Note: Descriptions are shown in the official language in which they were submitted.
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TELOMERASE-SPECIFIC CANCER VACCINE
BACKGROUND OF THE INVENTION
Telomeres, the DNA at the chromosome ends, are made up of simple tandem
repeats. In most somatic cells, telomere sequences are lost during DNA
replication due to the
need of DNA-dependent DNA polymerases for an RNA primer annealed to the
template
strand. Because the RNA primer cannot anneal beyond the 5' end of the DNA
strand, each
time a cell's DNA replicates, short bits of telomeric DNA are lost with each
generation. Cells
displaying such telomeric shortening go into senescence after a fixed number
of population
doublings, and senescence correlates directly with the erosion of telomeres to
a critical
minimum length.
Not all cells undergo such loss. however. While normal human somatic cells
lose telomeric repeats with each cycle of cell division, human germline and,
significantly,
cancer cells maintain a constant number of telomeric repeats. Telomere length
is maintained
in these cells by the action of telomerase, a ribonucleoprotein enzyme that
uses a short
endogenous RNA as a template for telomere addition. In fact, cancer cells
express high levels
of telomerase, whereas somatic cells express little, if any.
Because normal somatic cells do not appear to express or require telomerase,
whereas cancer cells express high levels of telomerase, the telomerase enzyme
presents an
attractive therapeutic target. Due to the fact that telomerase is a normal
"self' antigen,
however, conventional vaccination strategies are unavailable. Thus, the focus
of telomerase-
based therapeutics has been enzyme inhibitors of various sorts, rather than
vaccine-based
approaches.
A need exists, therefore, for new telomerase-based therapeutic approaches.
This need extends to vaccines, based on telomerase antigens.
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SUMMARY OF THE INVENTION
It is, therefore, an object of the invention to provide antigens that are
useful in
generating an immune response against telomerase. According to this object of
the invention,
telomerase antigens are provided that are capable of marshalling the immune
system against
telomerase-expressing cells. In one embodiment, telomerase antigens are
provided, which are
based on peptide sequences of the protein portion of telomerase. In another
embodiment,
telomerase antigens are provided as nucleic acids that are capable of being
used to express
peptide-based telomerase antigens.
It is another object of the invention to provide vaccine compositions which
include at least one telomerase antigen. Thus, in one aspect, the invention
provides vaccine
compositions containing telomerase peptide antigens. In another aspect,
polynucleotides are
provided, which encode protein-based telomerase antigens.
It is still another object of the invention to provide methods of treating or
preventing cancer. According to this object, the telomerase antigens of the
invention may be
directly administered in beneficial amounts to a patient. Also according to
this object, the
present telomerase antigens may be administered to a patient encoded in a
nucleic acid. This
object is also met by ex vivo methods that im~olve contacting a cell with a
telomerase antigen,
and administering that cell to a patient. In one aspect, the contacted cell
may be an antigen
presenting cell, which may be used to generate a primed T-cell ex vivo, at
which time the
primed T-cell may be administered to a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows fluorescence activated cell sorting (FACS) analysis of T-cells
activated by
telomerase-specific antigens. Panels A and B are negative and positive
controls, respectively,
and panels C, D and E are antigen candidates.
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DETAILED DESCRIPTION
The present invention relates to a telomerase-specific vaccines, which are
useful generating telomerase-directed activated T-cells. More particularly the
inventive
vaccines are useful in generating antigen-specific major histocompatability-
(MHC-)
restricted T-cell responses against telomerase presented by antigen presenting
cells. It is
believed that the present vaccines act to relieve the tolerance or anergy
induced through self
tolerance mechanisms to telomerase in normal individuals. Since telomerase
represents a
cancer-specific therapeutic target, in one embodiment, the vaccines of the
invention are useful
in the treatment or prevention of a variety of cancers.
A. Definitions
As used in this specification. an "activated T-cell" is one that is in the
following phases of the cell cycle: the G, phase, the S phase, the G, phase or
the M (mitosis)
phase. Thus, an "activated T-cell" is undergoing mitosis and/or cell division.
An activated T-
cell may be a T helper (TH) cell or a cytotoxic T-cell (cytotoxic T lymphocyte
(CTL or T~)).
Activation of a naive T-cell may be initiated by exposure of such a cell to an
antigen-
presenting cell (APC) (which contains anti~en%MHC complexes) and to a molecule
such as
IL-1, IL-2, IL-12, IL-13, y-IFN, and similar lsmphokines. The antigen/MHC
complex
interacts with a receptor on the surface of the T-cell (T-cell receptor
(TCR)). Golub et al.,
eds. IMMUNOLOGY: A SYNTHESIS, Chapter ~: "The T-cell Receptor" (1991).
As used in this specification. "priming" is used to mean exposing an animal
(including a human) or cultured cells to antigen, in a manner that results in
activation and/or
memory. The generation of CD4' and CDS- T-cell responses against a target
antigen is
usually dependent upon in vivo priming, either through natural infection or
through deliberate
immunization.
