Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CYSTEINE-CONTAINING OR METHIOINE-CONTAINING PEPTIDES ~ITH
IMMUNOMODULATORY EFFECTS
EXPRESSION OF RECOMBINANT PEPTIDES
The field of the invention is non-antigen-specific
s immunomodulation, including both immunosuppression and
immunostimulation.
Background of the Invention
The immune system, when working properly, protects
the individual from infection and from the growth of cancers.
In order to carry out these functions, it must be able to
recognize and mount an attack against foreign antigens,
including cancer-specific antigens, but not against antigens
that are normally present on cells throughout the body.
It is possible to stimulate the immune system in
order to improve the level of protection it affords. Immune
stimulation is potentially beneficial where the individual is
under attack from a chronic or an acute infection, or a
malignant disease. Vaccines, including single-protein
antigens such as diphtheria toxoid, are widely used to
generate immunity against a specific antigen and thus against
a specific disease. Where global stimulation of the immune
system is desired, this can sometimes be achieved with
nonspecific agents such as adjuvants, interleukins,
interferons, and colony-stimulating factors.
Occasionally, the immune system loses its ability to
distinguish self from non-self. As a result, the individual
may develop an autoimmune disease such as systemic lupus
erythrematosis, Type I diabetes, or rheumatoid arthritis.
These individuals would benefit greatly from suppression of
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the immune response, as would recipients of a transplanted
organ or tissue.
The immune response may be generally suppressed by
treatment with corticosteroids, azathioprine, cyclosporine,
s tacrolimus (FK506), rapamycin, or mycophenolate mofetil. In
addition, certain immunoglobulins, including the monoclonal
antibody OKT3, have been used for this purpose. It may also
be possible to suppress the immune response to a specific
antigen. This procedure, which has been called "tolerance
induction," can be achieved by intravenous or repeated
topical administration of the antigen in dilute form,
treatment with a very high dose of the antigen, or oral
administration of the antigen.
Summary of the Invention
It has been discovered that DNA molecules encoding
certain immunoactive peptides can be used to treat animals in
need of immunomodulation. When introduced into cells of the
animal, the DNA molecules are transcribed, and a therapeutic
amount of the peptide is produced at an appropriate site.
Some of the peptides are immunostimulatory, and so are useful
for treating conditions such as cancer. Other peptides are
immunosuppressive, and so would be used to treat, e.g.,
autoimmune diseases or transplant rejection. The
immunomodulatory effect appears to be generalized rather than
antigen-specific, and is believed to be related to the
function of T lymphocytes.
The DNA molecules of the invention encode
Cys-containing or Met-containing peptides that fall within
one of five motifs described by the formulas below. The
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peptide is optionally linked to a signal peptide that is
cleaved off by cellular proteases.
In one embodiment, the DNA molecule of the invention
encodes a peptide consisting of 4-30 amino acid residues
(preferably 4-10, and more preferably 4-8, e.g., 4-7 or 4-6
residues) that conforms to the motif represented by
Fonmula I:
An-X-Cys-Cys-Y-Bm (Formula I)
where
o each A and each B is independently selected from any
of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val,
Leu, Ile, Gly, Asp, Glu, Asn, Gln, His, and Pro;
Y is selected from the group consisting Ala, Val,
Leu, Ile, Gly, Ser, Thr, Asp, Glu, Asp, Gln, Tyr, Phe, and
Pro;
n and m are whole integers chosen with the proviso
that the sum of n and m is zero to twenty-six, inclusive;
and the nucleic acid molecule optionally further
encodes a mammalian signal peptide linked to the immunoactive
peptide at An.
Preferably: X is Gly, Pro, Ile, Val, Asp, Leu, Glu,
Gln, or Ala; Y is Gly, Pro, Ile, Val, Asp, Leu, Glu, Ser,
Phe, ~yr, or Thr; the sum of n and m is zero to eleven,
inclusive; and the nucleic acid molecule optionally further
encodes a mammalian signal peptide linked to the immunoactive
peptide at An.
More preferably, the DNA molecule of the invention
encodes a peptide conforming to the motif of Formula I,
optionally linked to a signal peptide, wherein
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X is Gly and Y is Gly,
X is Pro and Y is Pro,
X is Pro and Y is Val,
X is Ile and Y is Leu,
X is Pro and Y is Glu,
X is Glu and Y is ~yr,
X is Pro and Y is Phe,
X is Glu and Y is Phe,
X is Ala and Y is Val,
o X is Val and Y is Ile,
X is Gln and Y is Ser,
X is Ile and Y is Thr,
X is Leu and Y is Asp, or
X is Asp and Y is Ile; A and B vary according to the
s parameters above; and the sum of n and m is zero to eleven,
inclusive.
Most preferably, the peptide represented by Formula I
consists of 6-8 amino acid residues.
Examples of such peptides of Formula I include the
following:
Glu-Glu-Cys-Cys-Phe-Tyr (SEQ ID NO.:l; D22041AX),
Pro-Gly-Cys-Cys-Gly-Pro (SEQ ID NO.:2),
Pro-Gly-Cys-Cys-Pro-Gly (SEQ ID NO.:3i D22139AA),
Gly-Pro-Cys-Cys-Pro-Gly (SEQ ID NO.:4),
Ala-Pro-Cys-Cys-Val-Pro (SEQ ID NO.:5; D22037AX),
Val-Ile-Cys-Cys-Leu-Thr (SEQ ID NO.:6),
Lys-Pro-Cys-Cys-Glu-Arg (SEQ ID NO.:7),
Lys-Glu-Cys-Cys-~yr-Val (SEQ ID NO.:8),
Thr-Pro-Cys-Cys-Phe-Ala (SEQ ID NO.:9; D22040AX),
Leu-Ala-Cys-Cys-Val-Val (SEQ ID NO.:10),
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Pro-Val-Cys-Cys-Ile-Gly (SEQ ID NO.:ll),
Ser-Gln-Cys-Cys-Ser-Leu (SEQ ID NO.:12),
Ser-Ile-Cys-Cys-Thr-Lys (SEQ ID NO.:13),
Lys-Leu-Cys-Cys-Asp-Ile (SEQ ID NO.:14),
s Pro-Ala-Cys-Cys-Gly-Pro (SEQ ID NO.:15),
Pro-Asp-Cys-Cys-Ile-Pro (SEQ ID NO.:16), and
Arg-Cys-Ser-Gly-Cys-Cys-Asn (SEQ ID NO.:17).
The nucleic acid molecule of the invention could
encode a peptide where, for example, A is Gly, Lys, Arg, Cys,
o Ser, Val, Ala, Thr, Glu, Pro, Trp, Leu, Asp, Phe, or Ile; B
is Leu, Arg, Ile, Val, Pro, Ala, ~yr, Gly, Trp, Thr, Lys,
Met, Asp, Glu, or Phe; and the sum of n and m is two to four,
inclusive. The nucleic acid molecule could also encode a
peptide where, for example, A is Pro, Gly, Glu, Ala, Val,
Lys, Thr, Leu, or Ser; B is ~yr, Pro, Gly, Thr, Arg, Val,
Ala, Leu, Lys, or Ile; n is one; and m is one.
In a second embodiment, the DNA molecule of the
invention may encode a peptide consisting of 5-30 amino acid
residues (preferably 5-10, more preferably 5-9, e.g., 5, 6,
7, or 8 residues) that conforms to the motif represented by
Formula II:
An-X-Cys-Z-Cys~Y~Bm (Formula II)
where
each A and each B is independently selected from any
of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val,
Leu, Ile, Gly, Asp, Glu, Asn, Gln, Lys, Phe, His, and Pro;
Z is selected from the group consisting of Ala, Val,
Leu, Ile, Gly, Ser, Thr, Lys, His, Phe, Tyr, Arg, and Pro;
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Y is selected from the group consisting of Ala, Val,
Leu, Ile, Gly, Asp, Glu, Lys, Arg, Gln, Tyr, Phe, Ser, Thr,
and Pro; and
n and m are whole integers chosen with the proviso
s that the sum of n and m is zero to twenty-five, inclusive;
and the nucleic acid molecule optionally further encodes a
mammalian signal peptide linked to the immunoactive peptide
at An-
Preferably: X is Gly, Pro, Ile, Val, Asp, Leu, Glu,
o Gln, or Ala; Y is selected from the group consisting of Gly,
Glu, Val, Gln, Arg, Leu, Tyr, Phe, Ile, Ser, Thr, Asp, and
Pro; Z is selected from the group consisting of Ile, Gly,
Thr, Ala, Arg, and Lys; the sum of n and m is zero to ten,
inclusive; and the nucleic acid molecule optionally further
encodes a mammalian signal peptide linked to the immunoactive
peptide at An.
More preferably, the DNA molecule of the invention
encodes a peptide conforming to the motif represented by
Formula II, optionally linked to a signal peptide, wherein
X iS Gly and Y is Gly,
X is Pro and Y is Pro,
X is Pro and Y is Val,
X is Ile and Y is Leu,
X is Pro and Y is Glu,
X is Glu and Y is Tyr,
X is Pro and Y is Phe,
X is Glu and Y is Phe,
X is Ala and Y is Val,
X is Val and Y is Ile,
X is Gln and Y is Ser,
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X is Ile and Y is Thr,
X is Leu and Y is Asp, or
X is Asp and Y is Ile; Z is Ile, Gly, Thr, Ala, or
Lys; A and B vary according to the parameters above; the sum
of n and m is zero to ten, inclusive; and the nucleic acid
molecule optionally further encodes a mammalian signal
peptide linked to the immunoactive peptide at An.
Examples of such peptides of Formula II include the
following:
lo Val-Cys-Ile-Cys-Gln (SEQ ID NO.:18),
Val-Cys-Gly-Cys-Arg (SEQ ID NO.:l9),
Lys-Cys-Arg-Cys-Lys ~SEQ ID NO.:20),
Asp-Cys-Ile-Cys-Gln (SEQ ID NO.:21),
Ile-Cys-Thr-Cys-Glu (SEQ ID NO.:22),
Ile-Cys-Thr-Cys-Arg (SEQ ID NO.:23),
Leu-Cys-Ala-Cys-Val (SEQ ID NO.:24),
Phe-Cys-Ile-Cys-Lys (SEQ ID NO.:25),
Ala-Cys-Lys-Cys-Gln (SEQ ID NO.:26), and
Gly-Pro-Cys-Ile-Cys-Pro-Gly (SEQ ID NO.:27).
The nucleic acid molecule of the invention could
encode a peptide where, for example, X is Val, Ala, Leu, Ile,
Lys, Asp, Phe or Pro; Y is Glu, Val, Gln, Arg, Lys, or Pro; Z
is Gly, Ala, Ile, Arg, Thr, or Lys; and the sum of n and m is
one to three, inclusive.
In a third embodiment, the DNA molecule of the
invention may encode a peptide consisting of 4-30 amino acid
residues that conforms to the motif represented by
Formula III:
An-X-Y-Cys-Z-Bm (Formula III)
30 where
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each A and each B is independently selected from any
of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val,
Leu, Ile, Gly, Ser, Thr, Asp, Glu, Lys, Arg, His, Trp, Tyr,
and Phe;
Y is selected from the group consisting of Ala, Val,
Leu, Ile, Gly and Pro;
Z is selected from the group consisting of Ala, Val,
Leu, Ile, Gly, Lys, Arg, His, Phe, and Pro;
o n and m are whole integers chosen with the proviso
that the sum of n and m is zero to twenty-six, inclusive; and
the nucleic acid molecule optionally further encodes a
m~m~lian signal peptide linked to the immunoactive peptide
at An-
Preferably: X is Gly, Ala, Ile, Asp, Thr, Ser, Arg,
or Trp; Y is Ile, Gly, or Pro; Z is Lys, Ile, Phe, Pro, Ala,
Tyr or Gly; and the sum of n and m is zero to eleven,
inclusive.
More preferably, the DNA molecule of the invention
20 encodes a peptide conforming to the motif represented by
Formula III, optionally linked to a signal peptide, where
X is Gly, Y is Pro, and Z is Ile,
X is Gly, Y is Pro, and Z is Gly,
X is Ala, Y is Pro, and Z is Ala,
X is Ile, Y is Pro, and Z is Tyr,
X is Ala, Y is Pro, and Z is Ile,
X is Arg, Y is Pro, and Z is Ile,
X is Ile, Y is Pro, and Z is Ile,
X is Asp, Y is Pro, and Z is Ile,
X iS Trp, Y is Pro, and Z is Ile,
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X is Trp, Y is Pro, and Z is Gly,
X is Gly, Y is Ile, and Z is Ile,
X is Thr, Y is Pro, and Z is Tyr,
X is Ala, Y is Pro, and Z is Phe,
s X is Ser, Y is Pro, and Z is Phe,
X is Gly, Y is Pro, and Z is Pro, or
X is Gly, Y is Pro, and Z is Tyr; A and B vary
according to the parameters above; and the sum of n and m is
zero to eleven, inclusive.
