,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
CCTGAATGCC CCAACAGCTC TC 22
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
/A' LENGTH: 21 ba~e pairs
IB TYPE: nucleic acid
,C STRANDEDNESS: Lingle
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
W 095/08572 ~ _77_ ~ PCTrUS94/10728
ACTTTAACAA CAACGl~CCG A . 21
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 23 base pairs
Bl TYPE: nucleic acid
,C STRANDEDNESS: single
,D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
.
(xi) ~k~N~ D~SC~TPTION: SEQ ID NO:40:
CTAAATCTCC A~-A~AAAr-CT CAC 23
(2) INFORMATION FOR SEQ ID NO:41:
( i ) ~yur N~: CHARACTERISTICS:
'A' LENGTH: 22 base pairs
Bl TYPE: nucleic acid
C ST~ANn~DNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~Q~N~: DESCRIPTION: SEQ ID NO:41:
TC~APAAACT CA~C~-~AC CT 22
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHA~ACTERISTICS:
'A'l LENGTH: 24 ba~e pairs
B TYPE: nucleic acid
,Cj STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
AAA CCATGGGCCA TGAC 24
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
/A'I LENGTH: 27 ba~e pairR
B TYPE: nucleic acid
C STRANDEDNESS: single
~Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GATACTGACA AAGGAGAAGT CTCAGAT 27
(2) INFORMATION FOR SEQ ID NO:44:
W 09~08572 2~7~ PCTrUS94110728
-78-
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 18 ba~e pair~
IBI TYPE: nucleic acid
,C STRANDEDNESS: single
l,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GGTGAGGGTA CAACTGCC 18
(2) INFORMATION FOR SEQ ID NO:45:
(i) ~yu~NC~ CHARACTERISTICS:
'Aj LENGTH: 21 base pairs
IB TYPE: nucleic acid
,C STRANDEDNESS: single
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~yU~N~: DESCRIPTION: SEQ ID NO:45:
ACCCAAGATA CCTCATCACA G 21
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 21 base pairs
B TYPE: nucleic acid
,C STRANDEDNESS: single
,D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
AC.~1~.~.C GACAGGCACA G 21
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 24 base pairs
,~8, TYPE: nucleic acid
,C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
CATGATAATC TTTATCGACG TGTT 24
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
~ WO9JI~ 7~Sl ~ 79 PCTrUS94/10728
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~Q~hl~ DESCRIPTION: SEQ ID NO:48:
AGCCCAATGA AAGrA~r'ArA GTCAT 25
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
rA~ LENGTH: 24 base pair~
,BI TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
DI TOPOLOGY: linear
(iL) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
AGCCCAATGA AAGr,A~r~G TCAT 24
(2) INFORMATION FOR SEQ ID NO:S0:
(i) SEQUENCE CHARACTERISTICS:
rA~I LENGTH: 21 ba~e pairs
BI TYPE: nucleic acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
A~Ccccr~AAA AAGGACATAC T 21
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
,'A' LENGTH: 18 ba~e pair~
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
D~ TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
CTCTGAGGTG CCCCAGAA 18
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
,'A' LENGTH: 18 ba~e pair~
,BI TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
D, TOPOLOGY: linear
W 095/08572 ~ S ~ ~ PCTrUS94/10728
-80-
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
TTCTGATGGC T~A~r. 18
(2) INFORMATION FOR SEQ ID NO:S3:
(i) S~:~U~N~ CHARACTERISTICS:
~A', LENGTH: 28 amino acids
B, TYPE: amino acid
C, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Leu Cys Ala Ser Ser Leu Pro Gly Thr Pro Tyr Gly Tyr Phe Gly Ser
1 5 10 15
Gly Thr Arg Leu Thr Val Val Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 29 amino acid~
B TYPE: amino acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
Leu Cy~ Ala Ser Ser Leu Pro Gly Thr Pro Tyr Gly Tyr Thr Phe Gly
1 5 10 15
Ser Gly Thr Arg Leu Thr Val Val Glu A~p Leu Asn Lys
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 29 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5S:
Leu Cys Ala Ser Ser Leu Arg Leu Ala Asn Ser Pro Leu Hi~ Phe Gly
1 5 10 lS
A~n Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys
~ W O 95/08572 2 1 7 ~ S 1 2 PC~rrUS94/10728
-81-
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
,'A'I LENGTH: 30 amino acidq
B TYPE: amino acid
,C STRANDEDNESS: ~ingle
~D) TOPOLOGY: linear
ii) MOLECULE TYPE: peptide
(xi) ~Qu~:~CE DESCRIPTION: SEQ ID NO:56:
Leu Cyn Ala Ser Ser Leu A~p Arg Leu Tyr Asn Ser Pro Leu Hi~ Phe
1 5 10 15
Gly A~n Gly Thr Arg Leu Thr Val Thr Glu A~p Leu A~n Ly~
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 29 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: ~ingle
,,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Leu Cys Ala Ser Gln Leu Arg Leu Ala A~n Ser Pro Leu Hi~ Phe Gly
1 5 10 15
ARn Gly Thr Arg Leu Thr Val Thr Glu A~p Leu A~n Lys
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 30 amino acids
,B TYPE: amino acid
,C STRANDEDNESS: ~ingle
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
Leu Cy~ Ala Ser Ser Gln Leu Arg Leu Ala ARn Ser Pro Leu Hi~ Phe
1 5 10 15
Gly A~n Gly Thr Arg Leu Thr Val Thr Glu Aqp Leu Aqn Ly