As used in this specification. a "naive" T-cell is one that has not been
exposed
to foreign antigen (non-autologous) antigen or one that has not been exposed
to cryptic
autologous antigen. A "naive" T-cell is sometimes referred to as an "unprimed"
T-cell. The
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skilled artisan will recognize that a "resting" cell is in the G~, phase of
the cell cycle and hence
is not dividing or undergoing mitosis. The skilled artisan will also recognize
that an "anergic"
T-cell is one that is unable to function properly; i.e., such as a cell that
lacks the ability to
mediate the normal immune response. T-cells from diseased patients may contain
T-cells that
have been primed, but are anergic.
As used in this specification "memory T-cells," also known as "memory
phenotype" T-cells, is used to designate a class of T-cells that have
previously encountered a
peptide antigen but are now resting and are capable of being activated. Memory
T-cells are T-
cells which have been exposed to antigen and then survive for extended periods
in the body
without the presence of stimulating antigen. However, these memory T-cells
respond to
"recall" antigens. In general, memory T-cells are more responsive to a
"recall" antigen, when
compared with the naive T-cell response to peptide antigen. Memory cells can
be recognized
by the presence of certain cell-surface antigens, such as CD45R0, CD58, CD1
la, CD29,
CD44 and CD26, which are markers for differentiated T-cells.
As used in this specification. an "telomerase-specific" T-cell response is a T-
cell response (for example, proliferative, c~-rtotoxic and/or cytokine
secretion) to telomerase
antigenic stimulus, for example a peptide. which is not evident with other
stimuli, such as
peptides with different amino acid sequences (control peptides). The
responsiveness of the T-
cell is measured by assessing the appearance of cell surface molecules that
are characteristic
of T-cell activation, including, but not limited to CD25 and CD69. Such assays
are known in
the art.
The term "treating" in its various grammatical forms in relation to the
present
invention refers to preventing, curing, reversing, attenuating, alleviating,
minimizing,
suppressing or halting the deleterious effects of a disease state, disease
progression, disease
causative agent or other abnormal condition.
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B. Telomerase Antigens
1. Generally Useful Antigens
Telomerase antigens according to the invention share the characteristic
ability
to generate a specific T-cell response. This response may be either class I-
or class II-specific.
In one aspect, this response is MHC class I-specific, and will comprise
antigen- and MHC-
restricted cytotoxicity. Class I molecules include HLA-A, HLA-B and HLA-C.
Thus,
preferred antigens bind class I molecules, e.g., HLA-Al, HLA-A2, HLA-A3 or HLA-
Al 1.
More preferred antigens bind all class I molecules. In contrast, where class
II-specific (e.g.,
helper fimetions) responses are desired, class-II-binding antigens will be
used. Class II
molecules include HLA-DR, HLA-DQ and HLA-DP. Useful antigens can be determined
as
set out below.
Telomerase antigens are typically derived from the sequence of the protein
portion of telomerase, which is disclosed in U.S. Patent No. 5,837,857 ( 1998)
and at GenBank
Accession Nos. AF015950 and AFOI 8167, which sequences are hereby incorporated
by
reference. They may be made, for example. by proteolytic digestions of the
telomerase
protein and/or by recombinant DNA means. Generally, the relatively short
peptide versions
will be prepared by synthetic means.
Although telomerase antigens according to the invention are not limited by
size, and they may be a portion or even all of the telomerase protein, they
are usually small
peptide antigens. A small size is preferred, due to ease of manufacture and
greater specificity.
Accordingly, unless they are multimeric (i.e., multiple copies of the same
epitope) most
telomere antigens will be less than about 50 amino acids in length. Preferred
antigens are less
than about 25 amino acids in length, with other preferred antigens being
between about 8 to
about 12 amino acids long, although sequences as short as 6 or 7 amino acids
are
contemplated. Nine-mers are typical of class I antigens, since they usually
retain the requisite
functional character; they include Ile-Leu-Ala-Lys-Phe-Leu-His-Trp-Leu
(ILAKFLHWL) as a
preferred species.
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Variants of telomerase antigens are also contemplated. It is only important
that
any variants retain the functional characteristics of a telomerase antigen: (
1 ) the ability to bind
an MHC molecule, e.g., HLA-A2, and (2) the ability to induce a telomerase
specific T-cell
response. Amino acid substitutions, i.e. "conservative substitutions" that
yield "conservative
variants," may be made, for instance, on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues
involved.
For example: (a) nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b)
polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; (c)
positively charged (basic) amino acids include arainine, lysine, and
histidine; and (d)
negatively charged (acidic) amino acids include aspartic acid and glutamic
acid. Substitutions
typically may be made within groups (a)-(d). In addition, glycine and proline
may be
substituted for one another based on their relatively small sizes and lack of
side-chains.
Similarly, certain amino acids, such as alanine, cysteine, leucine,
methionine,
glutamic acid, glutamine, histidine and lysine are more commonly found in a-
helices, while
valine, isoleucine, phenylalanine, tyrosine, tn~ptophan and threonine are more
commonly
found in (3-pleated sheets. Glycine, serine, aspartic acid, asparagine, and
proline are
commonly found in turns. The importance or substitution groups based on
structure, of
course, increases with the length of the antigen.
