U.S. patent application number 10/001546 was filed with the patent office on 2003-02-06 for methods and compositions for stimulating t-lymphocytes.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Fisk, Bryan A., Ioannides, Constantin G., Ioannides, Maria G..
Application Number | 20030027766 10/001546 |
Document ID | / |
Family ID | 23595862 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027766 |
Kind Code |
A1 |
Ioannides, Constantin G. ;
et al. |
February 6, 2003 |
Methods and compositions for stimulating T-lymphocytes
Abstract
Disclosed are methods, compositions, antibodies, and therapeutic
kits for use in stimulating cytotoxic T-lymphocytes and generating
immune responses against epitopes of protooncogenes. Novel peptides
are described which have been shown to stimulate cytotoxic
T-lymphocytes, and act as antigens in generation of oncogenic
epitope-recognizing antibodies. Methods are disclosed for use in
treating various proliferative disorders, and diagnosing
HER-2/neu-containing cells; also disclosed are therapeutic kits
useful in the treatment of cancer and production of potential
anti-cancer vaccines.
Inventors: |
Ioannides, Constantin G.;
(Houston, TX) ; Fisk, Bryan A.; (Houston, TX)
; Ioannides, Maria G.; (Athens, GR) |
Correspondence
Address: |
David L. Parker
FULBRIGHT & JAWORSKI L.L.P.
Suite 2400
600 Congress Avenue
Austin
TX
78701
US
|
Assignee: |
Board of Regents, The University of
Texas System
Austin
TX
|
Family ID: |
23595862 |
Appl. No.: |
10/001546 |
Filed: |
October 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10001546 |
Oct 31, 2001 |
|
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08403459 |
Mar 14, 1995 |
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Current U.S.
Class: |
424/185.1 ;
514/19.4; 514/21.4; 530/326; 530/327; 530/328 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 14/71 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/13 ; 514/14;
514/15; 514/16; 530/326; 530/327; 530/328 |
International
Class: |
A61K 038/10; A61K
038/08; C07K 007/08; C07K 007/06 |
Goverment Interests
[0001] The United States government owns rights to the present
invention pursuant to Grants CA 57293 and CA 16672 from the
National Cancer Institute.
Claims
What is claimed is:
1. A peptide of between 8 and about 20 amino acid residues in
length, and including within its sequence the amino acid sequence
of:
AA.sub.1--AA.sub.2--AA.sub.3--AA.sub.4--AA.sub.5--AA.sub.6--AA.sub.7--AA.-
sub.8; wherein AA.sub.1 is Leu or Ile; AA.sub.2 is Ala, Arg, Gln,
Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; AA.sub.3 is
Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val; AA.sub.4
is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or
Val; AA.sub.5 is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Thr, or Val; AA.sub.6 is Ala, Asn, Asp, Cys, Gln, Glu,
Gly, Leu, Lys, Ser, or Thr; AA.sub.7 is Ala, Arg, Gln, Gly, His,
Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and AA.sub.8 is Val, Leu,
Met, Gly, or Glu.
2. The peptide of claim 1, further defined as a peptide of between
8 and about 15 amino acid residues in length.
3. The peptide of claim 2, further defined as a peptide of between
8 and 10 amino acid residues in length.
4. The peptide of claim 3, further defined as having the amino acid
sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4;
SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO:
9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ
ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO:
18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ
ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO:
27; SEQ ID NO: 28; or SEQ ID NO: 29.
5. The peptide of claim 4, further defined as having the amino acid
sequence of SEQ ID NO: 1.
6. The peptide of claim 4, further defined as having the amino acid
sequence of SEQ ID NO: 2.
7. The peptide of claim 4, further defined as having the amino acid
sequence of SEQ ID NO: 3.
8. The peptide of claim 4, further defined as having the amino acid
sequence of SEQ ID NO: 4.
9. The peptide of claim 4, further defined as having the amino acid
sequence of SEQ ID NO: 5.
10. The peptide of claim 4, further defined as having the amino
acid sequence of SEQ ID NO: 6.
11. The peptide of claim 4, further defined as having the amino
acid sequence of SEQ ID NO: 7.
12. The peptide of claim 4, further defined as having the amino
acid sequence of SEQ ID NO: 8.
13. The peptide of claim 4, further defined as having the amino
acid sequence of SEQ ID NO: 9.
14. The peptide of claim 4, further defined as having the amino
acid sequence of SEQ ID NO: 18.
15. The peptide of claim 4, further defined as having the amino
acid sequence of SEQ ID NO: 19.
16. The peptide of claim 1, wherein AA.sub.2 is Val or Gln;
AA.sub.3 is Ser, Gln, Glu, Lys, or Pro; AA.sub.4 is Glu, Gly, Ile,
Leu, or Ser; AA.sub.6 is Asn, Gln, or Ser; and AA.sub.7 is Arg,
Leu, Gln, Tyr, Val, or Lys.
17. The peptide of claim 16, wherein AA.sub.2 i s Val; AA.sub.3 is
Ser; AA.sub.4 is Glu; AA.sub.6 is Ser; and AA.sub.7 is Arg or
Lys.
18. A peptide of between 8 and about 20 amino acid residues in
length, said peptide stimulating cytotoxic T-lymphocytes and
comprising the amino acid sequence:
AA.sub.1--AA.sub.2--AA.sub.3--AA.sub.4--AA.sub.5--AA.sub.6-
--AA.sub.7--AA.sub.8; wherein AA.sub.1 is Leu or Ile; AA.sub.2 is
Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val;
AA.sub.3 is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or
Val; AA.sub.4 is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr,
Trp, Tyr, or Val; AA.sub.5 is Ala, Asn, Cys, Gln, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Thr, or Val; AA.sub.6 is Ala, Asn, Asp,
Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr; AA.sub.7 is Ala, Arg,
Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and AA.sub.8
is Val, Leu, Met, Gly, or Glu.
19. The peptide of claim 18, further defined as being from 8 amino
acid residues in length to about 15 amino acid residues in
length.
20. The peptide of claim 19, further defined as being from 8 amino
acid residues in length to about 10 amino acid residues in
length.
21. The peptide of claim 20, further defined as being 8 amino acid
residues in length.
22. The peptide of claim 21, further defined as being 9 amino acid
residues in length.
23. The peptide of claim 21, further defined as being 10 amino acid
residues in length.
24. The peptide of claim 18, further defined as having the amino
acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID
NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ
ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO:
13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ
ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO:
22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ
ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.
25. The peptide of claim 24, further defined as having the amino
acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ
ID NO: 18, or SEQ ID NO: 19.
26. A peptide of between 8 and about 20 amino acid residues in
length, said peptide binding HLA and stimulating cytotoxic
T-lymphocytes, and including within its sequence an amino acid
sequence represented by:
AA.sub.1--AA.sub.2--AA.sub.3--AA.sub.4--AA.sub.5--AA.sub.6--AA.sub.7--AA.-
sub.8; wherein AA.sub.1 is Leu or Ile; AA.sub.2 is Ala, Arg, Gln,
Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val; AA.sub.3 is
Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val; AA.sub.4
is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or
Val; AA.sub.5 is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Thr, or Val; AA.sub.6 is Ala, Asn, Asp, Cys, Gln, Glu,
Gly, Leu, Lys, Ser, or Thr; AA.sub.7 is Ala, Arg, Gln, Gly, His,
Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and AA.sub.8 is Val, Leu,
Met, Gly, or Glu.
27. The peptide of claim 26, wherein AA.sub.2 is Val or Gln;
AA.sub.3 is Ser, Gln, Glu, Lys, or Pro; AA.sub.4 is Glu, Gly, Ile,
Leu, or Ser; AA.sub.6 is Asn, Gln, or Ser; and AA.sub.7 is Arg,
Leu, Gln, Tyr, Val, or Lys.
28. The peptide of claim 27, wherein AA.sub.2 is Val; AA.sub.3 is
Ser; AA.sub.4 is Glu; AA.sub.6 is Ser; and AA.sub.7 is Arg or
Lys.
29. The peptide of claim 26, further defined as having the amino
acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID
NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ
ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO:
13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ
ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO:
22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ
ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.
30. A method for stimulating cytotoxic T-lymphocytes, comprising
contacting said cytotoxic T-lymphocytes with an amount of a peptide
in accordance with claim 1 effective to stimulate said cytotoxic
T-lymphocytes.
31. The method of claim 30, wherein said cytotoxic T-lymphocytes
are located within an animal and said peptide or composition is
administered to said animal.
32. The method of claim 30, wherein said cytotoxic T-lymphocytes
are obtained from an animal, contacted with said peptide, and
re-administered to said animal.
33. The method of claim 30, wherein said peptide is formulated for
administration parenterally, topically, or as an inhalant, aerosol
or spray.
34. The method of claim 31, wherein said animal is a human
subject.
35. A pharmaceutical composition including the composition of claim
1 in a pharmaceutically acceptable excipient.
36. The pharmaceutical composition of claim 35, wherein said
composition comprises the peptide of SEQ ID NO: 1; SEQ ID NO: 2;
SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO:
7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID
NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16;
SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID
NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25;
SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.
37. A method of treating a proliferative cell disorder in an
animal, comprising administering to said animal a
therapeutically-effective amount of a pharmaceutical composition in
accordance with claim 35.
38. The method of claim 37, wherein said proliferative cell
disorder is cancer.
39. The method of claim 38, wherein said cancer is breast or
ovarian cancer.
40. A method for detecting cytotoxic T-lymphocytes in a sample,
comprising obtaining a sample suspected of containing cytotoxic
T-lymphocytes, contacting said sample with a peptide in accordance
with claim 1, under conditions effective to allow the formation of
cell-peptide complexes, and detecting the cell-peptide complexes so
formed.
41. The method of claim 40, wherein said sample is a biological
sample from an animal suspected of having a HER-2/neu-related
cancer.
42. The method of claim 40, wherein said peptide is linked to a
detectable label and the cell-peptide complexes are detected by
detecting the presence of the label.
43. The method of claim 40, wherein said cell-peptide complexes are
detected by means of an antibody linked to a detectable label, the
antibody having binding affinity for the peptide.
44. A method of generating an immune response, comprising
administering to an animal a pharmaceutical composition comprising
an immunologically effective amount of a composition comprising the
peptide of claim 1.
45. The method of claim 44, wherein said composition comprises an
immunologically effective amount of a composition comprising the
peptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4;
SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO:
9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ
ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO:
18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ
ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO:
27; SEQ ID NO: 28; or SEQ ID NO: 29.
46. A purified antibody that binds to the peptide of claim 1.
47. The antibody of claim 46, wherein the antibody is a monoclonal
antibody.
48. The antibody of claim 46, wherein the antibody is linked to a
detectable label.
49. The antibody of claim 48, wherein the antibody is linked to a
radioactive label, a fluorogenic label, a nuclear magnetic spin
resonance label, biotin or an enzyme that generates a colored
product upon contact with a chromogenic substrate.
50. The antibody of claim 49, wherein the antibody is linked to an
alkaline phosphatase, hydrogen peroxidase or glucose oxidase
enzyme.
51. A method for detecting a neu-containing cancer cell, a neu
protein, or neu peptide; the method comprising: (a) generating an
antibody that binds to the peptide of claim 1. (b) obtaining a
sample suspected of containing a neu-containing cancer cell, a neu
protein, or neu peptide; (c) contacting said sample with said
antibody, under conditions effective to allow the formation of
immune complexes; and (d) detecting the immune complexes so
formed.
52. The method of claim 51, wherein said antibody is a monoclonal
antibody.
53. An immunodetection kit comprising, in suitable container means,
the peptide of claim 1, or a first antibody that binds to the
peptide of claim 1, and an immunodetection reagent.
54. The immunodetection kit of claim 53, wherein the
immunodetection reagent is a detectable label that is linked to
said peptide or said first antibody.
55. The immunodetection kit of claim 54, wherein the
immunodetection reagent is a detectable label that is linked to a
second antibody that has binding affinity for said peptide or said
first antibody.
56. The immunodetection kit of claim 54, wherein the
immunodetection reagent is a detectable label that is linked to a
second antibody that has binding affinity for a human antibody.
57. A DNA segment encoding the peptide of claim 1.
58. The DNA segment of claim 57, further defined as encoding the
peptide of claim 3.
59. The DNA segment of claim 57, further defined as encoding the
peptide of claim 4.
60. The DNA segment of claim 57, further defined as comprising the
DNA sequence of SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID
NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58;
SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID
NO: 63; or SEQ ID NO: 64.
61. A recombinant vector comprising the DNA segment of claim
57.
62. The recombinant vector of claim 61, further defined as
comprising a DNA segment encoding a peptide which stimulates a
cytotoxic T-lymphocyte.
Description
BACKGROUND OF THE INVENTION
[0002] A. Field of the Invention
[0003] The present invention relates generally to the field of
molecular biology, and particularly to the area of natural and
synthetic peptides. More particularly, the invention discloses
HER-2/neu peptide, DNA segment, antibody compositions. Various
methods for making and using these compositions are disclosed, such
as, for example, the use of peptides and antibodies in various
pharmacological and immunological applications, including the
stimulation of cytotoxic T-lymphocytes and cancer therapies.
[0004] B. Description of the Related Art
[0005] 1. HER-2/neu Proto-oncogene
[0006] The HER-2/neu proto-oncogene (HER-2) encodes a transmembrane
protein whose expression is enhanced in a number of breast and
ovarian tumors and correlates with tumor aggressiveness. Because of
its expression on normal epithelial cells, HER-2 can be defined as
a tumor-associated antigen (Ag) and may be of interest as a target
of a therapeutic anti-tumor T-cell response. A
CD3.sup.+CD8.sup.+CD4.sup.- line isolated from cell cultures have
been shown to lyse HLA-A2.sup.+, HER-s.sup.+ ovarian tumors but not
natural killer (NK) target K562 cells, and showed significantly
higher lysis of HER-2.sup.high than of HER-2.sup.low ovarian
tumors. Some inhibition of lysis was inhibited by HER-2
peptide-pulsed HLA-A2.sup.+ targets, suggesting that some epitopes
may be present on tumor cells associated with HLA-A2.
[0007] 2. Tumor-reactive T-cells
[0008] Tumor reactive T-cells have been reported to mediate
therapeutic responses against human cancers (Rosenberg et al.,
1988). In certain instances, in human immunotherapy trials with
tumor infiltrating lymphocytes (TIL) or tumor vaccines, these
responses correlated either with in vitro cytotoxicity levels
against autologous tumors (Aebersold et al., 1991) or with
expression of certain HLA-A,B,C gene products (Marincola et al.,
1992). Recent studies (Ioannides et al., 1992) have proposed that
in addition to virally encoded and mutated oncogenes, overexpressed
self-proteins may elicit some degree of tumor-reactive cytotoxic
T-lymphocytes (CTLs) in patients with various malignancies
(Ioannides et al., 1992; Ioannides et al., 1993; Brichard et al.,
1993; Jerome et al., 1991). Autologous tumor reactive CTLs can be
generated from lymphocytes infiltrating ovarian malignant ascites
(Ioannides et al., 1991), and overexpressed proteins such as HER-2
may be targets for CTL recognition (Ioannides et al., 1992).
[0009] Information on epitopes of self-proteins recognized in the
context of MHC Class I molecules remain limited, despite a few
attempts to identify epitopes capable of in vitro priming and
Ag-specific expansion of human CTLs. For example, peptide epitopes
have been proposed which are likely candidates for binding on
particular MHC Class I Ag (Falk et al., 1991), and some studies
have attempted to define peptide epitopes which bind MHC Class I
antigens.
[0010] Short synthetic peptides have been used either as target
antigens for epitope mapping or for induction of in vitro primary
and secondary CTL responses to viral and parasitic Ags (Bednarek et
al., 1991; Gammon et al., 1992; Schmidt et al., 1992; Kos and
Mullbacher, 1992; Hill et al., 1992). Unfortunately, these studies
failed to show the ability of proto-oncogene peptide analogs to
stimulate in vitro human CTLs to lyse tumors endogenously
expressing these antigens.
[0011] 3. Synthetic Peptides and T-cell Epitope Mapping
[0012] Synthetic peptides have been shown to be a useful tool for
T-cell epitope mapping. However in vivo and in vitro priming of
specific CTLs has encountered difficulties (Alexander et al., 1991;
Schild et al., 1991; Carbone et al., 1988). It is generally
considered that in vitro CTL priming cannot necessarily be achieved
with peptide alone, and in fact, a high antigen density is thought
to be required for peptide priming (Alexander et al., 1991). Even
in the limited instances when specific priming was achieved, APC or
stimulators were also required at high densities (Alexander et al.,
1991).
[0013] It is not clear when CTL induction by HER-2 peptides in
vitro was observed whether this reflects secondary activation of
CTL specific for, or cross-reacting with, the Ag of interest.
Whether or not this cross-reactivity can constitute the foundation
for development of an in vitro CTL response to tumor remains to be
determined.
[0014] Therefore, what is lacking in the prior art are universal
epitopes which are both immunodominant and CTL-stimulating.
Moreover, methods for the use of such CTL-stimulating peptides
would be most desirable in the treatment of human cancers,
particularly of breast and ovarian etiology, and the development of
cancer vaccines. Identifying universal oncoprotein epitopes would
permit not only an increased understanding of tumor immunity and
autoimmunity in humans, but would also open the door to the design
of novel therapeutic strategies for proliferative cell disorders
such as human cancers, and particularly breast and ovarian
cancers.
SUMMARY OF THE INVENTION
[0015] The present invention seeks to overcome these and other
inherent deficiencies in the prior art by providing the
identification of native and synthetic proteins or peptides derived
from the HER-2/neu proto-oncogene gene product, and methods for
their use in stimulating cytotoxic T-lymphocytes.
[0016] These selected "universal" immunodominant epitopic peptides,
and their synthetically-optimized derivatives are envisioned to be
useful in the development of tumor vaccines, and anti-cancer
therapeutics. Pharmaceutical reagents resulting from these novel
peptides and the DNA segments which encode them will also likely
prove useful as test reagents for the detection of
HER-2/neu-related polypeptides, facilitate the production of
anti-peptide antibodies specific to a range of HER-2/neu-related
polypeptides, and result in the stimulation and production of
cytotoxic T-lymphocytes specific for a variety of proliferative
disorders including human cancer.
[0017] Synthetic peptide analogs can be used to define CTL epitopes
recognized by tumor reactive T-cells and to stimulate in vitro
peptide-specific CTLs. Such CTLs can be further evaluated for
recognition of targets endogenously expressing the particular
antigen (Ag) and for Ag-specific adoptive therapy.
[0018] Disclosed herein are compositions and methods for their
making and use in development of anti-cancer vaccines. The
generation in vitro of HLA-A2-restricted CTLs using HER-2 synthetic
peptide analogs as immunogens, and peripheral blood mononuclear
cells (PBMC) from healthy volunteers as responder cells is also
described. Lysis with isolated CD8.sup.+ T-cells from these CTL
cultures was observed using both HER-2 peptide-pulsed HLA-A2 from
these CTL cultures was observed using both HER-2 peptide-pulsed
HLA-A2 transfectants and HLA-A2.sup.+ ovarian tumors expressing
high levels of HER-2 as targets.
[0019] Another aspect of the invention is the development and
maintenance in long-term culture a CD3.sup.+CD8.sup.+CD4.sup.- line
by restimulation with HER-2 peptide-pulsed autologous PBMC. This
line lysed HLA-A2.sup.+, HER-2.sup.high ovarian tumors, but not
HLA-A2.sup.+, HER-2.sup.low ovarian tumors. Tumor lysis was
inhibited by HER-2 peptide-pulsed HLA-A2.sup.+ transfectants,
demonstrating that epitopes either similar or cross-reactive with
the ones recognized by CTLs on the peptide used as immunogen in
vitro are present on the tumor cells. These CTL showed lower lysis
of targets pulsed with unrelated peptides (analogs of Muc-1 core
peptide where HLA-A2 anchors were introduced).
[0020] A novel approach to developing tumor reactive CTLs is
disclosed which focuses on a target Ag expressed on the tumor of
interest and identifying CTLs induced in vivo or developed in vitro
that recognize this target Ag. In tumor cells the level of
expression of a particular protein may be 10.sup.2-10.sup.3 fold
higher than in normal tissue.
[0021] The inventors expect that a number of target T-cell Ags on
human tumors may be derived from proteins that are expressed at low
levels in normal cells, and at significantly higher concentration
in tumor cells, such as overexpressed proto-oncogene products
(Ioannides et al., 1992). The rationale for this hypothesis is:
first, peptides from self-proteins which fulfill the criteria of
MHC allele-specific motifs should be capable of binding to the Ag
binding pockets in the MHC class I heavy chain; and second,
positive and negative selection of T-cell repertoire may result in
elimination or tolerization of high-affinity self-reactive CTLs
(Parmianai, 1993), although such peptide-MHC complexes should have
lower affinity for the TCR than a de novo expressed epitope from a
self-protein (as a consequence either of mutations creating
HLA-anchors or modifying the core recognized by the TCR), their
presence in high concentration may engage a large number of
TCR.
[0022] The HER-2/neu proto-oncogene was identified because it is
overexpressed (in certain instances by several hundred fold) in a
number of breast and ovarian tumors (Slamon et al., 1989).
Moreover, it was found that several CTL-TAL lines isolated from
ovarian malignant ascites could lyse autologous ovarian tumors.