(2) INFORMATION FOR SEQ ID NO:59:
( i ) ~QD~N~ CHARACTERISTICS:
'A'I LENGTH: 29 amino acidq
,B TYPE: amino acid
,C,, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
W O95l08572 ~ PCTnUS94/10728
'82-
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Leu Cy~ Ala Ser Ser Phe Leu Gly Tyr Asn Ser Pro Leu His Phe Gly
1 5 10 15
AQn Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys
(2) lN~Ok~ATION FOR SEQ ID NO:60:
U~N~ CHARACTERISTICS:
~A', LENGTH: 30 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) ~:Q~N~ DESCRIPTION: SEQ ID NO:60:
Leu Cy~ Ala Ser Ser Gln Pro Thr Val Tyr Asn Asn Glu Gln Phe Phe
1 5 10 15
Gly Gln Arg Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn
25 30
(2) INFORMATION FOR SEQ ID NO:61:
(i) ~QU~N~ CHARACTERISTICS:
'A'l LENGTH: 29 amino acids
B TYPE: amino acid
C STRANDEDNESS: ~ingle
,D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Leu Cys Ala Ser Ser Ser Asp Gly Arg Met Ser Thr Gln Tyr Phe Gly
1 5 10 15
Pro Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:62:
( i ) ~QD~N~ CHARACTERISTICS:
'A' LENGTH: 30 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: Dingle
~D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Leu Cy5 Ala Ser Ser Leu Val Ala Gly Ser Ile Tyr Glu Gln Tyr Phe
W095/08572 2 1 7? s I ~PcTAus94/lo728
-83-
1 5 10 15
Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:63:
~i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 29 amino acids
BI TYPE: amino acid
Cl STRANDEDNESS: ~ingle
~DJ TOPOLOGY: linear
(ii) ~nrFCUr~ TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
Leu Cy~ Ala Ser Ser Ser Glu Arg Glu Gly Arg Ala Gln Tyr Phe Gly
5 10 15
Gln Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 28 amino acids
IB TYPE: amino acid
,C, STRANDEDNESS: ~ingle
~Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Leu Cy~ Ala Ser Ser Gly Gly Glu Gly Arg Ala Gln Tyr Phe Gly Gln
Gly Thr Arg Leu Thr Val Leu Glu A~p Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 29 amino acids
B TYPE: amino acid
~C STRANDEDNESS: single
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
Leu Cy~ Ala Ser Ser Leu Asp Gly Val Pro Tyr Gly Tyr Thr Phe Gly
Ser Gly Thr Gly Leu Thr Val Val Glu Asp Leu Asn Ly~
(2) INFORMATION FOR SEQ ID NO:66:
W 095/08572 PCTrUS94/10728 _
2 ~ 84-
(i) SriQuriN~r; CHARACTERISTICS:
tA~ LENGTH: 29 amino acids
B TYPE: amino acid
C STRANDEDNESS: cingle
,DI TOPOLOGY: linear
(Li) MoT~T~cuT.T~` TYPE: peptide
(xi) ~r;Quhn~r; DESCRIPTION: SEQ ID NO:66:
Leu Cy~ Ala Ser Ser Leu A~p Gly Val Pro Tyr Gly Tyr Thr Phe Gly
1 5 10 15
Ser Gly Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys
(2) lNrOR~ATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 29 amino acids
~ B TYPE: amino acid
,C STRANDEDNESS: fiingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) xr;~uriN~ri DESCRIPTION: SEQ ID NO:67:
Leu Cys Ala Ser Ser Leu Asp Gly Val Asn Tyr Gly Tyr Thr Phe Gly
1 5 10 15
Ser Gly Thr Arg Leu Thr Val Val Glu Asp Leu Asn Ly
(2) INFORMATION FOR SEQ ID NO:68:
( i ) ~r;~ur;N~r; CHARACTERISTICS:
~A LENGTH: 30 amino acids
l B I TYPE: amino acid
,C, STRANDEDNESS: single
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
Leu Cys Ala Ser Ser Leu Val Gly Arg Gly Pro Tyr Gly Tyr Thr Phe
1 5 10 15
Gly Ser Gly Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 29 amino acids
B I TYPE: amino acid
C, STRANDEDNESS: single
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ W 095/08572 2 ~ ~ ~ 5 1 ~ PCTrUS94/10728
-85-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
Leu Cy9 Ala Ser Ser Leu Gly Gly Val Pro Tyr Gly Tyr Thr Phe Gly
1 5 10 15
Ser Gly Thr Gly Leu Thr Val Val Glu Asp Leu A~n Ly~
(2) INFORMATION FOR SEQ ID NO:70:
(L) SEQUENCE CHARACTERISTICS:
,'A'I LENGTH: 29 amino acid~
,BI TYPE: amino acid
,C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) ~OLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Leu Cys Ala Ser Ser Leu Arg Gly Thr Pro Tyr Gly Tyr Thr Phe Gly
1 5 10 15
Ser Gly Thr Arg Leu Thr Val Val Glu A~p Leu A~n Lys
0(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 29 amino acid~
Bl TYPE: amino acid
,C STRANDEDNESS: single
l,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
Leu Cys Ala Ser Ser Gln Pro Ala Val Tyr Asn Glu Gln Phe Phe Gly
1 5 10 15
Pro Gly Thr Arg Leu Thr Val Leu Glu A~p Leu Lys A~n
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
,'A LENGTH: 30 amino acids
B TYPE: amino acid
,C, STRANDEDNESS: single
lD, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
Leu Cys Ala Ser Ser Leu Glu Leu Ala Gly Tyr Asn Glu Gln Phe Phe
1 5 10 15
W O9S/08S72 2 ~ ~ S ~ ~ -86- PCTAUS94110728 ~
Gly Pro Gly Thr Arg Leu Thr Val Leu Glu A~p Leu Lyq A~n
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 30 amino acid~
Bl TYPE: amino acid
C, STRANDEDNESS: single
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Leu CYR Ala Ser Ser Leu Gly Gly Ser Glu Glu Asp Thr Gln Tyr Phe
l 5 l0 15
Gly Pro Gly Thr Arg Leu Thr Val Leu Glu A~p Leu Lys AMn
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
'A~l LENGTH: 29 amino acids
IB TYPE: amino acid