Some preferred conservative substitutions may be made among the following
groups: (i) S and T; (ii) P and G; and (iii) A. ~', L and I. Given the known
genetic code, and
recombinant and synthetic DNA techniques. the skilled scientist readily can
construct DNAs
encoding the conservative amino acid variants. Of course, smaller variants may
be
synthesized. One such genus of consen~ative HLA-A2-binding variants includes
peptides of the
structure: (A/V/L/I)(A/V/L/I)(A/V/L/I)(A/V L; I)KF(A/V/L/I)HW(A/V/L/I). Thus,
some
preferred conservative variants include LLAhFLHWL, ILAKFLHWI, IIAKFLHWL,
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IIAKFLHWI, ILARFLHWL, and ILVKFLHWL, and permutations thereof, so long as the
requisite functional characteristics are retained.
Moreover, one or more of the amino acids of the foregoing HLA-A2-binding
peptides may be replaced with glycine. Parker et al., J. Immunol. 149:3580-87
(1992)
disclose that up to six amino acids in a nine-mer may be replaced with
glycine; thus,
GLFGGGGGV can bind HLA-A2. The onlv real conservation observed in 9-mer HLA-A2-
binding peptides was an Ile or a Leu at about position 2 (counting N- to C-
terminal) and a Val
or a Leu at about position 9. Some simple variants, therefore, include
GLAKFLHWL,
ILAGFLHWL, ILAKGLHWL, ILAKFGH~t'L, ILAKFLGWL and ILAKFLHGL, subject to
the presence of the requisite functional characteristics.
An important source for guidance in regard to designing class I and class II
antigens, and in making conservative substitutions is Rammensee et al.,
Immunogenetics
41:178-228 (1995), which is hereby incorporated by reference in its entirety.
As indicated in
the Rammensee reference, the motifs for both class I and class II molecules
have certain
"anchor" residues, that retain high degrees of conservation. For instance, HLA-
A0201 (an
HLA-A2 molecule), which is the molecule that the telomerase peptide ILAKFLHWL
was
designed to bind (and does bind), has anchor residues at positions 2 and 9,
corresponding to
the conservative positions noted above. This molecule also has an "auxiliary"
position at 6,
the relative conservation of which is important, but less so than the anchor
residues. Thus,
using the general guidance of Rammensee, the artisan will appreciate that,
while the anchor
residues and auxiliary residues are relatively conserved in HLA binding, the
remainder of the
antigen can vary widely, and is probably responsible for the particular
antigenic character of
the antigen, i.e., it differentiates telomerase from non-telomerase.
Other substitutions include replacing L-amino acids with the corresponding D-
amino
acids. This rationale, moreover can be combined with the foregoing
conservative substitution
rationales. For example, D-leucine may be substituted for L-isoleucine. In
addition, these D-
amino acid-containing peptides may be prepared which have an inverse sequence,
relative to the
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native sequence. Hence, ILAKFLHWL becomes LWHLFKALI. Such "retro-inverso"
peptides
are expected to have improved properties. such as increased in vivo half life.
This translates into
smaller doses and more economically viable production.
Some embodiments contemplate multimers of the foregoing peptides.
Multimers can contain multiple copies of the same peptide, or they can be
mixed and
matched. The multimers can be direct tandem repeats, and may contain short
spacers
sequences of amino acids (e.g., 2-5 residues) like Gly and/or Pro, or other
suitable spacers.
Multimers may be any length, but typically will be less than about 100 amino
acids. Preferred
multimers are less than about 60 amino acids and have between about 2 and 5
copies of
peptides of about 8 to about 12 amino acids long. Multimers may also comprise
several
different telomerase antigens.
The telomerase antigens may be glycosylated or partially glycosylated
according to methods known in the art. They also can be modified with large
molecular
weight polymers, such as polyethylene glycols. In addition, lipid
modifications are preferred
IS because they may facilitate the encapsulation or interaction of the
derivative with liposomes.
Exemplary lipid moieties useful for this purpose include, but are not limited
to, palmitoyl,
myristoyl, stearoyl and decanoyl groups or. more generally, any C, to C3o
saturated,
monounsaturated or polyunsaturated fatty acv) group.
For convenience in making chemical modifications, it is sometimes useful to
include in a telomerase antigen one or more amino acids having a side chain
amenable to
modification. A preferred amino acid is lysine, which may readily be modified
at the E-amino
group. Side-chain carboxyls of aspartate and glutamate are readily modified,
as are serine,
threonine and tyrosine hydroxyl groups, the cysteine sulfhydryl group and the
histidine amino
group. The introduction of two cysteine residues. at spaced locations in a
peptide antigen,
may sen~e to form a structural constraint through a disulfide bridge, which
may improve
binding to MHC molecules.