[0023] Surprisingly, the inventors also discovered that this lysis
could also be effectively inhibited by natural and synthetic
peptide analogs of HER-2. These results suggested that these novel
peptides acted as epitopes that were either derived from an
endogenously-processed HER-2 peptide, mimicked, or cross-reacted
with a peptide of related sequence derived from another
protein.
[0024] Novel synthetic peptide compositions have also been
developed which correspond to the HER-2:968-981 and 971-979
regions. The compositions disclosed herein, were found to stimulate
in vitro PBMCs from healthy HLA-A2.sup.+ human volunteers (Fisk et
al., 1994), and CTLs (induced by peptide stimulation) consequently
lysed tumors overexpressing HER-2 (Fisk et al., 1994). These
studies demonstrated that these CTLs can effectively recognize the
epitope peptides of the present invention, and that these
HER-2-derived peptides can stimulate in vitro PBMCs to induce
peptide reactive CTLs.
[0025] This possibility may be particularly relevant for induction
of Ag and tumor-specific CTLs because peripheral T-cells that can
recognize such peptides from non-mutated self proteins are those
that have either escaped elimination or may have become tolerant to
one or more of these antigenic epitopes due to low affinity TCR-MHC
interactions (Ioannides et al., 1992; Parmiani, 1993).
[0026] Other aspects of this invention include the identification
of candidate HER-2-derived T-cell epitopes based on the presence of
anchors for HLA-A2, the analysis of these peptides to affect the
conformation of HLA-A2 as an indication of peptide binding, and
finally, the demonstration that these peptides can stimulate in
vitro peptide reactive CTLs from human HLA-A2.sup.+ PBMC.
[0027] Methods are described herein for stimulation of CTLs (and
consequently, production of an immune response) employing the novel
compositions disclosed herein. In vitro induction of cellular
responses to the peptides of the present invention by PBMC from
healthy HLA-A2.sup.+ volunteers demonstrated their ability to
stimulate and/or restimulate pre-existing T-cell responses to
HER-2. The peptides induced proliferative responses in one of four
donors tested and CTL responses (one of three peptides tested in
two of three donors), and may be used to induce tumor-reactive
T-cells in vitro and in vivo through either peptide-, lipopeptide-,
or cell-mediated methods. These peptides therefore find utility in
both generating an immune response, and serving as antigens in the
preparation of peptide-specific antibodies.
[0028] The peptides of this invention also may be used in
embodiments involving treatment, diagnosis, and identification of
proliferative cell disorders such as cancer, and particularly
cancers such as, inter alia, breast and ovarian tumors. Methods of
identification of HER-2/neu-containing cells, and also neu-related
proto-oncogene and oncogene products are also disclosed.
[0029] Cancer treatment methods, including vaccine development are
another aspect of the present invention. Additionally, a variety of
in vitro and in vivo assay protocols are facilitated as a result of
the novel compositions disclosed herein. In addition to stimulating
CTLs, and generating an immune response in an animal, and
particularly in a human, the peptides may also be used as
immunogens to generate anti-peptide antibodies, which themselves
have many uses, not least of which is the detection of
oncogene-containing cells (e.g., detection of HER-2/neu, related
oncogenic polypeptides, or peptide fragments thereof, in diagnostic
tests and kits based upon immunological binding assays).
[0030] Also, since the peptides of the invention bind to T-cells,
they may be employed in assays to identify T-cells, and
particularly CTLs, for example, to assess the immunological
capacity of a given individual or animal, or even to purify CTLs
themselves. Such methods could utilize radioactively- or
enzymatically-labeled peptides or anti-peptide antibodies, such as
those described herein.
[0031] Therefore, one contemplated use for the described peptides
concerns their use in methods for detecting the presence of T-cells
within a sample. These methods include contacting a sample
suspected of containing T-cells with a peptide or composition in
accordance with the present invention under conditions effective to
allow the peptide(s) to form a complex with T-cells of the sample.
One then detects the presence of the complex by detecting the
presence of the peptide(s) within the complex, e.g., by either
originally using radiolabeled peptides or by subsequently employing
anti-peptide antibodies and standard secondary antibody detection
techniques.
[0032] Preferred peptides of the present invention will likely be
from about 6 to about 20 amino acids, in length, with peptides of
from 7 to about 15 amino acids in length being even more preferred.
Most preferred are peptides having lengths of from about 8 to about
10 amino acids in length, with nonameric and decameric peptides
being most preferred. These peptides may include one or more
D-amino acids, or may even be entirely composed of D-amino acids,
and may, of course, contain additional elements, as desired for
stability or even for targeting purposes.
[0033] The peptides, or multimers thereof, may be dispersed in any
one of the many pharmacologically-acceptable vehicles known in the
art and particularly exemplified herein. As such, the peptides may
be encapsulated within liposomes or incorporated in a biocompatible
coating designed for slow-release. The preparation and use of
appropriate therapeutic formulations will be known to those of
skill in the art in light of the present disclosure. The peptides
may also be used as part of a prophylactic regimen designed to
prevent, or protect against, possible cancer progression and/or
metastasis and may thus be formulated as a vaccine, particularly as
a method of stimulating anti-tumor CTLs.
[0034] The present invention also provides methods for identifying
HER-2/neu and related proto-oncogene products, which methods
comprise contacting the cells suspected of containing such
polypeptides with an immunologically effective amount of a
composition comprising one or more specific anti-peptide antibodies
disclosed herein. Peptides that include the amino acid sequence of
any of SEQ ID NO: 1 through SEQ ID NO: 29 and their derivatives
will be preferred for use in generating such anti-CTL-stimulating
peptide antibodies.
[0035] The invention thus also provides compositions, including
peptides, peptide multimers, and pharmaceutical compositions
derived therefrom, that contain one or more peptides of from 8 to
about 20 amino acids in length that include within their sequence
the peptide sequence identified by the formula:
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.-
sub.7-AA.sub.8; where AA.sub.1 is Leu, Met, Ile, or Val; AA.sub.2
is any amino acid; AA.sub.3 is any amino acid; AA.sub.4 is Ser,
Glu, Thr, or Tyr; AA.sub.5 is any amino acid; AA.sub.6 is any amino
acid; AA.sub.7 is any amino acid; and AA.sub.8 is Val, Leu, Met,
Ile, or Cys. These peptides are submitted to be capable of
stimulating CTLs and producing an immune response in vitro and in
vivo.
[0036] Another aspect of the present invention concerns the use of
the amino acid sequences disclosed herein in the determination of
molecular weights of low-molecular-weight polypeptides. These
peptides represent a significant improvement over
commercially-available protein standards in this area owing to
their small size, and the presence of known nonapeptide motifs.
Commercially-available standards typically have a range of 3,000 to
200,000 Da, and as such, are not useful in the characterization of
proteins having molecular weights of about 300 to about 3,000 Da
using either conventional or gradient SDS-PAGE.
[0037] In a similar fashion, the peptides, and more particularly
peptide oligomers, of the present invention are readily employed as
standards in the identification of small molecular-weight
polypeptides using chromatographic separation. In preferred
embodiments, paper chromatography is utilized and proteins are
subsequently visualized after reaction with ninhydrin. More
preferred is the use of thin-layer chromatography in either one or
two dimensions.
[0038] The use of the peptides and peptide motifs of the present
invention is also contemplated for the calibration and
standardization of chromatographic columns used in the separation
of low-molecular-weight polypeptides. These peptides, and multimers
thereof, find important use in the calibration of
low-molecular-weight-range columns. Such molecular sieve (or gel
filtration) chromatography columns may include a filtration medium
having the capacity to fractionate any protein of interest and the
peptides of the present invention. Preferred chromatographic media
would include any gel filtration medium having a molecular
fractionation range suitable for the particular protein of
interest. Preferred media would include the G-50 or G-25
Sephadex.RTM. resins which have an approximate fractionation range
of 1,500-30,000 and 100-5,000 Da, respectively. A more preferred
medium would be either the G-10 or G-15 Sephadex.RTM. resins which
have an approximate fractionation range of 0-700 and 0-1500 Da,
respectively.
[0039] Peptides of the present invention comprising aromatic amino
acids and multimers thereof may also be used as protein
concentration standards in reactions employing either the Folin
reagent (Lowry et al., 1951), the biuret reaction (Coakley and
James, 1978) or the bicinconinic acid assay (Pierce Chemical Corp.,
Rockford, Ill.). Peptides and multimers thereof lacking aromatic
amino acids may also be used as protein concentration standards in
the latter two reactions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1A. Lysis of C1R:A2 cells after sensitization with
peptides C43 (HER-2:968-981) (.box-solid.) and C84
(HER-2:921-979(Val) (.quadrature.) or in the absence of exogenously
added peptides (0) by CTL cultures induced in vitro with C43 and
C84 peptides. Headings indicate: donor number (e.g., 51), number of
stimulations with peptide (e.g., 2.times./3.times.), and the
peptide used for stimulation.
[0041] Donor 51 PBMC were tested 3 weeks after the second
stimulation with the C43 peptide (total 5 weeks in culture). The
studies were performed in triplicate. The differences between
individual determinations were less than 10%. The differences
between HER-2 peptide and control targets recognition are
significant in 20-hr assays (P<0.003 for C43 and P<0.027 for
C84) and are not significant (P<0.10) in 4-hr assays. The
effector to target ratio was 10:1.
[0042] FIG. 1B. Lysis of C1R:A2 cells after sensitization with
peptides C43 (HER-2:968-981) (.box-solid.) and C84
(HER-2:921-979(Val) (.quadrature.) or in the absence of exogenously
added peptides (0) by CTL cultures induced in vitro with C43 and
C84 peptides. Headings indicate: donor number (e.g., 51), number of
stimulations with peptide (e.g., 2.times./3.times.), and the
peptide used for stimulation. Shown is the donor 51 PBMC were
stimulated two times with the C84 peptide and tested 3 weeks
after.
[0043] FIG. 1C. Lysis of C1R:A2 cells after sensitization with
peptides C43 (HER-2:968-981) (.box-solid.) and C84
(HER-2:921-979(Val) (.quadrature.) or in the absence of exogenously
added peptides (0) by CTL cultures induced in vitro with C43 and
C84 peptides. Headings indicate: donor number (e.g., 51), number of
stimulations with peptide (e.g., 2.times./3.times.), and the
peptide used for stimulation. Shown is the donor 41 PBMC stimulated
two times with C84.
[0044] FIG. 2A. Ag specificity of the 41.CD8.sup.+ CTL line. C1R:A2
cells were pre-pulsed with either HER-2 peptides or control MUC-1
peptides before being incubated with effectors. The effector to
target ratio was 10:1. C1R:A1 and C1R:A3 targets were pre-pulsed
with the same peptides in the same conditions as C1R:A2 cells.
Results for C1R:A1 and C1R:A3 show the difference between specific
lysis of targets preincubated with peptides and control C1R:A1 and
C1R:A3 targets. Specific lysis of control C1R:A1 and C1R:A3 cells
was less than 10% at the same E:T ratio. Shown in FIG. 2A are
results after 4 hrs' incubation.
[0045] FIG. 2B. Ag specificity of the 41.CD8.sup.+ CTL line. C1R:A2
cells were pre-pulsed with either HER-2 peptides or control MUC-1
peptides before being incubated with effectors. The effector to
target ratio was 10:1. C1R:A1 and C1R:A3 targets were pre-pulsed
with the same peptides in the same conditions as C1R:A2 cells.
Results for C1R:A1 and C1R:A3 show the difference between specific
lysis of targets preincubated with peptides and control C1R:A1 and
C1R:A3 targets. Specific lysis of control C1R:A1 and C1R:A3 cells
was less than 10% at the same E:T ratio. Shown in FIG. 2B are
results after 20 hrs incubation.
[0046] FIG. 3A. Lysis of fresh isolated ovarian tumor OVA-16
(HLA-A2.sup.+, HER-2.sup.high) cells by the donor 41 CD8.sup.+ cell
line. Target lysis was determined in 5-hr (.quadrature.) and 20-hr
(.box-solid.) assays in the same study against both targets.
[0047] FIG. 3B. Lysis of fresh isolated ovarian tumor K562 cells by
the donor 41 CD8.sup.+ cell line. Target lysis was determined in
5-hr (.quadrature.) and 20-hr (.box-solid.) assays in the same
study against both targets.
[0048] FIG. 3C. Lysis by 41.CD8.sup.+ CTL of HLA-A2.sup.+
HER-2.sup.high, HER-2.sup.low ovarian tumors and HLA-A3.sup.+
HER-2.sup.high (SKOV3) ovarian and HLA-A11.sup.+ HER-2.sup.high
(SKBr3) breast tumor lines. C1R:A2 and XX Cr cells were negative
control targets.
[0049] FIG. 4A. Target specificity of the 41.CD8.sup.+ CTL were
tested for the ability to lyse .sup.51Cr-labeled OVA-16 at an E:T
ratio of 10:1. C1R:A2 cells (A2.1) were incubated with synthetic
peptides (D125, C43, C85), washed, and used in cold target
inhibition studies at a cold:hot ratio of 2:1. Cytotoxicity studies
were performed for 5 hr. Results represent the mean of three
determinations. The variability between samples was less than 10%.
The differences between determination are statistically significant
(P<0.03) as determined by Student's t test. Percentage
inhibition is indicated in parentheses.
[0050] FIG. 4B. Target specificity of the 41.CD8.sup.+ CTL were
tested for the ability to lyse .sup.51Cr-labeled OVA-16 at an E:T
ratio of 10:1. C1R:A2 cells (A2.1) were incubated with synthetic
peptides (D125, C43, C85), washed, and used in cold target
inhibition studies at a cold:hot ratio of 2:1. Cytotoxicity studies
were performed for 20 hr. Results represent the mean of three
determinations. The variability between samples was less than 10%.
The differences between determination are statistically significant
(P<0.03) as determined by Student's t test.
[0051] Percentage inhibition is indicated in parentheses.
[0052] FIG. 5A. Effects of HER-2 peptides on reactivity of MA2.1
mAb with T2 cells. Fluorescence analysis and determination of FL1
were performed as described (Stauss et al., 1992). Peptides were
added to T2 cells at 50 .mu.g/ml (final concentration). After
overnight culture, in IMDM-FCS, cells were washed and the levels of
HLA-A2 expression were determined using HLA-A2 specific mAb.
Control indicates that no exogenous peptides was added in the T2
cultures. D98, D160, and D169 are control peptides which do not
contain HLA-A2 anchors in correct positions.
[0053] FIG. 5B. Effects of HER-2 peptides on reactivity of BB7.2
mAb with T2 cells. Studies were performed as described in the
legend to FIG. 5A.
[0054] FIG. 5C. Effects of HER-2 peptides on reactivity of MA2.1
mAb with T2 cells. Studies were performed as described in the
legend to FIG. 5A.
[0055] FIG. 5D. Effects of HER-2 peptides on reactivity of BB7.2
mAb with T2 cells. Studies were performed as described in the
legend to FIG. 5A.
[0056] FIG. 6. Effects of Folate Binding Protein (FBP) peptides on
reactivity of MA2.1 mAb with T2 cells. Experimental conditions as
described in the legend to FIG. 5A. Control column indicates that
T2 cells were cultured in the absence of peptide.
[0057] FIG. 7A. Surface phenotype of T-cells from PBMC cultures
stimulated with HER-2 peptide D97. Fresh isolated PBMC from healthy
volunteers were induced in vitro with HER-2 peptides. T-cell
surface phenotypes were determined after one (1) and two (2)
stimulations with the same peptide. Immunofluorescence analysis was
performed as described in the Materials and methods. Symbols
indicate (O-O) CD3.sup.+ cells, (.box-solid.-.box-solid.) CD8.sup.+
cells, and (.quadrature.-.quadrature.- ) CD4.sup.+ cells.
[0058] FIG. 7B. Surface phenotype of T-cells from PBMC cultures
stimulated with HER-2 peptide D121. Studies were performed as
described in the legend to FIG. 7A.
[0059] FIG. 7C. Surface phenotype of T-cells from PBMC cultures
stimulated with HER-2 peptide C85. Studies were performed as
described in the legend to FIG. 7A.
[0060] FIG. 8A. CTL induction by HER-2 D97 peptide. PBMC from donor
20 were induced in vitro with mock stimulated medium only (20.C.2).
After two cycles of stimulation CTL activity was determined in a 4
h .sup.51Cr release assay using as targets C1R:A2 cells
pulse-labelled with the indicated peptides (D97, D9, D99) or in the
absence of peptide (none).
[0061] FIG. 8B. CTL induction by HER-2 D97 peptide. PBMC from donor
20 were induced in vitro with D97 (20.D97.2), at a ratio of 3:1.
Studies were performed as described in the legend to FIG. 8A.
[0062] FIG. 8C. CTL induction by HER-2 D97 peptide. PBMC from donor
20 were induced in vitro with D97 (20.D97.2), at a ratio of 6:1.
Studies were performed as described in the legend to FIG. 8A.
[0063] FIG. 9A. CTL induction by HER-2 peptides D96 and D97. PBMC
from donor 20 were stimulated two times with D96 (20.D96.2). CTL
activity was determined in a 4 h .sup.51Cr release assay using as
targets C1R:A2 cells without addition of exogenous peptide
(control) or pulse-labelled with D96 (.quadrature.), D97
(.box-solid.), or NK sensitive targets K562 cells were used as an
additional control.
[0064] FIG. 9B. CTL induction by HER-2 control peptide D95
(.tangle-solidup.) peptides. NK sensitive targets K562 cells were
used as an additional control.
[0065] FIG. 9C. CTL induction by HER-2 peptides. PBMC from donor 30
were induced with D97 peptide (30.D97.1). Seven days later CTL
activity of these cells was determined using as targets the peptide
used for stimulation (D97) or two HLA-A2 binding peptides with
unrelated sequence D113 and D119, as specificity controls.
[0066] FIG. 10A. CTL induction by HER-2 peptide D113. PBMC from
three healthy donors (20, 25 and 30) were induced with D113
peptide. Each culture was restimulated with D113 once. One week
later CTL activity was determined using as targets C1R:A2 cells
pulsed with D113. The effectors are designated as 20.113.2,
25.113.2 and 30.113.2 to indicate the donor number, the peptide
symbol and the number of stimulations with peptide. Experimental
conditions were as described in Example 2 and the legends to FIG.
8A and FIG. 9A. E:T ratios were 20:1 (heavy stripes) and 10:1
(medium stripes).
[0067] FIG. 10B. CTL induction by HER-2 peptide D113. PBMC from
three healthy donors (20, 25 and 30) were induced with D113
peptide. Each culture was restimulated with D113 once. One week
later CTL activity was determined using as targets C1R:A2 cells
pulsed with control D119 peptide. The effectors are designated as
20.113.2, 25.113.2 and 30.113.2 to indicate the donor number, the
peptide symbol and the number of stimulations with peptide.
Experimental conditions were as described in Example 2 and the
legends to FIG. 8A and FIG. 9A. E:T ratios were 20:1 (heavy
stripes) and 10:1 (medium stripes).
[0068] FIG. 11A. CTL induction by HER-2 peptides D121 and D119.
PBMC from a healthy donor (20) were induced with D121:HER-2:392-410
or D119:HER-2:402-410 by stimulating with the peptides twice
(20.121.2) or once (25.121.1 and 25.119.1 respectively). CTL
activity was determined using C1R:A2 targets pulsed either with the
Ag of interest (D119) or control peptides D95, D99, D97, C85 (Table
5). E:T ratios were 20:1 (heavy stripes), 10:1 (medium stripes) and
3:1 (light stripes).
[0069] FIG. 11B. CTL induction by HER-2 peptides D121 and D119.
PBMC from a healthy donor (25) were induced with D121:HER-2:392-410
or D119:HER-2:402-410 by stimulating with the peptides once
(25.121.1). CTL activity was determined using C1R:A2 targets pulsed
either with the Ag of interest (D119) or control peptides D95, D99,
D97, C85 (Table 5). E:T ratios were 20:1 (heavy stripes), 10:1
(medium stripes) and 3:1 (light stripes).
[0070] FIG. 11C. CTL induction by HER-2 peptides D121 and D119.
PBMC from a healthy donor (25) were induced with D121:HER-2:392-410
or D119:HER-2:402-410 by stimulating with the peptides once
(25.119.1). CTL activity was determined using C1R:A2 targets pulsed
either with the Ag of interest (D119) or control peptides D95, D99,
D97, C85 (Table 5). E:T ratios were 20:1 (heavy stripes), 10:1
(medium stripes) and 3:1 (light stripes).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0071] 1. Molecular Therapies for Cancer
[0072] Development of molecular therapies for cancer have
historically focused on specific recognition of Ags by cellular
immune effectors. The present invention discloses novel strategies
aimed at identification of peptide targets for CTLs, and generation
of T-cell immunity against specific epitopes (for a review of
T-cell specific immunity, see, e.g., Ioannides et al., 1992;
Houbiers et al., 1993).
[0073] To achieve this, the present invention provides novel
naturally- and synthetically-derived peptides which bind human
leucocyte antigen- (HLA) class I heavy chains. Appropriate criteria
for epitope selection in vitro have been defined. Using HER-2
protein (which has been proposed as a candidate for an anti-tumor
immune response in breast and ovarian cancer) these novel peptides
have been identified, isolated away from intact HER-2 protein and
characterized. Additionally, synthetic peptides based on
immunogenic epitopes of the HER-2 protein have also been
produced.