,C STRANDEDNESS: ~ingle
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
Leu CYR Ala Ser Ser Leu Gly Gly Ser Glu Glu Thr Gln Tyr Phe Gly
l 5 l0 15
Pro Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn
2S
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
'A~ LENGTH: 29 amino acid~
(B TYPE: amino acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
Leu Cys Ala Ser Ser Leu Gly Gly Ser Val Glu Thr Gln Tyr Phe Gly
l 5 l0 . 15
Pro Gly Thr Arg Leu Leu Val Leu Glu A~p Leu Lys A~n
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3l amino acids
~W095/08572 2 ~ 7 2 ~ f ~ t, ., , .~ PCTAUS94/10728
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) ~yu~:N~ DESCRIPTION: SEQ ID NO:76:
Leu Cy~ Ala Ser Ser Leu Ala Ser Gly Thr Leu Gln Glu Thr Gln Tyr
l 5 l0 lS
Phe Gly Pro Gly Thr Arg Leu Leu Val Leu Glu A5p Leu Lys ARn
(2) INFORMATION FOR SEQ ID NO:77:
( i ) ~yU~N~: CHARACTERISTICS:
~A'I LENGTH: 31 amino acids
~B TYPE: amino acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:77:
Leu Cys Ala Ser Ser Leu Ala Ser Gly Thr Leu Gln Glu Thr Gln Tyr
l S l0 lS
Phe Gly Pro Gly Thr Arg Leu Leu Val Leu Glu ARP Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 31 amino acidc
IB, TYPE: amino acid
,C STRANDEDNESS: single
DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
Leu Cys Ala Ser Ser Leu Ala Ser Gly Thr Leu Gln Glu Thr Gln Tyr
l 5 l0 15
Phe Gly Pro Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Lyfi Asn
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
,'A', LENGTH: 28 amino acids
B, TYPE: amino acid
C STRANDEDNESS: single
~D,, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W 095/08572 2 ~ 7 2 ~ 1 ~ PCTrUS94/10728
-88-
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:79:
Leu Cy~ Ala Ser Ser Pro Thr Gly Ala A~n Val Leu Thr Phe Gly Ala
1 5 10 15
Gly Ser Arg Leu Thr Val Leu Glu A~p Leu Ly~ A~n
(2) INFORMATION FOR SEQ ID NO:80:
( i ) ~Q~N~: CHARACTERISTICS:
I'A~ LENGTH: 28 amino acids
r B TYPE: amino acid
~C STRANDEDNESS: ~ingle
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~QD~N~: DESCRIPTION: SEQ ID NO:80:
Leu Cy~ Ala Ser Ser Pro Thr Gly Ala Asn Val Leu Thr Phe Gly Ala
1 5 10 15
Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Ly~ Asn
(2) INFORMATION FOR SEQ ID NO:81:
( $ ) S~QU~N~ CHARACTERISTICS:
'A'I LENGTH: 24 amino acids
B~ TYPE: amino acid
~C, STRANDEDNESS: single
~,D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
Leu Cy~ Ala Ser Ser Gln Gly Ser Thr Phe Gly Ala Gly Ser Arg Leu
1 5 10 15
Thr Val Leu Glu A~p Leu Ly~ A~n
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQu~N~ CHARACTERISTICS:
'A' LENGTH: 27 amino acids
~B TYPE: amino acid
C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
Leu Cy~ Ala Ser Ser Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly
1 5 10 ' lS
Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
20 25
~ W O95/08572 ~ 1 72~1 2 ` ` ~ PCTrUS94110728
-89-
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
'A'I LENGTH: 27 amino acid~
BI TYPE: amino acid
C, STRANDEDNESS: single
~D,l TOPOLOGY: linear
( ii ) MnT.T~'CUT.T~' TYPE: peptide
(xi) ~UhN~ DESCRIPTION: SEQ ID NO:83:
Leu Cy~ Ala Ser Ser Leu Gly Ala Asn Val Leu Thr Phe Gly Ala Gly
1 5 10 15
Ser Arg Leu Thr Val Leu Glu Asp Leu Lys A3n
(2) INFORMATION FOR SEQ ID NO:84:
( i ) ~QU~N~ CHARACTERISTICS:
/A~ LENGTH: 28 amino acidR
,BI TYPE: amino acid
~C, STRANDEDNESS: single
~D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
Leu Cys Ala Ser Ser Leu Arg Gly Ala A~n Val Leu Thr Phe Gly Ala
1 5 10 15
Gly Ser Arg Leu Thr Val Leu Glu ARP Leu Ly~ Asn
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A' LENGTH: 30 amino acid~
,BI TYPE: amino acid
CI STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
Leu Cys Ala Ser Ser Leu Val Ala Gly Ser Ile Tyr Glu Gln Tyr Phe
1 5 10 15
Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys A~n
25 30
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 30 amino acids
B TYPE: amino acid
,C STRANDEDNESS: ~ingle
,DJ TOPOLOGY: linear
W O95/08572 ~ ~ ~ 2 5 1 2 90 PCTNS94/10728
(ii) MOLECULE TYPE: peptide
~xi) ~yu~N~ DESCRIPTION: SEQ ID NO:86:
Leu Cys Ala Ser Ser Leu Val Ala Gly Ser Ile Tyr Glu Gln Tyr Phe
1 S 10 15
Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys A~n
(2) INFORMATION FOR SEQ ID NO:87:
(i) ~yU~N~ CHARACTERISTICS:
IA~, LENGTH: 30 amino acids
~B, TYPE: amino acid
,C STRANDEDNESS: single
D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
Leu Cy~ Ala Ser Ser Leu Val Ala Gly Ser Ile Tyr Glu Gln Tyr Phe
1 5 10 15
Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 29 amino acids
Bl TYPE: amino acid
,C, STRANDEDNESS: single
~Dl TOPOLOGY: linear
(i$) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
Leu Cy~ Ala Ser Thr Leu Arg Leu Gly Asn Ser Pro Leu Hi~ Phe Gly
1 5 10 15
A~n Gly Thr Arg Leu Thr Val Thr Glu Asp Leu A~n Lys
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
lA' LENGTH: 27 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: single
~D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:89:
