U.S. patent application number 09/835853 was filed with the patent office on 2002-11-07 for mhc peptides and methods of use.
This patent application is currently assigned to Thomas Jefferson University. Invention is credited to Baserga, Renato L., Huang, Ziwei, Resnicoff, Mariana.
Application Number | 20020165136 09/835853 |
Document ID | / |
Family ID | 24829084 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020165136 |
Kind Code |
A1 |
Baserga, Renato L. ; et
al. |
November 7, 2002 |
MHC peptides and methods of use
Abstract
MHC or HLA Class I peptides and compositions thereof are
provided for the specific induction of apoptosis of cancer cells in
a patient. Methods of treating cancer cells in patients suffering
from cancer employing the MHC or HLA Class I peptides of the
invention. Also provided are methods of identifying MHC or HLA
Class I peptides and variants thereof capable of killing cancerous
cells in vivo in a patient suffering from cancer.
Inventors: |
Baserga, Renato L.;
(Ardmore, PA) ; Resnicoff, Mariana; (Philadelphia,
PA) ; Huang, Ziwei; (Philadelphia, PA) |
Correspondence
Address: |
Paul K. Legaard
Woodcock Washburn LLP
One Liberty Place - 46th Floor
Philadelphia
PA
19103
US
|
Assignee: |
Thomas Jefferson University
Philadelphia
PA
|
Family ID: |
24829084 |
Appl. No.: |
09/835853 |
Filed: |
April 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09835853 |
Apr 17, 2001 |
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08704344 |
Aug 28, 1996 |
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6218363 |
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Current U.S.
Class: |
514/18.9 ;
514/19.3 |
Current CPC
Class: |
C07K 14/70539 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method of killing a cancer cell in a patient, comprising
administering to said patient an amount of an MHC or HLA Class I
peptide sufficient to kill said cancer cell.
2. The method of claim 1, wherein said peptide is selected by use
of an in vivo or in vitro apoptosis screening assay.
3. The method of claim 2, wherein said assay is an in vivo
diffusion chamber assay.
4. The method of claim 2, wherein the cells used for said assay are
cancer cells obtained from said patient.
5. The method of claims 1, 2, 3, or 4, wherein said MHC or HLA
Class I peptide is selected from the group consisting 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 and SEQ ID NO: 13.
6. A composition comprising an MHC or HLA Class I peptide for use
in the method of claims 1, 2, 3 or 4.
7. Use of the composition of claim 6 for the induction of apoptosis
in a cancer cell in vivo.
8. A method of arresting the growth of a cancerous tumor in a
patient, comprising administering to said patient an amount of an
MHC or HLA Class I peptide sufficient to arrest the growth of said
tumor.
9. The method of claim 8, wherein said tumor exhibits
regression.
10. The method of claim 8, wherein said tumor is killed.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of cell
physiology, and more particularly, to programmed cell death, or
apoptosis. The novel peptides, compositions and methods of the
invention are useful for modulating cell cycle events in cells,
particularly in cancer cells.
BACKGROUND OF THE INVENTION
[0002] It is estimated that nearly one-third of all individuals in
the United States will develop cancer. While early diagnosis and
treatment of this disease has increased the five year relative
survival rate to nearly 50%, cancer remains second only to cardiac
disease as the leading cause of death; approximately 20% of
Americans die from cancer each year.
[0003] Cancer cells (also referred to as neoplastic or malignant
cells) can be defined in terms of four characteristics as to which
they differ from normal cells. "Clonality" refers to the fact that
most cancer originates from a single stem cell which proliferates
to form a clone of malignant cells. "Autonomy" refers to the fact
that the growth of malignant cells is not properly regulated by
normal biochemical and physical influences in the environment.
"Anaplasia" refers to the lack of normal, coordinated cell
differentiation which characterizes malignant cells. "Metastasis"
is the characteristic capacity of cancer cells for discontinuous
growth and dissemination to other parts of the body.
[0004] While each of these characteristics can be expressed by
normal, non-malignant cells at certain appropriate times during
development, cancer cells exhibit these characteristics in an
inappropriate or excessive manner. Many approaches to the diagnosis
and treatment of cancer have sought to take advantage of these
characteristic differences. In particular, many forms of cancer
demonstrate unusual sensitivity to radiation and chemotherapy. In
addition to, or in combination with, traditional surgical excision,
gains in treatment of cancers, including acute leukemia,
lymphoproliferative malignancies, testicular and breast cancer,
have been realized.
[0005] Limitations of these forms of treatment, including
undesirable side effects, have lead medical investigators to seek
new treatment modalities, including immunotherapy. Malignant cells
are sufficiently different from normal cells to be recognized and
destroyed by the immune system.
[0006] One approach to immunotherapy against tumors exploits
cytolytic T lymphocytes (CTL), which are key immune cells in the
body believed to direct the attack on cancer cells. The basis of
this approach is that the identification of tumor cell-specific
antigens not found on normal cells should lead to the generation of
CTL capable of attacking and lysing the cells which make up a
cancerous tumor. (See, e.g., Boone, T., et al., Ann. Rev. Immunol.
12:337-65 (1994); Van Pel, A., et al., Immunol. Rev. 145:229-250
(1995); Boone, T., et al., Immunol. Today 16:334-36 (1995)).
[0007] Investigators have also recognized, however, that in many
instances. known tumor cell specific antigens are insufficiently
immunogenic to bring about CTL activation. Accordingly, some have
looked to generate a cellular immune response in patients (i.e., to
achieve vaccination against cancer) by presenting the patient's
immune system with the putative antigen, together with an adjuvant,
such as the B7 protein and various cytokines including
interleukin-2 and interferon .gamma., granulocyte-macrophage
colony-stimulating factor (GM-CSF) and interleukin-4. (See, e.g.,
Rosenberg, A., J. Clin. Oncol. 10:180-199 (1992); Marchand, M., et
al., Dermnatol. 186:278-280 (1993); Marchand, M., et al., Rnt. J.
Cancer 63:883-85 (1995)). A number of tumor cell-specific antigens,
including MAGE-1, MAGE-3, and MART for melanoma and E6 and E7 for
human papillomavirus (HPV)-associated cervical carcinoma, are in
preliminary human clinical trials.
[0008] It is also known that CTL recognize tumor cell-specific
antigens which are presented by Major Histocompatibility Complex
(MHC) Class I glycoproteins. These glycoproteins, along with MHC
Class II glycoproteins, immunoglobulins and T cell receptors
(TCRs), make up families of antigen binding molecules responsible
for specificity, repertoire and memory in the immune response.
Although the somatic rearrangement of their immunoglobulin or TCR
genes restricts individual B and T cells to a single specificity
for antigen, millions of specificities are possessed by the immune
system as a population of cells. Immunoglobutin receptors on B
cells bind to native protein antigens, whereas TCRs recognize short
peptide fragments bound by polymorphic MHC glycoproteins. Diverse
species, ranging from cattle to chickens to amphibians to humans,
have an MHC region. The human MHC, known as the Human Leukocyte
Antigen (HLA) region, is located on the short arm of chromosome 6.
Because of its importance in self/non-self discrimination for
tissue transplantation in humans, the bulk of knowledge about the
MHC region has come from studies on human and murine MHCs.
[0009] The size and other physical characteristics of MHC Class I
peptides and the structural basis for their direct binding to MHC
Class I glycoproteins has been elucidated based on crystallographic
studies of the HLA-A2 molecule. (See, e.g., Engelhard, V. H., Ann.
Rev. Immunol. 12:181-207 (1994)), and investigators have attempted
to achieve vaccination against cancer cells by injecting such
peptides (Id.; Marchand, M., et al., Int. J. Cancer 63:883-85
(1995)). However, this approach is also subject to the limitations
described above for other vaccination methods.
[0010] Accordingly, there exists a need for methods and
compositions for the therapeutic treatment of cancer in patients
suffering therefrom which would overcome or obviate the limitations
of available approaches.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Fluorescence Activated Cell Sorting Analysis of C6
Cells Treated Ini Vivo with MHC Class I Peptide Demonstrates
Specificity of Toxic Effect.
[0012] FIG. 1 (A). C6 cells treated with active peptide
YLEPGPVTA.
[0013] FIG. 1 (B). C6 cells treated with control peptide SMAPGNYSV.
C6 cells in both Figures were incubated in vivo for 90 minutes in a
diffusion chamber as described herein.
[0014] FIG. 2. Expression of Baculovirus p35 Protein that Inhibits
Apoptosis by Inhibiting ICE-like Proteases, Prevents MHC Class I
Peptide-Induced Apoptosis in C6 Cells hII Vivo.
[0015] Expression of p35 is shown in various cell lines. The
percentage of cells recovered from the diffusion chamber is shown
for cells treated with the synthetic peptide YLRPGPVTA (SEQ. ID NO:
4) (+) and control cells that were not exposed to the peptide (-).
Only C6 cells and C6 cells stably transfected with the empty vector
underwent apoptosis when treated with the peptide. Clones
expressing the p35 protein grew in the diffusion chamber equally
well, whether untreated or incubated with the synthetic
peptide.