[0074] Although the dominant anchors for peptide binding to HLA-A2
are Leu (P2) and Val (P9), a number of residues with similar charge
and side chains such as Ile and Met were identified in CTL epitopes
from viral proteins (Falk et al., 1991; Bednarek et al., 1991).
Analysis of the HER-2 polypeptide sequence identified a large
number of nonapeptides meeting these criteria (Table 1). With few
exceptions, all HLA-A2 binding peptides identified in the present
invention contain Rothbard's epitope-motifs. In a few instances,
however, the peptide sequence contained between HLA-A2 anchors
matched or overlapped with amphiphilic areas.
[0075] Using more stringent selection criteria, in which only
Leu/Ile were accepted at amino acid position 2 of the peptide
(AA.sub.2) and at least one additional anchor was required,
seventeen novel sequences were found, 10 of which contained Leu and
Val at AA.sub.2 and AA.sub.9 respectively. Most of these sequences
(shown in Table 2) were adjacent to potential amphiphilic
sites.
[0076] Because it is well-known that not all HLA-A2
anchor-containing peptides are antigenic, and that it was generally
considered not possible to generate antigens from very short
peptide sequences (such as, e.g., peptides shorter than eight amino
acids) the discovery by the inventors that these nonameric peptides
both recognized CTLs, stimulated them, and produced an immune
response was indeed a surprising discovery.
[0077] Three criteria of epitope selection and identified the
effects of peptide length and presence of anchors on reactivity of
HLA-A2 with MA2.1 mAb. MA2.1 mAb recognizes an epitope made of
residues 62-65 of the .alpha.1 helix which is left to the center of
the binding site on HLA-A2 (Santon-Aguado et al., 1988). Therefore
exogenous peptide binding to HLA-A2 may have three potential
consequences:
[0078] (a) induction of a conformational epitope by binding to an
`empty` HLA-A2 molecule, or displacing a pre-existing endogenous
peptide in which case MA2.1 mAb reactivity with HLA-A2 will
increase;
[0079] (b) prevention of reactivity of MA2.1 with its epitope
either by obscuring residues with which the mAb may interact or
interfering with mAb epitope interaction, in which case MA2.1 mAb
reactivity with its epitope will decrease (Hogquist et al., 1993);
and,
[0080] (c) no effect in reactivity of MA2.1 mAb with HLA-A2 in
which case the exogenous added peptide may displace the existing
endogenous peptide, but the conformation of the `face` made of
.alpha.-peptide-.alpha.2 will not change. In this case
conformational changes on the MHC heavy chain may be detected in a
different position using another mAb such as BB7.2 which interacts
with an epitope containing W(108) (Salter et al., 1987).
[0081] Another surprising aspect of the invention was the fact that
when long peptides (such as, e.g., peptides longer than 20 amino
acids) were used which contained within their sequences peptide
sequences which are disclosed herein, these >20 amino acid
peptides failed to induce changes in FL1 while the novel
compositions disclosed herein, effectively induced FL1 changes.
This suggests that peptides >20 amino acids (1) either fail to
bind to MHC heavy chain because of low affinity, (2) fail to be
processed to shorter peptides because of either absence of
extracellular proteases secreted by T2 cells or (3) lack the
correct sites in the substrate for processing by extracellular
proteases.
[0082] The highest increase in FL1 was induced by a D113 analog
containing G (P1) replacing the bulky and hydrophilic H (P1),
suggesting that residues at P1 may interfere either with mAb or
peptide binding. Val (P9) appeared to be important for MA2.1
epitope induction because substitution M.fwdarw.V (P9) induced an
increase in FL1 compared with the wild-type nonapeptide C85
(971-979).
[0083] The D97 reactive CTLs identified, as well as the previously
demonstrated C85 reactive CTLs, indicate that T-cells reactive with
these epitopes are not clonally deleted, while the possible anergic
state of self-reactive CTLs from peripheral blood may have been
overcome by using PBMC at high density as APC. The use of PBMC as
APC may have selective advantages over T2 or C1R:A2 used in other
studies (Fisk et al., 1994; Houbiers et al., 1993). First, a number
of cells from PBMC can either present Ag, or release lymphokines,
or in general provide help for CTL induction; second, they reflect
closer the situation encountered during in vivo vaccination with
tumor peptides than T2/C1R:A2 cells; and third, induction of
peptide reactive T-cell may not only identify epitopes able to
induce a response to a tumor Ag but also re-stimulate in vivo
primed T-cells. These cells can either recognize, or cross-react
with epitopes from HER-2 or from other proteins which mimic the
corresponding HER-2 epitopes; fourth, by determining the frequency
of such responses among healthy HLA-A2.sup.+ donors, this may allow
identification of changes in the responder frequency in breast and
ovarian cancer patients with HER-2 high and HER-2 low expression on
their tumors.
[0084] No direct correlation could be demonstrated between the
ability of these peptides to affect the MA2.1 epitopes and either
their ability to stimulate lymphocyte proliferation or to induce in
vitro CTLs specific for the peptide of interest. Both D113 and D119
as well as longer peptides when used as immunogen to stimulate PBMC
in vitro failed to induce a sustained Ag specific CTL response. In
cytotoxicity assays, the PBMC cultures stimulated with these
peptides failed to show preferential recognition of Ag used for
stimulation. In Example 1 it is shown that HER-2:968-981 or
HER-2:971-979 peptides can induce a CTL response in vitro.
Therefore the inability of peptides HER-2:2:48-56 and HER-2:402-410
to induce in vitro Ag specific CTL may reflect: (1) clonal deletion
of epitope reactive CD8.sup.+ CTL; (2) anergy or suppression of
specific CTL clones; or (3) inefficient Ag presentation in the
sense that the peptide although increases the number of MA2.1
epitopes and apparently stabilizes HLA-A2 its conformation does not
provide efficient signaling through TCR (Hogquist et al.,
1993).
[0085] 2. CTL Epitopes
[0086] CTL epitopes reported to date are mainly derived from
foreign (viral) proteins with little or no homology with
self-proteins. With respect to CTL responses to self-proteins, it
is expected that T-cells expressing TCR with high affinity for
self-peptide-MHC class I complexes are eliminated in the thymus
during development. Self-peptides eluted from HLA-A2.1 molecules of
various cell lines show residues at P3-P5 and P7-P8 which are
different from the sequences of viral epitopes recognized by human
CTLs. Since these residues are likely to contact and interact with
TCR, they may reflect peptides for which autologous T-cells are
already tolerant/anergic.
[0087] For T-cell recognizing self-epitopes to be eliminated or
anergized, a precondition exists that the peptide-MHC complex is
stable enough to engage a sufficient number of TCRs, or at least
more stable than other HLA-A2 peptide complexes, where one peptide
can be easily displaced by other peptides. Consequently this would
suggest that for self-proteins with extension to HER-2, the ones
that can bind TCR with high affinity during development will be
less likely to be recognized later when expressed on a tumor other
target, than peptides that bind HLA-A2 with low affinity, which
under appropriate conditions (e.g., high protein concentration) may
occupy a higher number of HLA-A2 molecules. For low-affinity
peptides, modification of the anchors resulting in stabilization of
peptide--HLA-A2 interaction by replacing weak with dominant anchor
residues (e.g., (P9) M.fwdarw.V, should facilitate the reactivity
of CTL with targets expressing such antigens, because TCR interacts
mainly with the sequence P4-P8.
[0088] Tumor progression and metastasis are often associated with
overexpression of specific cellular proteins. Epitopes of
non-mutated overexpressed proteins can be targets of a specific
cellular immune response against tumor mediated by T-cells.
Moreover, when T-cell epitopes are present, distinction between
tumor immunity/autoimmunity and unresponsiveness can be predicated
on the protein concentration as a limiting factor of epitope
supply. The present inventors have demonstrated that CTLs from
patients with ovarian tumors which over-express HER-2
proto-oncogene can recognize both autologous tumor and novel
synthetic analogs of a specific HER-2 epitopes. These epitopes were
identified in HER-2 containing nonapeptides with HLA-A2 anchors.
Analysis of potential amphiphilic sites identified natural peptides
and novel synthetic peptides which surprisingly affected the
reactivity of conformationally-dependent HLA-A2 specific monoclonal
antibodies (mAbs), and indicated specific binding of these peptides
similar to that seen for HER-2 epitopes.
[0089] 3. Screening Kits
[0090] In another aspect, the present invention contemplates a
diagnostic kit for screening samples suspected of containing
HER-2/neu or neu-related polypeptides, or cells producing such
polypeptides. Said kit can contain a peptide or antibody of the
present invention. The kit can contain reagents for detecting an
interaction between an agent and a peptide or antibody of the
present invention. The provided reagent can be radio-,
fluorescently- or enzymatically-labeled. The kit can contain a
known radiolabeled agent capable of binding or interacting with a
peptide or antibody of the present invention.
[0091] In another aspect, the present invention contemplates a
diagnostic kit for detecting CTLs. The kit comprises reagents
capable of detecting a peptide of the present invention and a CTL.
The provided reagent may also be radio-, enzymatically-, or
fluorescently-labeled. The kit can contain a radiolabeled peptide
capable of binding to or interacting with a CTL, or may contain a
radiolabeled antibody capable of binding to or interacting with a
peptide of the present invention which in turn interacts with a
CTL. The kit can contain a polynucleotide probe from about 15 to 60
nucleotides that encodes a peptide of the present invention or any
of their complements. The kit can contain an antibody
immunoreactive with a peptide of the present invention.
[0092] The reagent of the kit can be provided as a liquid solution,
attached to a solid support or as a dried powder. Preferably, when
the reagent is provided in a liquid solution, the liquid solution
is an aqueous solution. Preferably, when the reagent provided is
attached to a solid support, the solid support can be chromatograph
media, a test plate having a plurality of wells, or a microscope
slide. When the reagent provided is a dry powder, the powder can be
reconstituted by the addition of a suitable solvent, that may be
provided.
[0093] 4. Immunodetection Kits
[0094] In still further embodiments, the present invention concerns
immunodetection methods and associated kits. It is proposed that
the CTL-stimulating peptides of the present invention may be
employed to detect antibodies having reactivity therewith, or,
alternatively, antibodies prepared in accordance with the present
invention, may be employed to detect CTLs or neu-related
epitope-containing peptides. In general, these methods will include
first obtaining a sample suspected of containing such a protein,
peptide or antibody, contacting the sample with an antibody or
peptide in accordance with the present invention, as the case may
be, under conditions effective to allow the formation of an
immunocomplex, and then detecting the presence of the
immunocomplex.
[0095] In general, the detection of immunocomplex formation is
quite well known in the art and may be achieved through the
application of numerous approaches. For example, the present
invention contemplates the application of ELISA, RIA, immunoblot
(e.g., dot blot), indirect immunofluorescence techniques and the
like. Generally, immunocomplex formation will be detected through
the use of a label, such as a radiolabel or an enzyme tag (such as
alkaline phosphatase, horseradish peroxidase, or the like). Of
course, one may find additional advantages through the use of a
secondary binding ligand such as a second antibody or a
biotin/avidin ligand binding arrangement, as is known in the
art.
[0096] For diagnostic purposes, it is proposed that virtually any
sample suspected of comprising either the HER-2/neu peptide or
neu-related peptides or antibody sought to be detected, as the case
may be, may be employed. Exemplary samples include clinical samples
obtained from a patient such as blood or serum samples, ear swabs,
sputum samples, middle ear fluid or even perhaps urine samples may
be employed. Furthermore, it is contemplated that such embodiments
may have application to non-clinical samples, such as in the
titering of antigen or antibody samples, in the selection of
hybridomas, and the like.
[0097] In related embodiments, the present invention contemplates
the preparation of kits that may be employed to detect the presence
of HER-2/neu or neu-related proteins or peptides and/or antibodies
in a sample. Generally speaking, kits in accordance with the
present invention will include a suitable CTL-stimulating peptide
or an antibody directed against such a protein or peptide, together
with an immunodetection reagent and a means for containing the
antibody or antigen and reagent. The immunodetection reagent will
typically comprise a label associated with the antibody or antigen,
or associated with a secondary binding ligand. Exemplary ligands
might include a secondary antibody directed against the first
antibody or antigen or a biotin or avidin (or streptavidin) ligand
having an associated label. Of course, as noted above, a number of
exemplary labels are known in the art and all such labels may be
employed in connection with the present invention.
[0098] The container means will generally include a vial into which
the antibody, antigen or detection reagent may be placed, and
preferably suitably aliquotted. The kits of the present invention
will also typically include a means for containing the antibody,
antigen, and reagent containers in close confinement for commercial
sale. Such containers may include injection or blow-molded plastic
containers into which the desired vials are retained.
[0099] 5. ELISAs
[0100] ELISAs may be used in conjunction with the invention. In an
ELISA assay, proteins or peptides incorporating dgA antigen
sequences are immobilized onto a selected surface, preferably a
surface exhibiting a protein affinity such as the wells of a
polystyrene microtiter plate. After washing to remove incompletely
adsorbed material, it is desirable to bind or coat the assay plate
wells with a nonspecific protein that is known to be antigenically
neutral with regard to the test antisera such as bovine serum
albumin (BSA), casein or solutions of milk powder. This allows for
blocking of nonspecific adsorption sites on the immobilizing
surface and thus reduces the background caused by nonspecific
binding of antisera onto the surface.
[0101] After binding of antigenic material to the well, coating
with a non-reactive material to reduce background, and washing to
remove unbound material, the immobilizing surface is contacted with
the antisera or clinical or biological extract to be tested in a
manner conducive to immune complex (antigen/antibody) formation.
Such conditions preferably include diluting the antisera with
diluents such as BSA, bovine gamma globulin (BGG) and phosphate
buffered saline (PBS)/Tween.RTM.. These added agents also tend to
assist in the reduction of nonspecific background. The layered
antisera is then allowed to incubate for from 2 to 4 hours, at
temperatures preferably on the order of about 25.degree. to about
27.degree. C. Following incubation, the antisera-contacted surface
is washed so as to remove non-immunocomplexed material. A preferred
washing procedure includes washing with a solution such as
PBS/Tween.RTM., or borate buffer.
[0102] Following formation of specific immunocomplexes between the
test sample and the bound antigen, and subsequent washing, the
occurrence and even amount of immunocomplex formation may be
determined by subjecting same to a second antibody having
specificity for the first. To provide a detecting means, the second
antibody will preferably have an associated enzyme that will
generate a color development upon incubating with an appropriate
chromogenic substrate. Thus, for example, one will desire to
contact and incubate the antisera-bound surface with a urease or
peroxidase-conjugated anti-human IgG for a period of time and under
conditions which favor the development of immunocomplex formation
(e.g., incubation for 2 hours at room temperature in a
PBS-containing solution such as PBS-Tween.RTM.).
[0103] After incubation with the second enzyme-tagged antibody, and
subsequent to washing to remove unbound material, the amount of
label is quantified by incubation with a chromogenic substrate such
as urea and bromocresol purple or
2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantification is then achieved by measuring the degree of color
generation, e.g., using a visible spectra spectrophotometer.
[0104] 6. Epitopic Core Sequences
[0105] The present invention is also directed to protein or peptide
compositions, free from total cells and other peptides, which
comprise a purified protein or peptide which incorporates an
epitope that is immunologically cross-reactive with one or more
anti-HER-2/neu antibodies.
[0106] As used herein, the term "incorporating an epitope(s) that
is immunologically cross-reactive with one or more anti-HER-2/neu
antibodies" is intended to refer to a peptide or protein antigen
which includes a primary, secondary or tertiary structure similar
to an epitope located within a HER-2 proto-oncogene polypeptide.
The level of similarity will generally be to such a degree that
monoclonal or polyclonal antibodies directed against the HER-2
polypeptide will also bind to, react with, or otherwise recognize,
the cross-reactive peptide or protein antigen. Various immunoassay
methods may be employed in conjunction with such antibodies, such
as, for example, Western blotting, ELISA, RIA, and the like, all of
which are known to those of skill in the art.
[0107] The identification of CTL-stimulating immunodominant
epitopes, and/or their functional equivalents, suitable for use in
vaccines is a relatively straightforward matter. For example, one
may employ the methods of Hopp, as taught in U.S. Pat. No. 15
4,554,101, incorporated herein by reference, which teaches the
identification and preparation of epitopes from amino acid
sequences on the basis of hydrophilicity. The methods described in
several other papers, and software programs based thereon, can also
be used to identify epitopic core sequences (see, for example,
Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No.
4,554,101). The amino acid sequence of these "epitopic core
sequences" may then be readily incorporated into peptides, either
through the application of peptide synthesis or recombinant
technology.
[0108] Preferred peptides for use in accordance with the present
invention will generally be on the order of 8 to 20 amino acids in
length, and more preferably about 8 to about 15 amino acids in
length. It is proposed that shorter antigenic CTL-stimulating
peptides will provide advantages in certain circumstances, for
example, in the preparation of vaccines or in immunologic detection
assays. Exemplary advantages include the ease of preparation and
purification, the relatively low cost and improved reproducibility
of production, and advantageous biodistribution.
[0109] It is proposed that particular advantages of the present
invention may be realized through the preparation of synthetic
peptides which include modified and/or extended
epitopic/immunogenic core sequences which result in a "universal"
epitopic peptide directed to HER-2/neu and neu-related sequences.
These epitopic core sequences are identified herein in particular
aspects as hydrophilic regions of the HER-2/neu proto-oncogene
polypeptide antigen. It is proposed that these regions represent
those which are most likely to promote T-cell or B-cell
stimulation, and, hence, elicit specific antibody production.
[0110] An epitopic core sequence, as used herein, is a relatively
short stretch of amino acids that is "complementary" to, and
therefore will bind, antigen binding sites on transferrin-binding
protein antibodies. Additionally or alternatively, an epitopic core
sequence is one that will elicit antibodies that are cross-reactive
with antibodies directed against the peptide compositions of the
present invention. It will be understood that in the context of the
present disclosure, the term "complementary" refers to amino acids
or peptides that exhibit an attractive force towards each other.
Thus, certain epitope core sequences of the present invention may
be operationally defined in terms of their ability to compete with
or perhaps displace the binding of the desired protein antigen with
the corresponding protein-directed antisera.
[0111] In general, the size of the polypeptide antigen is not
believed to be particularly crucial, so long as it is at least
large enough to carry the identified core sequence or sequences.
The smallest useful core sequence anticipated by the present
disclosure would generally be on the order of about 8 amino acids
in length, with sequences on the order of 9 or 10 being more
preferred. Thus, this size will generally correspond to the
smallest peptide antigens prepared in accordance with the
invention. However, the size of the antigen may be larger where
desired, so long as it contains a basic epitopic core sequence.
[0112] The identification of epitopic core sequences is known to
those of skill in the art, for example, as described in U.S. Pat.
No. 4,554,101, incorporated herein by reference, which teaches the
identification and preparation of epitopes from amino acid
sequences on the basis of hydrophilicity. Moreover, numerous
computer programs are available for use in predicting antigenic
portions of proteins (see e.g., Jameson & Wolf, 1988; Wolf et
al., 1988). Computerized peptide sequence analysis programs (e.g.,
DNAStar Software, DNAStar, Inc., Madison, Wis.) may also be useful
in designing synthetic peptides in accordance with the present
disclosure.
[0113] Syntheses of epitopic sequences, or peptides which include
an antigenic epitope within their sequence, are readily achieved
using conventional synthetic techniques such as the solid phase
method (e.g., through the use of commercially available peptide
synthesizer such as an Applied Biosystems Model 430A Peptide
Synthesizer). Peptide antigens synthesized in this manner may then
be aliquotted in predetermined amounts and stored in conventional
manners, such as in aqueous solutions or, even more preferably, in
a powder or lyophilized state pending use.
[0114] In general, due to the relative stability of peptides, they
may be readily stored in aqueous solutions for fairly long periods
of time if desired, e.g., up to six months or more, in virtually
any aqueous solution without appreciable degradation or loss of
antigenic activity. However, where extended aqueous storage is
contemplated it will generally be desirable to include agents
including buffers such as Tris or phosphate buffers to maintain a
pH of about 7.0 to about 7.5. Moreover, it may be desirable to
include agents which will inhibit microbial growth, such as sodium
azide or Merthiolate. For extended storage in an aqueous state it
will be desirable to store the solutions at 4.degree. C., or more
preferably, frozen. Of course, where the peptides are stored in a
lyophilized or powdered state, they may be stored virtually
indefinitely, e.g., in metered aliquots that may be rehydrated with
a predetermined amount of water (preferably distilled) or buffer
prior to use.
[0115] 7. Immunoprecipitation
[0116] The antibodies of the present invention are particularly
useful for the isolation of antigens by immunoprecipitation.
Immunoprecipitation involves the separation of the target antigen
component from a complex mixture, and is used to discriminate or
isolate minute amounts of protein. For the isolation of membrane
proteins cells must be solubilized into detergent micelles.
Nonionic salts are preferred, since other agents such as bile
salts, precipitate at acid pH or in the presence of bivalent
cations.
[0117] In an alternative embodiment the antibodies of the present
invention are useful for the close juxtaposition of two antigens.
This is particularly useful for increasing the localized
concentration of antigens, e.g. enzyme-substrate pairs.