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Also illustrative of a telomerase antigen within the present invention is a
non-
peptide "mimetic," i.e., a compound that mimics one or more functional
characteristics of the
telomerase antigen. Mimetics are generally water-soluble, resistant to
proteolysis, and non-
immunogenic. Conformationally restricted, cyclic organic peptides which mimic
telomerase
antigens can be produced in accordance with known methods described, for
example, by
Saragovi, et al.. Science 253: 792 (1991).
Telomerase antigens may also be constructed as hybrids (and/or formulated as
distinct molecules together in liposomes, as described below) with immune-
stimulatory
molecules, like cytokines and adjuvants. Interleukin-2 (IL-2) is one such
cytokine. Other
cytokines include GM-CSF, IL-12 and flt-3 ligand. Telomerase antigens may be
made as
fusion proteins with IL-2, for example, by recombinant DNA or chemical
synthetic means, or
they may made as chemical conjugates using bi-functional chemical linkers. It
is anticipated
that such chimeric proteins would possess an increased ability to generate a T-
cell-specific
response against telomerase. Adjuvants include monophosphoryl lipid A (MPLA),
and
derivatives thereof, which also may be attached to a telomerase antigen by
conventional
linkers. Other conventional immune stimulatory molecules include keyhole
limpet
hemocyanin (KLH).
2. Identification of Other Useful Antigens
It is of interest to identify additional, and especially small telomerase
antigens,
which would be expected to generate a more specific response, associated with
a particular
epitope for example. Moreover, these small antigens may be more economically
produced.
It is advantageous to identify additional telomerase antigen and further to
refine the T-cell antigenicity of telomerase, even down to the epitopic level.
One classic
method involves proteolytic treatment of the large antigen to derive smaller
antigens. In
addition, fragments of protein antigens can be produced by recombinant DNA
techniques and
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assayed to identify particular epitopes. Moreover, small peptides can be
produced by in vitro
synthetic methods and assayed.
As an alternative to the random approach of making parts of the intact antigen
then assaying them, a more biologically relevant approach is possible.
Specifically, since
antigenic fragments which bind to MHC class I and/or class II molecules,
especially class I
molecules, are of particular importance, one exemplary approach is to isolate
the MHC
molecules themselves and then to isolate the peptides associated with them.
For a general
description of such a method, see PCT/US98%09288; Agrawal et al., Int'1
Immuno1.10:1907-
16 ( 1998); and Agrawal et al., Cancer Res. ~ ~ :51 ~ 1-56 ( 1998).
In a typical method, either primary tumor cells or a cell line expressing the
antigen of interest are provided. In addition. it will be recognized that
phagocytic antigen
presenting cells (or any APC), such as macrophages, may be fed large antigens
(or portions
thereof) and thus act as the starting material for these methods. The MHC
class I or class II
molecules can be isolated from these starting cells using known methods, such
as antibody
affinity (MHC-specific antibodies) and chromatographic techniques.
Isolated MHC molecules are then treated to release bound peptides. This may
be accomplished by treatment with agents that disrupt the interactions between
the bound
peptide and the MHC molecule, for example. detergent, urea, guanidinium
chloride, divalent
cations, various salts and extremes in pH. The peptides released can be
further purified using
conventional chromatographic and antibody affinity (using antigen-specific
antibody)
methodologies. The purified peptides may then be subjected to sequence and
structural
determinations, using for example peptide sequencing, gas chromatography
and/or mass
spectroscopy.
In this mariner the sequences. structures of the most prevalent peptide
epitopes
associated with class I and/or class II molecules may be determined. Supplied
with this
sequence/structural information, permutations of the determined sequence can
be made, as
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detailed above, and assayed using known T-cell assays. Rammensee et al.,
supra, provides
extensive methods and guidance related to identifying both class I and class
II motifs.
Yet another method of generating telomerase antigens may utilize algorithms
known in the art for predicting binding sequences. Publicly available
comparison programs
using these algorithms to compare known peptide sequences to different HLA-
binding motifs
may be found, for example, at http://www-bimas.dcrt.nih.gov/hla bind.
Different class I and
class II binding motifs may be found at that site or in publications like
Rammensee et al. and
Parker et al., both supra.
C. Vaccine Compositions
In general, any telomerase-specific antigen, as described above, will be
useful
in formulating telomerase-specific vaccines. Preferred antigens may be
associated with lipids,
usually either by direct lipid modification of the antigen and/or by liposomal
association, as
described below. The antigens may be administered as peptides or peptide
mimetics, or they
may be administered in nucleic acid form.
1. Liposonaal Formulation
In one embodiment of the in~~ention, the telomerase antigen is associated with
a liposome. Techniques for preparation of iiposomes and the formulation of
various
molecules, including peptides, with liposomes (e.g., encapsulation or complex
formation) are
well known to the skilled artisan. Liposomes are microscopic vesicles that
consist of one or
more lipid bilayers surrounding aqueous compartments. See, generally, Bakker-
Woudenberg
et al.. Eur. .l. Clin. Microbiol. Infect. Dis. 12 (Suppl. I): S61 (1993) and
Kim, Drugs 46: 618
(1993). Liposomes are similar in composition to cellular membranes and as a
result,
liposomes generally can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be unilamellar or
multilamellar, and can vary in size with diameters ranging from 0.02 ~m to
greater than 10
pm. A variety of agents can be encapsulated in liposomes. Hydrophobic agents
partition in
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the bilayers and hydrophilic agents partition within the inner aqueous
space(s). See, for
example, Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (dohn Libbey
1987), and Ostro et al., ( 1989) American J. Hosp. Pharnz. 46: 1576.