Examples of such peptides of Formula III include the
following:
Gly-Pro-Cys-Gly (SEQ ID NO.:28),
Ala-Pro-Cys-Ala (SEQ ID NO.:29~,
Ile-Pro-Cys-Tyr (SEQ ID NO.:30),
Trp-Pro-Cys-Gly (SEQ ID NO.:31),
Gly-Pro-Cys-Ile-Leu-Asn (SEQ ID NO.:32),
Gly-Pro-Cys-Ile (SEQ ID NO.:33; D22078AX),
Leu-Leu-Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:34),
Leu-Leu-Phe-Ala-Pro-Cys-Ile (SEQ ID NO.:35),
Leu-Leu-Phe-Arg-Pro-Cys-Ile (SEQ ID NO.:36),
Leu-Leu-Phe-Ile-Pro-Cys-Ile (SEQ ID NO.:37),
Leu-Leu-Phe-Asp-Pro-Cys-Ile (SEQ ID NO.:38),
Ala-Val-Trp-Thr-Pro-Cys-Tyr (SEQ ID NO.:39),
Phe-Val-Met-Ala-Pro-Cys-Phe (SEQ ID NO.:40),
2s Leu-Leu-~yr-Ser-Pro-Cys-Phe (SEQ ID NO.:41),
Ile-Ser-Gly-Pro-Cys-Pro-Lys (SEQ ID NO.:42),
Phe-Leu-Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:43),
Leu-Phe-Gly-Pro-Cys-Ile-Leu (SEQ ID NO.:44),
Glu-Lys-Gly-Pro-Cys-Tyr-Arg (SEQ ID NO.:45),
Phe-Cys-Leu-Gly-Pro-Cys-Pro (SEQ ID NO.:46),
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Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:47; D22077AX),
Phe-Leu-Phe-Gly-Pro~Cys-Ile-Leu-Asn (SEQ ID NO.:48),
Gly-Pro-Cys-Ile-Leu-Asn-Arg (SEQ ID NO.:49; D22087AX),
Leu-Leu-Phe-Trp-Pro-Cys-Ile (SEQ ID NO.:50i D22023AX),
s Leu-Leu-Phe-Gly-Ile-Cys-Ile (SEQ ID NO.:51; D22022AX),
Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn
(SEQ ID NO.:52; D22014AX),
Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg
(SEQ ID NO.:53; D22087AX),
n Trp-Cys-Gly-Pro-Cys-Lys-Met-Ile-Lys-Pro-Phe-Phe
(SEQ ID NO.:54; D7233),
Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg-Leu-Met-Glu
(SEQ ID NO.:55), and
Phe-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg-Leu-Met-Glu
s (SEQ ID NO.:56).
The nucleic acid molecule of the invention may encode,
for example, a peptide where X is Gly, Ala, Ile, Arg, Asp, Trp,
Thr, or Ser; Y is Pro, Gly, or Ile; Z is Gly, Ala, Ile, Tyr,
Phe, or Pro; and the sum of n and m is one to three, inclusive.
In a fourth embodiment, the DNA molecule of the
invention encodes an immunoactive peptide consisting of
3-30 amino acid residues that conforms to the motif
represented by Formula IV:
An-Xp-Y-Cys-Zq-Bm (Formula IV)
25 where
each A and each B is independently selected from any of
the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ser, Glu,
Gly, Ala, Leu, Pro, Thr, Val, Asn, and Lys;
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11
Y is selected from the group consisting of Leu, Arg,
Pro, Tyr, Ile, Val, Ser, Ala, and Phe;
Z is selected from the group consisting of Met, Trp,
Tyr, Phe, Gly, Pro, Arg, Asn, Gln, Ala, and Lys;
s n, m, p, and q are whole integers chosen with the
following provisos: p and q are independently zero or 1 but are
not both simultaneously zero; when q is zero, m is zero; and
the sum of n, m, p, and q is 1 to 28, inclusive.
The nucleic acid molecule encoding a peptide conforming
o to Formula IV optionally further encodes a mammalian signal
peptide linked to the amino terminus of the immunoactive
peptide.
Preferably, both p and q are 1, and the peptide consists
of 4-20 amino acid residues. More preferably, the peptide
consists of 4-15 amino acid residues (e.g., 4-9 or
4-10 amino acid residues). Most preferably, the peptide
consists of 4-7 amino acid residues, optionally linked to a
signal peptide.
More preferably, the DNA molecule of the invention
20 encodes a peptide conforming to the motif represented by
Formula IV, optionally linked to a signal peptide, where
X is Glu, Y is Pro, and Z is Met,
X is Gly, Y is Pro, and Z is Met,
X is Ala, Y is Pro, and Z is Trp,
2s X is Ala, Y is Pro, and Z is Met,
X is Glu, Y is Pro, and Z is Trp,
X is Ser, Y is Pro, and Z is Trp,
X is Leu, Y is Leu, and Z is Gly,
~ X is Pro, Y is Arg, and Z is Arg,
X is Gly, Y is Tyr, and Z is Pro,
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X is Val, Y is Val, and Z is Asn,
X is Leu, Y is Ser, and Z is Gln,
X is Ser, Y is Pro, and Z is Tyr,
X is Ala, Y is Leu, and Z is Arg,
X is Ala, Y is Pro, and Z is Tyr,
X is Gly, Y is Ala, and Z is Pro,
X is Lys, Y is Ser, and Z is Lys,
X is Glu, Y is Pro, and Z is Phe,
X is Glu, Y is Pro, and Z is Tyr,
X is Ser, Y is Pro, and Z is Met,
X is Ala, Y is Pro, and Z is Tyr,
X is absent, Y is Leu, and Z is Phe, or
X is Gly, Y is Pro, and Z is Trp; A and B are selected
independently from the 20 common, naturally occurring amino
acids; and n, m, p, and q are whole integers chosen with the
provisos specified above.
Preferably, peptides conforming to Formula IV are chosen
with the proviso that:
when Y is Pro or Ile, and ~ is 1, and Z is Tyr, Phe,
Gly, Pro, or Ala, then
(i) when p is 1, X is not Ser, Gly, Ala, or Thr, or
(ii) when p is 0, any amino acid residue of A adjacent
to Y is not Ser, Gly, Ala, or Thr.
Examples of peptides of Formula IV include the
2s following:
Gln-Cys-Ala-Leu-Cys-Arg (SEQ ID NO.:81),
Val-Ala-Leu-Ser-Cys-Gln (SEQ ID NO.:82),
Ile-Val-Lys-Ser-Cys-Lys (SEQ ID NO.:83),
Leu-Ala-Phe-Glu-Pro-Cys-Met (SEQ ID NO.:84),
Leu-Leu-Pro-(~ly-Pro-Cys-Met (SEQ ID NO.:85),
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Met-Ala-Pro-Ala-Pro-Cys-Trp (SEQ ID NO.:86),
Ala-Leu-Tyr-Ala-Pro-Cys-Met (SEQ ID NO.:87),
Val-Leu-Trp-Glu-Pro-Cys-Trp (SEQ ID NO.:88),
Met-Leu-Phe-Ser-Pro-Cys-Trp (SEQ ID NO.:89),
Leu-Leu-Cys-Gly-Pro-Ala-Ile (SEQ ID NO.:90),
Leu-Cys-Phe-Gly-Pro-Ala-Ile (SEQ ID NO.:91),
Val-Met-Pro-Ser-Pro-Cys-Tyr (SEQ ID NO.:92),
Val-Val-Phe-Ala-Pro-Cys-Tyr (SEQ ID NO.:93),
Ala-Val-Pro-Glu-Pro-Cys-Phe (SEQ ID NO.:94),
o Met-Met-Tyr-Glu-Pro-Cys-Tyr (SEQ ID NO.:95),
Ala-Ala-Trp-Ser-Pro-Cys-Met (SEQ ID NO.:96),
Val-Ala-Tyr-Gly-Pro-Cys-Trp (SEQ ID NO.:97)
Leu-Arg-Pro-Arg-Cys-Arg-Pro-Ile (SEQ ID NO.:98),
Ala-Gly-~yr-Cys-Pro-Thr-Met-Thr (SEQ ID NO.:99),
Pro-Gln-Val-Val-Cys-Asn-Tyr-Arg (SEQ ID NO.:100), or
Ala-Asn-Phe-Cys-Ala-Gly-Ala-Cys-Pro-Tyr-Leu-Trp
(SEQ ID NO.:101); A and B are selected independently
from the 20 common, naturally occurring amino acids; and n, m,
p, and q are whole integers chosen with the provisos specified
20 above.
Examples of peptides of Formula IV that contain a
mammalian signal peptide sequence include the following:
Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met-Leu-Leu-Val-
Ala-Leu-Ser-Pro-Cys-Leu-Pro-Cys-Arg-Ala-Leu-Ala-Phe-Glu-Pro-
25 Cys-Met (SEQ ID NO.:102; D22184AA),
Met-His-Leu-Ser-Leu-Ser-His-Gln-Trp-Ser-Ser-Trp-Thr-Val-
Leu-Leu-Leu-Leu-Val-Ser-Asn-Leu-Leu-Leu-Trp-Glu-Asn-Thr-Ala-
Ser-Ala-Met-Ala-Pro-Ala-Pro-Cys-Trp (SEQ ID NO.:103; D22183AA),
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Met-Gly-Phe-Leu-Lys-Phe-Ser-Pro-Phe-Leu-Val-Val-Ser-Ile-
Leu-Leu-Leu-Tyr-Gln-Ala-Cys-Gly-Leu-Gln-Ala-Val-Leu-Trp-Glu-
Pro-Cys-Trp (SEQ ID NO.:104; D22196AA),
Met-Gly-Phe-Leu-Lys-Phe-Ser-Pro-Phe-Leu-Val-Val-Ser-Ile-
Leu-Leu-Leu-Tyr-Gln-Ala-Cys-Gly-Leu-Gln-Ala-Val-Met-Pro-Ser-
Pro-Cys-Tyr (SEQ ID NO.:105; D22197AA),
Met-His-Leu-Ser-Leu-Ser-His-Gln-Trp-Ser-Ser-Trp-Thr-Val-
Leu-Leu-Leu-Leu-Val-Ser-Asn-Leu-Leu-Leu-Trp-Glu-Asn-Thr-Ala-
Ser-Ala-Met-Leu-Phe-Ser-Pro-Cys-Trp (SEQ ID NO.:106; D22217AA),
o and
Met-Gly-Phe-Leu-Lys-Phe-Ser-Pro-Phe-Leu-Val-Val-Ser-Ile-
Leu-Leu-Leu-Tyr-Gln-Ala-Cys-Gly-Leu-Gln-Ala-Val-Val-Phe-Ala-
Pro-Cys-Tyr (SEQ ID NO.:107; D22215AA).
In a fifth embodiment, the DNA molecule of the invention
encodes an immunoactive peptide consisting of 3-30 amino acid
residues that conforms to the motif represented by Formula V:
An-W-X-Y-Zp-Bm (Formula V)
where
each A and each B is independently selected from any of
the 20 common, naturally occurring amino acids;
W is selected from the group consisting of Gly, Pro,
Asp, Arg, Ala, Ile, Trp, Ser, Met, Cys, and Glu;
X is selected from the group consisting of Cys, Pro,
Ile, Met, Tyr, Thr, and Arg;
Y iS selected from the group consisting of Cys and Met;
Z is selected from the group consisting of Gly, Phe,
Val, Ile, Pro, Tyr, Trp, Glu, Leu, and Met;
W, X, and Y are chosen with the proviso that at least
one of W, X, or Y is Met, and not more than one of W, X, or Y
is Cys;
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n, m, and p are whole integers chosen with the provisos
that p is zero or l; when p is zero, m is zero; and the sum of
n, m, and p is zero to 27, inclusive.
The nucleic acid molecule encoding a peptide conforming
to Formula V optionally further encodes a mammalian signal
peptide linked to the amino terminus of the immunoactive
peptide.
Preferably, p is 1, and the peptide consists of 4-20
amino acid residues. More preferably, the peptide consists of
o 4-15 amino acid residues (e.g., 4-9 or 4-10 amino acid
residues). Most preferably, the peptide consists of 4-7 amino
acid residues.
More preferably, the DNA molecule of the invention
encodes a peptide conforming to the motif of Formula V,
optionally linked to a signal peptide, wherein
W is selected from the group consisting of Gly, Pro,
Asp, Arg, Ala, Ile, Trp, and Ser;
X is selected from the group consisting of Cys, Pro,
Ile, and Met;
Y iS selected from the group consisting of Cys and Met;
and
Z is selected from the group consisting of Gly, Phe,
Val, Ile, Pro, and Leu.
More preferably, at least one of X and Y is Met.
More preferably, the DNA molecule of the invention
encodes a peptide conforming to the motif of Formula V,
optionally linked to a signal peptide, wherein
W is selected from the group consisting of Gly, Asp,
Arg, Ala, Trp, and Ser;
X iS selected from the group consisting of Pro and Ile;
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16
Y is Met; and
Z is selected from the group consisting of Phe, Ile, and
Pro.
Most preferably, the DNA molecule of the invention
s encodes a peptide conforming to the motif of Formula V,
optionally linked to a signal peptide, wherein
W is selected from the group consisting of Gly and Ser;
X is Pro;
Y is Met; and
o Z is selected from the group consisting of Phe, Ile, and
Pro.
The nucleic acid molecule of the invention may encode a
peptide having Met and Cys or Met and Met aligned contiguously.
For example, the encoded peptide may conform in sequence to
A-W-Met-Met-Z-B,
A-W-Met-Cys-Z-B, or
A-W-Cys-Met-Z-B.
Alternatively, the nucleic acid molecule of the
invention may encode a peptide in which Met and Cys are
separated by no more than one amino acid. For example, the
encoded peptide may conform in sequence to
A-Met-X-Cys-Z-B or
A-Cys-X-Met-Z-B.