Leu Cys Ala Ser Ser Asp Ser Ser Glu Thr Gln Tyr Phe Gly Pro Gly
wo9~ " 21 72~ 4,l0728
--91--
1 5 10 15
Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:90:
(i) ~UL.. - E CHARACTERISTICS:
~A'I LENGTH: 28 amino acids
,B TYPE: amino acid
,C STRANDEDNESS: single
, ,D,, TOPOLOGY: linear
( ii ) Mnr~FcuT~ TYPE: peptide
(xi) SEQUENCE DFCCRTPTION: SEQ ID NO:90:
Leu Cy~ Ala Ser Ser Leu Arg Gly Ala Asn Val Leu Thr Phe Gly Ala
5 10 15
Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Afin
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 28 amino acids
,B TYPE: amino acid
,C, STRANDEDNESS: single
~DJ TOPOLOGY: linear
(~i) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
Leu Cys Ala Ser Ser Leu Arg Gly Ala A~n Val Leu Thr Phe Gly Ala
1 5 10 15
Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Ly~ Asn
(2) INFORMATION FOR SEQ ID NO:92:
( i ) ~QU~N~ CHARACTERISTICS:
~A'I LENGTH: 28 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
Leu Cys Ala Ser Ser Pro Thr Gly Ala Asn Val Leu Thr Phe Gly Ala
1 5 10 15
Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:93:
wo gs~ 2~ ~S ~ -92- PCTAUS94/10728 ~
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 29 amino acid~
I'B TYPE: amino acid
,'C, STRANDEDNESS: single
,,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQ~ DESCRIPTION: SEQ ID NO:93:
Leu Cys Ala Ser Ser Leu Val Ala Gly Ile Tyr Glu Gln Tyr Phe Gly
Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lyq Asn
(2) INFORMATION FOR SEQ ID NO:94:
(i) ~QD~:~ CHARACTERISTICS:
'A' LENGTH: 30 amino acids
IBI TYPE: amino acid
,C, STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
Leu Cys Ala Ser Ser Leu Val Ala Gly Ser Ile Tyr Glu Gln Tyr Phe
l 5 l0 15
Gly Pro Ser Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn
25 30
(2) INFORMATION FOR SEQ ID No:95:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 30 amino acids
B TYPE: amino acid
C, STRANDEDNESS: single
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
Leu Cy~ Ala Ser Ser Leu Val Ala Gly Ser Ile Tyr Glu Gln Tyr Phe
l 5 l0 15
Gly Pro Ser Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn
25 30
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 33 amino acids
B' TYPE: amino acid
C STRANDEDNESS: single
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ W 095/08572 ~ t ~ PCTrUS94/10728
-93- ~ 2 ~ 72~ ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
Leu Cy~ Ala Ser Ser Leu Gly Ser Pro Gly Tyr Arg Thr Asn Glu Lys
l 5 l0 15
Leu Phe Phe Gly Ser Gly Thr Gln Leu Ser Val Leu Glu A~p Leu Asn
Ly~
(2) INFORMATION FOR SEQ ID NO:97 5
(L) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 30 amino acids
B TYPE: amino acid
,C, STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
Leu CYB Ala Ser Ser Phe Thr Gly Ala Tyr Tyr Asn Glu Gln Phe Phe
l 5 l0 15
Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 32 amino acid~
,8, TYPE: amino acid
,C STRANDEDNESS: ~ingle
~Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
Leu Cy~ Ala Ser Ser Arg Arg Thr Ser Gly Phe Val HiQ Asp Thr Gln
l 5 l0 15
Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys A~n
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 32 amino acidQ
B TYPE: amino acid
,C, STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
wo 9~I~J ~ ~ 2 ~ 7 2 ~ ~ ~ PCTAUS94/10728 0
-94-
Leu CYB Ala Ser Ala Arg Arg Thr Ser Gly Phe Val Thr Asp Thr Gln
1 5 10 15
Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Ly~ A~n
(2) lNb-ORhATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 33 amino acids
,BI TYPE: ~mino acid
C sTRANn~n~cs: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
Leu Cys Ala Ser Thr Ala Arg Arg Thr Ser Gly Phe Val Thr Asp Thr
1 5 10 15
Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Ly~
A3n
(2) INFORMATION FOR SEQ ID NO:101:
( i ) ~Q~N~ CHARACTERISTICS:
~AJ LENGTH: 32 amino acid~
IB TYPE: amino acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
Leu Cy~ Ala Thr Ala Arg Arg Thr Ser Gly Phe Val Thr Asp Thr Gln
1 5 10 15
Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
'A'I LENGTH: 32 amino acids
B, TYPE: amino acid
C, STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
Leu Cy5 Ala Thr Ala Arg Arg Thr Ser Gly Phe Val Thr Asp Thr Gln
1 5 10 15
~ wogs~ t ~f ~ PCTnUS94/10728
-95-
Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Aqp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
,~A'I LENGTH: 32 amino acids
~B TYPE: amino acid
,C sTRANn~nNEss single
~D, TOPOLOGY: linear
b( ii ) ~OLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
Leu Cys Ala Thr Ala Arg Arg Thr Ser Gly Phe Val Thr Asp Thr Gln
1 5 10 15
Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 32 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: single
,DI TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
Leu Cys Ala Thr Ala Arg Arg Thr Ser Gly Phe Val Thr Asp Thr Gln
1 5 10 15
Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS~
IA'I LENGTH: 31 amino acids
Bl TYPE: amino acid
Cl STRANDEDNESS: single
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
Leu Cys Ala Ser Arg Gln Gly Ala Arg Thr Gly Ala Asn Val Leu Thr