[0118] 8. Western Blots
[0119] The compositions of the present invention will find great
use in immunoblot or western blot analysis. The anti-peptide
antibodies may be used as high-affinity primary reagents for the
identification of proteins immobilized onto a solid support matrix,
such as nitrocellulose, nylon or combinations thereof. In
conjunction with immunoprecipitation, followed by gel
electrophoresis, these may be used as a single step reagent for use
in detecting antigens against which secondary reagents used in the
detection of the antigen cause an adverse background. This is
especially useful when the antigens studied are immunoglobulins
(precluding the use of immunoglobulins binding bacterial cell wall
components), the antigens studied cross-react with the detecting
agent, or they migrate at the same relative molecular weight as a
cross-reacting signal.
[0120] Immunologically-based detection methods for use in
conjunction with Western blotting include enzymatically-,
radiolabel-, or fluorescently-tagged secondary antibodies against
the toxin moiety are considered to be of particular use in this
regard.
[0121] 9. Vaccines
[0122] The present invention contemplates vaccines for use in both
active and passive immunization embodiments. Immunogenic
compositions, proposed to be suitable for use as a vaccine, may be
prepared most readily directly from immunogenic CTL-stimulating
peptides prepared in a manner disclosed herein. Preferably the
antigenic material is extensively dialyzed to remove undesired
small molecular weight molecules and/or lyophilized for more ready
formulation into a desired vehicle.
[0123] The preparation of vaccines which contain peptide sequences
as active ingredients is generally well understood in the art, as
exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;
4,599,230; 4,596,792; and 4,578,770, all incorporated herein by
reference. Typically, such vaccines are prepared as injectables.
Either as liquid solutions or suspensions: solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation may also be emulsified. The active
immunogenic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, or adjuvants which enhance the effectiveness of
the vaccines.
[0124] Vaccines may be conventionally administered parenterally, by
injection, for example, either subcutaneously or intramuscularly.
Additional formulations which are suitable for other modes of
administration include suppositories and, in some cases, oral
formulations. For suppositories, traditional binders and carriers
may include, for example, polyalkalene glycols or triglycerides:
such suppositories may be formed from mixtures containing the
active ingredient in the range of about 0.5% to about 10%,
preferably about 1 to about 2%. Oral formulations include such
normally employed excipients as, for example, pharmaceutical grades
of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain about 10 to about 95% of active ingredient, preferably
about 25 to about 70%.
[0125] The peptides of the present invention may be formulated into
the vaccine as neutral or salt forms. Pharmaceutically-acceptable
salts, include the acid addition salts (formed with the free amino
groups of the peptide) and those which are formed with inorganic
acids such as, for example, hydrochloric or phosphoric acids, or
such organic acids as acetic, oxalic, tartaric, mandelic, and the
like. Salts formed with the free carboxyl groups may also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, procaine, and the like.
[0126] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to synthesize
antibodies, and the degree of protection desired. Precise amounts
of active ingredient required to be administered depend on the
judgment of the practitioner. However, suitable dosage ranges are
of the order of several hundred micrograms active ingredient per
vaccination. Suitable regimes for initial administration and
booster shots are also variable, but are typified by an initial
administration followed by subsequent inoculations or other
administrations.
[0127] The manner of application may be varied widely. Any of the
conventional methods for administration of a vaccine are
applicable. These are believed to include oral application on a
solid physiologically acceptable base or in a physiologically
acceptable dispersion, parenterally, by injection or the like. The
dosage of the vaccine will depend on the route of administration
and will vary according to the size of the host.
[0128] Various methods of achieving adjuvant effect for the vaccine
includes use of agents such as aluminum hydroxide or phosphate
(alum), commonly used as about 0.05 to about 0.1% solution in
phosphate buffered saline, admixture with synthetic polymers of
sugars (Carbopol.RTM.) used as an about 0.25% solution, aggregation
of the protein in the vaccine by heat treatment with temperatures
ranging between about 70.degree. to about 101.degree. C. for a
30-second to 2-minute period, respectively. Aggregation by
reactivating with pepsin treated (Fab) antibodies to albumin,
mixture with bacterial cells such as C. parvum or endotoxins or
lipopolysaccharide components of Gram-negative bacteria, emulsion
in physiologically acceptable oil vehicles such as mannide
mono-oleate (Aracel A) or emulsion with a 20% solution of a
perfluorocarbon (Fluosol-DA.RTM.) used as a block substitute may
also be employed.
[0129] In many instances, it will be desirable to have multiple
administrations of the vaccine, usually not exceeding six
vaccinations, more usually not exceeding four vaccinations and
preferably one or more, usually at least about three vaccinations.
The vaccinations will normally be at from two to twelve week
intervals, more usually from three to five week intervals. Periodic
boosters at intervals of 1-5 years, usually three years, will be
desirable to maintain protective levels of the antibodies. The
course of the immunization may be followed by assays for antibodies
for the supernatant antigens. The assays may be performed by
labeling with conventional labels, such as radionuclides, enzymes,
fluorescents, and the like. These techniques are well known and may
be found in a wide variety of patents, such as U.S. Pat. Nos.
3,791,932; 4,174,384 and 3,949,064, as illustrative of these types
of assays.
[0130] 10. DNA Segments Encoding Novel Peptides
[0131] The present invention also concerns DNA segments, that can
be isolated from virtually any mammalian source, that are free from
total genomic DNA and that encode the novel peptides disclosed
herein. DNA segments encoding these peptide species may prove to
encode proteins, polypeptides, subunits, functional domains, and
the like of HER-2/neu-related or other non-related gene products.
In addition these DNA segments may be synthesized entirely in vitro
using methods that are well-known to those of skill in the art.
[0132] As used herein, the term "DNA segment" refers to a DNA
molecule that has been isolated free of total genomic DNA of a
particular species. Therefore, a DNA segment encoding a
CTL-stimulating peptide refers to a DNA segment that contains
CTL-simulation coding sequences yet is isolated away from, or
purified free from, total genomic DNA of the species from which the
DNA segment is obtained. Included within the term "DNA segment",
are DNA segments and smaller fragments of such segments, and also
recombinant vectors, including, for example, plasmids, cosmids,
phagemids, phage, viruses, and the like.
[0133] Similarly, a DNA segment comprising an isolated or purified
CTL-stimulating peptide-encoding gene refers to a DNA segment which
may include in addition to peptide encoding sequences, certain
other elements such as, regulatory sequences, isolated
substantially away from other naturally occurring genes or
protein-encoding sequences. In this respect, the term "gene" is
used for simplicity to refer to a functional protein-, polypeptide-
or peptide-encoding unit. As will be understood by those in the
art, this functional term includes both genomic sequences, cDNA
sequences and smaller engineered gene segments that express, or may
be adapted to express, proteins, polypeptides or peptides.
[0134] "Isolated substantially away from other coding sequences"
means that the gene of interest, in this case, a gene encoding
CTL-stimulating peptides, forms the significant part of the coding
region of the DNA segment, and that the DNA segment does not
contain large portions of naturally-occurring coding DNA, such as
large chromosomal fragments or other functional genes or cDNA
coding regions. Of course, this refers to the DNA segment as
originally isolated, and does not exclude genes or coding regions
later added to the segment by the hand of man.
[0135] In particular embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
that encode a CTL-stimulating peptide species that includes within
its amino acid sequence an amino acid sequence essentially as set
forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ
ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:
22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ
ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.
[0136] The term "a sequence essentially as set forth in any of SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ
ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
28, and SEQ ID NO: 29" means that the sequence substantially
corresponds to a portion of the sequence of either SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID
NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,
or SEQ ID NO: 29 and has relatively few amino acids that are not
identical to, or a biologically functional equivalent of, the amino
acids of any of these sequences. The term "biologically functional
equivalent" is well understood in the art and is further defined in
detail herein (for example, see Preferred Embodiments).
Accordingly, sequences that have between about 70% and about 80%,
or more preferably between about 81% and about 90%, or even more
preferably between about 91% and about 99% amino acid sequence
identity or functional equivalence to the amino acids of any of SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ
ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
28, and SEQ ID NO: 29 will be sequences that are "essentially as
set forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID
NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,
SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29."
[0137] It will also be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of biological protein activity where
protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences that may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
internal sequences, i.e., introns, which are known to occur within
genes.
[0138] The nucleic acid segments of the present invention,
regardless of the length of the coding sequence itself, may be
combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, nucleic acid fragments may be prepared that
include a short contiguous stretch encoding either of the peptide
sequences disclosed in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
13, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, or that
are identical to or complementary to DNA sequences which encode any
of the peptides disclosed in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13,
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and
particularly those DNA segments disclosed in SEQ ID NO: 51, SEQ ID
NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,
SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID
NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64. For
example, DNA sequences such as about 14 nucleotides, and that are
up to about 1,000, about 500, about 200, about 100, about 50, and
about 25 base pairs in length (including all intermediate lengths)
are also contemplated to be useful.
[0139] It will be readily understood that "intermediate lengths",
in these contexts, means any length between the quoted ranges, such
as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32,
etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,
152, 153, etc.; including all integers through the 200-500;
500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to and
including sequences of about 10,000 nucleotides and the like.
[0140] It will also be understood that this invention is not
limited to the particular nucleic acid sequences which encode
peptides of the present invention, or which encode the amino acid
sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, including those DNA
sequences which are particularly disclosed in SEQ ID NO: 51, SEQ ID
NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,
SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID
NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64. Recombinant
vectors and isolated DNA segments may therefore variously include
the peptide-coding regions themselves, coding regions bearing
selected alterations or modifications in the basic coding region,
or they may encode larger polypeptides that nevertheless include
these peptide-coding regions or may encode biologically functional
equivalent proteins or peptides that have variant amino acids
sequences.
[0141] The DNA segments of the present invention encompass
biologically-functional equivalent peptides. Such sequences may
arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded. Alternatively,
functionally-equivalent proteins or peptides may be created via the
application of recombinant DNA technology, in which changes in the
protein structure may be engineered, based on considerations of the
properties of the amino acids being exchanged. Changes designed by
man may be introduced through the application of site-directed
mutagenesis techniques, e.g., to introduce improvements to the
antigenicity of the protein or to test mutants in order to examine
activity at the molecular level.
[0142] If desired, one may also prepare fusion proteins and
peptides, e.g., where the peptide-coding regions are aligned within
the same expression unit with other proteins or peptides having
desired functions, such as for purification or immunodetection
purposes (e.g., proteins that may be purified by affinity
chromatography and enzyme label coding regions, respectively).
[0143] Recombinant vectors form further aspects of the present
invention. Particularly useful vectors are contemplated to be those
vectors in which the coding portion of the DNA segment, whether
encoding a full length protein or smaller peptide, is positioned
under the control of a promoter. The promoter may be in the form of
the promoter that is naturally associated with a gene encoding
peptides of the present invention, as may be obtained by isolating
the 5' non-coding sequences located upstream of the coding segment
or exon, for example, using recombinant cloning and/or PCR.TM.
technology, in connection with the compositions disclosed
herein.
[0144] In other embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA segment
under the control of a recombinant, or heterologous, promoter. As
used herein, a recombinant or heterologous promoter is intended to
refer to a promoter that is not normally associated with a DNA
segment encoding a CTL-stimulating peptide in its natural
environment. Such promoters may include promoters normally
associated with other genes, and/or promoters isolated from any
bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will
be important to employ a promoter that effectively directs the
expression of the DNA segment in the cell type, organism, or even
animal, chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of
skill in the art of molecular biology, for example, see Sambrook et
al., 1989. The promoters employed may be constitutive, or
inducible, and can be used under the appropriate conditions to
direct high level expression of the introduced DNA segment, such as
is advantageous in the large-scale production of recombinant
proteins or peptides. Appropriate promoter systems contemplated for
use in high-level expression include, but are not limited to, the
Pichia expression vector system (Pharmacia LKB Biotechnology).
[0145] In connection with expression embodiments to prepare
recombinant proteins and peptides, it is contemplated that longer
DNA segments will most often be used, with DNA segments encoding
the entire peptide sequence being most preferred. However, it will
be appreciated that the use of shorter DNA segments to direct the
expression of CTL-stimulating peptides or epitopic core regions,
such as may be used to generate anti-peptide antibodies, also falls
within the scope of the invention. DNA segments that encode peptide
antigens from about 8 to about 50 amino acids in length, or more
preferably, from about 8 to about 30 amino acids in length, or even
more preferably, from about 8 to about 20 amino acids in length are
contemplated to be particularly useful.
[0146] In addition to their use in directing the expression of
CTL-stimulating peptides of the present invention, the nucleic acid
sequences contemplated herein also have a variety of other uses.
For example, they also have utility as probes or primers in nucleic
acid hybridization embodiments. As such, it is contemplated that
nucleic acid segments that comprise a sequence region that consists
of at least a 14 nucleotide long contiguous sequence that has the
same sequence as, or is complementary to, a 14 nucleotide long
contiguous DNA segment any of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID
NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,
SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID
NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64 will find particular
utility. Longer contiguous identical or complementary sequences,
e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including
all intermediate lengths) and even up to full length sequences will
also be of use in certain embodiments.
[0147] The ability of such nucleic acid probes to specifically
hybridize to peptide-encoding sequences will enable them to be of
use in detecting the presence of complementary sequences in a given
sample. However, other uses are envisioned, including the use of
the sequence information for the preparation of mutant species
primers, or primers for use in preparing other genetic
constructions.
[0148] Nucleic acid molecules having sequence regions consisting of
contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of
100-200 nucleotides or so, identical or complementary to DNA
sequences of any of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,
SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID
NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62,
SEQ ID NO: 63, and SEQ ID NO: 64, are particularly contemplated as
hybridization probes for use in, e.g., Southern and Northern
blotting. Smaller fragments will generally find use in
hybridization embodiments, wherein the length of the contiguous
complementary region may be varied, such as between about 10-14 and
about 100 nucleotides, but larger contiguous complementarity
stretches may be used, according to the length complementary
sequences one wishes to detect.
[0149] The use of a hybridization probe of about 10-14 nucleotides
in length allows the formation of a duplex molecule that is both
stable and selective. Molecules having contiguous complementary
sequences over stretches greater than 10 bases in length are
generally preferred, though, in order to increase stability and
selectivity of the hybrid, and thereby improve the quality and
degree of specific hybrid molecules obtained. One will generally
prefer to design nucleic acid molecules having gene-complementary
stretches of 15 to 20 contiguous nucleotides, or even longer where
desired.
[0150] Of course, fragments may also be obtained by other
techniques such as, e.g., by mechanical shearing or by restriction
enzyme digestion. Small nucleic acid segments or fragments may be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, as is commonly practiced using an
automated oligonucleotide synthesizer. Also, fragments may be
obtained by application of nucleic acid reproduction technology,
such as the PCR.TM. technology of U.S. Pat. Nos. 4,683,195 and
4,683,202 (each incorporated herein by reference), by introducing
selected sequences into recombinant vectors for recombinant
production, and by other recombinant DNA techniques generally known
to those of skill in the art of molecular biology.
[0151] Accordingly, the nucleotide sequences of the invention may
be used for their ability to selectively form duplex molecules with
complementary stretches of DNA fragments. Depending on the
application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. For applications
requiring high selectivity, one will typically desire to employ
relatively stringent conditions to form the hybrids, e.g., one will
select relatively low salt and/or high temperature conditions, such
as provided by about 0.02 M to about 0.15 M NaCl at temperatures of
50.degree. C. to 70.degree. C. Such selective conditions tolerate
little, if any, mismatch between the probe and the template or
target strand, and would be particularly suitable for isolating
CTL-stimulating peptide-encoding DNA segments. Detection of DNA
segments via hybridization is well-known to those of skill in the
art, and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995
(each incorporated herein by reference) are exemplary of the
methods of hybridization analyses. Teachings such as those found in
the texts of Maloy et al., 1993; Segal 1976; Proskop, 1991; and
Kuby, 1991, are particularly relevant.
[0152] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
CTL-stimulating peptide-encoding sequences from related species,
functional equivalents, or the like, less stringent hybridization
conditions will typically be needed in order to allow formation of
the heteroduplex. In these circumstances, one may desire to employ
conditions such as about 0.15 M to about 0.9 M salt, at
temperatures ranging from about 20.degree. C. to about 55.degree.
C. Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0153] In certain embodiments, it will be advantageous to employ
nucleic acid sequences of the present invention in combination with
an appropriate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are
known in the art, including fluorescent, radioactive, enzymatic or
other ligands, such as avidin/biotin, which are capable of giving a
detectable signal. In preferred embodiments, one will likely desire
to employ a fluorescent label or an enzyme tag, such as urease,
alkaline phosphatase or peroxidase, instead of radioactive or other
environmental undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known that can be employed to
provide a means visible to the human eye or spectrophotometrically,
to identify specific hybridization with complementary nucleic
acid-containing samples.
[0154] In general, it is envisioned that the hybridization probes
described herein will be useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the test DNA (or RNA) is
adsorbed or otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to specific
hybridization with selected probes under desired conditions. The
selected conditions will depend on the particular circumstances
based on the particular criteria required (depending, for example,
on the G+C content, type of target nucleic acid, source of nucleic
acid, size of hybridization probe, etc.). Following washing of the
hybridized surface so as to remove nonspecifically bound probe
molecules, specific hybridization is detected, or even quantitated,
by means of the label.
[0155] 11. Biological Functional Equivalents
[0156] Modification and changes may be made in the structure of the
peptides of the present invention and DNA segments which encode
them and still obtain a functional molecule that encodes a protein
or peptide with desirable characteristics. The following is a
discussion based upon changing the amino acids of a protein to
create an equivalent, or even an improved, second-generation
molecule. The amino acid changes may be achieved by changing the
codons of the DNA sequence, according to the following codon
table:
1TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0157] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties. It is thus contemplated by the inventors that various
changes may be made in the peptide sequences of the disclosed
compositions, or corresponding DNA sequences which encode said
peptides without appreciable loss of their biological utility or
activity.
[0158] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporate herein by reference). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like.
[0159] Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0160] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0161] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein.
[0162] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0163] It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still obtain a
biologically equivalent, and in particular, an immunologically
equivalent protein. In such changes, the substitution of amino
acids whose hydrophilicity values are within .+-.2 is preferred,
those which are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
[0164] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0165] 12. Site-Specific Mutagenesis
[0166] Site-specific mutagenesis is a technique useful in the
preparation of individual peptides, or biologically functional
equivalent proteins or peptides, through specific mutagenesis of
the underlying DNA. The technique further provides a ready ability
to prepare and test sequence variants, for example, incorporating
one or more of the foregoing considerations, by introducing one or
more nucleotide sequence changes into the DNA. Site-specific
mutagenesis allows the production of mutants through the use of
specific oligonucleotide sequences which encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to 25 nucleotides in length is preferred, with about 5 to 10
residues on both sides of the junction of the sequence being
altered.
[0167] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage are readily commercially
available and their use is generally well known to those skilled in
the art. Double stranded plasmids are also routinely employed in
site directed mutagenesis which eliminates the step of transferring
the gene of interest from a plasmid to a phage.
[0168] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed,wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement.
[0169] The preparation of sequence variants of the selected
peptide-encoding DNA segments using site-directed mutagenesis is
provided as a means of producing potentially useful species and is
not meant to be limiting as there are other ways in which sequence
variants of peptides and the DNA sequences encoding them may be
obtained. For example, recombinant vectors encoding the desired
peptide sequence may be treated with mutagenic agents, such as
hydroxylamine, to obtain sequence variants.
[0170] 13. Monoclonal Antibody Generation
[0171] Means for preparing and characterizing antibodies are well
known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988; incorporated herein by
reference).
[0172] The methods for generating monoclonal antibodies (mAbs)
generally begin along the same lines as those for preparing
polyclonal antibodies. Briefly, a polyclonal antibody is prepared
by immunizing an animal with an immunogenic composition in
accordance with the present invention and collecting antisera from
that immunized animal. A wide range of animal species can be used
for the production of antisera. Typically the animal used for
production of anti-antisera is a rabbit, a mouse, a rat, a hamster,
a guinea pig or a goat. Because of the relatively large blood
volume of rabbits, a rabbit is a preferred choice for production of
polyclonal antibodies.
[0173] As is well known in the art, a given composition may vary in
its immunogenicity. It is often necessary therefore to boost the
host immune system, as may be achieved by coupling a peptide or
polypeptide immunogen to a carrier. Exemplary and preferred
carriers are keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin can also be used as carriers. Means
for conjugating a polypeptide to a carrier protein are well known
in the art and include glutaraldehyde, m-maleimidobencoyl-N-hy-
droxysuccinimide ester, carbodiimide and bis-biazotized
benzidine.
[0174] As is also well known in the art, the immunogenicity of a
particular immunogen composition can be enhanced by the use of
non-specific stimulators of the immune response, known as
adjuvants. Exemplary and preferred adjuvants include complete
Freund's adjuvant (a non-specific stimulator of the immune response
containing killed Mycobacterium tuberculosis), incomplete Freund's
adjuvants and aluminum hydroxide adjuvant.
[0175] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal). The production of
polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points following immunization. A
second, booster, injection may also be given. The process of
boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the
immunized animal can be bled and the serum isolated and stored,
and/or the animal can be used to generate mAbs.
[0176] mAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with a selected immunogen
composition, e.g., a purified or partially purified LTBP-3 protein,
polypeptide or peptide. The immunizing composition is administered
in a manner effective to stimulate antibody producing cells.