Examples of such peptides of Formula V include the
following:
Gly-Pro-Met-Ile (SEQ ID NO.:114),
Lys-Met-Arg-Met-Lys (SEQ ID NO.:115)
Phe-Met-Ile-Met-Lys (SEQ ID NO.:116),
Ile-Cys-Thr-Met-Glu (SEQ ID NO.:117),
Leu-Met-Ala-Met-Val (SEQ ID NO.:118),
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Ile-Met-Tyr-Met-Glu (SEQ ID NO.:119),
Ala-Pro-Met-Met-Val-Pro (SEQ ID NO.:120),
Gly-Pro-Met-Met-Pro-Gly (SEQ ID NO.:121),
Gly-Pro-Cys-Met-Pro-Gly (SEQ ID NO.:122),
.,
s Gly-Pro-Met-Cys-Pro-Gly (SEQ ID NO.:123),
Pro-Gly-Met-Met-Gly-Pro (SEQ ID NO.:124),
Val-Ile-Met-Met-Leu-Thr (SEQ ID NO.:125),
Leu-Ala-Phe-Glu-Pro-Met-Met (SEQ ID NO.:126),
Met-Leu-Phe-Ser-Pro-Met-Trp (SEQ ID NO.:127),
Val-Val-Phe-Ala-Pro-Met-Tyr (SEQ ID NO.:128~,
Leu-Leu-Phe-Gly-Pro-Met-Ile (SEQ ID NO.:129),
Leu-Leu-Tyr-Ser-Pro-Met-Phe (SEQ ID NO.:130),
Leu-Leu-Phe-Asp-Pro-Met-Ile (SEQ ID NO.:131),
Leu-Leu-Phe-Trp-Pro-Met-Ile (SEQ ID NO.:132),
Leu-Leu-Phe-Arg-Pro-Met-Ile (SEQ ID NO.:133),
Leu-Leu-Phe-Ala-Pro-Met-Ile (SEQ ID NO.:134),
Leu-Leu-Phe-Gly-Ile-Met-Ile (SEQ ID NO.:135), and
Phe-Met-Leu-Gly-Pro-Met-Pro (SEQ ID NO.:136).
An example of a peptide of Forumula V that contains a
mammalian signal peptide is:
Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met-Leu-Leu-Val-
Ala-Leu-Ser-Pro-Cys-Leu-Pro-Cys-Arg-Ala-Leu-Leu-Phe-Gly-Pro-
Met-Ile (SEQ ID NO.:137; D22020AX).
Furthermore, each of the peptides conforming to the
motifs represented by Forumula III, IV, or V, are selected with
the proviso that the following peptides are excluded:
Arg-Asn-Arg-Cys-Lys-Gly-Thr-Asp-Val-Gln-Ala-Trp-Ile-Arg-
Gly-Cys-Arg-Leu (SEQ ID NO.:139),
Ile-Asn-Thr-Lys-Cys-Tyr-Lys-Leu-Glu-His-Pro-Val-Thr-Gly-
Cys-Gly (SEQ ID NO.:140),
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18
Asp-Asn-Tyr-Arg-Gly-~yr-Ser-Leu-Gly-Asn-Trp-Val-Cys-Ala-
Ala-Lys-Phe-Glu-Ser-Asn-Phe-Thr-Gln (SEQ ID NO.:141),
Ala-Pro-Ser-Pro-Leu-Pro-Glu-Thr-Thr-Glu-Asn-Val-Val-Cys-
Ala-Leu-Gly (SEQ ID NO.:176),
Ala-Pro-Ser-Pro-Leu-Pro-Glu-Thr-Thr-Glu-Asn-Val-Val-Cys-
Ala-Leu-Gly-Leu-Thr-Val (SEQ ID NO.:142),
Gly-Asp-Met-Tyr-Pro-Lys-Thr-Trp-Ser-Gly-Met-Leu-Val-Gly-
Ala-Leu-Cys-Ala-Leu-Ala-Gly-Val-Leu-Thr-Ile (SEQ ID NO.:143),
Val-Pro-Gly-Leu-Tyr-Ser-Pro-Cys-Arg-Ala-Phe-Phe-Asn-Lys
o (SEQ ID NO.:144),
Val-Pro-Gly-Leu-Tyr-Ser-Pro-Cys-Arg-Ala-Phe-Phe-Asn-Lys-
Glu-Glu-Leu-Leu (SEQ ID NO.:145),
Val-Pro-Gly-Leu-Tyr-Ser-Pro-Cys-Arg-Ala-Phe-Phe-Asn-Lys
(SEQ ID NO.:146),
Glu-Ala-Ile-Tyr-Asp-Ile-Cys-Arg-Arg-Asn-Leu-Asp-Ile-Glu-
Arg-Pro-Thr (SEQ ID NO.:147),
Glu-Ala-Ile-Tyr-Asp-Ile-Cys-Arg-Arg-Asn-Leu-Asp-Ile (SEQ
ID NO.:148),
Asp-Leu-Leu-Glu-Gln-Arg-Arg-Ala-Ala-Val-Asp-Thr-Tyr-Cys-
Arg-His-Asn-Tyr-Gly-Val-Gly-Glu-Ser-Phe-Thr (SEQ ID NO.:149),
Thr-Ser-Ile-Leu-Cys-Tyr-Arg-Lys-Arg-Glu-Trp-Ile-Lys (SEQ
ID NO.:150),
Leu-Pro-Phe-Phe-Leu-Phe-Arg-Gln-Ala-Tyr-His-Pro-Asn-Asn-
Ser-Ser-Pro-Val-Cys-Tyr (SEQ ID NO.:151),
Gln-Ala-Lys-Phe-Phe-Ala-Cys-Ile-Lys-Arg-Ser-Asp-Gly-Ser-
Cys-Ala-Trp-Tyr-Arg-Gly-Ala-Ala-Pro-Pro-Lys-Gln-Glu-Phe (SEQ ID
NO.:152~,
Gln-Ala-Lys-Phe-Phe-Ala-Cys-Ile-Lys-Arg-Ser-Asp-Gly-Ser-
Cys-Ala-Trp-Tyr-Arg (SEQ ID NO.:153),
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19
Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ala-Ala-Ala-Met-Lys-Arg-
His-Gly-Leu-Asp (SEQ ID NO.:154),
Ala-Glu-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Thr-Thr-Lys-
Thr ~SEQ ID NO.:155), and
s Lys-Asn-Ile-Phe-His-Phe-Lys-Val-Asn-Gln-GLu-Gly-Leu-Lys-
Leu-Ser-Asn-Asp-Met-Met (SEQ ID NO.:156).
Preferably, the following sequences are excluded:
Leu-Glu-Cys-Gly-Pro-Cys-Phe-Leu (SEQ ID NO.:157),
Leu-Cys-Ala-Gly-Pro-Cys-Phe-Leu (SEQ ID NO.:158),
o Tyr-Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu-Leu-Ile
(SEQ ID NO.:159),
Tyr-Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu-Ile (SEQ
ID NO.:160),
Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser
(SEQ ID NO.:161),
Thr-Pro-Pro-Thr-Pro-Cys-Pro-Ser (SEQ ID NO.:162),
Asp-Pro-Cys-Ile-Ile (SEQ ID NO.:163),
Cys-Gly-Gly-Ile-Cys-Ile-Ala-Arg (SEQ ID NO.:164),
Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser
20 (SEQ ID NO.:165),
Cys-His-Gly-Ser-Asp-Pro-Cys (SEQ ID NO.:166),
Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser
(SEQ ID NO.:167),
Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ
2s ID NO.:168),
Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Leu-Cys-Leu-
Ala-Arg (SEQ ID NO.:169),
Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly- Gly-Leu-Cys-Leu-Ala-Arg
(SEQ ID NO.:170),
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~yr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Leu-Cys-
Leu-Ala-Arg (SEQ ID NO.:171),
Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly-Gly-Gly-Leu-Cys-Leu-Ala-
Arg (SEQ ID NO.:172),
Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Gly-Leu-
Cys-Leu-Ala-Arg (SEQ ID NO.:173),
Cys-Gly-Gly-Leu-Cys-Ala-Arg (SEQ ID NO.:174), and
Ser-Pro-~yr-Met-Glu-Ala (SEQ ID NO.:175).
The nucleic acid molecule of the invention can be RNA
o (e.g., in a retrovirus) or DNA. It preferably encodes an
immunoactive peptide that is not a naturally occurring human
polypeptide nor a fragment of a naturally occurring human
polypeptide. Even where the sequence of the immunoactive
peptide happens to be that of a fragment of a naturally
occurring polypeptide, the nucleic acid molecule of the
invention differs from any naturally occurring nucleic acid
molecule in that the coding sequence encodes just that peptide,
optionally linked to a signal peptide. Of course, multiple
coding sequences can be linked in tandem, separated by stop
codons and potentially other noncoding sequence.
As stated above, the nucleic acid molecule may include a
sequence encoding a m~mm~ lian signal peptide. That signal
peptide would, when linked to the amino terminus of the
immunoactive peptide within a mammalian cell, direct the
secretion of the immunoactive peptide out of the cell. The
signal peptide is typically enzymatically cleaved from the
immunoactive peptide during the process of secretion. Selection
of a particular signal peptide depends upon the species of the
animal to be treated, and the amino-terminal amino acid
sequence of the immunoactive peptide to be expressed. Numerous
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21
examples of secretory signal sequences linked to immunoactive
gene sequences are shown in Figure 2. When a nucleic acid
molecule is to be administered to a human patient, as described
below, the signal peptide will usually be a human secretory
s signal peptide. The nucleic acid molecule of the invention will
generally also include a eukaryotic expression control
sequence, e.g. a mammalian expression control sequence,
operatively linked to the coding sequence. The expression
control sequence may be an inducible or constitutively active
o promoter that directs the expression of one or more
immunoactive peptides encoded on the nucleic acid molecule in a
tissue- or cell-specific manner. Selecting appropriate
secretory signal sequences and expression control sequences is
well within the abilities of skilled artisans, and further
guidance regarding this selection is given below.
A related aspect of the invention is a mammalian
expression vector, such as a viral, e.g. a retroviral,
adenoviral, or adeno-associated vector, that has been modified
by standard recombinant techniques to encode an immunoactive
peptide. ~hese viral vectors may be a part of a viral particle
that is capable of infecting mammalian cells. The expression
vector of the invention can be used to produce an immunoactive
peptide by, for example, introducing the expression vector into
a cultured mammalian cell, culturing the cell in vitro under
2s conditions that permit expression of the immunoactive peptide,
and harvesting the immunoactive peptide from the cell. If the
vector includes a secretory signal sequence, the immunoactive
peptide may instead be harvested from the medium surrounding
the cells. Alternatively, an immunoactive peptide may be
produced in a mammal (e.g., a human, simian, mouse, rat, guinea
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pig, hamster, rabbit, dog, cat, cow, pig, goat, sheep or horse)
by introducing into the mammal either (1) the nucleic acid
molecule of the invention, (2) an expression vector containing
the nucleic acid molecule of the invention, or (3) a cell that
contains and expresses the nucleic acid. In the latter case,
the cell or its descendent would be transduced with the nucleic
acid ex vivo. Such cells (particularly mammalian cells such as
human cells) are considered to be within the invention.
Other non-integrating viral vectors include herpes
simplex virus-based vectors, which have a broad cell
specificity and can accept up to 36 kb of nonviral sequence,
and the SV40 vector, which also targets a wide range of
tissues. Other viruses known to be useful for gene transfer
include adenoviruses, adeno associated virus, mumps virus,
poliovirus, retroviruses, Sindbis virus, and vaccinia virus
such as canary pox virus. Well-known methods of transducing
cells that do not re~uire a viral vector include calcium
phosphate precipitation, lipofection, electroporation, or
blolistic methods.
The immunoactive peptides discussed herein were so named
because of their ability to modulate an animal's immune
response, as demonstrated by the biological assays described
below. Thus, the invention features a method for modulating the
immune response in a patient by administering to the patient a
25 nucleic acid molecule encoding at least one peptide that
functions either as an immunosuppressant or as an
immunostimulant. The method may be carried out by administering
to the patient either (1) the isolated nucleic acid molecule
consisting essentially of the coding sequence linked to
30 expression control elements, (2) the nucleic acid molecule
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within an expression vector, or (3) a cell that secretes the
immunoactive peptide. In the latter case, cells of the patient
could be transduced ex vivo by standard techniques, such as
those described herein. The nucleic acid molecule, the vector
containing it, or a cell secreting the immunoactive peptide
could be administered to the patient by any route commonly
known to skilled pharmacologists. These include introduction
into the patient's bloodstream or cerebrospinal fluid, into the
synovial fluid, into a tumor, or into the vicinity of a tumor.
o It will be apparent to skilled artisans that the nucleic acid
molecule of the invention can be contained within a therapeutic
composition that is formulated with a pharmaceutically
acceptable carrier.