SUMMARY OF THE INVENTION
[0016] It has now been unexpectedly found that MHC Class I peptides
and compositions comprising these peptides exhibit in vivo and in
vitro specific toxicity against tumor cells. Although not intending
to be bound by any particular theory, it is believed that this
specific toxicity occurs via the induction of apoptosis in those
cells. Surprisingly, the toxic effect of MHC Class I peptides and
compositions comprising them is independent of the immune system.
Accordingly, the peptides, compositions and therapeutic methods of
the present invention are not subject to the limitations of
immunotherapeutic approaches to cancer treatment.
[0017] It has further surprisingly been found that the peptides and
peptide compositions of the present invention exhibit specific
toxicity in vivo in a variety of tumor cells, and are not limited
to a specific tumor type or mammalian species.
[0018] An advantage of the present invention is that the
therapeutic MHC Class I peptides and compositions will be effective
for resectable and non-resectable (i.e., metastasizing) cancers, as
well as for cancers which do not lend themselves easily to
treatment by traditional radiation and chemotherapeutic methods,
such as cancers of the lung. Further, because cancer cells are more
sensitive than normal cells to the toxic effects of the peptides of
the invention, the methods of the present invention allow for
aggressive treatment of cancers in patients without the undesirable
side effects often associated with conventional radiation and
chemotherapy treatment.
[0019] Accordingly, it is an object of the present invention to
provide MHC Class I peptides and compositions thereof capable of
exhibiting specific toxicity against cancer cells in a patient.
[0020] It is another object of the present invention to provide for
methods of treating cancer cells in patients suffering from
cancer.
[0021] A further object of the present invention is to provide
methods of identifying MHC Class I peptides and variants thereof
capable of exhibiting specific toxicity against cancerous cells in
vivo in a patient suffering from cancer. Yet a further object of
the present invention to provide a method for the treatment of
cancer which is independent of an immune mechanism.
[0022] Another object of the present invention is to provide MHC
Class I peptides, compositions thereof and methods for the
induction of apoptosis in vivo on a variety of tumor cell types and
in different mammalian species, including humans.
[0023] These and other objects of the present invention will be
apparent to those of skill from the description which follows,
including illustrative non-limiting embodiments of the compositions
and methods of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Technical and scientific terms used herein have the meanings
commonly understood by one of ordinary skill in the art to which
the present invention pertains, unless otherwise defined. Reference
is made herein to various methodologies known to those of skill in
the art. Publications and other materials setting forth such known
methodologies to which reference is made are incorporated herein by
reference in their entireties as though set forth in full. Standard
reference works setting forth the general principles of recombinant
DNA technology include Sambrook, J., et al., Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y. (1989); Kaufman, P. B., et al., Eds., Handbook of
Molecular and Cellular Methods in Biology and Medicine, CRC Press,
Boca Raton (1995); McPherson, M. J., Ed., Directed Mutagenesis: A
Practical Approach, IRL Press, Oxford (1991); Jones, J., Amino Acid
and Peptide Synthesis., Oxford Science Publications, Oxford (1992);
Austen, B. M. and Westwood, O. M. R., Protein Targetina and
Secretion, IRL Press, Oxford (1991). Any suitable materials and/or
methods known to those of skill can be utilized in carrying out the
present invention; however, preferred materials and/or methods are
described. Materials, reagents and the like to which reference is
made in the following description and examples are obtainable from
commercial sources, unless otherwise noted.
[0025] The present invention is directed broadly to methods for the
therapeutic treatment of cancer cells in vivo comprising
administering to a patient suffering from cancer an effective
amount of a composition comprising one or more MHC Class I peptides
to interfere with the growth and/or proliferation of all or a
portion of the target cancer cells in the patient. MHC Class I
peptides useful according to the methods of the invention are
preferably selected for use according to the invention by means of
an in vitro or in vivo apoptosis assay. Presently preferred is the
in vivo apoptosis assay based upon the diffusion chamber methods
described in detail by Resnicoff, M., et al., Cancer Res.
55:2463-2469 (1995). The cells used for the screening assay are
most preferably target cancer cells obtained from the patient by
conventional means including, but not limited to, tissue biopsy. In
particular, where a cancerous tumor has been substantially excised
from a patient and it is desired to insure that any cancer cells
which may remain in the patient following surgery are treated,
cells from the excised tumor may be employed in the screening assay
to identify one or more MHC Class I peptides for post-surgery
treatment of the patient.
[0026] Peptides useful in the methods of the invention are
preferably MHC Class I peptides, or variants or mutants thereof,
capable of exhibiting specific toxicity in vivo against the target
cancer cells of a patient, and may be selected, as described
herein, by means of in vitro or in vivo apoptosis assays. Peptides
so selected may be isolated and purified from natural sources. Such
sources may include the target cancer cells themselves as described
above. However, it has surprisingly been found that useful peptides
according to the invention are capable of exhibiting specific
toxicity in vivo against a variety of tumor cell lines, regardless
of tumor type or species.
[0027] Accordingly, where a sample of cells from a patient's tumor
or other cancerous tissue is not available, other peptides may be
selected for administration on the basis of their ability to kill
cells as demonstrated by an appropriate screening assay. In making
such a selection, it may be desirable to select peptides which have
demonstrated an ability to kill cancer cells or cell from cell
lines related to the cancer from which the patient suffers.
Nonlimiting examples of presently preferred cell lines for this
purpose are set forth in Table 10. Selected peptides may include
peptides which have demonstrated specific toxicity (which may or
may not occur via the induction of apoptosis) against the cells of
other patients suffering from the same or similar forms of cancer
as the patient whom it is desired to treat. Presently preferred
peptides include:
1 LLDGTATLRL SEQ ID NO: 1 YLEPGPVTA SEQ ID NO: 2 FECNTAQPG SEQ ID
NO: 3 YLRPGPVTA SEQ ID NO: 4 YLEXGXVTA (X: N-Methyl-A) SEQ ID NO: 5
YLEPGPVKA SEQ ID NO: 6 YLAPGPVTA SEQ ID NO: 7 YLEPGPVAA SEQ ID NO:
8 YLEPGPATA SEQ ID NO: 9 YLEPAPVTA SEQ ID NO: 10 YLRPGPVRA SEQ ID
NO: 11
[0028] and functional equivalents thereof.
[0029] Sequence ID No: 1, a MHC Class I peptide derived from gp100,
was described by Kawakami, et al., Proc. Natl. Acad. Sci. USA
91(14):6458-6462 (1994), as a human melanoma specific antigen.
Sequence ID No: 2, reported by Cox, et al., Science 294:716-719
(1994), is recognized by melanoma-specific human CTL lines.
Sequence ID No: 3, described by Mandelboim, et al., Nature
369:67-71 (1994), is derived from connexin 37, and induces CTL
responses against murine lung carcinoma. These peptides reduce
recovery of C6 cells by 95% or more in an in vivo apoptosis assay,
as compared with control peptides.
[0030] Mutation of MHC Class I peptides is one method by which
variants of these peptides may be produced which are suitable for
use according to the invention. Such variants may conveniently be
screened as described herein without undue experimentation, and
will provide a means by which additional useful peptides may be
produced. Presently preferred variants include the inverted D-amino
acid forms of known MHC Class I peptides. Data presented herein
show them to be surprisingly as effective as their respective
L-amino acid peptide forms in inducing apoptosis, and they may he
desirable for patient administration. Presently preferred D-amino
acids include:
2 ATVPGPELY SEQ ID NO: 12 LRLTATGDLL SEQ ID NO: 13
[0031] SEQ ID NO: 12 is the inverted D-amino acid peptide of SEQ ID
NO: 2 and SEQ ID NO: 13 is the inverted D-amino acid peptide of SEQ
ID NO: 1.
[0032] Similarly, point mutations may be introduced into known MHC
Class I peptides to produce variants thereof useful in the methods
of the present invention. The variants so produced may be evaluated
by means of a screening assay to determine whether the resulting
peptide may be suitable for use according to the invention. The
inventors found that certain mutations do not affect the ability of
the peptide variants to exhibit specific toxicity, while others
completely abrogate the effect. Illustrative examples of point
mutations and their effect on cytotoxic capacity are shown in Table
1; however, the key criterion is that the resulting peptide variant
be capable of exhibiting specific toxicity, which will be evident
from a screening assay which may be routinely run by those of
skill.
[0033] Other variations in the peptides of the invention may be
desirable, for example, to increase or decrease the half life of
the resulting peptide in the bloodstream or tissue. Thus, it is
within the contemplated scope of the present invention to produce
variants of peptides useful according to the methods of the
invention, by introducing therein alterations, such as are known in
the art, which may include the use of synthetic or non-traditional
amino acid residues, side chains, non-amide bonds as in peptoides,
and the like, which may act as blocking groups to protect the
peptide against degradation. These and other methods of modifying
peptides are well known in the art
[0034] Peptides useful according to the invention may be isolated
from natural sources and purified. However, it is preferred to
synthesize the peptides, which will typically be of relatively
short length, by means well known in the art. Preferred is solid
phase synthesis with Fmoc-strategy, although any suitable method of
synthesis may be employed.