Liposomes can adsorb to virtually any type of cell and then release the
encapsulated agent. Alternatively, the liposome fuses with the target cell,
whereby the
contents of the liposome empty into the target cell. Alternatively, an
absorbed liposome may
be endocytosed by cells that are phagocytic. Endocvtosis is followed by
intralysosomal
degradation of liposomal lipids and release of the encapsulated agents.
Scherphof et al.,
(1985) Ann. >\! Y. Acad. Sci. 446: 368.
Anionic liposomal vectors hare also been examined. These include pH
sensitive liposomes which disrupt or fuse with the endosomal membrane
following
endocytosis and endosome acidification. Among liposome vectors, however,
cationic
liposomes are the most studied, due to their effectiveness in mediating
mammalian cell
transfection in vitro.
Cationic lipids are not found in nature and can be cytotoxic, as these
complexes appear incompatible with the physiological environment in vivo which
is rich in
anionic molecules. Liposomes are preferentially phagocytosed into the
reticuloendothelial
system. However, the reticuloendothelial system can be circumvented by several
methods
including saturation with large doses of liposome particles, or selective
macrophage
inactivation by pharmacological means. Classen et al., (1984) Biochinr.
Biophys. Acta 802:
428. In addition, incorporation of glycolipid- or polyethylene glycol-
derivatised
phospholipids into liposome membranes has been shown to result in a
significantly reduced
uptake by the reticuloendothelial system. Allen et al., (1991) Biochirn.
Biophys. Acta 1068:
133; Allen et al., (1993) Biochint. Biophys. .-lctcr 1150: 9.
Cationic liposome preparations can be made by conventional methodologies.
See, for example. Felgner et al., Pnoc. Nat'l .-1 cad. Sci USA 84:7413 (
1987); Schreier, J. of
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Liposome Res. 2:145 (1992); Chang et al. ( 1988), supra. Commercial
preparations, such as
Lipofectin~ (Life Technologies, Inc., Gaithersburg, Maryland USA), also are
available. The
amount of liposomes and the amount of DICTA can be optimized for each cell
type based on a
dose response curve. Felgner et al., supra. For some recent reviews on methods
employed
see Wassef et al., Immunomethods 4: 217 - 222 ( 1994) and Weiner, A. L.,
Immunomethods 4:
217 - 222 ( 1994).
Other suitable liposomes that are used in the methods of the invention include
multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar
vesicles (UV), small
unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MLJV), large
unilamellar
vesicles (LUV), giant unilamellar vesicles 1 GL~), multivesicular vesicles
(MVV), single or
oligolamellar vesicles made by reverse-phase evaporation method (REV),
multilamellar
vesicles made by the reverse-phase evaporation method (MLV-REV), stable
plurilamellar
vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by
extrusion
methods (VET), vesicles prepared by French press (FPV), vesicles prepared by
fusion (FUV),
dehydration-rehydration vesicles (DRV), and bubblesomes (BSV). The skilled
artisan will
recognize that the techniques for preparing these liposomes are well known in
the art. See
COLLOIDAL DRUG DELIVERY SYSTEMS, vol. 66 (1. Kreuter, ed., Marcel Dekker, Inc.
1994).
2. Suitable Adjuva~ats and Excipients
The present vaccine formulations, liposomal or not, may be formulated
advantageously with some type of adjuvant. As conventionally known in the art,
adjuvants
are substances that act in conjunction with specific antigenic stimuli to
enhance the specific
response to the antigen. MPLA, for example. has been shown to serve as an
effective
adjuvant to cause increased presentation of liposomal antigen by the APCs to
specific T
Lymphocytes. Alving, C.R. 1993. Intmmaobiol. 187:430-446. Moreover, the
skilled artisan
will recognize that other such adjuvants, such as Detox, alum, QS21, complete
and/or
incomplete Freund's adjuvant, MDP, LipidA and derivatives thereof, are also
suitable.
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Another class of adjuvants include stimulatory cytokines, such as IL-2. Thus,
the present vaccines may be formulated with IL-2 or IL-2 may be administered
separately for
optimal antigenic response. IL-2 is beneficially formulated with liposomes.
Vaccines may also be formulated with a pharmaceutically acceptable excipient.
Such excipients are well known in the art, but typically should be
physiologically tolerable and
inert or enhancing with respect to the vaccine properties of the inventive
compositions.
Examples include liquid vehicles such as sterile, physiological saline. When
using an
excipient, it may be added at any point in formulating the vaccine or it may
be admixed with
the completed vaccine composition.
Vaccines may be formulated for multiple routes of administration. Specifically
preferred routes include intramuscular, percutaneous, subcutaneous, or
intradermal injection,
aerosol, oral or by a combination of these routes, at one time, or in a
plurality of unit dosages.