Nucleic acid molecules encoding peptides that function
as immunosuppressants may be administered to a patient who has
received a biological transplant e.g., of an organ such as a
kidney, heart, liver, eye, or lung; of a tissue such as skin or
bone marrow; or of cells such as fibroblasts, neural cells,
islet cells, hepatocytes, or chondrocytes. These
immunosuppressant-expressing nucleic acids can also be used to
treat a person suffering from an autoimmune disease, including
but not limited to the following: (1) a rheumatic disease such
as rheumatoid arthritis, systemic lupus erythematosis,
Sjogren's syndrome, scleroderma, mixed connective tissue
disease, dermatomyositis, polymyositis, Reiter's syndrome, or
Behcet's disease, (2) type I diabetes; (3) an autoimmune
disease of the thyroid, such as Hashimoto's thyroiditis or
Graves' Disease; (4) an autoimmune disease of the central
nervous system, such as multiple sclerosis, myasthenia gravis,
or encephalomyelitis; and (5) phemphigus such as phemphigus
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24
vulgaris, phemphigus vegetans, phemphigus foliaceus, Senear-
Usher syndrome, or Brazilian phemphigus.
Nucleic acids encoding peptides that function as
immunostimulants may be administered to a patient who is
thought to be suffering from a chronic infection, an acute
infection, or a cancer such as cancer of the breast, lung,
colon stomach, skin, brain, cervix, uterus, liver, bone,
pancreas, or hemotopoietic system.
Also within the invention is use of the nucleic acid
molecule of the invention in the preparation of a medicament
useful in treating any of the above conditions.
By "peptide" is meant any chain of more than two amino
acid residues, regardless of post-translational modification
such as glycosylation or phosphorylation.
As referred to herein, naturally occurring amino acids
are L-glycine (Gly; G), L-alanine (Ala; A), L-valine (Val; V),
L-~eucine (Leu; L), L-isoleucine (Ile; I), L-serine (Ser; S),
L-threonine (Thr; T), L-aspartic acid (Asp; D), L-glutamic acid
(Glu; E), L-lysine (Lys; K), L-arginine (Arg; R), L-histidine
(His; H), L-methionine (Met; M), L-cysteine (Cys; C), L-
asparagine (Asn; N), L-glutamine (Gln; Q), L-tyrosine (Tyr; Y),
L-tryptophan (Trp; W), L-phenylalanine ~Phe; F); and L-proline
(Pro; P).
All publications, patents, and other references cited
herein are incorporated by reference in their entirety.
The preferred methods, materials, and examples that will
now be described are illustrative only and are not intended to
be limiting. Other features and advantages of the invention
will be apparent from the following detailed description, from
the drawings, and from the claims.
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Brief Description of the Drawings
Fig. 1 is a list of examples of nucleic acid molecules
of the invention and the peptides they encode. An asterisk
demarks the boundary between a rat secretory signal sequence
and the sequence of each immunoactive peptide.
Fig. 2 is a schematic diagram of the Moloney murine
sarcoma virus retroviral vector p~XSN.
o Detailed Description
The experiments described herein utilize nucleic acid
molecules that encode peptides which can be used to modulate
the immune response. These nucleic acid molecules were cloned
into expression vectors, transduced into mammalian cells, and
shown to inhibit the formation of tumors in vivo, presumably by
upregulating the activity of T lymphocytes in the treated
animal.
Vector Construction
The following procedures were carried out in order to
construct vectors that could be used to transduce malignant
cells in vitro. Each vector described below includes a sequence
encoding (a) an immunoactive peptide that conforms to the
motif represented in Formula I, II, III, IV, or V, and (b) a
signal sequence that targets the peptide for export from the
transduced cell, and a eukaryotic expression control sequence.
Standard recombinant techniques were used to link these
sequences and clone them into the vector of choice.
A first single-stranded oligodeoxynucleotide was
synthesized, using standard techniques for DNA synthesis. This
oligodeoxynucleotide consisted of, from the 5' end:
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26
3-4 adenosine residues, a restriction enzyme site, a sequence
encoding a signal peptide, a sequence encoding an immunoactive
peptide, two stop codons, a second restriction enzyme site, and
another 3-4 adenosine residues. The restriction enzyme sites
were chosen to facilitate ligation between the
oligodeoxynucleotide and the vector of choice. In the examples
below, the restriction enzymes were chosen from the following:
EcoRI, BamHI, XhoI, and HpaI.
In each case, the signal sequence was chosen on the
o basis of the first N-terminal amino acid of the immunoactive
peptide. For example, immunoactive peptides having alanine as
their N-terminal amino acid were linked to signal sequences
that are naturally associated with peptides that have alanine
as the N-terminal amino acid. For experiments conducted with
rat cells, signal sequences were chosen from those which occur
naturally in rat cells. For use in other species, appropriate
signal sequences would be selected in an analogous way. Further
guidance in selecting these se~uences for use in humans is
given below. The DNA sequence encoding each signal peptide used
in the experiments described below was the naturally occurring
rat DNA sequence, except where it was necessary to modify it to
avoid including a restriction enzyme site that would complicate
the cloning strategy. Similarly, the DNA sequence encoding each
immunoactive peptide was chosen in part to avoid introducing
problematic restriction sites.
The single-stranded oligodeoxynucleotide prepared as
described above was made double-stranded as follows. An
antisense oligodeoxynucleotide complementary to
approximately 15 nucleotides at the 3' end of the first
oligodeoxynucleotide was synthesized by standard synthetic
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means. In order to anneal these two DNA molecules to one
another, they were placed in a solution of T7 DNA polymerase
buffer (United States Biochemicals), gradually heated to 60~C,
and held at that temperature for 30 minutes. Once annealed, a
s complete double-stranded molecule was enzymatically generated
with T7 DNA polymerase, according to the manufacturerls
instructions (United States Biochemicals). The double-stranded
DNA was purified by passing it over a Sephadex G50 ~Pharmacia,
Uppsala, Sweden) column in TE buffer (10 mM Tris, 1 mM EDTA at
pH 7.5) and digested with restriction enzymes corresponding to
the restriction sites that had been placed at each end of the
oligodeoxynucleotide. The DNA was extracted from the digest
with phenol-chloroform, precipitated with absolute ethanol at -
70~C, and collected by centrifugation, according to standard
S methods. The DNA pellet was washed with 70~ ethanol, dried
under vacuum, and redissolved in TE buffer.
DNA prepared as described above can be inserted into any
vector that has compatible restriction sites. In this case, the
DNA was inserted into the Moloney murine sarcoma virus
retroviral vector pLXSN (Fig. 2) which had been digested with
restriction enzymes to create cohesive ends complementary to
those created by digestion of the insert, i.e., with one of the
following pairs of restriction enzymes: (1) EcoRI-BamHI, (2)
EcoRI-XhoI, (3) EcoRI-HpaI, (4) HpaI-BamHI, (5) HpaI-XhoI, or
2s (6) XhoI-BamHI. To insert the DNA into the vector, a ligation
reaction containing approximately 20 ng of vector DNA and 4 ng
of insert DNA was carried out at 16~C with T4 DNA ligase
(Boehringer Mannheim). The ligation reaction was then used to
transform electrocompetent E. col 7 cells (DH5a strain).
Individual colonies that developed from transformed cells were
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28
picked at random and checked by the polymerase chain reaction
(PCR) for the presence of vectors that contained insert.
Colonies consisting of a clone of cells that contained the
desired construct (vector with insert) were amplified, and the
s DNA construct was isolated and sequenced by standard methods.
Once the presence and orientation of the insert was confirmed
by sequencing, large scale cultures were established in LB
medium supplemented with ampicillin (50 ~g/ml) and grown until
the OD at 600 nm was 0.8. Chloramphenicol was then added to a
final concentration of 180 ~g/ml, and the flasks were incubated
at 37~C, on a shaker, overnight. The DNA constructs were
isolated from the bacterial cultures using a QIAGEN aPlasimd
kit (QIAGEN, GmbH, Hilden, Germany), according to the
manufacturer's instructions.
Transduction of Cultured Cells
Cultured cells were obtained from two types of m~mm~ry
carcinomas: SPMWl cells were obtained from a tumor that
developed spontaneously in a female Wistar rat, and Ad9-101
cells were obtained from a tumor that developed after newborn
female Wistar rats were inoculated with adenovirus type 9
(Lindvall et al., 1991, Cancer Immunol. Immunother. 33:21-27).
The tumors were kept in serial passage in syngeneic rats.
Cell lines were established from the tumors as follows.
The tumor was excised from the animal, minced with a pair of
2s scissors, and treated with Dispase grade II for 30 minutes (2.4
mg/ml; Boehringer Mannheim) in RPMI 1640 medium to disaggregate
the cells. The SPMWl cell line was established from the
nineteenth in vivo passage of a tumor, and the Ad9-101 cell
line was established at the fifth in vivo passage. The
disaggregated cells were cultured in RPMI 1640 medium
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29
supplemented with 4 mM 1-glutamine, 1 mM pyruvate, 10 mM HEPES
buffer, 10 mM NaHCO3, and 5% fetal calf serum (FCS) in vessels
obtained from NUNC (Roskilde, Denmark).
To transduce the cells, DNA constructs were prepared as
described above and used to transfect a retroviral packaging
cell line GP+E or Psi2 with TRANSFECTAM~ (Promega, USA),
according to the manufacturer's instructions. Transfectants
were selected with G418 (300 ~g/ml) in RPMI 1640 with 10% FCS.
Virus-laden supernatant from the transfectants was then used to
o infect either SPMW1 cells or Ad9-101 cells. Successfully
transduced cells were selected in the presence of G418, and
clonal cell lines were developed.
An assay based on the polymerase chain reaction (PCR)
was used to demonstrate that the DNA encoding an immunoactive
s peptide was transcribed into mRNA within the transduced cells.
In addition, an in vitro biological assay of the effect
on proliferation of activated T lymphocytes of supernatant from
SPMW1 cells which had been transduced with a nucleic acid of
the invention is consistent with the conclusion that the
immunoactive peptide (D22175AX) is expressed and secreted by
the transduced cells, and overcomes the suppressive effect of
wild type SPMW1 cells on T lymphocyte proliferation.
Implantation of Transduced Cells
Transduced tumor cells were harvested from the culture
2s vessels by the addition of trypsin, collected by
centrifugation, and resuspended in phosphate buffered saline
(PBS) supplemented with 5% normal syngeneic rat serum. Each
rat in the experimental group received a subcutaneous
~ injection in the right hindlimb of approximately 200 ml of
resuspended cells. As a control, comparable rats received
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subcutaneous injections of the same type of tumor cells, but
which had not been transduced, and thus did not express or
secrete an immunoactive peptide.
Assessment of Tumor Size in vivo
s On the day the tumor cells were inoculated and at
various intervals ranging up to 36 days afterward, the hindlimb
was palpated at the site of injection. When a tumor could be
felt, the largest diameter was measured with a caliper. A
second measurement was taken perpendicular to the first. The
o volume of the tumor was calculated by using the formula
V=0.4(a-b2), where V = volume ~in mm3), a = the largest diameter
(in mm), and b = the diameter perpendicular to the largest
diameter (in mm).
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Example 1: Immunostimulation by D22175AX-expressing
SPMWl Cells
SPMWl mammary carcinoma cells in cell culture were
infected with a retroviral vector containing an insert encoding
s the immunoactive peptide D22175AX. Transduced cells were
selected in G418, as described above, but not cloned.
Approximately 25,000 transduced tumor cells were subcutaneously
injected into the hindlimbs of each of 8 rats. As a control, an
equivalent group of rats was similarly injected with
o approximately 25,000 wild type (i.e., non-transduced) SPMWl
cells. The size of the tumor that developed in vivo was
estimated according to the above formula in both groups for up
to fifteen days following injection. On any given day after
injection, the mean size of the tumor that developed from
15 D22175AX-expressing SPMWl cells was less than one-tenth the
size of the tumor that developed from wild type SPMWl cells
(Table 1). This result demonstrates that a construct encoding
and presumably expressing D22175AX substantially impedes tumor
growth in vivo.
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Table 1: Estimated Volume (mm3) of Palpated Tumor
Day SPMW1 wild type SPMW1 (D22175AX)
O O O
4 0 0
7 72 4
9 482 16
13 2791 205
4704 423
1435
24 2955
Example 2: Lack of Immunostimulation by D22175AX-
expressing SPMW1 Cells in Nude Rats
D22175AX-expressing SPMW1 cells were also injected
subcutaneously into the right hindlimb of "nude" rats. These
animals do not have a thymus and thus do not produce
T lymphocytes. Five animals were injected subcutaneously with
approximately 25,000 uncloned D22175AX-expressing SPMW1 cells
o and five were injected with a comparable number of wild type
SPMW1 cells. The tumors that developed in these two groups of
animals following inoculation grew at a comparable rate
(Table 2), suggesting that the inhibition of tumor growth seen
when immunocompetent rats are inoculated with D22175AX-
expressing SPMW1 cells involves a T cell-mediated immune
response.
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Table 2: Estimated Volume (mm3) of Palpated Tumor
Day SPMW1 wild type SPMW1 (D22175AX)
O O O
6 0 0
13 470 555
17 1820 1930
5805 6461
Example 3: Immunostimulation by D22175AX-expressing
Ad9-101 Cells
s Ad9-101 mammary carcinoma cells in cell culture were
infected with a retroviral vector containing an insert encoding
the immunoactive peptide D22175AX. As described in Example 1,
the cells were selected in G418, but they were not cloned.
Approximately 10,000 cells were subcutaneously injected into
o the hindlimbs of each of 5 rats. As a control, an e~uivalent
group of rats was injected with 10,000 wild type Ad9-101 cells.
On any given day after injection, the average size of the tumor
that had developed from D22175AX-expressing Ad9-101 cells was
less than one-tenth the size of the tumor that developed from
wild type Ad9-101 cells (Table 3). Therefore, expression of
D22175AX significantly impedes tumor growth in at least two
model systems.