Rodents such as mice and rats are preferred animals, however, the
use of rabbit, sheep frog cells is also possible. The use of rats
may provide certain advantages (Goding, 1986, pp. 60-61), but mice
are preferred, with the BALB/c mouse being most preferred as this
is most routinely used and generally gives a higher percentage of
stable fusions.
[0177] Following immunization, somatic cells with the potential for
producing antibodies, specifically B lymphocytes (B cells), are a
selected for use in the mAb generating protocol. These cells may be
obtained from biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast
stage, and the latter because peripheral blood is easily
accessible. Often, a panel of animals will have been immunized and
the spleen of animal with the highest antibody titer will be
removed and the spleen lymphocytes obtained by homogenizing the
spleen with a syringe. Typically, a spleen from an immunized mouse
contains approximately 5.times.10.sup.7 to 2.times.10.sup.8
lymphocytes.
[0178] The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell,
generally one of the same species as the animal that was immunized.
Myeloma cell lines suited for use in hybridoma-producing fusion
procedures preferably are non-antibody-producing, have high fusion
efficiency, and enzyme deficiencies that render then incapable of
growing in certain selective media which support the growth of only
the desired fused cells (hybridomas).
[0179] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, pp. 65-66, 1986;
Campbell, pp. 75-83, 1984). For example, where the immunized animal
is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/.Ag 4 1,
Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;
for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and
U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in
connection with human cell fusions.
[0180] One preferred murine myeloma cell is the NS-1 myeloma cell
line (also termed P3-NS-1-Ag4-1), which is readily available from
the NIGMS Human Genetic Mutant Cell Repository by requesting cell
line repository number GM3573. Another mouse myeloma cell line that
may be used is the 8-azaguanine-resistant mouse murine myeloma
SP2/0 non-producer cell line.
[0181] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 2:1 ratio, though the ratio
may vary from about 20:1 to about 1:1, respectively, in the
presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Sendai
virus have been described by Kohler and Milstein (1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by
Gefter et al., (1977). The use of electrically induced fusion
methods is also appropriate (Goding pp. 71-74, 1986).
[0182] Fusion procedures usually produce viable hybrids at low
frequencies, about 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are
differentiated from the parental, unfused cells (particularly the
unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0183] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B-cells can operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B-cells.
[0184] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, plaque assays, dot immuno-binding assays, and the like.
[0185] The selected hybridomas would then be serially diluted and
cloned into individual antibody-producing cell lines, which clones
can then be propagated indefinitely to provide mAbs. The cell lines
may be exploited for mAb production in two basic ways. A sample of
the hybridoma can be injected (often into the peritoneal cavity)
into a histocompatible animal of the type that was used to provide
the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific monoclonal antibody
produced by the fused cell hybrid. The body fluids of the animal,
such as serum or ascites fluid, can then be tapped to provide mAbs
in high concentration. The individual cell lines could also be
cultured in vitro, where the mAbs are naturally secreted into the
culture medium from which they can be readily obtained in high
concentrations. mAbs produced by either means may be further
purified, if desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity
chromatography.
[0186] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE 1
Oligopetide Induction of a Cytotoxic T Lymphocyte Response to
HER-2/neu Proto-oncogene in Vitro
[0187] A. Materials and Methods
[0188] 1. Peptides
[0189] HER-2 peptides were prepared by the Synthetic Antigen
Laboratory of M.D. Anderson Cancer Center (Houston, Tex.) using
Merrifield's solid-phase system and a peptide synthesizer
(Ioannides et al., 1993). All reagents were of high purity
(>99%) and obtained from Millipore Corporation. Eluted peptides
were transferred in aqueous solution by passing over Sephadex G-25
columns and lyophilized. Crude synthetic peptides were separated by
reverse-phase HPLC. Identity and purity of the final materials were
established by amino acid analysis. Purification yielded single
peaks by analytical HPLC and the purity of peptides used in these
studies was .gtoreq.97%.
[0190] 2. Immunofluorescence
[0191] mAbs to CD3 (OKT3-FITC), CD4 (OKT4-FITC), and CD8
(OKT8-FITC) were obtained from Ortho Diagnostic (Ortho, Raitan,
N.J.); mAb W6/32 (anti-HLA, -A, -B, -C) was from Dako
(Dako-Dakopatts, Denmark); and mAb Leu1 1a (anti CD16) was obtained
from Beckton-Dickinson (Mountain View, Calif.). mAb BB7.2 and MA2.1
(anti-HLA-A2)-producing clones were from ATCC, mAb Ab2 against
HER-2/neu was obtained from Oncogene Science (Manhasset, N.Y.).
Immunofluorescence studies were performed as described (Ioannides
et al., 1993).
[0192] 3. Cells and Cell Lines
[0193] Tumor lines and leukocytes of the donors of ovarian
malignant ascites were phenotyped for HLA-A, B, and C antigens by
the blood bank at M.D. Anderson Cancer Center, Leukocytes of PBMC
donors used as responder cells (HLA-A, B, C) were typed at the
Histocompatibility Laboratory of the Methodist Hospital (Houston,
Tex.). The HLA types of the donors are presented in Table 2.
Expression of HLA-A2 on ovarian tumors, fibroblasts, and EBV-B cell
lines (HLA-A2 transfectants) was confirmed by immunofluorescence
using culture supernatant from mAb MA2.1 (Ioannides et al.,
1993).
[0194] C1R:A2, C1R:A1, and C1R:A3 cells express transfected genomic
clones of HLA-A2.1, HLA-A1, and HLA-A3. These cells were obtained
from Dr. William E. Biddison, National Institute of Neurological
Disorders, Bethesda, Md. C1R (Class I reduced) is a mutant cell
line that does not express HLA-A2 (Bednarek et al., 1991; Gammon et
al., 1992). These cells were maintained in complete RPMI 1640
medium containing 100 .mu.g/ml L-glutamine, 40 .mu.g/ml gentamicin,
and 10% fetal calf serum (FCS) (RPMI-FCS). Ovarian tumors and lines
of known HLA phenotype used in these studies were: SKOV3 (HLA-A3,
28, B18, 35, Cw5), OVA-1 (HLA-A1, 24, B8, 35, Cw4), OVA-14 (HLA-A2,
30, B14, 44, Cw2.8), OVA-16 (A2, 19 B8, 35), OVA-24 (HLA-A2, 24,
B8, 51, Cw2, 7), and (VA-31 (HLA-A11, -, B60, 62, Cw3). Additional
targets used in this study were the EBV-B cell line XxCr (HLA-A2,
-, B7, 8, Cw7) and the breast carcinoma line SKBr3 (HLA-A11, -,
B18, 40, Bw22). SKBr3 overexpressing HER-2 was obtained from Dr.
Mien Chie-Hung, Department of Tumor Biology, M.D. Anderson Cancer
Center.
2TABLE 2 HLA TYPING OF LYMPHOCYTE DONORS HLA type No. Donor number
A B C 1. 30 2, 33 14, 35 w4 2. 41 1, 2 8, -- w7 3. 51 1, 2 8, -- w7
4. 46 2, 2 18, 60 w3 5. 86 2, 2 18, 61 w3 6. 14 32, -- 41, 51
N.D..sup.a 7. 15 1, 32 8, 35 w4, w7 .sup.aNot determined.
[0195] Ovarian tumors were separated from TIL/TAL by centrifugation
over Ficoll-Hypaque gradients, as previously described (Ioannides
et al., 1991), and stored frozen in aliquots in liquid nitrogen
until used. Ovarian tumor lines were maintained in culture in L-15
medium (Gibco, Life Technologies, Grand Island, N.Y.) supplemented
with 10% FCS and 20 .mu.g/ml gentamycin. Ovarian CTL-TAL lines
autologous with OVA-1, OVA-14, OVA-16, and OVA-31 have been
generated as described from lymphocytes infiltrating malignant
ascites (TAL) by coculture of tumors with TAL in RPMI-FCS in the
presence of 25-50 U/ml of IL-2 (Cetus, Emeryville, Calif.) and 250
U/ml of tumor necrosis factor-.alpha. (TNF-.alpha.) (Genentech, San
Francisco, Calif.) (Ioannides et al., 1991).
[0196] 4. Transfection of Ovarian Tumor Line SKOV3 with HLA-A2
[0197] The HLA-A2 expression vector RSV.5-neo containing HLA-A2.1
full-length cDNA was provided by Drs. Richard V. Turner and William
E. Biddison. The RSV.5-neo expression vector is a derivative of
RSV.3 (Jacobson et al., 1989). The SKOV3 cell line was cloned by
stringent limiting dilution (Ioannides et al., 1993), and
individual clones were transfected with the plasmid using the
Lipofectin reagent and procedure (Gibco-BRL, Gaithersburg, Md.) as
described by the manufacturer. Transfectants were selected in
culture with 800 .mu.g/ml of G418 (Sigma Chemical Co., St. Louis,
Mo.). Surface expression of HLA-A2 was determined by
immunofluorescence with MA 2.1 mAb as described (Ioannides et al.,
1993). Several clones that expressed high levels of HLA-A2 such as
2B6 (SKVO3.A2) were selected for cytotoxicity studies.
[0198] 5. Cytotoxicity Assays
[0199] Tumor cells and fibroblasts were labeled with 200 .mu.Ci of
.sup.51Cr (Na.sup.51CrO.sub.4; Amersham, Arlington Heights, Ill.)
for 90 min at 37.degree. C. (Ioannides et al., 1991).
Lymphoblastoid cells and HLA-A2 transfectants were labeled
overnight in RPMI-FCS, then washed three times and incubated with
effector cells in RPMI-FCS in an incubator with 5% CO.sub.2
(Bednarek et al., 1991; Gammon et al., 1992). When peptide
recognition was determined, targets were incubated with 25 .mu.M of
peptides overnight during .sup.51Cr labeling or with 10 .mu.M
peptide for 2 h at 37.degree. C. in RPMI-FCS then washed three
times before being incubated with effector cells. Separate controls
for spontaneous and total lysis were made for each peptide-pulsed
target (Ioannides et al., 1991; Bednarek et al., 1991; Gammon et
al., 1992). After 4-5 h, 100 .mu.l of supernatant was collected and
counted. To determine maximum lysis in 20-h assays, plates were
left undisturbed in the incubator and the supernatant was collected
after overnight incubation. For cold target inhibition studies,
C1R:A2 cells were preincubated with HER-2 or control peptides
overnight, then washed and admixed with .sup.51Cr-labeled targets
at 2:1 and 6:1 (cold:hot targets) ratios. Percentage lysis was
calculated from the formula: 100.times.[(E-S)/(T-S)], where E is
experimental release, S is release in the absence of CTL, and T is
release in 2 M HCl.
[0200] 6. Generation of in Vitro HER-2 Peptide-reactive CTL
[0201] CTL cultures reacting with HER-2 peptides were generated
following procedures described for in vitro induction of influenza
matrix and tum- peptide-specific CTL (Bednarek et al., 1991; Gammon
et al., 1992; Alexander et al., 1991) with several modifications.
In brief, PBMC from HLA-A2.sup.+ and HLA-A2.sup.- donors were
separated by Ficoll-Hypaque.TM. gradient centrifugation. PBMC
(5-10.times.10.sup.6) were washed, resuspended in a final volume of
100-250 .mu.l in PBS, and incubated with the stimulating peptide
for 90 min at 37.degree. C. The final concentration of the
stimulating peptide ranged between 5 and 50.times.10.sup.-6 M.
Afterwards, cells were irradiated (4000 rad), washed, and plated in
wells of 24-well plates (Costar, Cambridge, Mass.) in 2.0 ml at a
final concentration of 0.5-1.0.times.10.sup.6 cells/ml. As
responding cells, autologous PBMC were added at a final
concentration of 1.0-1.5.times.10.sup.6/ml. Sequences of HER-2
peptide analogs used for stimulation or specificity determination
are presented in Table 3.
[0202] Cultures were initiated in RPMI 1640 medium containing 100
.mu.g/ml L-glutamine, 40 .mu.g/ml gentamycin, and 5%
heat-inactivated and sterile-filtered human AB plasma (RPMI-HS).
After 3 days, 5 U of IL-2 (Cetus) was added in each well. One unit
of IL-2 (Cetus) equals 6 IU of IL-2 (Ioannides et al., 1991). After
2 additional days one-third of the medium from each well was
replaced with an equal volume of RPMI-HS containing 15 U/ml of
IL-2. Four days later, cells were removed from cultures, washed,
and restimulated either with irradiated fresh autologous PBMC or
C1R:A2 cells pulsed with HER-2 additional days the expanding
cultures were restimulated with peptides following the procedures
described above. Five to 6 days after the second stimulation and 7
to 8 days after the third and subsequent stimulations, cultures
were tested for cytotoxic activity against C1R:A2 cells pre-pulsed
with HER-2 peptides and unrelated control peptides containing
HLA-A2 anchor motifs.
[0203] Cultures that showed higher lysis of targets pulsed with
HER-2 peptides than control peptides were maintained for further
studies. These cultures were propagated and expanded by periodic
cycles of restimulation with peptide-pulsed fresh autologous PBMC
as antigen-presenting cells. After the fourth stimulation cells
were gradually adapted to growth in RPMI-FCS by replacing 25% of
the culture medium every 3 days with RPMI-FCS over a period of 2
weeks. CD3.sup.+CD8.sup.+CD4.sup.- cells were isolated from bulk
CTL cultures by positive selection on anti-CD8 mAb-coated culture
flasks (AIS Micro CELLector, Applied Immune Sciences, Menlo Park,
Calif.) as described (Letessier et al., 1991). Isolated CD8.sup.+
cells were restimulated with HER-2 peptide-pulsed PBMC, either
autologous or in some instances allogeneic that matched only HLA-A2
with the responding cells.
3TABLE 3 SEQUENCES OF PEPTIDES Sequence Peptide.sup.a 1 2 3 4 5 6 7
8 9 10 11 12 13 14 SEQ ID NO: 1. C43 R F R E L V S E F S R M A R 65
2. C85 E L V S E F S R M 7 3. C84 E L V S E F S R V 6 4. C44 R F R
E L I I E F S R M A R 66 5. D132 Muc-1:16-1 S +TL,14L A +TL,18D P A
H G V 67 6. D125 Muc-1:8-17 G L T S A P D T R V 68 .sup.aPeptides
1-4 are analogs of HER-2:968-981 (C43 and C44) and 971-979 (C85 and
C84). The substitution VS .fwdarw. II in C44 is found in the
equivalent sequence of the epidermal growth factor receptor. D132
is an analog of the Muc-1 core peptide where L (P2) and D (P4)
substitute for Thr and Pro, respectively, to create a P2 anchor and
a hydrophilic residue at P4, respectively. In the D125 peptide,
also an analog of the muc-1 core peptide, L (P2) and V (P10) also
substitute for Val and Pro, respectively. Substituted amino acids
are in bold and underlined.
[0204] B. Results
[0205] 1. Generation of in Vitro HER-2 Peptide Reacting CTLs
[0206] To define conditions for in vitro CTL induction by
stimulation with HER-2 peptides of PBMC from healthy volunteers,
two synthetic peptides were used for priming: (1) C43
(HER-2:968-981)=RFRELVSEFSRMAR (SEQ ID NO: 31), which contains as
HLA-A2 anchors Leu(972) at P2 and Met (979) at P9, includes two
Rothbard epitope motifs ELVS and RMAR and most of the amphiphilic
area 968-984; and (2) C84 (HER-2:971-979(Val)=ELVSEFSRV (SEQ ID NO:
6) where Met(P9) has been substituted by Val because Val is the
dominant anchor residue at P9 and although it does not contact the
TCR (Malden et al., 1993), it stabilizes the HLA-A2-HER-2 peptide
complex. Leu and Met were also found in CTL epitopes at P9, as
indicated by sequence information (Parker et al., 1992). These
peptides were selected because of our previous observations that
tumor reacting CTL-TAL isolated from lymphocytes infiltrating
ovarian malignant ascites can recognize synthetic peptides derived
from the highly amphiphilic area HER-2:968-984 on HLA-A2.sup.+
targets (Ioannides et al., 1993). All cultures were initiated in
RPMI-HS to avoid induction of T-cells reactive with determinants on
FCS proteins. In contrast, cytotoxicity assays were performed in
RPMI-FCS to minimize interferences from recognition by CTL of human
proteins (Wolfel et al., 1993).
[0207] The ability of PBMC cultures to recognize peptides used for
priming was determined by measuring the lysis of peptide-pulsed
C1R:A2 cells. Three out of five individual cultures tested lysed
C1R:A2 targets pulsed with C43, C84, or both. Results with two
representative donors (No. 41 and No. 51) are shown in FIG. 1. It
should be mentioned that these two donors were siblings and had
identical HLA phenotype. A common feature of C43- and C84-induced
cultures was that they showed minimal lysis of C1R:A2 cells in 4-h
cytotoxicity assays but significant differences were observed
between lysis of peptide pulsed and control C1R:A2 targets in 20-h
assays at 2-3 weeks after stimulation. When cytotoxicity was
determined early (1 week after stimulation), in certain instances,
they showed high background lysis.
[0208] Interestingly, all cultures stimulated with the C84 peptide
showed similar levels of lysis of either C43- or C84-pulsed targets
in 20-h cytotoxicity assays (FIG. 1A, FIG. 1B, FIG. 1C, FIG. 2A,
and FIG. 2B).
[0209] HER-2 peptide-stimulated PBMC cultures tend to lose
specificity over time and that the numbers of CD8.sup.+ cells tend
to decrease, due to overgrowth of CD4.sup.+ cells. CD8.sup.+ cells
were isolated from bulk CTL cultures from donor 41 by positive
selection on anti-CD8 mAb-coated plates. The resulting cells were
100% CD3.sup.+, 97% CD8.sup.+, and 1% CD4.sup.+. Separated
CD8.sup.+ cells were propagated in culture by repeated stimulations
with C43 and C84 peptide-pulsed PBMC and expanded in medium
containing 15-25 U/ml of IL-2 for more than 6 months. The
41.CD8.sup.+ CTL line recognized both C43 and C84 peptides and at a
much lesser extent, control D125 and D132 peptides. These peptides
contain HLA-A2 anchors introduced by us but differ in sequence form
HER-2 peptides (Table 3). The absence of HLA-A2 anchors in the
natural sequence of D125 and D132 suggests that they are not
presented to corresponding CTL in humans. The sequences of D125 and
D132 were chosen from Muc-1 core sequence (Gendler et al., 1988).
When C43 and C84 peptides were preincubated with C1R:A1 cells
(HLA-A1 transfectants which expressed only HLA-A1), 41.CD8.sup.+
CTL failed to elicit a higher lysis of peptide pulsed than of
control targets.
[0210] Similar results were observed with HLA-A3 transfectants
(FIG. 2A and FIG. 2B). These results suggested that 41.CD8.sup.+
CTL line is peptide Ag specific. Similar results were obtained with
PBMC from donor 30 stimulated with C43/C84 peptides. HLA-A2.sup.+
transfectants did not cross-present C43/C85/C84 to HLA-A2.sup.- CTL
from donors 14 and 15 (Table 3) induced with the same peptides.
This may suggest that recognition of C43/C84 is HLA-A2 restricted.
C43/C85/C84 lack anchor residues for HLA-A1:E(P3), P(P4) and Y(P9);
HLA-B8:K(P3), R(P5) and L(P9) (Dibrino et al., 1994), as well as
for HLA-B35:Pro(P2) and Tyr(P9) (Hill et al., 1992).
[0211] 2. HER-2 Peptide-Induced CD8.sup.+ Cells can Lyse Ovarian
Tumors Overexpressing HER-2 Proto-oncogene
[0212] In vitro peptide-induced CTL cultures can recognize HER-2
peptides used as immunogen. The major question with respect to the
specificity of in vitro-induced CTL is whether they can
specifically-lyse targets endogenously expressing the antigen of
interest. To address this question, the ability of HER-2
peptide-stimulated CTL to lyse ovarian tumors overexpressing HER-2
protein was investigated. The ability of 41.CD8.sup.+ CTL line to
lyse an ovarian tumor (OVA-16) overexpressing HER-2 was tested
using NK-sensitive targets as lysability controls. OVA-16 tumor
shared HLA-A2 with donor 41 effectors (Table 2). The ability of
41.CD8.sup.+ effectors to lyse OVA-16 was determined at 4 and 20 h.
The results are shown in FIG. 3A and FIG. 3B. As expected from the
results presented in FIG. 1A, FIG. 1B, FIG. 1C, FIG. 2A and FIG.
2B, lysis of OVA-16 by 41.CD8.sup.+ effectors in 4-h assays was
low, although higher than K562. In 20-h cytotoxicity assays, lysis
of OVA-16 was significantly higher than K562 cells.
[0213] To determine whether susceptibility of ovarian tumors to
lysis correlates with levels of HER-2 protein expression on tumor,
the ability of the 41.CD8.sup.+ CTL line to lyse two HLA-2.sup.+
fresh isolated ovarian tumors OVA-16 and OVA-14 was tested. The
results are presented in FIG. 3C. Both shared only HLA-A2 with the
donor 41, but they differed at the levels of expression of HER-2.