[0035] Preferred peptides and peptide compositions useful according
to the present invention include MHC Class I peptides and, more
preferably, HLA Class I peptides, and their variants. Such peptides
and variants thereof are generally defined by their capacity to
bind to MHC or HLA Class I glycoprotein molecules. Peptides bound
by HLA class I molecules are the products of cytoplasmic
degradation of cellular proteins that are transported into the
endoplasmic reticulum. There they associate with a polymorphic
HLA-A, -B, or -C heavy chain and the invariant
.beta..sub.2-microglobulin (.beta..sub.2-M) to form a stable trimer
that moves to the cell surface (S. Kvist and F. Lvy, Semin. Immunol
5:105 (1993). In healthy cells, the peptides are derived from
normal cellular proteins, and the immune system is rendered
tolerant to these peptides during development. Upon infection of
cells, Class I molecules loaded with pathogen-derived peptides are
generated and recognized by cytolytic CD8.sup.+T cells that then
kill the infected cells (A. Townsend and H. Bodmer, Annu. Rev.
Immunol. 7:601 (1989); R. M. Zinkernagel, Science 271:173
(1996).
[0036] The peptides bound by a particular Class I allotype are
defined by positions within the peptide sequence that are
restricted to one or a few amino acids. The preferred residues are
termed anchors because their side chains extend into pockets of the
binding site. In aggregate. HLA Class I allotypes have a range of
Class I peptide binding motifs that covers the spectrum of peptide
characteristics: acidic, basic, neutral, and hydrophobic. O.
Rotzschke, K. Falk, O. Stevanovic, G. Jung, H.G., Rammensee, Nature
351:290 (1991); K. Falk and Rotzschke. Semin. Immunol. 5:81 (1993).
(See, Parham, P., and Ohta, T., Science 272:67-74 (1996). Preferred
peptides according to the present invention will include those
which comprise amino acid residues having side chains which extend
into the pockets of the binding site. Such peptides and variants
thereof which are capable of achieving specific binding to MHC or
HLA Class I glycoproteins are contemplated as falling within the
scope of the invention. The suitability of a particular peptide or
peptide variant for the exhibition of specific toxicity against a
target cancer cell will be determined by those of skill by means,
for example, of the in vivo apoptosis assay as described herein, or
by other known methods, without the exercise of undue
experimentation. Sequencing of peptides derived from cancer or
other cells and which bind to MHC or HLA Class I glycoproteins may
be accomplished by known methods. Examples of such methods are
described, for example, in Hancock, W., ed., New Methods in Peptide
Mapping for the Characterization of Proteins, CRC Press, Boca
Raton, Fla., Publisher (1996).
[0037] An understanding of the structure of MHC Class I
glycoproteins is of importance in selecting binding peptides and
variants useful according to the invention. Generally, MHC Class I
glycoproteins comprise a glycosylated polypeptide chain of 45 kDa
(heavy chain) in close, non-covalent association with beta,
microglobulin (.beta..sub.2m), a 12 kDa polypeptide which is also
found unassociated in serum. Amino acid sequence analyses of both
human and murine Class I molecules have demonstrated that the heavy
chain is divided into distinct regions: three extracellular
domains, a connecting polypeptide, a transmembrane region and a
cytoplasmic domain.
[0038] The three main extracellular domains, designated .alpha.1
(N-terminal), .alpha.2 and .alpha.3, can be cleaved from cell
surfaces with the enzyme papain. These domains each comprise about
90 amino acids. The .alpha.2 and .alpha.3 domains both have
intrachain disulphide bonds and the .alpha.3 domain also folds like
an Ig constant region. Both human and mouse heavy chains have an
N-glycosylated asparagine residue 86 in the .alpha.1 domain. Murine
heavy chains are also N-glycosylated at residue 176 in .alpha.2 and
some (D.sup.b, K.sup.d, L.sup.d) have additional carbohydrate side
chains at residue 256 in .alpha.3. In addition to the major papain
cleavage site between .alpha.3 and the transmembrane region, there
is also a minor cleavage site between the second and third
domains.
[0039] The transmembrane region consists of about 25 hydrophobic
uncharged residues, which probably assume an .alpha.-helical
conformation and traverse the cell membrane. There is a cluster of
about five basic amino acids, arginine and lysine, immediately
C-terminal to the transmembrane region. Such highly charged regions
are typical of membrane bound proteins and are believed to help to
anchor the polypeptide chain in the membrane by interacting with
the negatively charged phospholipid headgroups of the inner
membrane.
[0040] The hydrophilic cytoplasmic domain is about 30 (human) to 40
(mouse) residues long and consists of approximately 50%, polar
amino acids, particularly serine. Some of these serine residues are
phosphorylated. For example, the HLA-A2 heavy chain is
phosphorylated by a cyclic AMP-dependent protein kinase at two
serine residues in the cytoplasmic domain. Such phosphorylation has
been postulated to be involved in transmitting signals from the MHC
molecule to appropriate intracytoplasmic mediators.
[0041] The Class I light chain, .beta..sub.2 m, forms a single
Ig-like domain which has strong sequence homology with Ig constant
regions. Although it was initially thought that the .beta..sub.2m
associated with the Class I heavy chain primarily through
interaction with the .alpha.3 domain, in the same way as
inter-domain Ig interactions, subsequent X-ray crystallographic
analysis has revealed a more complex mode of interaction.
.beta..sub.2m is encoded outside the MHC on human chromosome 15 and
on mouse chromosome 2. It is a non-polymorphic protein in humans,
dimorphic in mice (a single amino acid change at position 85), with
a high degree of sequence homology among species implying
evolutionary conservation. Association with .beta..sub.2m is
required for expression of Class I antigens at the cell surface and
for stabilization of Class I structure.
[0042] The three dimensional structure of the extracellular portion
obtained by cleavage with papain of several Class I structures has
been elucidated. This region contains the .alpha.1, .alpha.2 and
.alpha.3 domains and .beta..sub.2m. The .alpha.3 and .beta..sub.2m
domains have Ig-folds; that is, they are each composed of two
anti-parallel .beta.-pleated sheets, one with four .beta.-strands
and one with three .beta.-strands, connected by a disulphide bond.
However, the .alpha.3 and .beta..sub.2m domains interact in a
manner not found between pairs of constant domains in the known
antibody structures.
[0043] The .alpha.1 and .alpha.2 domains have an overall structural
similarity. Each consists of an anti-parallel .beta.-pleated sheet
spanned by a long .alpha.-helical region that is C-terminal to the
four .beta.-strands in the sheet. A disulphide bond in a .alpha.2
connects a cysteine residue in the N-terminal .beta.-strand to one
in the .alpha.-helix. The .alpha.1 and .alpha.2 domains are paired
in the HLA molecule such that the four .beta.-strands from each
domain form a single antiparallel .beta.-sheet with eight strands.
This .beta.-sheet is topped by the helical regions from each
domain. The large groove between the .alpha.-helices constitutes
the binding site for processed protein in the form of peptides.
Peptides and their variants capable of binding to this groove will
be candidates for evaluation as toxic agents for the treatment of
cancer cells according to the invention.
[0044] The crystallographic structure of the HLA-A2 molecule has
provided a structural basis for the direct binding of peptides to
Class I antigen. A binding groove formed by the .alpha.1 and
.alpha.2 domains of the Class I heavy chain was occupied by an
ill-defined electron-dense material which was interpreted as
peptide(s) filling the binding site. Comparison of the high
resolution crystallographic structures of HLA-A2 and HLA-Aw68 has
added further insights into the nature of the Class I antigen
binding site. The polypeptide backbones of these two Class I
antigens are extremely similar, the differences resulting from
amino acid side chain differences at 13 positions, six of which are
in .alpha.1, six in .alpha.2 and one in the .alpha.3 domain. The
single .alpha.3 domain difference (at residue 245) has been shown
to contribute to interactions with the CD8 glycoprotein. Ten of the
.alpha.1 and .alpha.2 differences are located at positions lining
the floor and side of the peptide-binding groove. The pattern of
the amino acid variation between HLA-A2, Aw68 as well as other
Class I molecules is similar to the distribution of variable
residues obtained from accumulated HLA-A, B and C sequences, which
must reflect the conformations with which peptides can occupy the
groove. The groove is not a smooth structure but has a number of
pockets with which amino acid side chains interact.
[0045] Based on this detailed analysis of the three dimensional
structures of HLA-A2 and Aw68, a picture has emerged that links the
amino acid polymorphism that is such a feature of MHC proteins with
limited structural changes within the peptide binding cleft, such
as shape, charge distribution and local pockets. These structural
changes presumably form the basis for differences in peptide
binding affinity which in turn govern responsiveness versus
non-responsiveness in the immune response.
[0046] In Class I molecules, clusters of conserved residues form
hydrogen bonds with the amino and carboxyl termini of bound
peptide. These bonds involve conserved residues in the Class I
structure, particularly tyrosines at the N-terminus of the peptide
and a conserved lysine, as well as other residues at the
C-terminus. Peptides of eight to ten residues can apparently be
accommodated by maintaining hydrogen bonds at these anchor
positions and by bulging out at the center. Peptides can therefore
have different conformations at their centers with ends buried in
the same pockets.