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Table 3: Estimated Volume (mm3) of Pal~ated Tumor
Day Ad9-101 wild type Ad9-101
(D22175AX)
O O O
8 0 0
14 34 0
16 87 2
465 23
23 1055 55
27 4165 366
997
Example 4: Immunostimulation by two D22175AX-
expressing Ad9-101 clones; clone 8 and clone 9
s Cells from four different D22175AX-expressing Ad9-101
clonal cell lines were injected into rats in order to determine
whether different transduced clones expressing the same peptide
were equally effective in impeding tumor growth. Wild type Ad9-
101 cells served as the control for this experiment. Five
o animals in each group received subcutaneous injections
containing approximately 50,000 cells of uncloned, clone 6,
clone 7, clone 8, clone 9, or wild type Ad9-101 lines. As shown
in Table 4, all transduced clones exhibited significantly
slower tumor growth than did wild type Ad9-101 cells, at least
after day 13.
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Table 4: Estimated Volume (mm3) of Palpated Tumor
Day Wild Uncloned Clone 6 Clone 7 Clone 8 Clone 9
~ype
O O O O O O O
7 0 0 0 0 0 0
2 0 2 28 0 0
13 64 7 4 80 0 2
17 646 28 37 230 14 3
21 3024 84 85 714 108 8
24 149 77 1679 500 9
28 757 279 2700 972 48
Example 5: Immunostimulation by D22175AX-expressing
Ad9-101 Cells is Mediated by T Lymphocytes
In order to determine whether T lymphocytes are
activated in response to in vivo expression of an immunoactive
peptide, the following experiment, which extends from
Example 4, was performed. Rats were injected subcutaneously
with either wild type Ad9-101 cells or D22175AX-expressing Ad9-
o 101 cells (clone 8 or clone 9). Each animal was killed when its
tumor reached 50-100 mm3 in size, and approximately 2.5 x 105
cells were harvested from the lymph nodes associated with the
tumor, i.e., the inguinal and para-aortal lymph nodes
ipsilateral to the tumor. Cells were also harvested from the
s lymph nodes of normal rats (which were free of tumors). The
lymphatic cells from each rat were placed in culture either as
- a homogeneous population, or in co-culture with approximately
7.5 x 103 lethally irradiated (8000 rad=80 Gray) wild type Ad9-
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101 cells. The homogeneous and heterogeneous cultures were
established in parallel in a total of 10 wells of a 96 well
plate (NUNC, Roskilde, Denmark), and grown for 5 days in RPMI
1640 medium supplemented with 4 mM L-glutamine, 1 mM pyruvate,
10 mM Hepes buffer, 15 mM NaHCO3, 50 ~I,13-mercaptoethanol, and
10% FCS. On the fifth day in culture, the cells were exposed to
~3H]-thymidine (0.5 ~Ci) for 6 hours. Although other cell
types, such as monocytes, natural killer cells, and B
lymphocytes are present, under the conditions of the culture,
n only T lymphocytes can respond with mitotic activity on day 5.
The radioactivity incorporated into acid-insoluble material,
which reflects the mitotic activity of the cells in culture,
was measured with a scintillation counter. The amount of
radioactivity incorporated into homogeneously cultured lymph
cells was subtracted from the amount of radioactivity
incorporated into heterogeneous cultures containing lymphatic
and irradiated tumor cells. (The incorporated radioactivity is
attributable solely to proliferation of the lymphatic cells,
because the tumor cells were lethally irradiated prior to co-
culture and so could not proliferate.) The data obtained fromtwo trials, and expressed as counts per minute (cpm), are
presented in Table 5.
, .
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Table 5: 3H-thymidine Incorporation by Cultured Lymphatic Cells
~ymphatic Cell~ from animal Trial 1 Trial 2
with:
No tumor 1,016 669
Wild type Tumor 680 0
Clone 8 Tumor 5,101 12,632
Clone 9 Tumor 18,394 59,026
These results demonstrate that, in the presence of
lethally irradiated (but presumably still antigenic) wild
s type Ad9-101 cells, the mitotic activity of T lymphocytes
harvested from animals that had been injected with D22175AX-
expressing Ad9-101 cells (clone 8 or clone 9) is
substantially greater than the mitotic activity of T
lymphocytes from either normal (non-injected) animals or
o animals that were injected with wild type Ad9-101 tumor
cells. The data suggest that "vaccination" with wild type
tumor cells actually decreases the ability of the animal's T
lymphocytes to respond to a subsequent challenge with wild
type tumor cells, while vaccination with D22175AX-expressing
s tumor cells not only overcomes this inhibition, but renders
the animal's T lymphocytes substantially more sensitive to
challenge with wild type tumor cells than the T lymphocytes
of an unimmunized animal.
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Example 6: Immunization with Irradiated D22175AX-
expressing Ad9-101 Cells Impedes Subsequent Tumor
formation by Wild Type Ad9-101 Cells
In this experiment, rats were inoculated with either
Ad9-101 wild type cells that had been irradiated with
10,000 rad (100 Gray), or clone 9 cells.that had been similarly
irradiated. (Irradiation prevents the cells from dividing more
than once, but it does not immediately affect protein
synthesis.) Animals received two subcutaneous injections of
o irradiated cells (1 x 106 cells/animal/dose), 14 days apart,
while a group of control animals received comparable injections
of phosphate buffered saline (PBS). Fourteen days following the
second injection, by which time it is expected the irradiated
cells had all been cleared from the injection site, the animals
were challenged with an injection of 10,000 non-irradiated Ad9-
101 wild type cells, and tumor growth was assessed. The tumors
that developed in animals that had been injected with
irradiated clone 9 cells were on average substantially smaller
than the tumors that developed in rats that were injected with
PBS or with irradiated wild type Ad9-101 cells (Table 6). Thus,
the antitumor effect of D22175AX persists even after the
D22175AX-expressing cells were presumably cleared from the test
animals' bodies.
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Table 6: Estimated Volume (mm3) of Palpated Tumor
DayPBS immunized Wild type Clone 9
immunized immunized
O O O O
7 0 0 0
12 0 0 0
1 7
19 158 347 102
21 395 618 193
26 2023 2136 882
28 4218 1481
Example 7: Immunomodulation by D22139AA-expressing,
D22069AX-expressing, or D7208-expressing Ad9-101
cells
To compare the effects of three different immunoactive
peptides on tumor growth, rats were inoculated with transduced
Ad9-101 cells that expressed either D22139AA, D22069AX, or
D7208 (see Fig. 1). The immunoactive peptide D22139AA (without
o signal sequence) has previously been shown to be
immunostimulatory when administered directly in a delayed-type
hypersensitivity (DTH) assay, while D22069AX and D7208 (each
without signal sequence) have shown activity consistent with
immunosuppression. Approximately 20,000 transduced cells were
s injected subcutaneously into each rat (5 animals/group), and
the tumors which developed were measured. As shown in Table 7,
~ uncloned cells expressing D22139AA formed tumors that grew
somewhat more slowly than those which developed from wild type
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Ad9-101 cells, consistent with the immunostimulatory activity
of this peptide observed in the DTH test. In contrast, the
tumors that developed from uncloned cells expressing either
D22069AX or D7208 grew faster than the tumors that developed
from wild type Ad9-101 cells (at least after day 22),
consistent with the immunosuppressant activity of these
peptides in the DTH test. This suggests that the delayed-type
hypersensitivity assay described below is a useful predictor of
the immunomodulatory effect of peptides expressed from the
nucleic acid molecules of the invention.
Table 7: ~stimated Volume (mm3) of Palpated Tumor
Day Wild type D22139AA D22069AX D7208
O O O O O
12 0 0 0 0
6 0 7 10
19 95 38 148 107
22 502 248 674 488
26 582 328 1035 767
29 1206 766 1546 1734
34 1936 1728 3842 2681
36 3047 2792 5306 3493
,, ~, ~ . . .
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Example 8: Inoculation of Various Clones of D22139AX-
expressing Cells
Several clones of D22139AX-expressing Ad9-101 cells were
established. Each rat (n=5 animals/group) received an injection
s of either wild type AD9-101 cells, uncloned D22139AX-expressing
Ad9-101 cells, or cells from one of the D22139AX-expressing
Ad9-101 clones designated 1, 2, 3, 5, 6, 7, and 9. Tumor growth
was assessed as described above. The data gathered from animals
injected with clone 6, clone 7, wild type, or uncloned
D22139AX-expressing cells are presented in Table 8. The tumors
that developed from uncloned D22139AX-expressing Ad9-101 cells
were on average somewhat smaller than the tumors that developed
from wild type Ad9-101 cells, consistent with the results of
the experiment shown in Table 7, while the tumors that
developed from clones 6 and 7 were substantially smaller. In
addition, clones 1, 2, 3, 5, and 9 showed essentially no tumor
outgrowth through day 35 (data from clone 2 is shown in Table
8).
Table 8: Estimated Volume (mm3) of Palpated Tumor
Day Uncloned Clone 6 Clone 7 Wild Type Clone 2
O O O O O O
7 0 0 0 0 0
18 27 0 139 0
19 77 59 3 289 0
- 26 692 131 88 906 0
1455 447 128 2376 0
2575 901 301 6973
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Example 9: Expression of IL-7 has
No Effect on Tumor Growth
In order to demonstrate that the immunomodulatory effect
observed in the experimental paradigms described above is due
s to the expression of nucleic acid molecules that encode
peptides conforming to Formula I, II, III, IV, or V, a nucleic
acid molecule encoding a peptide that does not conform to any
of these formulas was studied according to the paradigm
described in Example 1. Approximately 10,000 SPMW1 cells
n transduced with IL-7 (interleukin 7) were subcutaneously
injected into the hindlimbs of each of 7 rats. As a control, an
equivalent group of rats was similarly injected with
approximately 10,000 wild type (i.e., non-transduced) SPMW1
cells. The size of the tumor that developed in vivo was
estimated in both groups for up to seventeen days following
injection. As shown in Table 9, expression of IL-7 had no
effect on tumor growth, and therefore presumably no effect on
the animals' immune response.
Table 9: Estimated Volume (mm3) of Palpated Tumor
Day Wild Type IL-7 Infected
O O O
6 65 85
13 925 1543
17 2574 2380
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Additional Assays for Identifying Immunostimulants
The following assays can be used to identify
immunostimulatory peptides that can be delivered by gene
therapy in accordance with the invention.
Delayed Type Hypersensitivity (DTH) Test
The ability of a DNA molecule to encode a peptide that
modulates the immune response can be determined using the
delayed type hypersensitivity (DTH) test in mice. The detailed
protocol for this assay can be found, for example, in Carlsten
et al. (1986, Int. Arch. Allergy Appl. Immunol. 81:322).
Briefly, male or female mice, such as Balb/c mice, are
sensitized by exposure to 4-ethoxymethylene-2-phenyloxazolin-5-
one (OXA; Sigma Chemical Co.). On Day 0, 150 ~1 of an absolute
ethanol-acetone (3:1) solution containing 3% OXA is applied to
s the animal's shaved abdomen. Treatment with the immunoactive
peptide itself (e.g., by topical or IV administration), or with
gene therapy using a nucleic acid encoding the peptide, is then
begun (methods of administration are discussed below).
Approximately one week after sensitization, the thickness of
the animal's ears is measured with an Oditest spring caliper
before both ears are challenged by topical application of 20 ~1
of 1% OXA dissolved in an oil, such as peanut oil. Ear
thickness is measured again 24 and 48 hours after the
challenge. To minimize discomfort, challenges and measurements
are performed under light anesthesia.
The intensity of the DTH reaction is measured as described
by van Loveren et al. (1984, J. Immunol. Methods 67:311), and
expressed according to the formula: Tt24/48 - TtO (in mm
units) where t0, t24, and t48 represent ear thickness before,
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24 hours after, and 48 hours after the challenge,
respectively. The ability of the peptide or gene therapy to
modulate ear thickness is an indication of its ability to
modulate the immune response: a relative increase in
thickness indicates a heightened response, while a relative
decrease in thickness indicates immune suppression.
Inhibition of Tumor Growth
Nucleic acids encoding peptides that stimulate the
immune response can be identified using any model system
analogous to the mammary carcinoma models described above.
These additional model systems could be developed with
immortalized cells from an established cell line or an induced
or spontaneous tumor of an animal. Other cell lines that would
be amenable to an assay for tumor growth are readily available
from the American Type Culture Collection (A.T.C.C.), which
maintains cell lines established from a wide variety of tumors
that developed in many different species. Transgenic animals
that develop tumors due to, for example, overexpression of an
oncogene or inhibition of a tumor suppressor gene provide a
second source of tumor cells suitable for tumor growth assays.
Alternatively, a spontaneous or induced tumor from an animal
(e.g., a spontaneous tumor from a human) could be used. Cells
from any of these sources could be placed in culture,
transduced with a nucleic acid encoding a candidate
immunoactive peptide, and transplanted into a test animal.