Immunofluorescence staining with the anti-HER-2-specific mAb showed
77.5% HER-2.sup.+ cells with a mean fluorescence intensity (MFI) of
28.67 for OVA-16 and 17.4% HER-2.sup.+ cells with a MFI of 6.2 for
OVA-14. They were designated as HER-2.sup.high and HER-2.sup.low
respectively. The control HLA-A target, ovarian tumor line SKOV3
(99% HER-2.sup.+) was also designated as HER-2.sup.high.
[0214] The 41.CD8.sup.+ line showed significantly higher lysis of
HER-2.sup.high than HER-2.sup.low targets, suggesting that lysis of
HER-2 expressing ovarian tumors may be dependent on Ag density.
Lysis of the OVA-14 tumor was similar to that of control XxCr and
C1R:A2 cells (HLA-A2.sup.+, HER-2.sup.-) and SKBr3 (HLA-A2.sup.-,
HER-2.sup.high). The SKOV3 tumor (HLA-A2.sup.-, HLA-A3.sup.+,
HER-2.sup.high) was lysed at levels comparable with control lines,
suggesting that HER-2 recognition requires presentation by HLA-A2,
because SKOV3.A2 targets were recognized. Both OVA-14 and OVA-16
expressed comparable levels of HLA-A2 antigens on the surface as
determined by immunofluorescence with MA2.1 mAb (92.1% HLA-A2
positive cells and 49.0 mean fluorescence for OVA-14 and 85.3%
HLA-A2 positive cells and 42.0 mean fluorescence for OVA-16,
respectively). In separate studies, OVA-16, OVA-14, and SKOV3
tumors were efficiently lysed by LAK cells, suggesting that there
were no major differences in their lysability by cytolytic
effectors.
[0215] It is unlikely that tumor killing by 41.CD8.sup.+ CTL
reflects LAK type activity. LAK cells lyse K562 with higher
efficiency than they lyse human tumors (Grimm et al., 1982). Also,
both C1R:A2 and SKOV3.A2 were transfected with the same HLA-A2
plasmid expression vector. Therefore, lysis of SKOV3.A2 but not of
C1R:A2 suggests that 41.CD8.sup.+ CTL are not only HLA-A2 reactive
but also Ag reactive. In separate studies, LAK cells lysed
effectively both C1R:A2 and T2 cells. This lysis was not affected
by C43/C84 or mutated peptides based on this sequence. These
results show that HER-2 peptide-induced CD8.sup.+ cells from human
PBMC can recognize targets endogenously expressing HER-2
protein.
[0216] To confirm that the 41.CD8.sup.+ CTL line recognizes
epitopes on HER-2.sup.high tumors contained on peptides used for
stimulation, cold target inhibition studies were performed. In an
attempt to inhibit lysis of the OVA-16 tumor by 41.CD8.sup.+ CTL
with either C43- or C85-(the wild-type HER-2 peptide 971-979)
pulsed C1R:A2 cells were used with C1R:A2 cells alone or pulsed
with the D125 peptide as controls. The results are shown in FIG. 4A
and FIG. 4B. Inhibition of OVA-16 lysis by the 41.CD8.sup.+ line
was observed in both 4- and 20-h assays. C43- and C85-pulsed C1R:A2
cells but not specificity controls, C1R:A2 cells alone or pulsed
with the D125 peptide,inhibited the lysis of the OVA-16 tumor. As
expected, levels of lysis were lower in 4-h versus 2-h assays.
Increasing the cold:hot ratio to 6:1 did not significantly increase
the inhibitory effects of the HER-2 peptide-pulsed C1R:A2. That
highly specific, but incomplete, inhibition was observed here and
in other human CTL systems (Jerome et al., 1993) reflect low Ag
(peptide) density on targets use for inhibition or an increase in
background nonspecific lysis as observed in FIG. 3C.
[0217] These peptides were recognized by autologous tumor reactive
CTL-TAl, suggesting the presence on the tumor of similar or
cross-reactive CTL epitopes (Ioannides et al., 1993). To address
whether these peptides interfere with tumor lysis by autologous
tumor reactive CTL-TAL in HLA-A2.sup.- systems, the lysis of OVA-1,
HER-2.sup.high (HLA-A1, 24, B8, 35, Cw4) and OVA-31, HER-2.sup.high
was determined by pre-pulsing with either C43 or as a control, C44
peptide (VS.fwdarw.II). Target lysis by CTL-1 was: OVA-1 (68%),
K562 (18%), OVA-1 plus C43 (37%), and OVA-1 plus C44 (51%). C43
significantly inhibited by 45% lysis of OVA-1 by CTL-1, while less
inhibition (25%) was observed with C44. However, C43 and C44 had no
effect on lysis of OVA-31 by CTL-31. This suggested that these
peptides can bind certain MHC Class I heavy chains other than
HLA-A2 and can interfere with lysis of certain HLA-A2.sup.- tumors
by autologous CTLs.
[0218] Therefore CD8.sup.+ CTL lines can be induced in vitro with
HER-2 peptide analogs and lyse ovarian tumors overexpressing HER-2.
It is also likely that a T-cell epitope with a sequence similar or
cross-reacting with peptide analogs from the area HER-2:968-981 is
associated with HLA-A2 on the tumor cell surface.
[0219] C. Discussion
[0220] Evidence has been presented showing that human PBMC from 10
healthy volunteers can be primed in vitro with HER-2 peptide
analogs to develop lymphocyte cultures with Ag-specific CTL
activity. A CD8.sup.+ CTL line developed from bulk cultures
recognized not only peptides used as immunogen but also ovarian
tumors endogenously expressing HER-2. Peptide-induced CD8.sup.+ CTL
lysed targets endogenously expressing HER-2 but not K562 cells, an
ovarian tumor expressing low levels of HER-2. Furthermore, based on
the ability of C1R:A2 cells pulsed with C43, C85, or C84 to inhibit
HER-2.sup.high tumor lysis compared with the inability of C1R:A2
cells alone or pulsed with D125 to mediate the same inhibition, the
findings demonstrate that HER-2 peptide-induced CD8.sup.+ CTL
recognizes similar or cross-reactive epitopes on tumors expressing
HER-2. At similar levels of HLA-A2 expression efficiency of tumor
lysis was dependent on the levels of HER-2 expression.
[0221] The weak lysis observed in 4-hr assays does not reflect
"slow" lysis. Slow lysis rarely achieves target lysis above 500% at
E:T ratios of 60:1 in 20- to 24- hr assays (Ratner and Clark,
1993). CTL showed levels of lysis in the range of 60-80% at 10:1 or
even 5:1 E:T ratios. One possibility to be considered is that the
frequency of HER-2 reactive clones in peptide-induced CTLs is
relatively low and they diluted among non-cytotoxic cells. The
41.DD8.sup.+ line secreted TNF-.alpha. when cocultured overnight
with C1R:A2 cells in the presence, but not in the absence, of HER-2
peptides. TNF-.alpha. secretion was inhibited by HLA-A2 specific
MA2.1 mAb, suggesting that peptide recognition associated with
HLA-A2 is needed for lymphokine secretion. With respect to the
efficiency of these peptides for target sensitization for maximum
lysis this was observed when targets were preincubated with 5 .mu.M
peptide for 1 hr or cultured with 25 .mu.M peptide overnight. The
amount of peptide bound on HLA-A2.sup.+ molecules cannot be
estimated, however, by comparing with other reports on human CTL
assays performed in the presence of peptide in solution, these CTLs
needed 2-3.times.10.sup.2-fold more peptide for similar levels of
target recognition, but in 20-hr assays (Gammon et al., 1992;
Schmidt et al., 1991; Kos and Mullbacher, 1992; Stauss et al.,
1992; Anderson et al., 1992). This peptide concentration is
significantly less than the 10.sup.7-fold difference in peptide
concentration needed for efficient Ag recognition reported for
murine CTL induced in vivo and in vitro by peptides (Schild et al.,
1991).
[0222] It may be possible that if HLA-A2 acts as a restriction
element for specific HER-2 peptides, TCR with high affinity for
these natural peptides may be eliminated during thymic selection,
leaving only TCR with low affinity (Bowness et al., 1993). The only
conservative substitution introduced to strengthen the P9 anchor
(Met.fwdarw.Val) had no inhibitory effects in peptide-stimulating
ability or CTL specificity. TCR contacts mainly residue in the
sequence P4-P7, while P2 and P9 are buried in the HLA-A2 binding
pockets (Madden et al., 1993). Of interest, the 14mer peptide C43
had similar sensitizing ability for lysis of targets as the shorter
peptide C84. Although it may be possible that activity in C43 is
associated with the presence of contaminating peptides at levels
lower than the ability of detection, several other possibilities
need to be taken in consideration: proteolytic degradation as
extracellular processing occurs and the longer peptides are better
substrates than shorter than peptides for proteolysis (Sherman et
al., 1992). This may also suggest a role for the group RFR and/or
the carboxy-terminal R in Ag processing before HLA-A2 binding.
[0223] Since targets were always pulsed with the same
concentrations of peptides, the kinetics of target recognition may
also reflect different effects of factors involved in in vitro
priming of T-cells with Ag. It has been previously shown that by
increasing both responder cell and Ag (peptide) density, murine
Ag-specific CTL can be induced in vitro. These CTL recognized
targets which endogenously expressed the Ag of interest (Winter et
al., 1991).
[0224] The experience with in vitro induction of human CTLs by
peptide is limited. Recent reports have shown that Ag-specific CTLs
can be induced in vitro using peptide analogs of EBV nuclear
antigens (EBNA) (Schmidt et al., 1992), influenza matrix (Bednarek
et al., 1991; Gammon et al., 1992), or Plasmodium falciparum
pre-erythrocytic stage antigens (Hill et al., 1992). Given the
frequency of EBV and influenza infections it is possible that they
represented, at least in some instances, secondary CTL responses of
in vivo-primed T-cells. Based on molecular mimicry between self and
foreign proteins at the three and tetrapeptide levels (Ohno, 1991),
it is not unlikely that naturally processed T-cell epitopes from
self-proteins may be cross-reactive (Anderson et al., 1992).
[0225] Since HER-2 is a self-antigen, HER-2 reactive T-cells may be
primed in vivo and non-deletional mechanisms of tolerance in the
periphery may render HER-2-primed T-cells anergic or suppressed.
However, a recent report demonstrated that Ag-reactive T-cells
transferred in Ag-reactive T-cells transferred in Ag tolerant
transgenic mice can be recovered, suggesting that tolerance
induction in the periphery may not affect primed T-cells and that
the lack of auto-reactivity may be because of the low levels of
antigen expressed on normal cells (Hu et al., 1993). The HER-2
proto-oncogene product is expressed at low levels in normal cells
of origin. Results suggest that in vivo priming to HER-2 epitopes
is possible when HER-2 is expressed at 100- to 200-fold higher than
normal levels (Ioannides et al., 1993). In contrast with viral
infections which essentially turn off the host protein synthesis to
favor the expression of virally coded polypeptides, overexpression
of HER-2 does not generally inhibit the tumor's protein synthesis.
Thus, additional antigens are expected to compete with HER-2 for
HLA-A2 binding and presentation to TCR.
[0226] PBMC from 5 of 11 healthy HLA-A2.sup.+ volunteers tested
showed CTL responses to HER-2 peptides used for priming, and CTLs
and tumor clones have been developed to identify HER-2 epitopes
recognized by tumor reactive CTLs.
EXAMPLE 2
Sequence Motifs of Human HER-2 Proto-Oncongene Important for
Peptide Binding to HLA-A2
[0227] A. Materials and Methods
[0228] 1. Peptides
[0229] HER-2 peptides were synthesized as described in Example 1.
The purity of peptides used in these studies was .gtoreq.97.
[0230] 2. Immunofluorescence
[0231] mAbs to CD3 (OKT3-FITC), CD4 (OKT4-FITC) and CD8 (OKT8-FITC)
were obtained from Ortho Diagnostic (Ortho, Raritan, N.J.), mAb
W6/32 (anti-HLA, -A, -B, -C) from Dako (Dako-Dakopatts, Denmark);
HLA-A2 reacting mAb BB7.2 and MA2.1 from ATCC. Immunofluorescence
studies were performed as described in Example 1.
[0232] 3. HLA-typing
[0233] Leukocytes of the PBMC donors used as responder cells were
typed by the Blood Bank at M.D. Anderson Cancer Center. The
HLA-types were as follows: donor 20: HLA-A 2, 11, B35, 51, Cw7,
donor 25: HLA-A 2, 3, B44, 60; donor 30: HLA-A2, 33 B14, 35, Cw4.
Expression of HLA-A2 on HLA-A2 transfectants was confirmed by
immunofluorescence using culture supernatant from mAb MA2.1
(Ioannides et al., 1993).
[0234] 4. Cytotoxicity Assays
[0235] Target cells were labeled with .sup.51Cr
(Na.sup.51CrO.sub.4; Amersham, Arlington Heights, Ill.) for 90 min
at 37.degree. C. (Ioannides et al., 1991; Ioannides et al., 1991),
or overnight in RPMI medium, containing 10% FCS, 100 82 g/ml
L-glutamine and 40 .mu.g/ml gentamycin (RPMI-FCS), then washed and
incubated with the effector cells in complete RPMI-FCS in an
incubator with 5% CO.sub.2. Targets were incubated either with 25
.mu.M of peptide overnight during labeling, or with 10 .mu.M
peptide for 2 h at 37.degree. C. in RPMI-FCS, then washed three
times before being incubated with effector cells. Separate controls
for spontaneous and total lysis of targets were made for each
peptide pulsed target (Ioannides et al., 1993; Fisk et al., 1994;
Gammon et al., 1992). After 4-5 h of incubation 100 .mu.l of
supernatant were collected and counted. Percent lysis was
calculated from the formula: 100.times.[(E-S)/(T-S)], were
E=experimental release, S=release in the absence of CTL, T=release
in 2 M HCl.
[0236] 5. Target Cells and Cell Lines
[0237] The human lymphoblastoid cell lines C1R and T2 have been
previously described (Gammon et al., 1992; Bednarek et al., 1991;
Salter and Creswell, 1986; Anderson et al., 1993). C1R (Class I
reduced) is a mutant cell line that does not express HLA-A2, C1R:A2
cells express transfected genomic clones of HLA-A2.1. These cells
were obtained from Dr. William E. Biddison (National Institute of
Neurological Disorders, Bethesda, Md.). T2 (transport deletion
mutant) cells were obtained from Dr. Peter Creswell (Yale
University School of Medicine, New Haven, Conn.). C1R:A2 cells were
maintained in RPMI-FCS. T2 cells were maintained in Iscove's
Modified Dulbecco Medium (IMDM) containing 5% Fetal Calf Serum
(IMDM-FCS).
[0238] 6. Generation of in vitro HER-2 Peptide-reactive CTL
[0239] CTL cultures were generated following the procedures
described for in vitro induction of influenza matrix and tum
peptide specific CTL (Gammon et al., 1992; Bednarek et al., 1991;
Salter and Creswell, 1986) with several modifications. In brief,
PBMC from HLA-A2.sup.+ donors were separated by Ficoll-Hypaque.TM.
gradient centrifugation. 5-10'10.sup.6 PBMC were resuspended in a
final volume of 100-250 .mu.l in PBS and incubated with the
stimulating peptide at a final concentration between
5-50.times.10.sup.-6 M for 90 min at 37.degree. C. Afterwards,
cells were irradiated (4000 rad), washed, and plated in wells of 24
well plates (Costar, Cambridge, Mass.) in 2.0 ml at a final
concentration of 0.5-1.0.times.10.sup.6 cells/ml. As responders,
autologous PBMC were added at a final concentration of
1.0-1.5.times.10.sup.6/ml.
[0240] Cultures were initiated in RPMI 1640 medium containing 100
.mu.g/ml L-glutamine, 40 .mu.g/ml gentamycin and 5%
heat-inactivated and sterile filtered human AB plasma (RPMI-HS).
The use of human serum during stimulation and culture and of FCS
during CTL assays was intended to avoid induction of FCS peptide
reactive CTLs. After three days, 5 U of IL-2 (Cetus) equals 6 IU of
IL-2 (Ioannides et al., 1991). Two days later, one third of the
medium was replaced with an equal volume of RPMI-HS containing 15
U/ml of IL-2. Four days later, cells were restimulated with
irradiated fresh autologous PBMC pulsed with the same peptides.
Three days later, 5 U of IL-2 (Cetus) was added to each well. The
expanding cultures were subjected to a second round of
restimulation as described above. Six days after the first and
second stimulations and seven to eight days after the third
stimulation, cultures were tested for cytotoxic activity against
C1R:A2 cells pulsed with either stimulating peptides or unrelated
control peptides. Control cultures were established without HER-2
peptides, containing the same number of autologous stimulators and
responders PBMC.
[0241] 7. Proliferation Assays
[0242] Fresh PBMC from healthy volunteers isolated by
Ficoll-Hypaque.TM. were distributed into 96-well round-bottomed
plates (Falcon, Becton-Dickinson) at 2.times.10.sup.5/well in
RPMI-FCS. Peptides were added at 50 .mu.g/ml. The studies were
performed at least twice using PBMC from the same donor, in
quadruplicate. After 5 days, for the last 16 h in culture, wells
were pulsed with 1 .mu.Ci of [.sup.3H]-thymidine (.sup.3H-Tdr) and
counted. Proliferation was determined as .sup.3H-Tdr incorporation
and c.p.m. determined in the samples of PBMC cultured with peptides
and were divided by c.p.m. determined same cultures in the absence
of peptides, to determine the stimulation index (S.I.).
[0243] B. Results
[0244] 1. Selection of Candidate Antigenic Peptides from HER-2
Peptides Predicted by Algorithmic Methods
[0245] Sequence analysis for the presence of potentially
amphiphilic areas revealed a small number (<20) of potential
sites capable of forming long amphiphilic .alpha.-helices over 3-4
turns (12 residues) in the 1255 residue sequence of HER-2
(Ioannides et al., 1993). A number of shorter sequences have also
been identified. Most of these sequences contained Rothbard's
epitope motifs (Rothbard and Taylor, 1988). Since the focus was
peptides presented by HLA-A2, these regions as well as the entire
HER-2 sequence were searched for areas containing the predicted, as
well as the alternatively reported, HLA-A2 anchors: i.e. L/M/I/V
(P2) and V/L/M/I (P9) (Parmiani, 1993; Bednarek et al., 1991;
Parker et al., 1992). Several areas were found to meet all three
criteria of selection. These areas are as follows:
[0246] (a) HER-2: 968-984, which not only forms a perfect
amphiphilic helix but also contains two Rothbard's epitope motifs
and a nonapeptide with predicted HLA-A2 anchors. This area has been
previously found to be recognized by tumor reactive CTL (Ioannides
et al., 1993).
[0247] (b) The area HER-2:41-56 contains L(43), L(49) and the group
VV(55-56). This corresponds to two overlapping potentially HLA-A2
binding peptides: an octapeptide HER-2:42-49 followed by a
nonapeptide HER-2:48-56. The presence of HL and VV groups renders
these peptides highly hydrophobic, and consequently they have low
solubility in PBS or culture medium. With respect to the sequence
HER-2:48-56, the corresponding synthetic peptide (D113) had low
solubility in PBS. DMSO up to 50% was used for rapid
solubilization. The analogs D114=HER-2:47-56(48H.fwdarw.L) and
D115=HER-2:48-56(48H.fwdarw.G) were designed in an attempt to
improve solubility and increase the ability of exogenously supplied
peptides to bind to HLA-A2. To overcome these problems at least in
part, peptides D96=HER-2:4-54 and D97=HER-2:42-51 were synthesized,
which, although longer than the minimum HLA-A2 binding peptide, are
water-soluble.
[0248] (c) The area HER-2:391-411 contains two potentially HLA-A2
binding nonapeptides: HER-2:391-399 (PLQPEQLQV) (SEQ ID NO: 12) and
HER-2:402-410 (TLEEITGYL) (SEQ ID NO: 13). An octapeptide
HER-2:396-403 with HLA-A2 anchors at P2 and P8:QLQVFETL (SEQ ID NO:
14) is nested in the sequence and overlaps with the carboxy- and
amino terminal regions of the HER-2:391-399 and 402-410.
[0249] Two other areas containing decapeptides:
HER-2:344-353=GLGMEHLREV (SEQ ID NO: 15) and
HER-2:1089-1098=DLGMGAAKGL (SEQ ID NO: 16) both include predicted
HLS-A2 anchors but not overlapping or continuous epitopes. Several
other areas also show potential amphiphilic sites and include
Rothbard's epitope motifs. While these areas do not include HLA-A2
anchor motifs, they may, however, include anchors for other
HLA-types.
[0250] 2. Peptides Identified by the Presence of HLA-A2 Anchors
[0251] In addition to the sites identified by the overlap of
potentially amphiphilic sites and Rothbard's epitope motifs, a
number of peptides can be identified in the sequence of HER-2 by
the presence of HLS-A2 anchors at positions 2 and 9. A large number
of sites (>35) containing nonapeptides with: dominant, strong or
weak P2 and P9 anchors predicted or reported for HLA-A2 (Falk et
al., 1991) were found in the HER-2 sequence. The sequences of most
of these peptides are presented in Table 4. Additional nonapeptides
are found in the Leu and Val rich transmembrane domain (655-675).
In addition to nonamers, a large number of octa- and decamers were
found in the HER-2 sequence containing L/I/V/M as HLA-A2 anchors.