[0047] Techniques for purifying MHC molecules and eluting the bound
peptides with acid are known in the art and are described, for
example, by Rammensee, H-G., Die Medizinische Verlagsgesellschaft
mbH:Marburg (1994). In addition, tandem mass spectrometry allows
the rapid, convenient and accurate elucidation of smaller peptides,
as is known in the art and described, for example, in Hancock, W.,
ed., New Methods in Peptide Mapping for the Characterization of
Proteins, CRC Press, Boca Raton, Fla., Publisher (1996).
[0048] The many peptide sequences now obtained illustrate that the
optimum size for Class I peptides is nine amino acids. While nine
amino acid Class I peptides are currently preferred for use
according to the invention, shorter or longer sequences may also be
useful according to the invention and their usefulness will be
determinable by those of ordinary skill employing screening methods
such as are described herein. Class II peptides, on the other hand,
tend to be longer, at over 15 residues. The anchor residues for
Class I peptides, from different allelic products, are clearly
identifiable since they lie at fixed points (e.g., position 2 and
position 9). By searching protein databases it is possible to
determine the origins of the eluted peptides. Peptides from Class I
molecules have been estimated to number over 10,000 different
sequences. Comparison of different cell types, such as B cells and
melanoma, shows that over 90% of the prominent peptides are shared
between the two tissues. Many of the peptides eluted from HLA-A2
and HLA-B7 are derived from signal sequences. Those of skill will
be able to obtain and evaluate Class I peptide or other peptide
sequences for use according to the invention from available
sources, including databases such as MHCPEP, maintained by The
Walter and Eliza Hall Institute, Parkville, Victoria 3050,
Australia. MHCPEP is a curated database comprising peptide
sequences known to bind MHC molecules, compiled from published
reports as well as from direct submissions of experimental data.
The database can be accessed via Internet using Gopher, FFP or WWW
by methods known to those of skill.
[0049] The present invention also provides for other peptides
comprising fragments of the proteins of the invention and
polypeptides substantially homologous thereto. The protein peptides
of the invention will generally exhibit at least about 80% homology
with naturally occurring sequences of the MHC or HLA Class I
peptides, typically at least about 85% homology, and more usually
at least about 97% homology.
[0050] The present invention also includes fusion polypeptides
between the MHC or HLA Class I peptides, which may be truncated,
and other proteins. For example, homologous polypeptides may be
fused with other proteins, or other apoptosis-modulating proteins,
resulting in fusion proteins having mixed functionalities. Examples
of suitable proteins are members of the Bcl-2 family of proteins,
Bak, Bax and the like. Similarly, fusions may be generated with
heterologous proteins, for example, the first 16 amino acids of
SRC. Such polypeptides may also have amino acid residues which have
been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition of other moieties, using methods
known in the art. In some embodiments, the modification will be
useful labelling reagents, or serve as purification targets, for
example, affinity ligands. Fusion polypeptides will typically be
made by either recombinant nucleic acid methods or by synthetic
polypeptide methods, as are generally described in Sambrook, et
al., supra; Merrifield, J. Amer. Chem. Soc. 85: 2149-2156 (1963)
Merrifield, Science 232: 341-347 (1986); and Atherton, et al.,
Solid Phase Peptide Synthesis; A Practical Approach, IRL Press,
Oxford (1989).
[0051] The peptides, functional equivalents thereof and
compositions of the present invention have utility for modulating
the growth and differentiation of cells, and preferably of cancer
cells, through the apoptotic process. Modulation of the apoptotic
process includes deceleration of the rate of apoptosis in a
population of cells, or elimination of the cellular apoptotic
response to apoptosis inducing agents. Modulation of the apoptotic
process also includes induction or acceleration of apoptosis where
it is desirable to increase the rate of cell death or to
specifically target a population of cells. For example, the
induction of apoptosis in tumor cells or in other cells showing
increased proliferation and growth provides an effective therapy to
decrease or abolish the growth of these cells, and is a
particularly preferred method according to the invention. Of
course, while the peptides of the invention exhibit specific
toxicity against cancer cells, the actual mechanism of this toxic
effect may be a function of a number of pathways; while not
intending to be bound by a particular theory, it is believed that
modulation of cell cycle events via specific apoptosis induction is
responsible for the observed specific toxicity of the peptides,
functional equivalents thereof and compositions of the present
invention. However, treatment with the peptides of the invention
may also involve, for example, necrotic or other events. Those of
skill will appreciate that when treating populations of cells, such
as cancerous tumor cells, a therapeutic effect may be observed by
any number of known clinical endpoints, non-limiting examples of
which will include a reduction in the growth rate of the tumor cell
population (which may be recognized as a reduction in the rate at
which a palpable tumor or group of tumors increases in size),
complete arrest of the growth rate, tumor regression, and the
complete elimination or killing of the tumor. The compounds of the
present invention also have utility in combatting drug resistance,
which is a common problem with current cancer treatments. Drug
resistance may be a resistance to apoptosis in general, and thus,
the proteins of the present invention may be used to decrease drug
resistance. In this embodiment, the compounds of the invention may
be used in conjunction with other anti-neoplastic agents.
Mechanisms of drug resistance are described, for example, in
Remington's Pharmaceutical Sciences, 18th Edition, supra. In some
embodiments, the compositions of the invention may be used to assay
tissue injury and regeneration. A suitable model system for the
assay of tissue injury is the thymus of dexamethasone-treated rats,
as described in Schwartzman, R., et al., Endoctinol. 128(2):
1190-1197 (1991).
[0052] The compositions of the present invention thus have utility
for a variety of therapeutic indications, including as anti-viral,
anti-microbial, or anti-parasitic agents, and particularly as
anti-neoplastic agents for the treatment of tumors, including but
not limited to tumors of the lung, breast, pancreas and liver, as
well as for acute lymphoblastic or myeloid leukemia, chronic
myeloid, myelogenous, granulocytic, or lymphatic leukemia, acquired
immune deficiency syndrome (AIDS), neurodegenerative diseases,
myelodysplasic syndrome, Hodckin's lymphoma, malignant lymphomas
such as non-Hodgkin's lymphoma, or Burkitt's lymphoma, neoplasms
and the like. With respect to treatment of cancer, general
principles of cancer therapy employing apoptotic agents are known
and are set forth, for example, in Green, D. R. et al., Apoptosis
and Cancer, in Principles and Practice of Oncology Updates Volume
8, J. B. Lippincott Company, January 1994 Number 1, and
Gerschenson, L.E., et al. FASEB J. 6: 2450-2455 (1992).
[0053] The quantities of active ingredient necessary for effective
therapy will depend on many different factors, including means of
administration, target site, physiological state of the patient,
and other medicaments administered. Thus, treatment dosages should
be titrated to optimize safety and efficacy. Typically, dosages
used in vitro may provide useful guidance in the amounts useful for
in situ administration of the active ingredients. Animal testin of
effective doses for treatment of particular disorders will provide
further predictive indication of human dosage. Various
considerations are described, for example, Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 7th Ed., MacMillan
Publishing Co., New York (1985), and Remington's Pharmaceutical
Sciences 18th Ed., Mack Publishing Co., Easton, Penn (1990).
Methods for administration are discussed therein, including oral,
intravenous, intraperitoneal, intramuscular, transdermal, nasal,
iontophoretic administration, and the like.
[0054] A key finding of the present inventors is that the toxic
effect of the peptides according to the invention is specific.
Accordingly, in selecting a dose for use in treating a patient
according to the present invention, a treatment regiment will be
selected which will achieve a sufficient concentration of the
peptide(s) to achieve a toxic effect in the target cells. Because
of the specificity of the toxic effect, effective target cell
concentrations as low as 10.sup.-12-10.sup.-13 M are possible.
Those of skill will be able to determine an effective dose, which
will vary depending upon the manner and mode of administration, in
order to achieve such concentrations, although higher and lower
concentrations may prove effective for the treatment of cancer
cells in vivo. In selecting an appropriate dose, another surprising
finding of the present invention will be considered by those of
skill; that is, that the toxic effect of the peptides administered
according to the present invention is independent of an immune
mechanism, and it may be desirable to administer the peptides of
the invention at doses and time frames which will avoid an immune
response against the peptide being administered. This objective
will be achieved, to some extent, by the fact that the small size
of many of the peptides useful according to the invention renders
them non-immunogenic. Accordingly, it may be desirable to
administer the peptides of the present invention at higher doses,
in order to achieve a higher effective concentration in the target
cancer cell or tissue to achieve the desired toxicity. However, in
contrast to immunotherapeutic approaches to the treatment of
cancer, the methods of the present invention do not have as their
objective the induction of an immune response of CTL against the
target cancer cells.
[0055] One particularly attractive method for administering the
peptides and compositions of the present is inhalation of
dry-powder formulations comprising the peptide(s) or peptide
composition(s). In order to allow for absorption of the active
components through the alveoli into the bloodstream, the powder
must be very fine, on the order of 1-5 micron particles. The highly
disbursable powder is delivered via an inhaler which generates an
aerosol cloud containing the bolus of drug at the top of the
inhalation chamber.