1 5 10 15
Phe Gly Ala Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
W 095/08572 ~25~ -96- PCTrUS94/10728
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 30 amino acids
~B TYPE: amino acid
,C, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
(ii) MOT.T~`CUT.~ TYPE: peptide
(Xi) S~:Q~N~: DESCRIPTION: SEQ ID NO:106:
Leu Cy~ Ala Ser Ser Val Ala Leu Gln Asp Arg Tyr Gly Tyr Thr Phe
1 5 10 15
Gly Ser Gly Thr Gly Leu Thr Val Val Glu A~p Leu Asn Ly~
(2) INFORMATION FOR SEQ ID NO:107:
(i) ~QD~N~ CHARACTERISTICS:
'A' LENGTH: 29 amino acids
BI TYPE: amino acid
C STRANDEDNESS: ~ingle
,D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~yu~Ne~: DESCRIPTION: SEQ ID NO:107:
Leu Cys Ala Ser Ser Thr Val Arg Gly Ser Gln Pro Gln His Phe Gly
1 5 10 15
A~p Gly Thr Arg Leu Ser Ile Leu Glu Asp Leu Asn Lys
(2) INFORMATION FOR SEQ ID NO:108:
( i ) S~Q~N~ CHARACTERISTICS:
~A~I LENGTH: 28 amino acids
BI TYPE: amino acid
C, STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
Leu Cys Ala Ser Ser Pro Gly Met Lys Asn Ile Gln Tyr Phe Gly Ala
1 5 10 15
Gly Thr Arg Leu Ser Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:109:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 3û amino acids
B TYPE: amino acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ Wo 9~/08572 PCT/US94/10728
~ 7~ 97-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
Leu Cy8 Ala Ser Ser Asp Ser Pro Ser Gly Gln Glu Thr Gln Tyr Phe
1 5 10 15
Gly Pro Gly Thr Arg Leu Thr Val Leu Glu A~p Leu Lys A~n
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
,~A~I LENGTH: 30 amino acids
~B TYPE: amino acid
C sTRANnr~nNr~ss: single
~D,l TOPOLOGY: linear
(ii) ~nL~curr~ TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
Leu Cy~ Ala Ser Ser Arg Pro Gly Asn Ile Arg Glu Thr Gln Tyr Phe
1 5 10 15
Gly Pro Gly Thr Arg Leu Ser Val Leu Glu A~p Leu A~n Lys
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
Aj LENGTH: 33 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
Leu Cy~ Ala Ser Ser Arg Ser Gln Gly Ala Arg Thr Gly Ala Asn Val
1 5 10 15
Leu Thr Phe Gly Ala Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys
Asn
(2) INFORMATION FOR SEQ ID NO:112:
(i) S~yu~N~ CHARACTERISTICS:
'A~ LENGTH: 29 amino acid~
IB TYPE: amino acid
,CI STRANDEDNESS: single
D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
W O95/08572 2 ~ 2 ~ ~ ~ PCTrUS94/10728
-98-
Leu Cys Ala Ser Ser Asp Ala Gly Tyr Asn Ser Pro Leu His Phe Gly
1 5 10 15
Agn Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Lys
(2) INFORMATION FOR SEQ ID NO:113:
(i) ~-y~N~ CHARACTERISTICS:
'A'I LENGTH: 30 amino acids
B, TYPE: amino acid
C STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~:yu~N~ DESCRIPTION: SEQ ID NO:113:
Leu Cy~ Ala Ser Ser Tyr Arg Thr Gln Leu Asn Ser Pro Leu Hiu Phe
1 5 10 15
Gly Agn Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Asn Ly~
25 30
(2) INFORMATION FOR SEQ ID NO:114:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 32 amino acids
IB TYPE: amino acid
,'C, STRANDEDNESS: Ringle
lD,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
Leu Cys Ala Ser Ser Leu Glu His Arg Pro Thr Ala Ly~ Asn Ile Gln
1 5 10 15
Tyr Phe Gly Ala Gly Thr Arg Leu Ser Val Leu Glu Lys Leu Lys A~n
25 30
(2) INFORMATION FOR SEQ ID NO:115:
(i) SEQUENCE CHARACTERISTICS:
'A'I LENGTH: 29 amino acids
IB TYPE: amino acid
,C STR~NDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:
Leu Cy~ Ala Ser Ser Pro Glu Arg Gly Ala Asn Val Leu Thr Phe Gly
1 5 10 15
Ala Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn
(2) INFORMATION FOR SEQ ID NO:116:
~ wo gs/08s72 - 2 1 7 ~ PCT~US94/10728
_99_
(L) SEQUENCE CHARACTERISTICS:
A' LENGTH: 28 amino acids
'B TYPE: amino acid
C STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~EQ~:N~'~ DESCRIPTION: SEQ ID NO:116:
Leu Cy~ Ala Ser Ser Gln Glu Ala Ser Tyr Glu Gln Tyr Phe Gly Pro
1 5 10 15
Gly Thr Arg Leu Thr Val Thr Glu Lys Leu Lyn A~n
(2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 30 amino acid~
IBI TYPE: amino acid
,C, STRANDEDNESS: single
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
Leu Cy~ Ala Ser Arg Leu Val Arg Asp Leu Ser Hi~ Glu Gln Tyr Phe
1 5 10 15
Gly Pro Ser Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn
25 30
(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 21 ba~e pair~
'B' TYPE: nucleic acid
C,, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:118:
AGCAGCCTAC GCGGGGCCAA C 21
(2) INFORMATION FOR SEQ ID NO:ll9:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 21 ba~e pairs
~B TYPE: nucleic acid
,C, STRANDEDNESS: ~ingle
,,DJ TOPOLOGY: linear
(ii) ~OLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll9:
W0~5/08572 PCT/US94110728 ~
100- --
AGCAGCTTAC GCGGGACACC C 21
(2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 21 ba~e pairs
B TYPE: nucleic acid
, C, STRANDEDNESS: ~3ingle
~DJ TOPOLOGY: linear
( ii ) M~T~P~CUT-~ TYPE: cDNA
(xi) ~;yu~iNc~; DESCRIPTION: SEQ ID NO:120:
AGCAGCTTGC GCTTGGCTAA T 21
(2) INFORMATION FOR SEQ ID NO:121:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 21 base pairs
B TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
,,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~ !;QI~!iNC ' DESCRIPTION: SEQ ID NO:121:
AGCCAGTTGC GCTTGGCTAA T 21
(2) INFORM~TION FOR SEQ ID NO:122:
( i ) ``;~;QUI~;N~ !; CHARACTERISTICS:
'A~ LENGTH: 24 base pairs
B TYPE: nucleic acid
, C, STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
AGCAGCCAGT TGCGCTTGGC TAAT 24
(2) INFORMATION FOR SEQ ID NO:123:
;yu~ ; CHARACTERISTICS:
'A~ LENGTH: 24 base pairs
~B TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
AGCAGCTTGG ATCGCTTGTA TAAT 24
(2) INFORMATION FOR SEQ ID NO:124:
~ W 095/08~72 2 1 ~ 2 ~ 1 2 PCTrUS94/10728
1101--
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 18 base pairs
IBI TYPE: nucleic acid
,C, STRANDEDNESS: single
~D, TOPOLOGY: linear
( $$ ) M~n~CUT.~ TYPE: cDNA
(x$) SEQUENCE DESCRIPTION: SEQ ID NO:124:
AGCACGTTGC GCTTGGGT 18
(2) INFORMATION FOR SEQ ID NO:125:
(i) ~u~ CHARACTERISTICS:
'A' LENGTH: 21 base pa$rs
Bl TYPE: nucle$c ac$d
C STR~NDEDNESS: single
~D, TOPOLOGY: linear
(ii) ~OLECULE TYPE: cDNA
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:125:
AGCAGCCTAC GGGGGGCCAA C 21
(2) INFORMATION FOR SEQ ID NO:126:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 21 base pairs
~B, TYPE: nucleic ac$d
C STRANDEDNESS: single
,DJ TOPOLOGY: l$near
($$) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:
AGCAGCCTAC GGGGGGCCAA C 21
(2) INFORMATION FOR SEQ ID NO:127:
($) SEQUENCE CHARACTERISTICS:
~A; LENGTH: 21 base pairs
'B~ TYPE: nucleic acid
,C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
ACGACGTTGA GGGGGGCGCT A 21
(2) INFORMATION FOR SEQ ID NO:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
W O 95/08572 2~ 102- PC~rAUS94/10728
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:
AGCAGCCTCA GGGGG 15
(2) lN~OR~ATION FOR SEQ ID NO:129:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 18 base pair~
B TYPE: nucleic acid
C, STRANDEDNESS: single
,DJ TOPOLOGY: linear
( ii ) ~nT~T'`CuT~T~ TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:
AGCAGCATAA GGGGAAGC 18
(2) INFORMATION FOR SEQ ID NO:130:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 21 base pair~
IBI TYPE: nucleic acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:
AGCAGCATCG TCAGGGGATC G ' 21
(2) INFORMATION FOR SEQ ID NO:131:
(i) SEQUENCE CHARACTERISTICS:
,'A' LENGTH: 18 base pairs
8 TYPE: nucleic acid
,C, STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(Xi) ~QU~'N~ DESCRIPTION: SEQ ID NO:131:
AGCAGTTTAA GGGCGGGA 18
(2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A' LENGTH: 18 base pairs
(Bl TYPE: nucleic acid
(C, STRANDEDNESS: single
(D) TOPOLOGY: linear
WO 9S/08572 ~ ~ 72S12 PCTrUS94/10728
-103-
(ii) ~OLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:
AGCAGCCTCC GGGACTTT 18
(2) INFORMATION FOR SEQ ID NO:133:
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 24 ba~e pair~
~B, TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
( ii ) M~T-T~CUT~T'` TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:
AGCAGCTTGG GAGGGGTACC CTAT 24
(2) INFORMATION FOR SEQ ID NO:134:
(i) SEQUENCE CHARACTERISTICS:
,'A'I LENGTH: 24 ba~e pair~
BI TYPE: nucleic acid
C STRANDEDNESS: ~ingle
l,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:
AGCAGCTTGG GAGGG.CCGA AGAG 24
(2) INFORMATION FOR SEQ ID NO:135:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 24 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:
AGCAGCTTGG GAGGGTCCGA AGAG 24
(2) INFORMATION FOR SEQ ID NO:136:
( i ) ~QU~N~: CHARACTERISTICS:
'A' LENGTH: 24 base pair~
~B' TYPE: nucleic acid
,C STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
W O 9S/08S72 2~7~ PC~rrUS94/10728
-104-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:
AGCAGCTTGG GAGGG-CC~ TGAG 24
(2) INFORMATION FOR SEQ ID NO:137:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 18 base pair~
Bl TYPE: nucleic acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(Li) ~OLECULE TYPE: cDNA
(xL) ~QU~:N~ DESCRIPTION: SEQ ID NO:137:
AGCAGCCTGG GGGGCGAA 18
(2) INFORMATION FOR SEQ ID NO:138:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 7 amino acids
'B, TYPE: amino acid
C STRANDEDNESS: ~ingle
,DI TOPOLOGY: linear
( ii ) MnT~T~CUT~T~ TYPE: peptide
(xi) ~:Qu~N~ DESCRIPTION: SEQ ID NO:138:
Ser Ser Leu Arg Gly Ala A~n
1 5
(2) INFORMATION FOR SEQ ID NO:139:
(i) SEQUENCE CHARACTERISTICS:
~A~I LENGTH: 6 amino acids
IB TYPE: amino acid
,C. STRANDEDNESS: ~ingle
~,D, TOPOLOGY: linear
(ii) ~OLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:
Ser Leu Arg Gly Thr Pro
1 5
(2) INFORMATION FOR SEQ ID NO:140:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 7 amino acids
B TYPE: amino acid
,C STR~NDEDNESS: single
,D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:
W 095/08572 ~ 1 7~ 5 1 2 PCTrUS94/10728
-105-
Ser Ser Leu Arg Leu Ala Asn
1 5
~2) INFORMATION FOR SEQ ID NO:141:
U~N~ CHARACTERISTICS:
'A'l LENGTH: 6 amino acidn
,BI TYPE: amino acid
,C, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~yu~N~: DESCRIPTION: SEQ ID NO:141:
Ser Gln Leu Arg Leu Ala
(2) INFORMATION FOR SEQ ID NO:142:
( i ) ~:yUhl~ CHARACTERISTICS:
'A' LENGTH: 7 amino acid~
B' TYPE: amino acid
,C, STRANDEDNESS: ffingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:
Ser Ser Gln Leu Arg Leu Ala
1 5
(2) INFORMATION FOR SEQ ID NO:143:
(i) ~yu~:N~ CHARACTERISTICS:
A'l LENGTH: 7 amino acid~
B TYPE: amino acid
C~ STRANDEDNESS: ~ingle
,D. TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:
Ser Ser Leu ALP Arg Leu Ala
1 5
(2) INFORMATION FOR SEQ ID NO:144:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 6 amino acid~
B TYPE: amino acid
,C, STRANDEDNESS: cingle
,D, TOPOLOGY: linear
(ii) XOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:
W O95/08~72 2~7 ~ PCTrUS94/10728
-106-
Ser Thr Leu Arg Leu Gly
(2) INFORMATION FOR SEQ ID NO:145:
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 7 amino acids
,B TYPE: amino acid
C~ STRANDEDNESS: single
~D,I TOPOLOGY: linear
( ii ) ~nT~CuT-F TYPE: peptide
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:145:
Ser Ser Leu Arg Gly Ala A~n
1 5
(2) INFORMATION FOR SEQ ID NO:146:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 7 amino acids
BI TYPE: amino acid
C STRANDEDNESS: single
D,, TOPOLOGY: linear
(iL) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:
Ser Ser Leu Arg Gly Ala Asn
1 5
(2) INFORMATION FOR SEQ ID NO:147:
(i) SEQUENCE CHARACTERISTICS:
/A~I LENGTH: 7 amino acids
IB' TYPE: amino acid
,C, STRANDEDNESS: single
,,D,, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:
Ser Ser Leu Arg Gly Ala Leu
(2) INFORMATION FOR SEQ ID NO:148:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 5 amino acids
Bl TYPE: amino acid
C, STP~NDEDNESS: single
,,D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:
w ogs~ 2 ~ 7 2 ~ 1 2 , PCTrUS94/10728
-107-
Ser Ser Leu Arg Gly
1 5
(2) INFORMATION FOR SEQ ID NO:149:
(i) SEQUENCE CHARACTERISTICS:
,~A' LENGTH: 6 amino acids
,B TYPE: amino acid
,C STRANDEDNESS: single
~DJ TOPOLOGY: linear
(ii) MnTFCU~ TYPE: peptide
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:149:
Ser Ser Ile Arg Gly Ser
(2) INFORMATION FOR SEQ ID NO:150:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 7 amino acids
,BI TYPE: amino acid
C, STRANDEDNESS: qingle
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:
Ser Ser Ile Val Arg Gly Ser
1 5
(2) INFORMATION FOR SEQ ID NO:151:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 6 amino acids
,~BI TYPE: amino acid
,C, STRANDEDNESS: single
,,D,, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:
Ser Ser Leu Arg Ala Gly
1 5
(2) INFORMATION FOR SEQ ID NO:152:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 6 amino acidq
B TYPE: amino acid
C STRANDEDNESS: single
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W 095l08572 ~ 5 ~ 2 PCTrUS94/10728
-108-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:
Ser Ser Leu Arg Asp Phe
1 5
(2) INFORMATION FOR SEQ ID NO:153:
(i) ~Qu~NCE CHARACTERISTICS:
,'A~I LENGTH: 8 amino acids
B TYPE: amino acid
,C STRANDEDNESS: single
~Dl TOPOLOGY: linear
(ii) M~T.~CUT.T~` TYPE: peptide
(Xi) S~:~D~N~ D~CCRTPTION: SEQ ID NO:153:
Ser Ser Leu Gly Gly Val Pro Tyr
1 5
(2) INFORMATION FOR SEQ ID NO:154:
(i) SEQUENCE CHARACTERISTICS:
'A~l LENGTH: 8 amino acids
Bl TYPE: amino acid
,C, STRANDEDNESS: ~ingle
~DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~QD~N~ DESCRIPTION: SEQ ID NO:154:
Ser Ser Leu Gly Gly Ser Glu Glu
1 5
(2) INFORMATION FOR SEQ ID NO:155:
(i) SEQUENCE CHARACTERISTICS:
rA~l LENGTH: 8 amino acids
Bl TYPE: amino acid
C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:
Ser Ser Leu Gly Gly Ser Glu Glu
1 5
(2) INFORMATION FOR SEQ ID NO:156:
(i) SEQUENCE CHARACTERISTICS:
rA LENGTH: 8 amino acids
B TYPE: amino acid
'C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ W 095/08572 ~ 5t~ PCTrUS94110728
--109--
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:156:
Ser Ser Leu Gly Gly Ser Val Glu
(2) INFORMATION FOR SEQ ID NO:157:
(i) ~QD ~:N~: CHARACTERISTICS:
'A' LENGTH: 6 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: single
,D~ TOPOLOGY: linear
($~) MOLECULE TYPE: peptide
(xi) SEQUENCE D~CCRTPTION: SEQ ID NO:157:
Ser Ser Leu Gly Gly Glu
1 5
(2) INFORMATION FOR SEQ ID NO:158:
(i) SEQUENCE CHAR~CTERISTICS:
'A' LENGTH: 13 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: ~ingle
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:
Pro Ser Gln Arg Hi~ Gly Ser Lys Tyr Leu Ala Thr Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:159:
(i) SEQUENCE CHARACTERISTICS:
~A~l LENGTH: 17 amino acid~
Bl TYPE: amino acid
C, STRANDEDNESS: ~ingle
,,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
'(xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:
Gly Ile Leu A~p Ser Ile Gly Arg Phe Phe Ser Gly Asp Arg Gly Ala
Pro
(2) INFORMATION FOR SEQ ID NO:160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acid~
(B) TYPE: amino acid
2~
W 095/08572 ~ 94llo7z8 ~
--110--
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:
Leu Asp Ser Ile Gly Arg Phe Phe Ser Gly Asp Arg Gly Ala Pro
. 1 5 10 15