These sequences are not included in Table 4 except in a few cases
where octa- and decamers are part of epitope clusters. In addition
to clustered potential HLA-A2 binding peptides from the signal
(Ioannides et al., 1992; Ioannides et al., 1993; Parmiani, 1993;
Slamon et al., 1989; Fisk et al., 1994; Falk et al., 1991; Rothbard
and Taylor, 1988; DeLisi and Berzofsky, 1985; Stauss et al., 1992)
and transmembrane (655-675) areas, putative HLA-A2 binding peptides
are clustered either as continuous or overlapping peptides as
follows: 42-91 (two 8- and three 9-mers), 141-179 (three 9-mers),
391-419 (three 9-mers), continued with 423-474 (six 9-mers),
781-807 (three 9-mers and one 10-mer), 828-859 (one 8-mer and four
9-mers).
[0252] In certain areas, the last two carboxyterminal residues of a
putative HLA-A2 binding peptide overlap with the first two
aminoterminal residues of the next peptide because P2 and P9
anchors are the same or similar (L/V). Most of the peptides include
Rothbard's epitope motifs. However most of the nonapeptides either
do not derive from long amphiphilic areas, or are highly
hydrophobic according to their sequence; when their sequence is
viewed on axial projection (Edmundson's wheel) (Kaiser and Kezdy,
1984) the majority of the peptides (28/38) show limited segregation
of hydrophilic and hydrophobic residues.
[0253] Crystallographic analysis of the LSA-A2 peptide complex
reveals an additional binding pocket in HLA-A2 accommodating a
hydrophobic residue in position 6 and the likelihood that residues
in positions 4 and 8 are hydrophilic and oriented upwards (towards
TDR) (Saper et al., 1991; Madden et al., 1993). None of the
nonapeptides of sequences shown in the Table 4 contains all the
additional strong anchors in the positions 4, 6, and 8 identified
by Rammensee and collaborators (Falk et al., 1991). However at
least 3/17 nonamers contain one additional strong HLA-A2 anchor and
11/18 nonamers contains at least two additional weak HLA-A2 anchors
(Table 5). For peptide selection the following groups were
considered equivalent: L and I at P2, R and K at Pa, P4, P5 and P8,
L and M at P9, either because of
4TABLE 4 HER-2/NEU PEPTIDES CONTAINING P2 AND P9 HLA-A2
ANCHORS.sup.a From Amino Acid To Amino Acid Peptide No. Position #
Position # 1 5 13 2 42 49 3 48 56 4 76 84 5 84 91 6 141 149 7 160
168 8 171 179 9 369 377 10 391 399 11 402 410 12 411 419 13 423 431
14 435 443 15 442 450 16 447 455 17 457 465 18 466 474 19 596 604
20 603 611 21 627 635 22 650 658 23 689 697 24 747 755 25 781 790
26 789 797 27 793 801 28 799 801 29 828 836 30 835 842 31 838 846
32 845 853 33 851 859 34 883 891 35 904 912 36 971 979 37 986 994
38 1172 1180 .sup.aselection of anchors was made from the cDNA
sequence of human HER-2/neu (Yamamoto et al., 1986). To accommodate
HER-2 peptides that may bind HLA-A2 with low affinity both I and V
were accepted at position 2. Based on reported epitopes, I can be
tolerated at P2. V binds with much lower affinity to HLA-A2. CTL
epitopes containing V at P2 have not been reported yet. CTL
epitopes containing M at P9 have been reported (Parker et al.,
1992). Although M is tolerated at P2 only octa- and deca-peptides
were found containg M (p2) in the HER-2 sequence, and they are not
included in this table. For the sequences of each of the motifs,
see Fisk et al., 1994b.
[0254] structural similarities, or because they have been reported
to be part of CTL epitopes (Parker et al., 1992).
[0255] Sequence analysis of HLA-A2 bound peptides shows an
alternation of hydrophobic and hydrophilic residues. P2 and P3 are
generally made of hydrophobic residues, P4 of hydrophilic residues,
while the charge and hydropathy of residues in P5-P9 alternate,
following in general the pattern: P5 (variable/neutral)--P6
(hydrophobic)--P7 (variable)--P8 (hydrophilic)--P9 (always
hydrophobic) (Falk et al., 1991). Although the peptide is bound to
HLA in an extended conformation stabilized with hydrogen bonds, the
alternation between hydrophobic and hydrophilic residues is in
general agreement with Rothbard's epitope motifs and with
hypotheses that certain T-cell epitopes are derived from
amphiphilic sites.
[0256] Examination of the physicochemical properties of HER-2
peptides may assist in predicting which of the peptides shown in
Table 4 will bind HLA-A2. However this would not address whether
these self-peptides are capable of activating T-cells. To gain
insight into these questions, specific areas were targeted: 41-56,
392-411, and 968-984. Each area contains a nonapeptide: 48-56
(D113), 402-410 (D119) and 971-979 (C85). All share Leu as P2
anchor. Peptide 43-56 contains the dominant P9 anchor Val, while
402-410 contains Leu and 971-979 contains Met which are expected to
be weak anchors. All share a hydrophilic residue at P4: D97 (Q),
D119 (E) and C85 (S). Differences are evident in the residues in
the other positions, where only C85 has a strong P8 anchor (R). The
sequences of peptides from these regions are shown in the Table
4.
[0257] 3. Effects of HER-2 Synthetic Analogs on Conformational
Epitopes on HLA-A2
[0258] To determine whether these peptides and longer analogs
affect HLA-A2 conformation as an indication of HLA-A2 binding, the
effects of HER-2 peptides on the reactivity of conformationally
dependent mAb MA2.1 and BB.7.2 were examined with HLA-A2 of T2
cells. The human cell line T2 has a defect affecting endogenous
peptide loading of MHC class I molecules. As a consequence, cell
surface expression of HLA-A2 is lower (30-40%) than in normal LBL
lines transfected with HLA-A2 (e.g., C1R) but the reactivity of
MA2.1 and BB.7.2 mAb is increased when certain HLA-A2 binding
peptides are added to the culture medium (Anderson et al., 1993).
Although most human MHC class I molecules cannot be induced at the
low temperatures used for their murine counterparts because of
fundamental structural differences between human and mouse class I
(Anderson et al., 1993), the fact that they express few endogenous
(mainly signal) peptides (Zweerink et al., 1993) increases the
sensitivity of detection of peptide-HLA-A2 interaction.
[0259] The results of immunofluorescence studies are presented in
FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D. The nonapeptide D113
induced a significant increase in FL1. As expected, its analogs
D114 and D115 increased FL1 even further (FIG. 5A). However since
they have shown low solubility, the study was repeated with
peptides dissolved in DMSO. The nonapeptide D113 induced a
significant decrease in the reactivity of MA2.1 mAb with HLA-A2.
Both D114 and D115 were unable to increase reactivity of MA2.1 with
HLA-A2. In contrast, D97 which has identical P1-2 anchors with D113
but nests an octapeptide, D96 which covers the entire area 41-54
with the exception of the VV group (P8-9) of D113 and decapeptide
D99=DLGMGAAKGL (HER-2:1089-1098) (SEQ ID NO: 5) showed a slight
increase in FL1 of T2 cells in comparison with control peptides
(D98 and D100) or in the absence of peptide.
5TABLE 5 HER-2 PEPTIDES CONTAINING ADDITIONAL HLA-A2 ANCHORS TO P2
AND P9 Peptide Sequence No. of Anchors SEQ ID No. Position 1 2 3 4
5 6 7 8 9 Strong Weak NO: 1 48-56 H L Y Q G C Q V V 2 2 17 2 76-84
D I Q E V Q G Y V 3 1 20 3 141-149 Q L R S L T E I L 1 4 21 4
171-179 D I F H K N N Q L 1 3 22 5 369-377 K I F G S L A F L 1 6 11
6 391-399 P L Q P E Q L Q V 2 1 12 7 402-410 T L E E I T G Y L 2 3
13 8 411-419 Y I S A W P D S L 1 4 23 9 457-465 S L R E L G S G L 2
3 24 10 650-658 P L T S I I S A V 2 2 25 11 689-697 R L L Q E T E L
V 2 2 26 12 747-755 K I P V A I K V L 1 4 27 13 789-797 C L T S T V
Q L V 3 0 10 14 828-836 Q I A K G M S Y L 2 3 28 15 851-859 V L V K
S P N H V 3 1 8 16 971-979 E L V S E F S R M 2 1 7 17 1172-1180 T L
S P G K N G V 2 2 29
[0260]
6TABLE 6 SEQUENCES OF HER-2 PEPTIDES USED IN THIS STUDY HER-2
Peptides: SEQ ID Peptide Position Sequence NO: D97 42-51 H L D M L
R H L Y Q 30 D96 41-54 T H L D M L R H L Y Q G C Q 31 D113 48-56 H
L Y Q G C Q V V 17 D114 47-56 R L L Y Q G C Q V V.sup.a 18 D115
48-56 G L Y Q G C Q V V 19 D98 N Q E V T A W D G T Q R 32 D119
402-410 T L E E I T G Y L 13 D120 397-410 L Q V F E T L E E I T G Y
L 33 D121 392-411 L Q P E Q L Q V F E T L E E I T G Y L Y 34 D122
396-406 Q L Q V F E T L E E I 35 D95 392-404 L Q P E Q L Q V F E T
L E 36 C85 971-979 E L V S E F S R M 7 C86 971-981 E L V S E F S R
M A R 37 C43 968-981 R F R E L V S E F S R M A R 38 C44 968-981 R F
R E L I I E F S R M A R 39 B69 972-984 L V S E F S R M A R D P Q 40
C61 968-977 R F R E L V S E F S 41 D169 964-972 E C R P R F R E
L.sup.b 42 D170 968-984 R F R E L V S 43 D99 1089-1098 D L G M G A
A K G L 16 D100 1086-1098 F D G D L G M G A A K G L 45 FBP
Peptides: E37 25-33 R I A W A R T E L 46 E38 112-120 N L G P W I Q
Q V 47 E39 191-199 E I W T H S T K V 48 E40 247-255 S L A L M L L W
L 49 E41 245-253 L L S L A L M L L 50 .sup.aUnderlined residues
represent mutations from the natural sequence of HER-2; .sup.bD169
was selected to contain HLA-B8 anchors, shown underlined.
[0261] Of peptides from the area 392-410, the analog D119
corresponding to a nonapeptide with dominant P2 and weak P9 anchor
showed a significant increase in FL1 over control T2 cells
preincubated without peptide. Interestingly, aminoterminal
elongation of the peptide (D120) and elongation followed by
truncation (D122) increased the FL1 only slightly over the base
level. Similarly, a peptide (D121) containing the entire area
failed to significantly affect the FL1, suggesting that it is
probably not processed by external proteases to shorter fragments
of correct length.
[0262] The model nonapeptide C85 (=HER-2:971-979) from the third
area failed to significantly increase FL1 of T2 cells reacting with
MA2.1. C85 contains a dominant P2, a strong P8 and weak P9 anchor.
This was also true for longer analogs B69 and C43. To address the
question whether this reflects the weakness of the P9, and P6
anchors analogs C84(M.fwdarw.V), C83(RM.fwdarw.KV) and
C81(F.fwdarw.V, RM.fwdarw.KV) were synthesized. Peptide C84 induced
a significant increase in FL1 that was comparable with D119 (FIG.
5C), suggesting that the presence of a strong anchor at P9 in this
peptide is important for induction of a MA2.1 conformational
epitope on T2. C83 did not increase further the FL1, suggesting
that the substitution R.fwdarw.K may not be critical for reactivity
of MA2.1 with HLA-A2. Of note, C81 significantly increased the FL1,
suggesting that the presence of V at P6 is important for induction
of MA2.1 conformational epitopes. Since the previous data suggest
that C85 may interact with HLA-A2, the reactivity of BB7.2 mAb with
T2 cells preincubated with the same peptides was examined. The
results show that C85 induces an increase in FL1 of cells stained
with BB.7.2 (FIG. 5D). The analogs C84 and C81 induced an even
higher increase in FL1 of cells reacted with BB.7.2.
[0263] To clarify whether (L/I (P2) and V/L (P9) as critical
elements for induction of MA2.1 conformational epitopes is
restricted to HER-2, a control study analyzed the effect on FL1 of
T2 cells stained with MA2.1 of five nonapeptide analogs from the
sequence of folate binding protein (FBP) which is also
overexpressed in ovarian cancer. The results are presented in FIG.
6. Of five peptides, one (E37) failed to affect MA2.1 epitope
expression, three showed a moderate increase similar with C84
regardless that either Leu or Val were present P9, and only one
showed a very high increase in FL1. This peptide (E38) has a
different P2 (L vs I) from E39 which showed only a moderate
increase in FL1 and included I(P6). Two other peptides (E40-E41)
containing the groups ALM and MLL at P5-P7 failed to induce an
increase in FL1. These results indicate that in addition to the
presence of predicted dominant P2 and P9 anchors, induction of
conformational MA2.1 epitopes on HLA-A2 also depends on the peptide
sequence at P3-P8. It is likely that the presence of certain
residues affects the reactivity of MA2.1 mAb with HLA-A2. Therefore
MA2.1 epitope expression alone does not necessarily reflect the
affinity of peptide binding to MA2.1.
[0264] 4. Stimulation of Peptide Reactive CTL in Vitro
[0265] HER-2 peptides (Table 6) were tested for their ability to
stimulate HLA-A2.sup.+ PBMC to proliferate in vitro. PBMC from
healthy donors were incubated with HER-2 peptides from the groups:
41-56, 392-410 and 968-984 for 5 days. With few exceptions,
significant cell proliferation was not observed in all 4 PBMC
samples from individual donors of different HLA-types including
HLA-A2. (S.I. ranged between 0.8-1.5) suggesting that these short
peptides were not mitogenic. The exception to these observations
was Donor 20.S.I. for D95 was 6.4, for D121 was 4.3, but for the
nonamer D119 was only 1.1. Similarly, the S.I. for the longer
peptide D96 (41-54) was 2.8 but for the shorter peptide D97 was
only 1.5. These differences were statistically significant.
Proliferation of PBMC stimulated with peptides in the presence of
IL-2 failed to clearly distinguish between peptides that induced
lymphocyte proliferation and those that did not, because of the
overall increase in the levels of proliferation of both control and
peptide stimulated samples.
[0266] To address the question of whether in vitro stimulation of
PBMC with HER-2 peptides followed by culture in the presence of
IL-2 leads to T-cell phenotype change, the % CD3, CD4, and CD8
expression on the surface of HER-2 peptide-stimulated PBMC were
determined. The results of a typical study are shown in FIG. 7A,
FIG. 7B and FIG. 7C. Nine days after the first stimulation with
either peptide D97 (a decamer containing a nested octapeptide),
D121 (a 20-mer containing nested an octapeptide and a nonapeptide)
and C85 (nonapeptide) and expansion in IL-2, all cultures showed a
significant increase in CD8.sup.+ cells and a decrease in CD4.sup.+
cells was observed associated with overall cell expansion and
growth. This trend continued in all cultures and 10-15 days after a
third stimulation, with the same peptide in all cultures, CD4.sup.+
cells constituted the dominant (>80%) cell population.
[0267] 5. Lytic Activity and Specificity of HER-2
Peptide-stimulated PBMC
[0268] To elucidate the ability of HER-2 peptides to induce CTLs in
vitro, the ability of HLA-A2.sup.+ PBMC cultured in the presence of
HER-2 peptides and IL-2 to recognize peptides used as stimulators
was determined. HER-2 peptides with different sequences were used
as specificity controls. C1R:A2 cells were used as targets because
they express only HLA-A2. A first group of peptides selected as
stimulators were from the area HER-2:41-56 as follows: D96 and D97
containing the octapeptide: 42-49, and D113 and D114 corresponding
to the overlapping peptides 48-56 and 47-56. Stimulation and
restimulation with irradiated autologous PBMC pulsed with peptide
showed mixed results. In certain cases, higher peptide recognition
was determined, in others lack of peptide specificity was observed.
In most cultures, after the second stimulation with HER-2 peptides,
CD4.sup.+ cells became the dominant population, and they expressed
either LAK type lytic activity or failed to recognize the Ag used
for stimulation.
[0269] The results of a typical study that used as targets three
HER-2 peptides and as effectors PBMC from donor 20 stimulated
either once with D97 or cultured in the same conditions in the
absence of HER-2 peptide (as control) are shown in FIG. 8A, FIG.
8B, and FIG. 8C. Control cultures showed low levels of similar
lysis of all targets. In contrast, cultures stimulated with D97
showed at 6:1 E/T ratio somewhat higher lysis of targets pulsed
with the peptides used for stimulation, than of control peptides
D98 (no HLA-A2 anchors) and D99 but the background lysis was
relatively high. Similarly when PBMC from the same donor were
stimulated with D96 which includes the area HER-2:42-51, higher
lysis of targets pulsed with D97 than D96 was observed (FIG. 9A,
FIG. 9B, and FIG. 9C). The same cultures showed lower lysis of
targets pulsed either with control D95 peptide (not used for
stimulation), or control C1R:A2 cells, or the NK sensitive targets
K562 cells. D97 stimulated PBMC from the donor 30 showed higher
lysis of targets pulsed with D97 than with the overlapping 48-56
and control D119 nonapeptides. The results are shown in FIG. 9A,
FIG. 9B, and FIG. 9C. In 2/3, donors peptides D96/D97 induced in
vitro CTLs can preferentially recognize the peptide used as
stimulator. This suggests that a potential epitope capable of
stimulating T-cells in vitro is nested in the area 42-51.
[0270] Peptide D113 and its mutated analog D114 induced a CTL
response which apparently lacked Ag specificity (FIG. 10A and FIG.
10B). Although in 2/3 HLA-A2.sup.+ donors, at certain E:T ratios
peptide induced CTL showed higher recognition of targets pulsed
with D113 than of control D119 peptide, the differences were
minimal. These peptide-induced CTL were designated as non-specific.
D113, D114 and D115 showed higher increase in reactivity of MA2.1
mAb with HLA-A2 than D96/D97. However they induced less specific
CTL than D96/D97. The reasons for these differences are unknown,
however the results should be interpreted with caution because of
the difficulties in solubilizing D113 and its analogs.
[0271] The peptide D121 (HER-22:392-411) induced a CTL response
that lacked Ag specificity (FIG. 11A). However, D121 stimulated
PBMC from donor 20 showed somewhat higher lysis of targets pulsed
with the nonapeptide D119 than D121, but this response was
short-lived. PBMC from two other donors (25 and 30) stimulated with
D121 and D119, were used as effectors to determine the specificity
of D119 recognition of every peptide. Similar results were obtained
with peptide stimulated PBMC from donor 30. Peptide recognition was
also determined in 20 h cytotoxicity assays. No major differences
in recognition of targets pulsed with peptides used as stimulator
versus control peptides were observed. Similarly D119 was found to
increase the reactivity of MA2.1 mAb with HLA-A2 on T2 cells but
failed to induce peptide specific CTLs (FIG. 11B and FIG. 11C).
EXAMPLE 3
Synthesis of Novel Universal Immunodominant Peptide Epitopes
[0272] A large number of nonapeptides (synthetic analogs) have been
constructed, and it has been determined which ones are recognized
by CTLs associated with and lysing ovarian tumors. Of more than
peptides tested for recognition by three HLA-A2+ CTL lines, the
following peptides have been recognized more often. Based on the
levels of lysis induced they were designated as high: C85 (2/3);
E90 (2/3), E75 (2/3) E71 (2/3), E89 (2/3); and moderate E77
(2/3).
[0273] The sequences of these peptides are as follows:
7 C85 = HER-2:971-979 - E L V S E F S R M (SEQ ID NO:7) E89 =
HER-2:851-859 - V L V K S P N H V (SEQ ID NO:8) E71 = HER-2:798-806
- Q L M P Y G C L L (SEQ ID NO:9) E90 = HER-2:788-796 - C L T S T V
Q L V (SEQ ID NO:10) E75 = HER-2:370-378 - K I F G S L A F L (SEQ
ID NO:11) E77 = HER-2:391-399 - P L Q P E Q L Q V (SEQ ID
NO:12)
[0274] The ability of these peptides to sensitize targets for lysis
by tumor associated CTLs (relative to positive control C85) is
shown in Table 7.
[0275] These sequences, being immunodominant, can provide universal
HER-2 targets and antigens for CTLs in the HLA-A2 system expressed
by over 450 of North American population.
[0276] Since HER-2 is a self-antigen, during thymic selection, a
number of T-cells carrying receptors with high affinity for the
HLA-peptide complex are silenced either by elimination ro finer
tolerization. A pre-condition for induction of a high affinity
TCR-(MHC+peptide) interactions, is a stable (MHC+peptide) complex.
Therefore T-cells reacting with peptides that bind HLA with low
affinity and have weak stabilizing effect, are not likely to be
eliminated in vivo but they can become CTL targets. However,
stabilization of HLA-Class I binding by exogenously added peptide
is dependent on introduction of dominant anchors in positions P2
and P9 which are not recognized by TCR. In addition to patenting
this concept we found that replacement of Met (P9) stabilize HLA-A2
expression on an indicator line T2 used for these types of
studies.