[0056] The peptides and peptide compositions of the present
invention will lend themselves to injection into the bloodstream of
a patient suffering from cancer. The half life of the active
compositions so administered may be manipulated for best
therapeutic effect by employing known drug technologies. One
example of such technologies is known as DEPOFOAM phospholipid
spheres (Depo Tech Corp., San Diego, Calif. ), which gradually
release the active component(s) over a period of days to weeks.
This allows for a constant level of systemic concentration with
lower initial drug levels and injection frequency.
[0057] Known methods of entrapping and stabilizing the peptides of
the present invention may be employed to accommodate several
different routes of drug delivery. One example of such technologies
is the TECHNOSPHERE powder (Pharmaceutical Discovery Corp.,
Elmsford, N.Y.), which reliably forms two micron diameter spheres
under conditions preserving the structural and functional integrity
of the active peptide component. The pH-sensitive spheres, when
injected into the blood, dissolve and release the active component,
which is rapidly absorbed. Fine powders such as these are suitable
for pulmonary, oral, intravenous and intraperitoneal
administration.
[0058] The site of administration and cells will be selected by one
of ordinary skill in the art based upon an understanding of the
particular degenerative disorder being treated. In addition, the
dosage, dosage frequency, and length of course of treatment, can be
determined and optimized by one of ordinary skill in the art
depending upon the particular degenerative disorder being treated.
The particular mode of administration can also be readily selected
by one of ordinary skill in the art and can include, for example,
oral, intravenous, subcutaneous, intramuscular, etc. Principles of
pharmaceutical dosage and drug delivery are known and are
described, for example, in Ansel, H. C. and Popovich, N. G.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Ed, Lea
& Febiger, Pub., Philadelphia, Pa., (1990). It is possible, for
example, to utilize liposomes to specifically deliver the agents of
the invention. Such liposomes can be produced so that they contain
additional bioactive compounds and the like such as drugs,
radioisotopes, lectins and toxins, which would act at the target
site.
[0059] Nucleic acid compositions encoding the peptides and peptide
variants useful according to the invention will generally be in RNA
or DNA forms, mixed polymeric forms, or any synthetic nucleotide
structure capable of binding in a base-specific manner to a
complementary strand of nucleic acid. Such a nucleic acid
embodiment is typically derived from genomic DNA or cDNA, prepared
by synthesis, or derived from combinations thereof. The DNA
compositions generally include the complete coding region encoding
the MHC or HLA Class I peptides, or fragments thereof, e.g.,
comprising about 4-12 codons, and typically about 9 codons. One or
more introns may be present.
[0060] The nucleic acids encoding the MHC or HLA Class I peptides
or fragments thereof such as C-terminal fragments, may be used to
prepare an expression construct for the MHC or HLA Class I
peptides. The expression construct normally comprises one or more
DNA sequences encoding the MHC or HLA Class I peptides operably
linked and under the transcriptional control of a native or other
promoter. Usually the promoter will be a eukaryotic promoter for
expression in a mammalian cell. The transcriptional regulatory
sequences will typically include a heterologous promoter or
enhancer which is recognized by the host cell. The selection of an
appropriate promoter will depend on the host cell. Convenient
expression vectors are commercially available.
[0061] By "functional equivalent" is meant a peptide possessing a
biological activity or immunological characteristic substantially
similar to that of a composition of the invention, and is intended
to include "fragments," "variants," "analogs," "homologs," or
"chemical derivatives" possessing such activity or characteristic.
Functional equivalents of a peptide according to the invention,
then, may not share an identical amino acid sequence, and
conservative or non-conservative amino acid substitutions of
conventional or unconventional amino acids are possible.
[0062] Reference herein to "conservative" amino acid substitution
is intended to mean the interchangeability of amino acid residues
having similar side chains. For example, glycine, alanine, valine,
leucine and isoleucine make up a group of amino acids having
aliphatic side chains; serine and threonine are amino acids having
aliphatic-hydroxyl side chains; asparagine and glutamine are amino
acids having amide-containing side chains; phenylalanine, tyrosine
and tryptophan are amino acids having aromatic side chains; lysine,
arginine and histidine are amino acids having basic side chains;
and cysteine and methionine are amino acids having
sulfur-containing side chains. Interchanging one amino acid from a
given group with another amino acid from that same group would be
considered a conservative substitution. Preferred conservative
substitution groups include asparagine-glutamine, alanine-valine,
lysine-arginine, phenylalanine-tyrosine and
valine-leucine-isoleucine.
[0063] A "mutant" as used herein refers to a peptide having an
amino acid sequence which differs from that of a known peptide or
protein by at least one amino acid. Mutants may have the same
biological and immunological activity as the known protein.
However, the biological or immunological activity of mutants may
differ or be lacking. For example, a mutant may lack the biological
activity which characterizes a known MHC or HLA Class I peptide,
but may be useful as an antigen for raising antibodies against the
peptide or for the detection or purification of antibodies
thereagainst, or as an agonist (competitive or non-competitive),
antagonist, or partial agonist of the toxic function of the
peptide.
[0064] Modulation of MHC or HLA Class I peptide mediated functions
may be effected by agonists or antagonists of MHC or HLA Class I
peptides as well. Screening of peptide libraries, compound
libraries and other information banks to identify agonists or
antagonists of the function of proteins comprising an MHC or HLA
Class I peptide is accomplished with assays for detecting the
ability of potential agonists or antagonists to inhibit or augment
MHC or HLA Class I peptide binding. Suitable labels for use in
screening assays according to the invention include a detectable
label such as an enzyme, radioactive isotope, fluorescent compound,
chemiluminescent compound, or bioluminescent compound. Those of
ordinary skill in the art will know of other suitable labels or
will be able to ascertain such using routine experimentation.
Furthermore, the binding of these labels to the peptides is
accomplished using standard techniques known in the art.
[0065] The further isolation, purification and sequencing of MHC or
HLA Class I peptides from cancer cells for use according to the
invention may be accomplished by standard biochemical methods such
as, for example, those described in Cantor, C., ed., Protein
purification: Principles and Practice, Springer Verlag, Heidelberg,
Publisher (1982); Hancock, W., ed., New Methods in Peptide Mapping
for the Characterization of Proteins, CRC Press, Boca Raton, Fla.,
Publisher (1996).
[0066] MHC or HLA Class I peptidomimetic agents are of use in the
therapeutic treatment of cancer and viral disease. Peptidomimetics
of an MHC or HLA Class I peptide are also provided by the present
invention, and can act as drugs for the modulation of cell cycle
events in a target cell population by, for example, enhancing the
function of proteins, preferably of MHC or HLA Class I peptides.
Peptidomimetics are commonly understood in the pharmaceutical
industry to include non-peptide drugs having properties analogous
to those of those of the mimicked peptide. The principles and
practices of peptidomimetic design are known in the art and are
described, for example, in Fauchere J., Adv. Drug Res. 15: 29
(1986); and Evans, et al., J. Med. Chem. 30: 1229 (1987).
Peptidomimetics which bear structural similarity to therapeutically
useful peptides may be used to produce an equivalent therapeutic or
prophylactic effect. Typically, such peptidomimetics have one or
more peptide linkages optionally replaced by a linkage which may
convert desirable properties such as resistance to chemical
breakdown in vivo. Such linkages may include --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH--, --COCH.sub.2,
--CH(OH)CH.sub.2--, and --CH.sub.2SO--. Peptidomimetics may exhibit
enhanced pharmacological properties (biological half life,
absorption rates, etc.), different specificity, increased
stability, production economies, lessened antigenicity and the like
which makes their use as therapeutics particularly desirable.
[0067] It will be appreciated by those of skill that the precise
chemical structure of peptides comprising MHC or HLA Class I
peptides will vary depending upon a number of factors. For example,
a given protein may be obtained as an acidic or basic salt, or in
neutral form, since ionizable carboxyl and amino groups are found
in the molecule. For the purposes of the invention, then, any form
of peptide comprising an MHC or HLA Class I peptide which retains
the therapeutic or diagnostic activity of the naturally occurring
peptide is intended to be within the scope of the present
invention.
[0068] The MHC or HLA Class I peptides and other compositions of
the present invention may be produced by recombinant DNA techniques
known in the art. For example, nucleotide sequences encoding MHC or
HLA Class I peptides of the invention may be inserted into a
suitable DNA vector, such as a plasmid, and the vector used to
transform a suitable host. The recombinant MHC or HLA Class I
peptide is produced in the host by expression. The transformed host
may be a prokaryotic or eukaryotic cell. Preferred nucleotide
sequences for this purpose encoding an MHC or HLA Class I peptide
are NUCLEOTIDE SEQ ID NOs: 1-13.
[0069] Polynucleotides encoding MHC or HLA Class I peptides may be
genomic or cDNA, isolated from clone libraries by conventional
methods including hybridization screening methods. Alternatively,
synthetic polynucleotide sequences may be constructed by known
chemical synthetic methods for the synthesis of oligonucleotides.
Such synthetic methods are described, for example, in Blackburn, G.
M. and Gait, M. J., Eds., Nucleic Acids in Chemistry and Biology,
IRL Press, Oxford, England (1990), and it will be evident that
commercially available oligonucleotide synthesizers also may be
used according to the manufacturer's instructions. One such
manufacturer is Applied Bio Systems.