Administration of vaccines is well known and ultimately will depend upon the
particular
formulation and the judgement of the attending physician.
Vaccine formulations can be maintained as a suspension, or they may be
lyophilized and hydrated later to generate a useable vaccine.
D. Targeting the Inventive Antigens and Vaccines
In order to provide greater specificity, thus reducing the risk of toxic or
other
unwanted effects during in vivo administration, it is advantageous to target
the inventive
compositions to the cells through which they are designed to act, namely
antigen-presenting
cells. This may conveniently be accomplished using conventional targeting
technology. One
exemplary form of targeting using antibodies, or similar specifically-binding
molecules,
associated in some fashion with the antigen and/or vaccine composition.
Targeting molecules have the characteristic of being able to distinguish to
some degree, target APCs over background. non-APCs. Targeting molecules
include
mannose and the Fc portion of antibodies, and the like. which will target
antigen presenting
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cells. Targeting molecules may be directly associated with telomerase
antigens, for example,
by chemical conjugation or by fusion protein production, in the case of
protein-based
targeting sequences.
Due to their convenience and extensive familiarity in the art, antibodies and
antibody derivatives are preferred targeting molecules. Antibodies and their
derivatives
include, but are not limited to polyclonal antibodies, monoclonal antibodies
(mAbs),
humanized or chimeric antibodies, single chain antibodies including single
chain Fv (scFv)
fragments, Fab fragments, F(ab'), fragments. fragments produced by a Fab
expression library,
epitope-binding fragments, and humanized forms of any of the above. Of course,
the smaller
versions of these molecules are preferred, based on the fact that they will
more readily target
to an APC.
In general, techniques for preparing polyclonal and monoclonal antibodies as
well as hybridomas capable of producing the desired antibody are well known in
the art
(Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry
and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands
(1984); St.
Groth et al., J. Immunol. Methods 35:1-21 (1980); Kohler and Milstein, Nature
256:495-497
(1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et
al.,
Immunology Today 4:72 (1983); Cole et al.. in :hlonoclonul Antibodies and
Cancer Tlzerapy,
Alan R. Liss, Inc. (1985), pp. 77-96). Affinity of the antisera for the
antigen may be
determined by preparing competitive binding cur<~es, as described, for
example, by Fisher,
Chap. 42 in: Manual of Clinical Immasnoloy, second edition, Rose and Friedman,
eds., Amer.
Soc. For Microbiology, Washington, D.C. ( 1980).
Fragments or derivatives of antibodies include any portion of the antibody
which is capable of binding an APC target molecule, typically a surface
antigen. Antibody
fragments specifically include F(ab')=, Fab, Fab' and Fv fragments. These can
be generated
from any class of antibody, but typically are made from IgG or IgM. They may
be made by
conventional recombinant DNA techniques or. using the classical method, by
proteolytic
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digestion with papain or pepsin. See CURRENT PROTOCOLS IN IMMUNOLOGY, chapter
2, Coligan et al., eds., (John Wiley & Sons 1991-92).
F(ab')2 fragments are typically about 110 kDa (IgG) or about 150 kDa (IgM) and
contain two antigen-binding regions, joined at the hinge by disulfide bond(s).
Virtually all, if not
all, of the Fc is absent in these fragments. Fab' fragments are typically
about 55 kDa (IgG) or about
75 kDa (IgM) and can be formed, for example, by reducing the disulfide bonds)
of an F(ab')2
fragment. The resulting free sulfhydryl groupls) may be used to conveniently
conjugate Fab'
fragments to other molecules, such as telomerase antigens or adjuvant
molecules.
Fab fragments are monovalent and usually are about 50 kDa (from any source).
Fab
fragments include the light (L) and heavy (H) chain, variable (VL and VH,
respectively) and
constant (CL CH, respectively) regions of the antigen-binding portion of the
antibody. The H and L
portions are linked by one or more intramolecular disulfide bridges.
Fv fragments are typically about 25 kDa (regardless of source) and contain the
variable regions of both the light and heavy chains (VL and VH, respectively).
Usually, the VL and
VH chains are held together only by non-covalent interactions and, thus, they
readily dissociate.
They do, however, have the advantage of small size and they retain the same
binding properties of
the larger Fab fragments. Accordingly, methods have been developed to
crosslink the VL and VH
chains, using, for example, glutaraldehyde (or other chemical crosslinkers),
intermolecular disulfide
bonds (by incorporation of cysteines) and peptide linkers. The resulting Fv is
now a single chain
(i.e., scFv).
Other antibody derivatives include single chain antibodies (U.S. Patent
4,946,778;
Bird, Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988);
and Ward et al., Nature 334:544-546 ( 1989)). Single chain antibodies are
formed by linking the
heavy and light chain fragments of the Fv region via an amino acid bridge,
resulting in a single
chain FV (scFv).
Derivatives also include "chimeric antibodies" (Morrison et al., Proc. Natl.