Cells cultured in parallel, but untransduced or transduced only
with the "empty" vector or a vector encoding a non-immunoactive
peptide, would be transplanted into control animals. If the
transplanted cells that expressed the candidate peptide failed
to produce a tumor, or produced a tumor that was significantly
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smaller than that found in control animals, the encoded peptide
would be deemed an effective immunostimulant. In a variation on
this assay, the transduced cells could be irradiated and mixed
with non-irradiated wild type cells prior to injection, so that
the effect of the secreted peptide on growth of wild type tumor
cells in vivo would be measured. Model systems developed from
different sources of immortalized cells may provide the means
to determine whether peptides are broadly effective or
particularly effective in treating a certain type of cancer.
o Immunization Assay
Genes encoding peptides that stimulate the immune
response can also be identified in various model systems by the
~immunization" procedure described in Example 6. Immortalized
cells obtained from the A.T.C.C. or established from primary
s tumor tissue as described above, would be transduced with the
gene of interest, lethally irradiated, and injected into
animals. As a control, animals could be injected with
irradiated wild type tumor cells. Subsequently, both groups of
animals would be challenged with wild type tumor cells and
examined for tumor formation. If the peptide is an
immunostimulant with therapeutic potential, the growth of the
tumor following the challenge with wild type cells would be
impeded in animals that had been immunized with peptide-
expressing cells. By performing the analysis of a given gene in
model systems that employ immortalized cells from an array of
tumors, it should be possible to determine whether the gene
encodes a peptide that could serve as a broad-based vaccine, or
whether it is primarily effective against a particular form of
cancer. In a variation on this method, one could use as the
test animals transgenic animals (e.g., mice) that spontaneously
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46
develop tumors. Transduced, irradiated tumor cells from such an
animal can be used to imml]nize other animals of the same line,
before they begin to develop any tumors. Subsequent spontaneous
or induced tumor formation and growth are then assessed in the
s immunized animals.
Assay for Tumor Regression
Another way to assay a peptide-encoding gene is to
administer it to an animal after a tumor has formed. The animal
can be one that is a transgenic model for tumor formation, or
o one which has developed a tumor following injection of wild
type tumor cells. When a tumor of a given size has formed in a
test animal, the animal is injected with the peptide-encoding
vector or with peptide-expressing cells, which could either be
viable or lethally irradiated. A reduction in tumor size or
number compared to control, or an extended time to death, would
provide very strong evidence for the utility of genes encoding
immunostimulatory peptides in the treatment of cancer.
In each of the assays described above, it would be
possible to show that the immune system was suppressed by the
gene product in question by performing the experiment presented
in Example 5. This experiment would demonstrate that the gene
product exerted its influence by stimulating the activity of T
lymphocytes, rather than by a mechanism that is independent of
the immune system.
2s Peptides that are ineffective in impeding tumor
outgrowth, or that enhance tumor outgrowth, could be tested
further, as described below, in order to determine whether they
would be useful immunosuppressants.
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Assays for Identifying Immunosuppressants
The following procedures could be employed in order to
determine whether a given peptide is an immunosuppressant that
could be usefully delivered by genetic therapy.
s Transplantation Paradigms
In order to determine whether a peptide is capable of
functioning as an immunosuppressant, it can be administered,
directly or by genetic therapy, in the context of well-
established transplantation paradigms.
o A putative immunosuppressing peptide, or a nucleic acid
molecule encoding it, could be systemically or locally
administered by standard means to any conventional laboratory
animal, such as a rat, mouse, rabbit, guinea pig, or dog,
before an allogeneic or xenogeneic skin graft, organ
transplant, or cel~ implantation is performed on the animal.
Alternatively, the graft itself could be transduced with the
nucleic acid of the invention. Strains of mice such as
C57Bl-10, B10.BR, and B10.AKM (Jackson Laboratory, Bar Harbor,
ME), which have the same genetic background but are mismatched
for the H-2 locus, are well suited for assessing various organ
grafts.
A method for performing cardiac grafts by anastomosis of
the donor heart to the great vessels in the abdomen of the host
was first published by Ono et al. (1969, J. Thorac. Cardiovasc.
Surg. 57:225; see also Corry et al., 1973, Transplantation
16:343). According to this surgical procedure, the aorta of a
donor heart is anastomosed to the abdominal aorta of the host,
and the pulmonary artery of the donor heart is anastomosed to
the adjacent vena cava using standard microvascular techniques.
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Once the heart is grafted in place, and warmed to 37~C with
Ringer's lactate solution, normal sinus rhythm will resume.
Function of the transplanted heart can be assessed frequently
by palpation of ventricular contractions through the abdominal
s wall. Rejection is defined as the cessation of myocardial
contractions. With regard to the current invention, a given
peptide would be considered a successful immunosuppressant if
it prolonged the time the grafted organ was tolerated by the
host.
o The effectiveness of an immunoactive peptide can also be
assessed following a skin graft. To perform skin grafts on a
rodent, a donor animal is anesthetized and the full thickness
skin is removed from a part of the tail. The recipient animal
is also anesthetized, and a graft bed is prepared by removing a
portion of skin from the shaved flank. Generally, the patch is
approximately 0.5 x 0.5 cm. The skin from the donor is shaped
to fit the graft bed, positioned, covered with gauze, and
bandaged. The grafts can be inspected daily beginning on the
sixth post-operative day, and are considered rejected when more
than half of the transplanted epithelium appears non-viable.
Another technique for assaying immunosuppression is with
cells that have been tranduced with a nucleic acid of the
invention, then implanted into an allogeneic or xenogeneic
animal. If the transduced implanted cells survive longer than
control implanted cells which have not been transduced, then
the nucleic acid molecule presumably encodes an
immunosuppressive peptide.
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Models of Autoimmune Disease
Models of autoimmune disease provide another means to
assess peptides in vivo. These models are well known to skilled
artisans and can be used to determine whether a given peptide
s is an immunosuppressant that would be therapeutically useful in
treating a specific autoimmune disease when delivered via
genetic therapy.
Autoimmune diseases that have been modeled in animals
include rheumatic diseases, such as rheumatoid arthritis and
o systemic lupus erythematosis (SLE), type I diabetes, and
autoimmune diseases of the thyroid and central nervous system.
For example, animal models of SLE include MRL mice, BXSB mice,
and NZB mice and their Fl hybrids. These animals can be crossed
in order to study particular aspects of the rheumatic disease
process; the NZB strain develops severe lupus
glomerulonephritis when crossed with NZW mice (Bielschowsky
et al., 1959, Proc. Univ. Otago Med. Sch. 37:9; see also
Fundamental Immunology, 1989, Paul, Ed., Raven Press, New York,
NY). Similarly, a shift to lethal nephritis is seen in the
progeny of NBZ X SWR matings (Data et al., 1976, Nature
263:412). The histological appearance of renal lesions in SNFl
mice has been well characterized (Eastcott et al., 1983, J.
Immunol. 131:2232; Paul, supra) . Therefore, the general health
of the animal as well as the histological appearance of renal
2s tissue can be used to determine whether the administration of
genes encoding immunoactive peptides can effectively suppress
the immune response in an animal model of SLE.
One of the MRL strains of mice that develops SLE, MRL,-
lpr/lpr, also develops a form of arthritis that resembles
rheumatoid arthritis in hurnans (Theofilopoulos et al., 1985,
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Adv. Immunol. 37:269). Alternatively, an experimental arthritis
can be induced in rodents by injecting rat type II collagen ~2
mg/ml) mixed 1:1 in Freund~s complete adjuvant (100 ~1 total)
into the base of the tail. Arthritis develops 2-3 weeks after
s immunlzatlon.
The ability of genes encoding immunoactive peptides to
combat the arthritic condition can be assessed by targeting the
genes to T lymphocytes and/or to synovial cells of the joint.
One way to target T lymphocytes is the following: spleen cell
o suspensions are prepared 2-3 days after the onset of arthritis
and incubated with collagen (100 ~g/ml) for 48 hours to induce
proliferation of collagen-activated T lymphocytes. During this
time, the cells are transduced with a vector encoding the
peptide of interest. As a control, parallel cultures are
untransduced or transduced with the "empty" vector. The cells
are then injected intraperiotoneally (5 x 107 cells/animal).
The effectiveness of the treatment is assessed by following the
disease symptoms during the subsequent 2 weeks, as described by
Chernajovsky et al. (1995, Gene Therapy 2:731-735). A decrease
in symptoms compared to control indicates that the peptide of
interest, and the gene encoding it, function as an
immunosuppressant potentially useful in treating autoimmune
disease.
Alternatively, one could introduce the peptide-encoding
vector, e.g., an adenoviral vector, directly into the cells of
the joint synovium. An effective control in this instance would
entail injecting a joint on the opposite side of the same
animal or the joint of a second animal, with an adenovirus that
carries the vector sequence only (Evans et al., 1995, Trends in
30 Mol. Med. 27:543-546).
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The ability of genes encoding immunoactive peptides to
suppress the immune response in the case of Type I diabetes can
be tested in the BB rat strain, which was developed from a
commercial colony of Wistar rats at the Bio-Breeding
s Laboratories in Ottawa. These rats spontaneously develop
autoantibodies against pancreatic islet cells and insulin, just
as occurs in human Type I diabetes. Prior to development of
full-scale diabetes in these animals, the vector of the
invention could be targeted to their T lymphocytes, as
o discussed above, or to islet cells.
Human Therapy
The application of peptide-encoding genes to the
treatment of cancer, infection, autoimmune disease, or graft
rejection in humans can utilize either in vivo or ex vivo based
therapeutic approaches.
Ex vivo-based Human Therapies
This approach would entail harvesting cells (e.g., tumor
cells, synovial cells, or T lymphocytes) from a patient,
establishing them in culture, and transducing them with a
nucleic acid of the invention. The transduction step could be
accomplished by any standard means used for ex vivo gene
therapy, including calcium phosphate, lipofection,
electroporation, viral infection, and biolistic gene transfer.
Cells that have been successfully transduced are then selected,
2s for example via a drug resistance gene. The cells may then be
lethally irradiated if desired (e.g., for tumor cells) and
injected or implanted into the patient.
For treatment of rheumatoid arthritis, T lymphocytes
obtained from the synovial fluid of an affected joint are
stimulated ex vivo with IL-2 or anti-human CD3 monoclonal
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antibody and simultaneously infected with a relevant gene
construct. The cells are reintroduced into the patient,
e.g., by intravenous administration, where they should home to
the diseased joints and produce the peptide at the site of the
s inflammation. A retroviral or adeno-associated viral vector
would be appropriate for this ex vivo infection procedure.
In vivo-based Human Therapies
The in vivo approach re~uires delivery of the construct
of the invention directly into the patient, targeting it to the
o cells or tissue of interest. For example, after surgical
removal of a primary tumor, residual cells may be targeted by
treating the vicinity of the tumor with a composition
containing a retroviral vector encoding an immunostimulatory
peptide. Instead of surgery, the primary tumor could be treated
by in si tu injection of the vector directly into the tumor.
Malignant cells distal to the primary tumor site may be reached
by delivering the vector intravenously. Targeting of tumor
cells can be accomplished by the use of a retrovirus, which
targets proliferating cells. Alternatively, one could utilize
an engineered cell attachment ligand on the vector or the viral
particle to accomplish preferential targeting of specific cells
in accordance with standard methods.
An example of a non-viral vector system is a molecular
conjugate composed of a plasmid attached to poly-L-lysine by
electrostatic forces. Poly-L-lysine covalently binds to a
ligand that can bind to a receptor on tumor cells (Cristiano et
al., 1995, J. Mol. Med 73:479-486). A promoter inducing
relatively tumor-specific expression can be used to achieve a
further level of targeting: for example, a-fetoprotein promoter
for hepatocellular carcinoma (Huber et al., 1991, Proc. Natl.
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Acad. Sci. USA 88:8039-8043) or the tyrosinase promoter for
melanoma (Hart et al., 1995, Br. Med. Bull. 51(3):647-655).
Alternatively, a constitutively active promoter could be used,
e.g., the SV40 promoter or CMV promoter.
One could treat rheumatoid arthritis via direct
inoculation of the vector into the affected joint in order to
infect the synovial cells in situ. Adeno-associated viral
vectors may be used if long-term expression is desired, or an
adenoviral vector for shorter-term expression. Both of these
o vectors are known to infect synovial cells in rabbits (Evans et
al. Gene therapy for arthritis. In Wolff, J.A. Ed.
Therapeutics: Methods and Applications of Direct Gene Transfer.
Birkhauser, Boston, MA, 1994 320-343).
Other embodiments are within the following claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
s (i) APPLICANT: Bergstrand et al., Hakan
(ii) TITLE OF INVENTION: EXPRESSION OF RECOMBINANT PEPTIDES
(iii) NUMBER OF SEQUENCES: 176
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Astra AB
(B) CITY: SODERTALJE
(C) COUNTRY: SWEDEN
(D) ZIP: S-151 85
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
CA 02250707 l998-l0-07
W O 97/39023 rCT/SE97/00574
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: D 1530
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: +46 8 553 26000
(B) TELEFAX: +46 8 553 28820
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Glu Glu Cys Cys Phe Tyr
1 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
s (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Pro Gly Cys Cys Gly Pro
l 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Pro Gly Cys Cys Pro Gly
l 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Gly Pro Cys Cys Pro Gly
l 5
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ala Pro Cys Cys Val Pro
l 5
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(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
txi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Val Ile Cys Cys Leu Thr
l 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Lys Pro Cys Cys Glu Arg
l 5
CA 022~0707 1998-10-07
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59
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Lys Glu Cys Cys Tyr Val
l 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Thr Pro Cy5 Cys Phe Ala
l 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
Leu Ala Cys Cys Val Val
l 5
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
20 (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Pro Val Cys Cys Ile Gly
l 5
... . . ...