[0070] Polymerase chain reaction (PCR) using primers based on the
nucleotide sequence data disclosed herein may be used to amplify
DNA fragments from mRNA pools, cDNA clone libraries or genomic DNA.
PCR nucleotide amplification methods are known in the art and are
described, for example, in Erlich, H. A., Ed., PCR Technology:
Principles and Applications for DNA Amplification, Stockton Press,
New York, N.Y. (1989); U.S. Pat. Nos. 4,683,202; 4,800,159; and
4,683,195. Various nucleotide deletions, additions and
substitutions may be incorporated into the polynucleotides of the
invention as will be recognized by those of skill, who will also
recognize that variation in the nucleotide sequence encoding MHC or
HLA Class I peptides may occur as a result of, for example, allelic
polymorphisms, minor sequencing errors, and the like. The
polynucleotides encoding MHC or HLA Class I peptides of the
invention may include short oligonucleotides which are useful, for
example, as hybridization probes and PCR primers. The
polynucleotide sequences of the invention also may comprise a
portion of a larger polynucleotide and, through polynucleotide
linkage, they may be fused, in frame, with one or more
polynucleotide sequences encoding different proteins. In this
event, the expressed protein may comprise a fusion protein. Of
course, the polynucleotide sequences of the invention may be used
in the PCR method to detect the presence of mRNA encoding MHC or
HLA Class I peptides in the diagnosis of disease or in forensic
analysis.
[0071] The sequence of amino acid residues in a protein or peptide
comprising an MHC or HLA Class I peptide is designated herein
either through the use of their commonly employed three-letter
designations or by their single-letter designations. A listing of
these three-letter and one-letter designations may be found in
textbooks such as Lehninger, A., Biochemistry 2d Ed, Worth
Publishers, New York, N.Y. (1975). When the amino acid sequence is
listed horizontally, the amino terminus is intended to be on the
left end whereas the carboxy terminus is intended to be at the
right end. The residues of amino acids in a peptide may be
separated by hyphens. Such hyphens are intended solely to
facilitate the presentation of a sequence.
[0072] Suitable agents for use according to the invention include
MHC or HLA Class I peptides and mimetics, fragments, functional
equivalents and/or hybrids or mutants thereof, as well as mutants,
and vectors containing cDNA encoding any of the foregoing. Agents
can be administered alone or in combination with and/or
concurrently with other suitable drugs and/or courses of
therapy.
[0073] The agents of the present invention are suitable for the
treatment of degenerative disorders, including disorders
characterized by inappropriate cell proliferation or inappropriate
cell death or in some cases, both. Inappropriate cell proliferation
will include the statistically significant increase in cell number
as compared to the proliferation of that particular cell type in
the normal population. Also included are disorders whereby a cell
is present and/or persists in an inappropriate location, e.g., the
presence of fibroblasts in lung tissue after acute lung injury. For
example, such cells include cancer cells which exhibit the
properties of invasion and metastasis and are highly an aplastic.
Such cells include but are not limited to, cancer cells including,
for example, tumor cells. Inappropriate cell death will include a
statistically significant decrease in cell number as compared to
the presence of that particular cell type in the normal population.
Such underrepresentation may be due to a particular degenerative
disorder, including, for example, AIDS (HIV), which results in the
inappropriate death of T-cells, and autoimmune diseases which are
characterized by inappropriate cell death. Autoimmune diseases are
disorders caused by an immune response directed against self
antigens. Such diseases are characterized by the presence of
circulating autoantibodies or cell-mediated immunity against
autoantigens in conjunction with inflammatory lesions caused by
immunologically competent cells or immune complexes in tissues
containing the autoantigens. Such diseases include systemic lupus
erythematosus (SLE), rheumatoid arthritis.
[0074] Standard reference works setting forth the general
principles of immunology include Sell, S., Immunology,
Immunopathology& Immunity, 5th Ed., Appleton & Lange,
Publ., Stamford, CT (1996); Male, D., et al., Advanced Immunology,
3d Ed., Times Mirror Int'l Publishers Ltd., Publ., London (1996);
Stites, D. P., and Terr, A. I., Basic and Clinical Immunology, 7th
Ed., Appleton & Lange, Publ., Norwalk, Conn., (1991); and
Abbas, A. K., et al., Cellular and Molecular Immunology, W. B.
Saunders Co., Publ., Philadelphia, Pa., (1991).
[0075] The MHC or HLA Class I peptides, mimetics, agents and the
like disclosed herein, as well as vectors comprising nucleotide
sequences encoding them or their corresponding antisense sequences,
and hosts comprising such vectors, may be used in the manufacture
of medicaments for the treatment of diseases including cancer.
[0076] The invention may be appreciated in certain aspects with
reference to the following examples, offered by way of
illustration, not by way of limitation.
EXAMPLES
[0077] Methods and Materials
[0078] In vivo Apoptosis Assay
[0079] The test for apoptosis in vivo was based on the use of the
diffusion chamber described in detail by Resnicoff, M., et al.,
Cancer Res. 55: 2463-2469 (1995). According to this method, the
cells are placed in a diffusion chamber that allows the passage of
nutrients, antigens, antibodies, etc., but not of intact cells. The
sterilized chamber is loaded with cells and then inserted into the
subcutaneous tissue of rats or mice; after 24 hours, (or at other
times, depending on the experiment) the chamber is removed and the
number of cells in the chamber is determined with a hemocytometer,
and expressed as a percentage of the original inoculum. The cells
are also stained with trypan blue for viability; in the experience
of the inventors, surviving cells are >95% viable.
[0080] For in vivo effects, 5.times.10.sup.5 cells (C6 and CaOV-3
were both used for these studies) were placed in diffusion chambers
and implanted into the s.c. tissue of the animals. Once the skin
was sutured, the peptides were injected s.c. next to the chambers
at the indicated concentrations in a final volume of 0.2 ml.
Twenty-four hours later, the chambers were removed from the animals
and the cells were quantitatively recovered.
[0081] Synthesis of Peptides
[0082] All chemicals were of analytical grade and were used without
further purifications. Various solvents were purchased from Fisher
Scientific Co. and Aldrich Co. Na-Fmoc amino acids and other
chemical reagents were purchased from Fisher Scientific Co,
Novabiochem, Fluka and Perseptive Biosystems.
Fmoc-tris(alkoxy)benzylamide linked polystyrene resin (PAL-support)
with a substitution level of 0.38 mmol/g and polyethylene glycol
graft polystyrene resin (PAL-PEG-support) with substitution level
of 0.16 mmol/g were both purchased from Perseptive Biosystems.
[0083] Peptides were prepared by solid phase synthesis with
Fmoc-strategy using an Applied Biosystems model 430A peptide and
Perseptive Biosystems 9050 Pepsynthesizer Plus. The side
chain-protecting groups of N(-Fmoc) amino acids were: Boc for Lys,
OtBu for Asp and Glu, Pmc for Arg, tBu for Ser and Thr, and Trt for
Asn, Gln, His and Cys. The four-fold excess of N(Fmoc) amino acid,
HBTU and HOBt, and ten-fold excess of DIPEA, were used in every
coupling reaction step. Removal of the N-terminal Fmoc group was
accomplished by 20% piperidine in DMF. The coupling and
deprotection steps were repeated for all the amino acid residues.
The cleavage of a peptide from the resin was carried out with
reagent K (TFA:
Phenol:thioanisole:ethandithiol:H20/10:0.75:0.5:0.25:0.5) for 2
hours at room temperature with gentle stirring. The mixture was
then filtered directly into ice-cold methyl t-butyl ether. The
resulting suspension was transferred into a centrifuge tube and
centrifuged for 10 minutes at 2000.times.g at room temperature. The
supernatant was discarded and the precipitate was resuspended in
methyl t-butyl ether, and again centrifuged for 10 minutes. The
procedure was repeated twice before the precipitate was dissolved
in aqueous solvent and lyophilized. The crude peptide was then
purified by preparative RP-HPLC using Dynamax-300 .ANG.C18 25
cm.times.21.4 mm I.D. column with flow rate of 9 ml/min. The
fractions containing the peptide were pooled together and
lyophilized.
[0084] The purity of the final products was assessed by analytical
RP-HPLC, capillary electrophoresis and MALD-TOF MS. A gradient
analytical RP-HPLC system, comprising a Waters 600E multisolvent
delivery system, Waters 490E programmable multiwavelength detector
setting at 206 nm and 280 nm, Waters 715 Ultra wisp autosampler and
Maxima 820 chromatography workstation for data acquisition, was
used. For a preparative RP-HPLC, a system comprising two Applied
Biosystems 400 solvent delivery systems, Applied Biosystems 783A
programmable absorbance detector and pump controller, Pharmacia,
LKB REC120 chart recorder and Waters fraction collector was used.
In both HPLC systems, solvent A was 99.9% water and 0.1% TFA, and
solvent B was 99.9% acetonitrile and 0.1% TFA. Capillary
electrophoresis was performed by using Applied Biosystems 270A-HT
systems equipped with 72 cm.times.0.05 mm I.D. fused silica
capillary at 30.degree. C. Injection was hydrodynamic for 3 seconds
at 5" Hg and the applied voltage during the run was 20 kV. Matrix
assisted laser desorption time of flight mass spectra (MALD-TOF MS)
were taken using Biomolecular Separations LDI1700 with sinapinic
acid and .alpha.-cyano-4-hydroxycinnamic acid solutions as
matrices.