8TABLE 7 RECOGNITION OF HER-2 PEPTIDES BY OVARIAN TUMOR ASSOCIATED
CYTOTOXIC T LYMPHOCYTES CTL-24 % of C85.sup.a CTL-34 % of C85 High
High C85.sup.b 1.000 C85 1.000 E90 0.885 E90 1.149 E75 0.850 E89
1.149 Moderate Moderate E77 0.759 E71 0.600 E89 0.734 E77 0.300 E71
0.625 Negative CTL-16 D113 0.095 D99 0.050 High D97 -0.025 E75
>10.00.sup.c .sup.aThe levels of targets lysis by CTL in the
presence of each HER-2 peptide are shown as % of positive control
(C85 peptide) recognition by CTL. .sup.bE75 was the only peptide
significantly recognized by this CTL line.
[0277] C85=ELVSEFSRM (SEQ ID NO: 7) is the natural nonapeptide
recognized by CTL. Peptides C84=ELVSEFSRV, (SEQ ID NO: 6) and
C83=ELVSEFSKV (SEQ ID NO: 5) are analogs with strengthened P9 and
P8. C84 also can specifically inhibit tumor lysis by peptide
induced CTL. Furthermore, Leu (P2) is a dominant anchor, but E (P1)
may be electrostatically rejected by residues that form the MHC
class I binding pocket. Thus replacement of E.fwdarw.G
(P1)(neutral) or E.fwdarw.K (P1, positive charge) are also expected
to stabilize the interaction, while the residues being buried in
the pocket, are expected not to affect CTL recognition.
[0278] The analogs with sequences C91=GLVSEFSRV, (SEQ ID NO: 4) and
C92 =KLVSEFSRV (SEQ ID NO: 3) are also compositions of the present
invention. In addition, substitutions at P4 (S.fwdarw.K) and P6
(F.fwdarw.V) affect residues that are expected to interact with
TCR. The analog C81=ELVSEVSKV (SEQ ID NO: 2) stabilized HLA-A2 more
than C84, while C82: ELVKEVSKV (SEQ ID NO: 1) although binds HLA-A2
is no longer recognized by C84 reactive CTL. Both C81 and C82 can
form the core for antagonists of HER-2 reactive CTLs (to control
and stop CTL reactions), and as such represent the first
"universal" antagonists reported for stimulating CTLs.
[0279] Peptide D113, HLYQGCQVV (SEQ ID NO: 17), is the natural
nonapeptide HER-2:42-51. D113 stabilizes HLA-A2 on indicator T2
cells. The novel synthetic peptide analog, D114, RLLYQGCQVV (SEQ ID
NO: 18), shows little improvement on stabilization of HLA-A2, but
the novel peptide, D115, GLYQGCQW (SEQ ID NO: 19), shows
significantly higher improvement which confirmed the predictions
above.
EXAMPLE 4
Peptide Formulations
[0280] Peptides containing the epitope motifs described herein are
contemplated for use in therapeutics to provide universal HER-2
targets and antigens for CTLs in the HLA-A2 system expressed by
over 45% of the North American population. The development of
therapeutics based on these novel sequences provides induction of
tumor reactive immune cells in vivo through the formulation of
synthetic cancer vaccines, as well as induction of tumor-reactive
T-cells in vitro through either peptide-mediated (e.g.,
lipopeptide) or cell-mediated (e.g., EBV-B lines using either
autologous or HLA-A2 transfectants where the gene for the peptide
of interest is introduced, and the peptide is expressed associated
with HLA-A2 on the surface). The use of these novel peptides as
components of vaccines to prevent, or lessen the chance of cancer
progression is also contemplated.
[0281] The peptides contemplated for use, being smaller than other
compositions, such as envelope proteins, will have improved
bioavailability and half lives. If desired, stability examinations
may be performed on the peptides, including, e.g., pre-incubation
in human serum and plasma; treatment with various proteases; and
also temperature- and pH-stability analyses. If found to be
necessary, the stability of the synthetic peptides may be enhanced
by any one of a variety of methods such as, for example, employing
D-amino acids in place of L-amino acids for peptide synthesis;
using blocking groups like t-boc and the like; or encapsulating the
peptides within liposomes. The bio-availability of select mixtures
of peptides may also be determined by injecting radio-labeled
peptides into experimental animals, such as mice and/or Rhesus
monkeys, and subsequently analyzing their tissue distribution.
[0282] If stability enhancement was desired, it is contemplated
that the use of dextrorotary amino acids (D-amino acids) would be
advantageous as this would result in even longer bioavailability
due to the inability of proteases to attack these types of
structures. The peptides of the present invention may also be
further stabilized, for example, by the addition of groups to the
N- or C-termini, such as by acylation or amination. If desired, the
peptides could even be in the form of lipid-tailed peptides,
formulated into surfactant-like micelles, or other peptide
multimers. The preparation of peptide multimers and surfactant-like
micelles is described in detail in U.S. Ser. No. 07/945,865,
incorporated herein by reference. The compositions of the present
invention are contemplated to be particularly advantageous for use
in economical and safe anti-tumor/anti-cancer therapeutics, and
specific therapeutic formulations may be tested in experimental
animal models, such as mice, rats, rabbits, guinea pigs, cats,
goats, Rhesus monkeys, chimpanzees, and the like, in order to
determine more precisely the dosage forms required.
[0283] In addition to the peptidyl compounds described herein, the
inventors also contemplate that other sterically similar compounds
may be formulated to mimic the key portions of the peptide
structure and that such compounds may also be used in the same
manner as the peptides of the invention. This may be achieved by
the techniques of modelling and chemical design known to those of
skill in the art. For example, esterification and other alkylations
may be employed to modify the terminus of a peptide to mimic a
particular terminal motif structure. It will be understood that all
such sterically similar constructs fall within the scope of the
present invention.
[0284] Therapeutic or pharmacological compositions of the present
invention will generally comprise an effective amount of a
CTL-stimulating peptide or peptides, dissolved or dispersed in a
pharmaceutically acceptable medium. The phrase "pharmaceutically
acceptable" refers to molecular entities and compositions that do
not produce an allergic, toxic, or otherwise adverse reaction when
administered to a human. Pharmaceutically acceptable media or
carriers include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated.
[0285] Supplementary active ingredients can also be incorporated
into the therapeutic compositions of the present invention. For
example, the stimulatory peptides may also be combined with
peptides including cytotoxic T-cell- or T-helper-cell-inducing
epitopes (as disclosed in U.S. Ser. No. 07/945,865; incorporated
herein by reference) to create peptide cocktails for immunization
and treatment.
[0286] The preparation of pharmaceutical or pharmacological
compositions containing a CTL-stimulating peptide or peptides,
including dextrorotatory peptides, as active ingredients will be
known to those of skill in the art in light of the present
disclosure. Typically, such compositions may be prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid prior to
injection; as tablets or other solids for oral administration; as
time release capsules; or in any other form currently used,
including cremes, lotions, mouthwashes, inhalents and the like.
[0287] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0288] Sterile solutions suitable for intravenous administration
are preferred in certain embodiments and are contemplated to be
particularly effective in stimulating CTLs and/or producing an
immune response in an animal. The pharmaceutical forms suitable for
injectable use include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0289] A peptide or peptides can be formulated into a composition
in a neutral or salt form. Pharmaceutically acceptable salts,
include the acid addition salts (formed with the free amino groups
of the peptide) and which are formed with inorganic acids such as,
e.g., hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine, and the like.
[0290] The carrier can also be a solvent or dispersion medium
containing, e.g., water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and vegetable oils. The proper fluidity
can be maintained by inter alia the use of a coating, such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. The prevention of
the action of microorganisms can be brought inter alia by various
antibacterial ad antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, e.g.,
sugars or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0291] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0292] The preparation of more- or highly-concentrated solutions
for intramuscular injection is also contemplated. This is
envisioned to have particular utility in facilitating the treatment
of needle stick injuries to animals or even humans. In this regard,
the use of DMSO as solvent is preferred as this will result in
extremely rapid penetration, delivering high concentrations of the
active peptide, peptides or agents to a small area.
[0293] The use of sterile formulations, such as saline-based
washes, by veterinarians, technicians, surgeons, physicians or
health care workers to cleanse a particular area in the operating
field may also be particularly useful. Therapeutic formulations in
accordance with the present invention may also be reconstituted in
the form of mouthwashes, including the peptides alone, or in
conjunction with antifungal reagents. Inhalant forms are also
envisioned, which again, may contain active peptides or agents
alone, or in conjunction with other agents, such as, e.g.,
pentamidine. The therapeutic formulations of the invention may also
be prepared in forms suitable for topical administration, such as
in cremes and lotions.
[0294] Buffered ophthalmic solutions also fall within the scope of
the invention, and may be created in accordance with conventional
pharmaceutical practice, see for example "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1488 to 1501 (Mack
Publishing Co., Easton, Pa.). Suitable ophthalmic preparations will
generally contain a novel dipeptide, peptide or agent as disclosed
herein in a concentration from about 0.01 to about 1% by weight,
and preferably from about 0.05 to about 0.5%, in a pharmaceutically
acceptable solution, suspension or ointment. The ophthalmic
preparation will preferably be in the form of a sterile buffered
solution containing, if desired, additional ingredients, for
example preservatives, buffers, tonicity agents, antioxidants and
stabilizers, nonionic wetting or clarifying agents,
viscosity-increasing agents and the like.
[0295] Suitable preservatives for use in such a solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9.+-.0.2%. Suitable
antioxidants and stabilizers include sodium bisulfite, sodium
metabisulfite, sodium thiosulfate, thiourea and the like. Suitable
wetting and clarifying agents include polysorbate 80, polysorbate
20, poloxamer 282 and tyloxapol. Suitable viscosity-increasing
agents include dextran 40, dextran 70, gelatin, glycerin,
hydroxyethylcellulose, hydroxmethyl-propylcellulose, lanolin,
methylcellulose, petrolatum, polyethylene glycol, polyvinyl
alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the
like.
[0296] Upon formulation, therapeutics will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed. As used herein, "pharmacologically
effective amount" means an amount of composition is used that
contains an amount of a peptide or peptides sufficient to
significantly stimulate a CTL or generate an immune response in an
animal.
[0297] In this context, the quantity of peptide(s) and volume of
composition to be administered depends on the host animal to be
treated, such as, the capacity of the host animal's immune system
to produce an immune response. Precise amounts of active peptide
required to be administered depend on the judgment of the
practitioner and are peculiar to each individual.
[0298] A minimal volume of a composition required to disperse the
peptide is typically utilized. Suitable regimes for administration
are also variable, but would be typified by initially administering
the compound and monitoring the results and then giving further
controlled doses at further intervals. For example, for parenteral
administration, a suitably buffered, and if necessary, isotonic
aqueous solution would be prepared and used for intravenous,
intramuscular, subcutaneous or even intraperitoneal administration.
One dosage could be dissolved in 1 ml of isotonic NaCl solution and
either added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580).
[0299] In certain embodiments, active compounds may be administered
orally. This is contemplated for agents that are generally
resistant, or have been rendered resistant, to proteolysis by
digestive enzymes. Such compounds are contemplated to include
chemically designed or modified agents; dextrorotatory peptides;
and peptide and liposomal formulations in timed-release capsules to
avoid peptidase, protease and/or lipase degradation.
[0300] Oral formulations may include compounds in combination with
an inert diluent or an edible carrier which may be assimilated;
those enclosed in hard- or soft-shell gelatin capsules; those
compressed into tablets; or those incorporated directly with the
food of the diet. For oral therapeutic administration, the active
compounds may be incorporated with excipients and used in the form
of ingestible tablets, buccal tables, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. Such compositions and
preparations should generally contain at least 0.1% of active
compound. The percentage of the compositions and preparations may,
of course, be varied and may conveniently be between about 2 to
about 60%. of the weight of the unit. The amount of active
compounds in such therapeutically useful compositions is such that
a suitable dosage will be obtained.
[0301] Tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia, corn
starch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparaben as
preservatives, a dye and flavoring, such as cherry or orange
flavor. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compounds may be
incorporated into sustained-release preparation and
formulations.
[0302] The peptides may be used in their immunizing capacity by
administering an amount effective to generate an immune response in
an animal. In this sense, such an "amount effective to generate an
immune response" means an amount of composition that contains a
peptide or peptide mixture sufficient to significantly produce an
antigenic response in said animal.
EXAMPLE 5
Methods for Protein Size Determination and Gel Chromatography
[0303] The amino acid sequences disclosed herein, and particularly
the tripeptide motifs and multimers thereof, find particular use in
the determination of molecular weights of small polypeptides. These
peptides represent a significant improvement over
commercially-available protein standards in this area owing to
their small size and, since their amino acid sequence is known,
their precise molecular weight is readily determined.
[0304] 1. SDS-PAGE Analysis of Proteins
[0305] Commercially-available protein standards for SDS-PAGE or gel
filtration chromatography typically have a range of 3,000 to
200,000 Da (Gibco BRL, Bethesda, Md.), and as such, are not useful
in the characterization of proteins having molecular weights of
about 300 to about 3,000 Da. By employing peptides of the present
invention (e.g., SEQ ID NOS: 1-15) and multimers thereof, a range
of suitable low-molecular weight standards may be readily prepared.
Such a molecular weight ladder mixture may be employed either in
SDS-PAGE or gel filtration protocols which are well-known to those
of skill in the art (see e.g., Wood, 1981).
[0306] 2. Paper and Thin-Layer Chromatography
[0307] In a similar fashion, the polypeptides, and more
particularly the tripeptide motifs, of the present invention are
readily employed as standards in the identification of small
molecular-weight polypeptides using chromatographic separation. In
preferred embodiments, paper chromatography is utilized and
proteins are subsequently visualized after reaction with ninhydrin.
More preferred is the use of thin-layer chromatography in either
one or two dimensions.
[0308] 3. Gel Filtration Chromatography
[0309] The polypeptides of the present invention provide excellent
standards for the calibration of chromatographic columns used in
the separation of low molecular-weight polypeptides. In particular,
the tripeptide motifs, and multimers thereof, find important use in
the standardization of low-molecular weight-range columns (Rawn,
1983). These chromatography columns may include a filtration medium
having the capacity to fractionate any protein of interest and the
polypeptides of the present invention. Chromatographic media such
as G-50 or G-25 Sephadex.RTM. resins (approximate fractionation
range of 1,500-30,000 and 100-5,000 Da, respectively) may be used
for generalized separation, or in cases where the approximate
molecular weight of the protein of interest is known, a medium
having a narrower fractionation range (e.g., G-10 Sephadex.RTM.
[0-700 Da separation range] or G-15 Sephadex.RTM. [0-1,500 Da
separation range]) may be employed. A regression line of the
elution position versus the log of the molecular weight is
established using the peptides of the present invention, and the
molecular weight of the protein of interest is then determined from
this graph. Detailed protocols for preparation, calibration, and
execution of these columns is well-known to those of skill in the
art (see e.g., Wood, 1981).
[0310] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the composition, methods and in the
steps or in the sequence of steps of the method described herein
without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
[0311] References
[0312] The following literature citations as well as those cited
above are incorporated in pertinent part by reference herein for
the reasons cited in the above text.
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[0330] Fisk et al., "Sequence motifs of human HER-2 proto-oncogene
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Sequence CWU 1
1
68 1 9 PRT Artificial Sequence Description of Artificial Sequence
Synthetic Peptide 1 Glu Leu Val Lys Glu Val Ser Lys Val 1 5 2 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 2 Glu Leu Val Ser Glu Val Ser Lys Val 1 5 3 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 3 Lys Leu Val Ser Glu Phe Ser Arg Val 1 5 4 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 4 Gly Leu Val Ser Glu Phe Ser Arg Val 1 5 5 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 5 Glu Leu Val Ser Glu Phe Ser Lys Val 1 5 6 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 6 Glu Leu Val Ser Glu Phe Ser Arg Val 1 5 7 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 7 Glu Leu Val Ser Glu Phe Ser Arg Met 1 5 8 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 8 Val Leu Val Lys Ser Pro Asn His Val 1 5 9 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 9 Gln Leu Met Pro Tyr Gly Cys Leu Leu 1 5 10 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 10 Cys Leu Thr Ser Thr Val Gln Leu Val 1 5 11 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 11 Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5 12 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 12 Pro Leu Gln Pro Glu Gln Leu Gln Val 1 5 13 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 13 Thr Leu Glu Glu Ile Thr Gly Tyr Leu 1 5 14 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 14 Gln Leu Gln Val Phe Glu Thr Leu Glu 1 5 15 10 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 15 Gly Leu Gly Met Glu His Leu Arg Glu Val 1 5 10 16 10 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 16 Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1 5 10 17 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 17 His Leu Tyr Gln Gly Cys Gln Val Val 1 5 18 10 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 18 Arg Leu Leu Tyr Gln Gly Cys Gln Val Val 1 5 10 19 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 19 Gly Leu Tyr Gln Gly Cys Gln Val Val 1 5 20 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 20 Asp Ile Gln Glu Val Gln Gly Tyr Val 1 5 21 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 21 Gln Leu Arg Ser Leu Thr Glu Ile Leu 1 5 22 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 22 Asp Ile Phe His Lys Asn Asn Gln Leu 1 5 23 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 23 Tyr Ile Ser Ala Trp Pro Asp Ser Leu 1 5 24 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 24 Ser Leu Arg Glu Leu Gly Ser Gly Leu 1 5 25 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 25 Pro Leu Thr Ser Ile Ile Ser Ala Val 1 5 26 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 26 Arg Leu Leu Gln Glu Thr Glu Leu Val 1 5 27 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 27 Lys Ile Pro Val Ala Ile Lys Val Leu 1 5 28 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 28 Gln Ile Ala Lys Gly Met Ser Tyr Leu 1 5 29 9 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 29 Thr Leu Ser Pro Gly Lys Asn Gly Val 1 5 30 10 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 30 His Leu Asp Met Leu Arg His Leu Tyr Gln 1 5 10 31 14 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 31 Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln
1 5 10 32 12 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 32 Asn Gln Glu Val Thr Ala Trp Asp Gly
Thr Gln Arg 1 5 10 33 14 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Peptide 33 Leu Gln Val Phe Glu Thr
Leu Glu Glu Ile Thr Gly Tyr Leu 1 5 10 34 20 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 34
Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile Thr 1 5
10 15 Gly Tyr Leu Tyr 20 35 11 PRT Artificial Sequence Description
of Artificial Sequence Synthetic Peptide 35 Gln Leu Gln Val Phe Glu
Thr Leu Glu Glu Ile 1 5 10 36 13 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 36 Leu Gln Pro
Glu Gln Leu Gln Val Phe Glu Thr Leu Glu 1 5 10 37 11 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 37
Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg 1 5 10 38 14 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 38 Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg
1 5 10 39 14 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 39 Arg Phe Arg Glu Leu Ile Ile Glu Phe
Ser Arg Met Ala Arg 1 5 10 40 13 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 40 Leu Val Ser
Glu Phe Ser Arg Met Ala Arg Asp Pro Gln 1 5 10 41 10 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 41
Arg Phe Arg Glu Leu Val Ser Glu Phe Ser 1 5 10 42 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 42
Glu Cys Arg Pro Arg Phe Arg Glu Leu 1 5 43 7 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 43
Arg Phe Arg Glu Leu Val Ser 1 5 44 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 44 Asp Leu Gly
Met Gly Ala Ala Lys Gly Leu 1 5 10 45 13 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 45 Phe Asp Gly
Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1 5 10 46 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 46
Arg Ile Ala Trp Ala Arg Thr Glu Leu 1 5 47 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 47
Asn Leu Gly Pro Trp Ile Gln Gln Val 1 5 48 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 48
Glu Ile Trp Thr His Ser Thr Lys Val 1 5 49 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 49
Ser Leu Ala Leu Met Leu Leu Trp Leu 1 5 50 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 50
Leu Leu Ser Leu Ala Leu Met Leu Leu 1 5 51 27 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 51
gaaytngtna argaagtnws naargtn 27 52 27 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 52 gaaytngtnw
sngaagtnws naargtn 27 53 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 53 aarytngtnw sngaattyws
nmgngtn 27 54 27 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Primer 54 ggnytngtnw sngaattyws nmgngtn 27 55 27
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 55 gaaytngtnw sngaattyws naargtn 27 56 27 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 56 gaaytngtnw sngaattyws nmgngtn 27 57 27 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 57
gaaytngtnw sngaattyws nmgnatg 27 58 27 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 58 gtnytngtna
arwsnccnaa ycaygtn 27 59 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 59 carytnatgc cntaygartg
yytnytn 27 60 27 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Primer 60 tgyytnacnw snacngtnca rytngtn 27 61 27
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 61 aarathttyg gnwsnytngc nttyytn 27 62 27 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 62 ccnytncarc cngarcaryt ncargtn 27 63 27 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 63
acnytngarg arathacngg ntayytn 27 64 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 64 carytncarg
tnttygarac nytn 24 65 14 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Peptide 65 Arg Phe Arg Glu Leu Val
Ser Glu Phe Ser Arg Met Ala Arg 1 5 10 66 14 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 66
Arg Phe Arg Glu Leu Ile Ile Glu Phe Ser Arg Met Ala Arg 1 5 10 67 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic Peptide 67 Ser Leu Ala Asp Pro Ala His Gly Val 1 5 68 10
PRT Artificial Sequence Description of Artificial Sequence
Synthetic Peptide 68 Gly Leu Thr Ser Ala Pro Asp Thr Arg Val 1 5
10
* * * * *