CA 02250707 l998-l0-07
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(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ser Gln Cys Cys Ser Leu
1 5
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ser Ile Cys Cys Thr Lys
1 5
CA 022~0707 l998-l0-07
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62
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Lys Leu Cys Cys Asp Ile
1 5
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Pro Ala Cys Cys Gly Pro
1 5
CA 022~0707 l998-l0-07
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63
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Pro Asp Cys Cys Ile Pro
1 5
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Arg Cys Ser Gly Cys Cys Asn
1 5
CA 022~0707 l998-l0-07
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64
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Val Cys Ile Cys Gln
1 5
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Val Cys Gly Cys Arg
1 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Lys Cys Arg Cys Lys
1 5
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Asp Cys Ile Cys Gln
1 5
CA 022~0707 1998-10-07
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66
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ile Cys Thr Cys Glu
l 5
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Ile Cys Thr Cys Arg
l 5
CA 022~0707 1998-10-07
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67
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Leu Cys Ala Cys Val
1 5
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Phe Cys Ile Cys Lys
1 5
CA 02250707 1998-10-07
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68
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
s (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ala Cys Lys Cys Gln
l 5
(2) INFORMATION FOR SEQ ID No 27
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Gly Pro Cys Ile Cys Pro Gly
l 5
. , . , , ~
CA 02250707 1998-10-07
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69
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Gly Pro Cys Gly
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ala Pro Cys Ala
CA 022~0707 1998-10-07
W 097t39023 PCT/SE97/00574
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Ile Pro Cys Tyr
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Trp Pro Cys Gly
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Gly Pro Cys Ile Leu Asn
1 5
(2) INFO~MATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Gly Pro Cys Ile
CA 022~0707 1998-10-07
W O 97139023 PCT/SE97100574
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Leu Leu Phe Gly Pro Cys Ile
l 5
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Leu Leu Phe Ala Pro Cys Ile
l 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
s (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Leu Leu Phe Arg Pro Cys Ile
1 5
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Leu Leu Phe Ile Pro Cys Ile
1 5
CA 022~0707 1998-10-07
W 097t39023 PCT/SE97/00574
74
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Leu Leu Phe Asp Pro Cys Ile
1 5
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Ala Val Trp Thr Pro Cys Tyr
1 5
CA 022~0707 l998-l0-07
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(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Phe Val Met Ala Pro Cys Phe
1 5
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Leu Leu Tyr Ser Pro Cys Phe
- 1 5
CA 022~0707 1998-10-07
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76
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
tD) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Ile Ser Gly Pro Cys Pro Lys
1 5
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Phe Leu Phe Gly Pro Cys Ile
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Leu Phe Gly Pro Cys Ile Leu
1 5
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Glu Lys Gly Pro Cys Tyr Arg
1 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID No:46:
Phe Cys Leu Gly Pro Cys Pro
l 5
~2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Phe Gly Pro Cys Ile
l 5
CA 022~0707 1998-10-07
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79
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
s (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Phe Leu Phe Gly Pro Cys Ile Leu Asn
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Gly Pro Cys Ile Leu Asn Arg
1 5
CA 022~0707 l998-l0-07
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(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Leu Leu Phe Trp Pro Cys Ile
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Leu Leu Phe Gly Ile Cys Ile
1 5
CA 022~0707 1998-10-07
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(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID No:52:
Leu Leu Phe Gly Pro Cys Ile Leu Asn
1 5
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
-
Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg
~ 1 5 10
CA 022~0707 1998-10-07
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82
(2) INFORMATION FOR SEQ ID No:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe
l 5 l0
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
ZS
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu Met Glu
l 5 l0
CA 022~0707 l998-l0-07
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83
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Phe Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu Met Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Met Lys Phe Leu Ser Ala Arg Asp Phe His Pro Val Ala Phe Leu Gly
1 5 10 15
CA 022~0707 l998-l0-07
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84
Leu Met Leu Val Thr Thr Thr Ala Phe Gly Pro Cys Ile Leu Asn Arg
(2) INFORMATION FOR SEQ ID NO:58:
s
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
Ser Pro Cys Leu Pro Cys Arg Ala Leu Leu Phe Gly Pro Cys Ile
20 25 30
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02250707 l998-l0-07
W O 97/39023 PCT/SE97/00574
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
Ser Pro Cys Leu Pro Cys Arg Ala Leu Leu Tyr Ser Pro Cys Phe
lo (2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Met Trp Phe Leu Ile Leu Phe Leu Ala Leu Ser Leu Gly Gln Ile Asp
1 5 10 15
Ala Ala Pro Gly Cys Cys Pro Gly
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
CA 02250707 l998-l0-07
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86
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Met Lys Phe Leu Ser Ala Arg Asp Phe His Pro Val Ala Phe Leu Gly
1 5 10 15
Leu Met Leu Val Thr Thr Thr Ala Phe Cys Leu Gly Pro Cys Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Met Trp Phe Leu Ile Leu Phe Leu Ala Leu Ser Leu Gly Gln Ile Asp
1 5 10 15
, .. . .
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
87
Ala Ala Pro Gly Cys Cys Gly Pro
(2) INFORMATION FOR SEQ ID NO:63:
s
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
Met Lys Val Ala Ile Ile Phe Leu Leu Ser Ala Leu Ala Leu Leu Ser
1 5 10 15
Leu Ala Gly Pro Cys Cys Pro Gly
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
- (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
CA 022~0707 l998-l0-07
WO 97/39023 PCT/SE97/00574
88
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
Ser Pro Cys Leu Pro Cys Arg Ala Leu Leu Phe Ala Pro Cys Ile
20 25 30
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
Met Gly Phe Leu Lys Phe Ser Pro Phe Leu Val Val Ser Ile Leu Leu
1 5 10 15
Leu Tyr Gln Ala Cys Gly Leu Gln Ala Val Ile Cys Cys Leu Thr
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
89
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
Ser Pro Cys Leu Pro Cys Arg Ala Leu Leu Phe Gly Pro Cys Ile Leu
20 25 30
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67O
CA 02250707 l998-l0-07
W O g7/39023 PCT/SE97/00574
Met Trp Phe Leu Ile Leu Phe Leu Ala Leu Ser Leu Gly Gln Ile Asp
1 5 10 15
Ala Ala Pro Asp Cys Cys Ile Pro
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
lS (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
Ser Pro Cys Leu Pro Cys Arg Ala Leu Leu Phe Asp Pro Cys Ile
CA 022~0707 1998-10-07
W O 97/39023 PCT/SE97/00574
91
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs
S (B) TYPE: nucl~ic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID No:69:
ATGAAGTTCC TCTCTGCAAG AGACTTCCAT CCAGTTGCCT TCTTGGGACT GATGCTGGTG 60
ACAACCACGG CCTTCGGACC CTGCATTCTT AATCGA 96
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGGCTCTGTC GCC~~ l~ 60
CCTTGCAGGG CCCTGCTGTT CGGACCGTGC ATT 93
(2) INFORMATION FOR SEQ ID NO:71:
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
92
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
S (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGGCTCTGTC GCC~~ lG 60
CCTTGCAGGG CCCTGCTGTA TTCCCCGTGC 90
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
ATGTGGTTCC TGATCCTGTT CCTCGCCCTG TCCCTGGGAC AGATTGATGC TGCACCTGGC 60
T TGC CT 72
GC C G GC
CA 022~0707 1998-10-07
W O 97/39023 PCTISE97/00574
93
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
ATGAAGTTCC TCTCTGCAAG AGACTTCCAT CCAGTTGCCT TCTTGGGACT GATGCTGGTG 60
ACAACCACGG CCTTCTGCCT CGGACCATGC 90
(2) INFORMATION FOR SEQ ID NO:74:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
ATGTGGTTCC TGATCCTGTT CCTCGCCCTG TCCCTGGGAC AGATTGATGC TGCACCAGGA 60
TGCTGCGGAC CA 72
(2) INFORMATION FOR SEQ ID NO:75:
CA 022~0707 1998-10-07
W 0 97/39023 PCT/SE97/00574
94
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
ATGAAAGTGG CCATCATCTT CCTGCTGTCC GCACTGGCAC TGCTGTCCCT GGCAGGACCA 60
TGCTGCCCAG GA 72
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGG~~ ~lC GCCCTGTCTG 60
25 CCTTGCAGGG CCCTGCTGTT CGCGCCATGC 90
CA 022~0707 1998-10-07
WO 97/39023 PCT/SE97/00574
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:77:
ATGGGCTTCC TGAAATTCTC CCCTTTCCTG GTGGTGTCCA TCCTGCTGCT GTATCAAGCG 60
TGTGGCCTGC AAGCGGTGAT CTGCTGCCTG 90
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGGCTCTGTC GCC~ lG 60
CCTTGCAGGG CCCTGCTGTT CGGACCGTGC ATTCTG 96
(2) INFORMATION FOR SEQ ID No:79
CA 022~0707 1998-10-07
W 097/39023 PCT/SE97/00574
96
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 72 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
S (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
ATGTGGTTCC TGATCCTGTT CCTCGCCCTG TCCCTGGGAC AGATTGATGC TGCACCAGAC 60
TGCTGCATCC CA 72
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGG~ ~lC GCCCTGTCTG 60
25 CCTTGCAAGG CCCTGCTGTT CGACCCGTGC ATT 93
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
97
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
Gln Cys Ala Leu Cys Arg
l 5
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
Val Ala Leu Ser Cys Gln
1 5
CA 022~0707 1998-10-07
W O 97/39023 PCT/SE97/00574
98
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
~B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
Ile Val Lys Ser Cys Lys
l 5
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
Leu Ala Phe Glu Pro Cys Met
l 5
CA 022~0707 1998-10-07
W O 97/39023 PCT/SE97/00574
99
(2) INFORMATION FOR SEQ ID No:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID No 8s
Leu Leu Pro Gly Pro Cys Met
l 5
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
Met Ala Pro Ala Pro Cys Trp
l 5
CA 022~0707 1998-10-07
W 097t39023 PCT/SE97/00574
100
~2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
Ala Leu Tyr Ala Pro Cys Met
1 5
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
Val Leu Trp Glu Pro Cys Trp
1 5
CA 022~0707 1998-10-07
W O 97/39023 rCT/SE97/00574
101
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
Met Leu Phe Ser Pro Cys Trp
l 5
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
Leu Leu Cys Gly Pro Ala Ile
CA 022~0707 1998-10-07
WO 97/39023 PCT/SE97/00574
102
(2) INFORMATION FOR SEQ ID NO:9l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9l:
Leu Cys Phe Gly Pro Ala Ile
l 5
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
Val Met Pro Ser Pro Cys Tyr
l 5
CA 022~0707 1998-10-07
W 097/39023 PCT/SE97/OOS74
103
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
Val Val Phe Ala Pro Cys Tyr
l 5
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
Ala Val Pro Glu Pro Cys Phe
CA 022~0707 1998-10-07
W O 97/39023 PCT/SE97/00574
104
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
Met Met Tyr Glu Pro Cys Tyr
l 5
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
Ala Ala Trp Ser Pro Cys Met
l 5
CA 022~0707 1998-10-07
W O 97/39023 PCT1SE97/00574
10~
(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptlde
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
Val Ala Tyr Gly Pro Cys Trp
l 5
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
Leu Arg Pro Arg Cys Arg Pro Ile
l 5
CA 022~0707 1998-10-07
W 097/39023 PCT/SE97/OOS74
106
(2) INEORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
Ala Gly Tyr Cys Pro Thr Met Thr
l 5
(2) INFORMATION FOR SEQ ID NO:l00:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l00:
Pro Gln Val Val Cys Asn Tyr Arg
l 5
CA 022~0707 l998-l0-07
W O 97t39023 PCT/SE97/00574
107
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
lo
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr Leu Trp
1 5 10
(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
CA 022~0707 l998-l0-07
W O 97t39023 PCT/SE97/00574
108
Ser Pro Cys Leu Pro Cys Arg Ala Leu Ala Phe Glu Pro Cys Met
(2) INFORMATION FOR SEQ ID NO:103:
s
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
Met His Leu Ser Leu Ser His Gln Trp Ser Ser Trp Thr Val Leu Leu
1 5 10 15
Leu Leu Val Ser Asn Leu Leu Leu Trp Glu Asn Thr Ala Ser Ala Met
20 25 30
Ala Pro Ala Pro Cys Trp
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
109
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
Met Gly Phe Leu Lys Phe Ser Pro Phe Leu Val Val Ser Ile Leu Leu
1 5 10 15
lo Leu Tyr Gln Ala Cys Gly Leu Gln Ala Val Leu Trp Glu Pro Cys Trp
20 25 30
(2) INFORMATION FOR SEQ ID NO:105:
lS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
Met Gly Phe Leu Lys Phe Ser Pro Phe Leu Val Val Ser Ile Leu Leu
1 5 10 15
Leu Tyr Gln Ala Cys Gly Leu Gln Ala Val Met Pro Ser Pro Cys Tyr
20 25 30
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
110
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
S (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
Met His Leu Ser Leu Ser His Gln Trp Ser Ser Trp Thr Val Leu Leu
1 5 10 15