[0085] Results
[0086] Cytotoxicity of MHC-Associated Peptides
[0087] To demonstrate the use of MHC or HLA Class I peptides for
induction of apoptosis in cancer cells, three peptides were tested
for their cytotoxicity in the diffusion chamber assay: 1)
LLDGTATLRL (from gp100), involved in regression of human melanoma
(Kawakami et al. 1994); 2) YLEPGPVTA, recognized by
melanoma-specific human CTL lines (Cox et al, 1994); and 3)
FECNTAQPG, derived from connexin 37, that induces CTL responses
against murine lung carcinoma (Mandelboim, et al., 1994). For each
of these peptides, a control peptide having the same amino acid
composition, but in scrambled order (controls), was also tested. C6
cells were preincubated with the peptides at a concentration of
10.sup.-5M and transferred to the diffusion chamber (see Methods
and Materials). Cell number was determined after 24 hours in the
diffusion chamber transplanted into the subcutaneous tissue of
rats. With the 3 test peptides, recovery ranged from 5-7.5% of the
inoculated cells, indicating that these peptides exhibited potent
cytotoxicity under those conditions. Recovery with the control
peptides was >200%, indicating that cells were not killed but
proliferated. These results suggest that the cytotoxic activity of
the test peptides was specific.
[0088] Effect of Mutations on the Ability of MHC-associated
Peptides to Induce Apoptosis in Vivo
[0089] Two of the three peptides were selected for further studies
of the effects of mutations on the ability to induce apoptosis.
Several point mutations were introduced into the peptide YLEPGPVTA.
These mutant peptides were tested on C6 cells in the diffusion
chamber assay following preincubation at a concentration of
10.sup.-5 M. The results (Table I) indicate that some mutations do
not affect the ability of these peptides to induce apoptosis, while
others completely abrogate this capacity. Mutations at tyrosine in
position 1, or the two prolines in positions 4 and 6, effectively
inactivated peptide no. 2. An exception to this observation was
seen when the prolines were replaced by an N-methyl amino acid.
Mutations at E3 or T8 had little effect, but when both residues
were mutated, recovery increased to 77%. D-amino acid versions of
the LLDGTATLRL and YLEPGPVTA peptides, in which the amino acid
sequence is reversed (i.e., LRLTATGDLL and ATVPGPELY, respectively)
were as effective as the L-amino acid sequences.
[0090] Dose-response for Peptide-induced Apoptosis
[0091] In order to determine the minimum concentration of these
peptides that still would induce apoptosis in vivo, C6 cells were
preincubated with different concentrations of the YLRPGPVTA and
D-amino acid LRLTATGDLL peptides and recovery of viable cells was
determined after 24 hours in the diffusion chamber. Table 4 shows
the results. Recovery of inoculated cells was only 18% when
pretreated with peptide YLRPGPVTA at a concentration of 10.sup.-12
M. Pretreatment with the D-amino acid sequence LRLTATGDLL resulted
in similar recovery of C6 cells at a concentration of 10.sup.-13M.
These concentrations are 7-8 orders of magnitude lower than the
concentration at which the inactive peptides were tested.
[0092] Evidence For Induction Of Apoptosis By Active Peptides
[0093] In order to determine that the loss of cells upon treatment
with active peptides was due to the induction of apoptosis, the
presence of apoptotic cells within the diffusion chamber was
determined at several time intervals by FACS analysis (Sell, et al.
(1995)). FIG. 1 represents a typical experiment using C6 cells. C6
cells in FIG. 1 (A) were treated with an active peptide
(YLEPGPVTA); FIG. 1 (B) shows results for C6 cells treated with a
control peptide (SMAPGNYSV). In both cases, the cells had been
incubated in vivo for 90 minutes in a diffusion chamber as
described herein. Apoptotic cells are detectable in FIG. 1 (A), but
not in FIG. 1 (B). Similar results were obtained at several time
intervals thereafter.
[0094] In addition, one of the common pathways of apoptotic injury
is through the ICE proteins (Henkart (1996)). The activation of ICE
and ICE-like proteins is inhibited by the p35 protein of
baculovirus (Clem and Miller (1994)), which can also be functional
in mammalian cells (Rabizadeh, et al. (1993). To determine whether
the synthetic peptides caused apoptosis through the ICE pathway, a
plasmid expressing the p35 protein was stably introduced into C6
cells. C6 and C6/p35 cells were then tested for apoptosis in the
diffusion chambers, using the synthetic peptide YLRPGPVTA. FIG. 2
is a composite picture showing the expression of p35 in the various
cell lines, and, below each lane, the percentage of cells recovered
from the diffusion chamber. Only C6 cells and C6 cells stably
transfected with the empty vector underwent apoptosis when treated
with the peptide; the two clones expressing the p35 protein grew in
the diffusion chamber equally well whether untreated or incubated
with the synthetic peptide.
[0095] Selected Peptides Induce Apoptosis when Injected in Vivo
[0096] The purpose of this experiment was to show that the
synthetic peptides induced apoptosis when injected in vivo, and
that their effect extended to human tumor cells. Human ovarian
carcinoma cells CaOV3 (Resnicoff, et al. (1993)) were loaded into a
diffusion chamber, and the chamber was inserted into the
subcutaneous tissue of mice. The animals were then injected, also
subcutaneously next to the chamber, with 0.2 ml of a 0.05 mM
solution of four different peptides, and the number of cells was
determined 24 hours later. The results (Table 2) clearly show that
active peptides can induce apoptosis of tumor cells, even when
injected into mice. The control peptide had no effect, and the
number of cells more than doubled. CaOV3 cells also undergo
apoptosis, as determined by FACS analysis.
[0097] Selected Peptides Inhibit Tumorigenesis in Syngeneic
Rats
[0098] C6 cells pre-incubated with various concentrations
(10.sup.-12, 10.sup.-10, 10.sup.-8, and 10.sup.-5 M) of the peptide
YLRPGPVTA were injected simultaneously into nude mice and the time
of appearance of subcutaneous tumors was determined. In parallel
experiments, the percentage of cells killed in the diffusion
chamber assay was determined. From the percentage of cells killed,
the expected delay in the appearance of tumors in mice was
calculated as described previously (Resnicoff, et al., Cancer Res.
55:3739 (1995)). The results are summarized in Table 3. There is a
concentration-dependent inhibition of tumorigenesis in C6 cells
pre-treated with the active peptide. An inactive peptide had no
effect. The time of appearance of tumors is slightly more delayed
than expected from the percentage of cells killed in 24 hours in
the diffusion chamber assay, a phenomenon previously observed also
with antisense oligodeoxynucleotides to the IGF-IR RNA (Resnicoff,
et al., Cancer Res. 55:3739 (1995)).
[0099] A similar inhibition of tumorigenesis was observed using the
D-amino acid peptide LRLTATGDLL. In this case, the peptide (all
D-aa, sequence from Kawakami, et al. (1994)) was injected s.c. into
nude mice (0.1 ml, at the indicated concentrations), simultaneously
with C6 cells (10.sup.5 cells in 0.1 ml) and next to them. The
animals were followed for tumor development. The results were as
follows: For C6 cells without peptide injection, the tumors
appeared after 4 days; for a peptide injection at 10.sup.-10 to
10.sup.-12 M, tumors appeared after 11 days.
[0100] The induction of apoptosis by MHC-associated peptides is a
specific phenomenon that cannot be explained by an aspecific
toxicity of the synthetic peptides. Active synthetic peptides are
capable of inducing apoptosis at concentrations of
10.sup.-12-10.sup.-13 M, while control peptides are inactive even
at concentrations as great as 10.sup.-5 M. In addition, selected
point mutations in the active synthetic peptides completely
inactivate them. The direct induction of apoptosis by these
peptides was a surprising finding.
[0101] Two different approaches were used to demonstrate apoptosis.
In some experiments, the cells were pre-incubated with the
peptides, then loaded in a diffusion chamber ( Resnicoff, M., et
al., Cancer Res. 55: 2463-2469 (1995)), which was then implanted
into the subcutaneous tissue of rats or mice. The apoptotic effect
of MHC-associated peptides is independent of an immune mechanism.
This statement is based on the following considerations: I) in the
experiments of Tables 1 and 2, the peptides induce apoptosis in
vivo in the short period of 24 hours, (onset of apoptosis was seen
in a period as short as 90 minutes, see FIG. 1) which seems to
exclude an immune response, since the animals were naive; 2) the
cells were in a diffusion chamber, which is impermeable to cells;
and 3) although the synthetic peptides are of human origin, they
were equally active on rat and on human tumor cells. In fact,
experiments indicate that these peptides induce apoptosis in vivo
on a variety of tumor cell lines, regardless of the type of tumor
or species, a surprising and unexpected finding. In summary, the
present experiments conclusively demonstrate that MHC-associated
peptides can induce apoptosis of tumor cells in vivo by a
non-immune mechanism.