Acad. Sci., 81:6851-6855 (1984); Neuberger et crl., Nata~re, 312:604-608
(1984); Takeda et al.,
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Nature, 314:452-454 (1985)). These chimeras are made by splicing the DNA
encoding a
mouse antibody molecule of appropriate specificity with, for instance, DNA
encoding a
human antibody molecule of appropriate specificity. Thus, a chimeric antibody
is a molecule
in which different portions are derived from different animal species, such as
those having a
variable region derived from a murine mAb and a human immunoglobulin constant
region.
Recombinant molecules having a human framework region and murine
complementarity
determining regions (CDRs) also are made using well-known techniques. These
are also
known sometimes as "humanized" antibodies and they and chimeric antibodies or
antibody
fragments offer the added advantage of at least partial shielding from the
human immune
system. They are, therefore, particularly useful in therapeutic in vivo
applications.
E. Nucleic Acid-Based Vaccines
Recently, there has been increased interest in polynucleotide-based vaccines,
and such applications are contemplated here. These vaccines generally rely on
either a DNA
vector that encodes the antigen of interests under operable control of
transcription and
translation signals, or a RNA vector that encodes the antigen of interests
under operable
control of translation signals. When these vaccines are administered, they are
thought to be
taken up by the surrounding cells, which then express the target antigen. The
expressed
antigen apparently becomes associated with the cell's major histocompatability
(MHC)
antigens and are thus localized to the surface of the cell and presented to
immune cells. See,
for example, Corr et al., J. Exp. Med. 184: 1 »~-60 (1996). Such vaccines may
employ
naked DNA (Id.) or the DNA may be liposomally associated or trapped
(Gregoriadis et al.,
FEBS Lett. 402: 107-10 ( 1997)).
These so-called "naked DNA' vaccines or vaccines comprising RNA have
broad applicability. They may be employed, for example, as anti-cancer
vaccines (Scheurs et
al., Cancer Res. ~8: 2509-14 (1998); Hurpin et ul., Vaccine 16: 208-15 (1998))
and anti-viral
vaccines (Bohm et al., Vaccine 16: 949-54 (1998); Lekutis et al., J. Immunol.
158: 4471-77
( 1997)), among others. Naked DNA vaccines have been shown to elicit both
class I- (Scheurs
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et al., supra; Bohm et al, supra; Hurpin et al, supra) and class II-restricted
responses
(Lekutis et cal., szzpra; Manickan et czl., J. Leukoc. Biol. 61: 125-32
(1997))).
Accordingly, any of the foregoing telomerase antigens may be administered as
a naked DNA vaccine. These vaccines will comprise a nucleic acid vector that
encodes a
telomerase antigen under the control of transcription and translation signals
that operate in a
mammal, preferably a human. They may be administered associated with or
encapsulated by
(usually cationic) liposomes, as detailed above, or they may be administered
in any other
physiologically tolerable excipient.
F. Methods of the Invention
According to one aspect of the invention, the foregoing telomerase antigens
(or
vaccine compositions) may be used as a conventional vaccine directly to induce
an immune
response against telomerase. In some cases, however, for an improved
therapeutically or
prophylactically suitable T-cell response, the antigen may be liposome-
associated, as
indicated above.
Since telomerase is expressed at high levels in cancer cells, the present
antigens and vaccines are particularly suited for methods of treating and/or
preventing cancer.
A representative method involves administering to a cancer patient an
effective amount of one
or more of the foregoing telomerase antigens. which may be formulated as a
vaccine. Again,
smaller peptide antigens are preferred.
In addition, it is contemplated that the present telomerase antigens and
vaccines will be particularly useful in ex vino techniques. In general, these
techniques entail
isolating cells from a patient, contacting them with a telomerase antigen (or
a vaccine,
including nucleic acid vaccines) and administering the contacted cells back to
the patient. In
some cases (subject, for example, to MHC matching) the cells may be taken from
one patient
for administration to another.
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In one embodiment, autologous or compatible antigen presenting cells (usually
dendritic cells or peripheral blood lymphocwtes) are primed ex vivo, with a
telomerase antigen.
These "telomerase-primed" cells may then be transferred in beneficial amounts
into a patient
in need of therapy or prophylaxis. As with all aspects of the invention, the
ex vivo priming
step may be accomplished using lipid- and or liposome-associated small peptide
antigen.
Yet another adoptive approach is contemplated, whereby antigen presenting
cells are generated, as above, and used to generate autologous or compatible T-
cells effectors
ex vivo. T-cells so generated may be adoptively transferred in beneficial
amounts to a patient
in need. For a description of art-recognized techniques for adoptive T-cell
transfer therapy,
see Bartels, et al. Annals of Surgical Oncology, 3(1):67 (1996), which is
hereby incorporated
by reference.
Co-treatment with other immunostimmulatory, listed above, is also
contemplated. Molecules like IL-2, GM-CSF, IL-12, flt-3 ligand, CD 40, and the
like, are
envisioned as quite useful. IL-2, for example. may be administered
concurrently, separately
or in a combined formulation, or it may be administered in an alternative
dosing regime with
the telomerase antigen or vaccine. In a one method, the IL-2 is formulated
with liposomes.