~2) INFORMATION FOR SEQ ID NO:161:
~i) ~U~:N~ CHARACTERISTICS:
~A, LENGTH: 18 amino acids
Bl TYPE: amino acid
C STT~ANnP'nNF..~:S: 8 ingle
D TOPOLOGY: linear
~ii) M~TT~`CUT-~ TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:
Asp Thr Pro Tyr Leu Asp Ile Thr Tyr His Phe Val Met Gln Arg Leu
1 5 10 15
Pro Leu
~2) lNrORMATION FOR SEQ ID NO:162:
yu~N~ CHARACTERISTICS:
A LENGTH: 21 amino acidQ
~B TYPE: amino acid
C, STRANDEDNESS: single
~D TOPOLOGY: linear
ii ) MnT.~CUT.~ TYPE: peptide
~ix) FEATURE:
A) NAME/REY: Peptide
B) LOCATION: 1..11
D) OTHER INFORMATION: /note= "where Xaa=AcNH"
~xi) S.:QUENCE DESCRIPTION: SEQ ID NO:162:
Xaa Ala Ser Gln Lys Arg Pro Ser Gln Arg His Gly Ser Lys Tyr Leu
1 5 10 15
Ala Thr Ala Ser Thr
(2) INFORMATION FOR SEQ ID NO:163:
(i) ~yU~N~ CHARACTERISTICS:
~A' LENGTH: 13 amino acids
IB TYPE: amino acid
,C STR~NDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W 095/08572 2 1 7 2 5 I 2 PCTNS94110728
--111--
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:
Val Hi~ Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro
1 5 10
~2) INFORMATION FOR SEQ ID NO:164:
( i ) ~r;uur;N~r; CHARACTERISTICS:
~A' LENGTH: 13 amino acids
B TYPE: amino acid
ic, STRANn~nNESS: 5ingle
t ~ D,, TOPOLOGY: linear
~i$) MOLECULE TYPE: peptide
(xL) SEQUENCE DESCRIPTION: SEQ ID NO:164:
Ala Hi~ Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro
(2) lNrORMATION FOR SEQ ID NO:165:
(i) SEQUENCE CHARACTERISTICS:
,~A~ LENGTH: 13 amino acids
,B TYPE: amino acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:
Val Ala Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:166:
(i) SEQUENCE CHARACTERISTICS:
,'A' LENGTH: 13 amino acids
IB TYPE: amino acid
,C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:
Val His Ala Phe Lys Asn Ile Val Thr Pro Arg Thr Pro
(2) INFORMATION FOR SEQ ID NO:167:
(i) SEQUENCE CHARACTERISTICS:
~A~I LENGTH: 13 amino acids
,BJ TYPE: amino acid
,C, STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W 095/08572 2 ~ 112- PCTNS94/10728
(xL) ~Q~N~ DESCRIPTION: SEQ ID NO:167:
Val Hi~ Phe Ala Lys Asn Ile Val Thr Pro Arg Thr Pro
1 5 10
~2) INFORMATION FOR SEQ ID NO:168:
(i) ~yu~nCE CHARACTERISTICS:
~A~I LENGTH: 13 amino acids
Bl TYPE: amino acid
C, STR~NDEDNESS: single
~D,l TOPOLOGY: linear
( ii ) M~T~CUT~T~ TYPE: peptide
(xi) ~u~.. ~ DESCRIPTION: SEQ ID NO:168:
Val His Phe Phe Ala Asn Ile Val Thr Pro Arg Thr Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:169:
(i) SEQUENCE CHARACTERISTICS:
'A'I LENGTH: 13 amino acids
8 TYPE: amino acid
C STR~NDEDNESS: single
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( Xi ) XE~U~N~ DESCRIPTION: SEQ ID NO:169:
Val His Phe Phe Lys Ala Ile Val Thr Pro Arg Thr Pro
1 5 10
~2) INFORMATION FOR SEQ ID NO:170:
(i) SEQUENCE CH~RACTERISTICS:
'A~ LENGTH: 13 amino acids
Bl TYPE: amino acid
C STRANDEDNESS: single
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~u~:~ DESCRIPTION: SEQ ID NO:170:
Val His Phe Phe Lys Asn Ala Val Thr Pro Arg Thr Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:171:
(i) SEQUENCE CHARACTERISTICS:
'A~ LENGTH: 13 amino acids
~B TYPE: amino acid
,C, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
W09~08572 ~ 1 7`~ S lj? - . PCTrUS94/10728
-113-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:
Val His Phe Phe Lys Asn Ile Ala Thr Pro Arg Thr Pro
(2) INFORMATION FOR SEQ ID NO:172:
( i ) S~U~N~ CHARACTERISTICS:
'A'I LENGTH: 13 amino acids
B TYPE: amino acid
C, STRANDEDNESS: ~ingle
~D,l TOPOLOGY: linear
( ii ) N ~T~T''CuT~ TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:
Val HiQ Phe Phe Lys AQn Ile Val Ala Pro Arg Thr Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:173:
(i) S~Q~N~ CHARACTERISTICS:
'A', LENGTH: 13 amino acids
Bl TYPE: amino acid
C, STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:
Val His Phe Phe Lys Asn Ile Val Thr Ala Arg Thr Pro
(2) INFORM~TION FOR SEQ ID NO:174:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 13 amino acids
BI TYPE: amino acid
,C STRANDEDNESS: ~ingle
l,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE D~SCRTPTION: SEQ ID NO:174:
Val His Phe Phe Lys Asn Ile Val Thr Pro Ala Thr Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:175:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 13 amino acids
,8 TYPE: amino acid
C, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) M~T~cur~ TYPE: peptide
W 095/08572 ~ 114- PCTrUS94/10728
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:
Val ~is Phe Phe Lys Asn Ile Val Thr Pro Arg Ala Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:176:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 13 amino acids
'Bl TYPE: amino acLd
C, STRANDEDNESS: single
l,D, TOPOLOGY: linear
( ii ) ~OT-~cur ~ TYPE: peptide
(Xi) ~EQD~:N~ DESCRIPTION: SEQ ID NO:176:
Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:177:
u~N~r~ CHARACTERISTICS:
,'A' LENGT~: 5 amino acids
B TYPE: amino acid
C, STR~NDEDNESS: ~ingle
~D, TOPOLOGY: linear
( ii ) M~T~r~`CUr~r~` TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:
Leu Arg Gly Ala Asn