3TABLE 1 Effect of Point Mutations on Induction of Apoptosis In
Vivo in C6 Cells by MHC Class I Peptides Protection from wild type
Peptide % Recovery (24 hs) cell challenge YLEPGPVTA 3.0 yes
YLRPGPVTA 3.5 yes YLEXGXVTA (X: N-Methyl-A) 6.5 yes YLAPGPVTA 7.4
yes YLEPGPVAA 9.3 yes YLEPGPATA 11.0 yes YLEPGPVKA 12.0 yes
YLEPAPVTA 70.0 no YLRPGPVRA 77.0 no YAEPGPVTA 186.0 no YLEAGPVTA
224.0 no ALEPGPVTA 224.0 no YLEPGAVTA 260.0 no LLDGTATLRL 5.0 yes
ATVPGPELY (D-amino acid) 6.0 yes LRLTATGDLL (D-amino acid) 4.9
yes
[0102] Synthetic peptide sequences listed above were tested at
5.times.10.sup.-M on C6 cells. Tests for apoptosis and for
protection from subsequent challenge with C6 cells are described
herein. Point mutations are shown in bold characters.
4TABLE 2 Selected Peptides Induce Apoptosis In Vivo in Human
Ovarian Carcinoma Cells. Peptide Percent Recovery YLRPGPVTA 3.0
YLEPGPVTA 0.2 LRLTATGDLL 6.6 YLEPGAVTA (control) 280
[0103] CaOV3 cells (human ovarian carcinoma cells, 5.times.10.sup.5
cells) were placed without any previous treatment into diffusion
chambers that were then implanted into the subcutaneous tissue of
Balb/c mice. Animals were then injected subcutaneously next to the
chambers either with 0.2 ml of a 0.05 mM solution of one of the
three test peptides or with 0.2 ml of a 0.05 mM solution of the
control peptide. The results summarized demonstrate that the three
test peptides induce apoptosis in vivo of human tumor cells.
5TABLE 3 Tumorigenesis in nude mice. Concentration of Peptide %
Expected Delay Palpable Tumors YLRPGPVTA Recovery (days) (days)
None >200 4 4 10.sup.-12 M 18 8 11 10.sup.-10 M 4.5 10 14
10.sup.-8 M 2.1 11 15 10.sup.-5 M 0.3 14 21
[0104] C6 cells were incubated with the synthetic peptide at the
indicated final concentrations for 24 hours in serum-free medium
before injection into the subcutaneous tissue of seven week-old
male Dalb/c nude mice. Percentage recovery was determined using the
diffusion chamber assay as described herein. Expected Delay is the
number of days after injection before the tumors should become
palpable, based on survival in vivo estimated by percentage of
cells recovered. The last column shows the actual number of days
after injection when tumors became palpable. Three nude mice were
used in each experimental condition.
[0105] Dose Response of Peptide-induced Apoptosis
[0106] The concentration of peptides required to induce apoptosis
of C6 tumor cells in the diffusion chamber was tested with two of
the peptides, and the results are given in Table 4.
6 TABLE 4 Peptide Concentration Percent Recovery YLRPGPVTA
10.sup.-5 M 0.3 10.sup.-8 M 2.1 10.sup.-10 M.sup. 4.5 10.sup.-12
M.sup. 18.0 LRLTATGDLL (D-aa) 10.sup.-5 M 0.5 10.sup.-8 M 3.0
10.sup.-12 M.sup. 6.6 10.sup.-13 M.sup. 18.0
[0107] Table 4 shows that these two peptides are essentially equal
in their ability to induce apoptosis of tumor cells.
[0108] Viral MHC Class I Peptides Induce Apoptosis In Vivo
[0109] Experiments were carried out using synthetic MHC Class I
Peptides other than tumor antigens to illustrate the broad scope of
the present invention and the effective use of screening methods as
described to identify useful peptides for the induction of
apoptosis in cancer cells.
[0110] In the first of these experiments, MHC Class I peptides
derived from Human Papilloma Virus Type 16 (HPV-16) E7 protein were
selected for screening with C6 glioblastoma cells using the
diffusion chamber method described herein. These viral peptides
were designated HPV-16 E7.sub.86-93 (amino acid sequence: TLGIVCPI)
and HPV-16 E7.sub.11- 20 (amino acid sequence: YMLDLQPETT).
Ressing, et al., Cancer Res. 56:582-588 (1996). C6 rat glioblastoma
cells were pre-treated with these peptides at a final concentration
of 50 .mu.M for 24 hours in serum-free medium. Control cells were
pre-treated without peptide. The assay was carried out using the
diffusion chamber implanted in the subcutaneous tissue of rats for
24 hours, as described. The results are shown in Table 6.
7 TABLE 6 Condition Recovery (%) C6 with no peptide treatment 204
C6 + HPV-16 E7.sub.86-93 27 C6 + HPV-16 E7.sub.11-20 13
[0111] In a second experiment, a synthetic peptide corresponding to
one derived from influenza virus, designated M1.sub.58-66 (amino
acid sequence: GILGFVPTL), was used to pre-treat C6 glioblastoma
cells for 24 hours at a final concentration of 10.sup.1M in
serum-free medium. Controls were treated with medium, but without
the M1.sub.58-66 peptide. Following implantation of the cells in
mice, using the diffusion chamber method described herein, and in
vivo growth for 25 hours, the chambers were removed and the cells
were quantitatively recovered. The results are shown in Table
7.
8 TABLE 7 Condition Recovery (%) C6 with no peptide treatment 260
C6 + M1.sub.58-66 23.4
[0112] The results of these two experiments demonstrate that
peptides not identified as tumor antigens induce apoptosis in viva
in cancer cells, and illustrate that the screening methods
disclosed herein, or, indeed, other in vivo and in vitro cell death
detection methods known in the art (including, but not limited to,
TdT-mediated dUTP Nick End Labeling (TUNEL) and In Situ Nick
Translation (ISNT) (Pihlgren, M., et al., Biochemica 3[1996]: 12-14
(1996))) may be employed by those of skill, having the benefit of
knowledge of the present invention, to identify MHC Class I
peptides and other peptides useful according to the invention for
the treatment of cancer.
9TABLE 8 TISSUE OF ORIGIN CELL LINE CELL TYPE SOURCE Neuronal
IMR-32 Neuroblastoma ATCC #CCL 127 Tissue SK-N-SH Neuroblastoma
ATCC #HTB 11 Bladder SCaBER Squamous cell ATCC #HTB 3 Bladder T24
Transitional cell ATTC #HTB 4 Bone RD-ES Ewing's Sarcoma ATCC #HTB
166 Brain U-373 MG Glioblastoma ATCC #HTB 17 U-87 MG Glioblastoma
ATCC #HTB 14 Breast BT-549 Ductal Carcinoma ATCC #HTB 122 MCF7
Adenocarcinoma ATCC #HTB 22 MDA-MB- Adenocarcinoma ATCC #HTB 132
468 Colon COLO 201 Adenocarcinoma ATCC #CCL 224 LoVo Adenocarcinoma
ATCC #CCL 229 LS 174T Adenocarcinoma ATCC #CL 188 HT-29
Adenocarcinoma ATCC #HTB 38 Cervix HeLa Epitheloid Carcinoma ATCC
#CCL 2 Kidney A-704 Adenocarcinoma ATCC #HTB 45 CAKI-1 Clear cell
Carcinoma ATCC #HTB 46 Lung LU-99 Large cell Carcinoma SynPhar
Laboratories Alberta, Canada SW-1573 Small Cell Carcinoma Dr.
Robert Arcea DFCI, Boston, MA SW2 Small Cell Carcinoma Dr. Sam
Bernal DFCI, Boston, MA Lymph Namalwa B Cell Lymphoma ATCC #CRL
1432 Node Mouth KB (2D1) Epidermoid ATCC #CCL 17 Carcinoma Ovary
OVCAR-3 Adenocarcinoma ATCC #HTB 161 SK-OV-3 Adenocarcinoma ATCC
#HTB 77 Pancreas PANC-1 Epitheloid Carcinoma ATCC #CRL 1469
Peripheral HL-60 Promyelocytic ATCC #CCL 240 Blood leukemia MOLT-4
T Cell leukemia ATCC #CRL 1582 Prostate DU 145 Carcinoma ATCC #HTB
81 PC-3 Adenocarcinoma ATCC #CRL 1435 LNCaP.FEC Adenocarcinoma ATCC
#CRL 1740 Skin SK-MEL-37 Melanoma Dr. Aaron Lerner Yale Medical
School Hs 695T Melanoma ATCC #HTB 137 A-375 Melanoma ATCC #CRL 1619
SqCc/Y1 Squamous cell Dr. Alan Sartorelli Yale Medical School
Stomach AGS Adenocarcinoma ATCC #CRL 1739
[0113] All publications mentioned in this specification are herein
incorporated by reference, to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference. It will be understood that the invention
is capable of further modifications and this application is
intended to cover any variations, uses, or adoptions of the
invention including such departures from the present disclosure as
come within known or customary practice within the art to which the
invention pertains, and is intended to be limited only by the
appended claims.
Sequence CWU 1
1
* * * * *