Leu Leu Val Ser Asn Leu Leu Leu Trp Glu Asn Thr Ala Ser Ala Met
20 25 30
Leu Phe Ser Pro Cys Trp
(2) INFORMATION FOR SEQ ID NO:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
~C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
111
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:
Met Gly Phe Leu Lys Phe Ser Pro Phe Leu Val Val Ser Ile Leu Leu
1 5 10 15
Leu Tyr Gln Ala Cys Gly Leu Gln Ala Val Val Phe Ala Pro Cys Tyr
(2) INFORMATION FOR SEQ ID NO:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
lS (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGGCTCTGTC GCC~ G 60
CCTTGCAGGG CCCTGGCCTT CGAACCGTGC ATG 93
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/OOS74
112
(2) INFORMATION FOR SEQ ID NO:l09:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 114 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l09:
ATGCACCTGA GCCTGAGCCA CCAATGGAGC AGCTGGACAG TACTGCTGCT GCTGGTAAGC 60
AACTTATTAT TATGGGAAAA CACAGCAAGC GCAATGGCAC CAGCACCATG CTGG ll4
(2) INFORMATION FOR SEQ ID NO:ll0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll0:
ATGGGCTTCC TGAAATTCTC CCCTTTCCTG GTGGTGTCCA TCCTGCTGCT GTATCAAGCG 60
TGTGGCCTGC CAGCGGTATT ATGGGAACCA TGCTGG 96
(2) INFORMATION FOR SEQ ID NO:lll:
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
113
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:lll:
ATGGGCTTCC TGAAATTCTC CCCTTTCCTG GTGGTGTCCA TCCTGCTGCT GTATCAAGCG 60
TGTGGCCTGC AAGCGGTAAT GCCTTCCCCT TGCTAC 96
(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 115 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
ATGCACCTGA GCCTGAGCCA CCAATGGAGC AGCTGGACAG TACTACTGCT GCTGGTAAGC 60
AACTTATTAT TATGGGAAAA CACAGCAAGC AGCAATGTTA TTCAGCCCAT GCTGG 115
CA 022~0707 l998-l0-07
W 097l39023 PCTISE97/OOS74
114
(2) IN~ORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs
s (B) TYPE: nuclei~ acid
(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
ATGGGCTTCC TGAAATTCTC CCCTTTCCTG GTGGTGTCCA TCCTGCTGCT GTATCAAGCG 60
TGTGGCCTGC AAGCGGTAGT ATTCGCGCCT TGCTAC 96
(2) INFORMATION FOR SEQ ID NO:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
Gly Pro Met Ile
(2) INFORMATION FOR SEQ ID NO:115:
CA 022~0707 l998-l0-07
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115
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
S (C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:115:
Lys Met Arg Met Lys
1 5
(2) INFORMATION FOR SEQ ID NO:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
Phe Met Ile Met Lys
1 5
(2) INFORMATION FOR SEQ ID NO:117:
CA 02250707 1998-10-07
W O 97/39023 PCT/SE97/00574
116
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
Ile Cys Thr Met Glu
l 5
(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:
Leu Met Ala Met Val
l 5
(2) INFORMATION FOR SEQ ID NO:ll9:
CA 022~0707 l998-l0-07
WO 97/39023 PCT/SE97/00574
117
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
s (C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:
Ile Met Tyr Met Glu
1 5
(2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:
Ala Pro Met Met Val Pro
1 5
(2) INFORMATION FOR SEQ ID NO:121:
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
118
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
s (C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:
Gly Pro Met Met Pro Gly
1 5
lS (2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
Gly Pro Cys Met Pro Gly
(2) INFORMATION FOR SEQ ID NO:123:
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
119
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
Gly Pro Met Cys Pro Gly
1 5
(2) INFORMATION FOR SEQ ID NO:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:
Pro Gly Met Met Gly Pro
1 5
(2) INFORMATION FOR SEQ ID NO:125:
CA 02250707 l998-l0-07
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120
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
~C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:
Val Ile Met Met Leu Thr
1 5
(2) INFORMATION FOR SEQ ID NO:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:
Leu Ala Phe Glu Pro Met Met
1 5
(2) INFORMATION FOR SEQ ID NO:127:
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
12
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
Met Leu Phe Ser Pro Met Trp
1 5
(2) INFORMATION FOR SEQ ID NO:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:
Val Val Phe Ala Pro Met Tyr
1 5
(2) INFORMATION FOR SEQ ID NO:129:
CA 022~0707 l998-l0-07
WO 97/39023 PCT/SE97/00574
122
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:
Leu Leu Phe G}y Pro Met Ile
1 5
lS (2) INFORMATION FOR SEQ ID NO:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:
Leu Leu Tyr Ser Pro Met Phe
1 5
(2) INFORMATION FOR SEQ ID NO:131:
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
123
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amlno acids
(B) TYPE: amino acid
s (C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
lo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:
Leu Leu Phe Asp Pro Met Ile
1 5
lS (2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:
Leu Leu Phe Trp Pro Met Ile
1 5
(2) INFORMATION FOR SEQ ID NO:133:
CA 02250707 l998-l0-07
WO 97/39023 PCT/SE97/00574
124
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 7 amino acids
(B) TYPE: amino acid
S (C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:
Leu Leu Phe Arg Pro Met Ile
1 5
(2) INFORMATION FOR SEQ ID NO:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:
Leu Leu Phe Ala Pro Met Ile
1 5
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
125
(2) INFORMATION FOR SEQ ID NO:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:
Leu Leu Phe Gly Ile Met Ile
1 5
(2) INFORMATION FOR SEQ ID NO:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:
Phe Met Leu Gly Pro Met Pro
CA 02250707 l998-l0-07
W O 97/39023 PCT/SE97/OOS74
126
(2) INFORMATION FOR SEQ ID NO:137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:
Met Arg Leu Arg Leu Leu Val Ser Ala Gly Met Leu Leu Val Ala Leu
1 5 10 15
Ser Pro Cys Leu Pro Cys Arg Ala Leu Leu Phe Gly Pro Met Ile
20 25 30
(2) INFORMATION FOR SEQ ID NO:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 022~0707 l998-l0-07
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127
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:
ATGCGGCTGC GGCTGCTGGT GTCCGCGGGC ATGCTGCTGG TGGCTCTGTC GCC~ lG 60
5 CCTTGCAGGG CCCTGCTGTT CGGACCGATG ATT 93
(2~ INFORMATION FOR SEQ ID NO:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:
Arg Asn Arg Cys Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly Cys
1 5 10 15
Arg Leu
(2) INFORMATION FOR SEQ ID NO:140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
~ (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
CA 02250707 l998-l0-07
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128
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:
Ile Asn Thr Lys Cys Tyr Lys Leu Glu His Pro Val Thr Gly Cys Gly
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
Is (C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:
Asp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn Trp Val Cys Ala Ala Lys
1 5 10 15
Phe Glu Ser Asn Phe Thr Gln
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
129
(2) INFORMATION FOR SEQ ID NO:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
s (B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:
Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu
1 5 10 15
Gly Leu Thr Val
(2) INFORMATION FOR SEQ ID NO:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
130
Gly Asp Met Tyr Pro Lys Thr Trp Ser Gly Met Leu Val Gly Ala Leu
1 5 10 15
Cys Ala Leu Ala Gly Val Leu Thr Ile
20 25
(2) INFORMATION FOR SEQ ID NO:144:
o ~i) SEQUENCE CH M ACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:
Val Pro Gly Leu Tyr Ser Pro Cys Arg Ala Phe Phe Asn Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:145:
(i) SEQUENCE CH M ACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
CA 02250707 l998-l0-07
W 097/39023 PCT/SE97/00574
13
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:
S Val Pro Gly Leu Tyr Ser Pro Cys Arg Ala Phe Phe Asn Lys Glu Glu
1 5 10 15
Leu Leu
(2) INFORMATION FOR SEQ ID NO:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:
Val Pro Gly Leu Tyr Ser Pro Cys Arg Ala Phe Phe Asn Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
CA 02250707 l998-l0-07
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132
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
s
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:
Glu Ala Ile Tyr Asp Ile Cys Arg Arg Asn Leu Asp Ile Glu Arg Pro
1 5 10 15
Thr
(2) INFORMATION FOR SEQ ID NO:148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:
Glu Ala Ile Tyr Asp Ile Cys Arg Arg Asn Leu Asp Ile
1 5 10
(2) INFORMATION FOR SEQ ID NO:149:
CA 022~0707 l998-l0-07
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133
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:
Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His
1 5 10 15
Asn Tyr Gly Val Gly Glu Ser Phe Thr
20 25
(2) INFORMATION FOR SEQ ID NO:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:
Thr Ser Ile Leu Cys Tyr Arg Lys Arg Glu Trp Ile Lys
30 1 5 10
CA 022~0707 l998-l0-07
W O 97l39023 PCT/SE97/00574
134
(2) INFORMATION FOR SEQ ID NO:151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:
Leu Pro Phe Phe Leu Phe Arg Gln Ala Tyr His Pro Asn Asn Ser Ser
1 5 10 15
Pro Val Cys Tyr
(2) INFORMATION FOR SEQ ID NO:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:
CA 022~0707 l998-l0-07
W097/39023 PCT/~E97/00574
135
Gln Ala Lys Phe Phe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala
1 5 10 15
S Trp Tyr Arg Gly Ala Ala Pro Pro Lys Gln Glu Phe
20 25
(2) INFORMATION FOR SEQ ID NO:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:
Gln Ala Lys Phe Phe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala
1 5 10 15
Trp Tyr Arg
(2) INFORMATION FOR SEQ ID NO:154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
136
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:
Lys Val Phe Gly Arg Cys Glu Leu Ala Ala Ala Met Lys Arg His Gly
1 5 10 15
Leu Asp
(2) INFORMATION FOR SEQ ID NO:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:
Ala Glu Ala Leu Glu Arg Met Phe Leu Ser Phe Thr Thr Lys Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:156:
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
137
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:
Lys Asn Ile Phe His Phe Lys Val Asn Gln Glu Gly Leu Lys Leu Ser
1 5 10 15
Asn Asp Met Met
lS 20
(2) INFORMATION FOR SEQ ID NO:157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:
Leu Glu Cys Gly Pro Cys Phe Leu
30 1 5
CA 022~0707 l998-l0-07
W O 97l39023 PCT/SE97100574
138
(2) INFORMATION FOR SEQ ID NO:158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:
Leu Cys Ala Gly Pro Cys Phe Leu
1 5
(2) INFORMATION FOR SEQ ID NO:159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:
Tyr Ile Pro Cys Phe Pro Ser Ser Leu Lys Arg Leu Leu Ile
30 1 5 10
CA 022~0707 l998-l0-07
W 097/39023 PCT/SE97/00574
139
(2) INFORMATION FOR SEQ ID NO:160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:
Tyr Ile Pro Cys Phe Pro Ser Ser Leu Lys Arg Leu Ile
lS 1 5 10
(2) INFORMATION FOR SEQ ID NO:161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:
Ser Gly Pro Cys Pro Lys Asp Gly Gln Pro Ser
30 1 5 10
CA 022~0707 1998-10-07
W O 97/39023 PCT/SE97/00574
140
(2) INFORMATION FOR SEQ ID NO:162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:
Thr Pro Pro Thr Pro Cys Pro Ser
1 5
(2) INFORMATION FOR SEQ ID NO:163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:
Asp Pro Cys Ile Ile
30 1 5
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
14
(2) INFORMATION FOR SEQ ID NO:164:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:
Cys Gly Gly Ile Cys Ile Ala Arg
1 5
(2) INFORMATION FOR SEQ ID NO:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:
Ser Gly Pro Cys Pro Lys Asp Gly Gln Pro Ser
30 1 5 10
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97100574
142
(2) INFORMATION FOR SEQ ID NO:166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:
Cys His Gly Ser Asp Pro Cys
1 5
(2) INFORMATION FOR SEQ ID NO:167:
~i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: ll amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:
Ser Gly Pro Cys Pro Lys Asp Gly Gln Pro Ser
30 1 5 10
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
143
(2) INFORMATION FOR SEQ ID NO:168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
lo (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:
Tyr Arg Arg Gly Arg Cys Gly Gly Leu Cys Leu Ala Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02250707 l998-l0-07
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144
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:
Tyr Arg Arg Gly Arg Ala Ala Ala Cys Gly Gly Leu Cys Leu Ala Arg
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:170:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
lS ~ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:
Tyr Arg Arg Gly Arg Cys Gly Gly Gly Leu Cys Leu Ala Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02250707 l998-l0-07
W O 97/39023 PCT/SE97/00574
145
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:
Tyr Arg Arg Gly Arg Ala Ala Ala Cys Gly Gly Gly Leu Cys Leu Ala
1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID NO:172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:
~0
Tyr Arg Arg Gly Arg Cys Gly Gly Gly Gly Leu Cys Leu Ala Arg
1 5 10 15
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
146
(2) INFORMATION FOR SEQ ID NO:173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:
Tyr Arg Arg Gly Arg Ala Ala Ala Cys Gly Gly Gly Gly Leu Cys Leu
1 5 10 15
Ala Arg
(2) INFORMATION FOR SEQ ID NO:174:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
2~
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:
Cys Gly Gly Leu Cys Ala Arg
CA 022~0707 l998-l0-07
W O 97/39023 PCT/SE97/00574
147
1 5
(2) INFORMATION FOR SEQ ID NO:175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:
Ser Pro Tyr Met Glu Ala
1 5
(2) INFORMATION FOR SEQ ID NO:176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02250707 1998-10-07
W O 97139023 PCT/SE97100574
148
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:
Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu
1 5 10 15
Gly