In a further embodiment of the invention, any of the foregoing antigens or
vaccines may be used in conjunction with known anti-cancer agents. One example
includes
MUC-1-based therapeutics. Numerous additional examples of these are well-known
in the art
Conventional chemotherapeutic agents include alkylating agents,
antimetabolites, various
natural products (e.g., vinca alkaloids, epipodophyllotoxins, antibiotics, and
amino acid-
depleting enzymes), hormones and hormone antagonists. Specific classes of
agents include
nitrogen mustards, alkyl sulfonates, nitrosoureas. triazenes, folic acid
analogues, pyrimidine
analogues, purine analogs, platinum complexes, adrenocortical suppressants,
adrenocorticosteroids, progestins, estrogens, antiestrogens and androgens.
Some exemplary
compounds include cyclophosphamide, chlorambucil, methotrexate, fluorouracil,
cytarabine,
thioguanine, vinblastine, vincristine, doxorubincin, daunorubicin, mitomycin,
cisplatin,
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hydroxyurea, prednisone, hydroxyprogesterone caproate, medroxyprogesterone,
megestrol
acetate, diethyl stilbestrol, ethinyl estradiol. tomoxifen, testosterone
propionate and
fluoxymesterone.
A therapeutically or prophylactically beneficial or effective amount is an
amount sufficient to induce a clinically relevant telomerase-specific T-cell
response, as
defined above. Clinical relevance can be determined by clinician.
Administration may be by any number of routes, including parenteral and oral.
Cell-based vaccines are advantageously administered intravenously. Other
vaccines typically
will be administered intramuscularly, intradermally, subcutaneously or orally.
The skilled
artisan will recognize that the route of administration will vary depending on
the nature of the
vaccine formulation. Determining the optimal route of vaccination may be
determined
empirically and is well within the level of ordinary skill in the art.
Nucleic acid vaccines may also be administered by a variety of routes, the
optimal route being determined empirically. For instance, some antigens have
been found to
elicit a superior cytotoxic response when administered intravenously. Hurpin
et al, sarpra.
For a superior immune response to oral administration, it may be advantageous
to co-
administer with the vaccine a mucosal adjuvant, like cholera toxin or cationic
lipids. Ethchart
et al., J. Gen. Virol. 78: 1577-80 (1997). Intramuscular, intradermal and
subcutaneous
administration are also preferred.
EXA_VIPLES
EXAMPLE 1
This example illustrates the use of the telomerase-specific peptides to
generate
an antigen-specific cytotoxic T-cell response.
A. Materials and Methods
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In general, PCT/US98/09288; Agrawal et al., Int'1 Immuno1.10:1907-16
(1998); and Agrawal et al., Cancer Res. ~~:~ 1 ~ 1-~6 (1998) provide suitable
methods, and
those disclosures are hereby incorporated by reference, in their entirety.
Peptides. Peptides were selected using the HLA peptide search program at
http://www-bimas.dcrt.nih.gov. HLA-A2 specificity was selected, with default
parameters.
Three of the top twenty scores were synthesized and tested. These peptides has
the
sequences: RLVDDFLLV, ELLRSFFYV and ILAKFLHWL.
Preparation of Liposomes. The bulk liquid composition of liposomes consisted
of dipalmitoyl phosphatidyl choline (DPPC), cholesterol (Chol) and dimyristoyl
phosphatidyl
glycerol (DMPG) in a molar ratio of 3:1:0.2 and contained Lipid A at a
concentration of 1%
(w/w) of bulk lipid. Synthetic telomerase peptides were present in the aqueous
phase during
liposome formation at a concentration of 0.7 mgiml, and approximately 28% of
the input
peptide was captured within the liposome structures. The formulated product
contained 2 mg
of bulk lipid, 20 ~g Lipid A and about 20 ~g of peptide per injected dose of
100 ~l.
Bulk lipids and Lipid A were dissolved in chloroform/methanol (methanol was
used initially to solubilize DMPG). The lipid mixturefor each 4 ml
preparationof liposomes
consisted of 64 mg DPPC, 11 mg Chol, 5 my DMPG, 0.8 mg Lipid A in 12 ml of
chloroform/methanol in amolar ratio of 3:1:0.25 at a final lipid concentration
of 30 mM. Each
12 ml of the lipid mixture was dried to a film by rotary evaporation at
53°C in a 250 ml round
bottom flask, and residual solvent was removed under high vacuum. The lipid
film was
hydrated by addition of 4 ml PBS containing the peptide and slow rotation of
the flasks at
53°C followed by 5 cycles of vortexing and warming to 53°C.
Liposome structures were reformed to a more uniform size by a series of 5
freeze/thaw cycles consisting of freezing in a dry ice bath, thawing, warming
to 41 °C and
vortexing before beginning the next cycle. Liposomes then were collected by
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ultracentrifugation at 1500,000 x g at 4°C for 20 minutes, washed twice
by addition of PBS
and ultracentrifugation again. Liposomes were finally reconstituted to the
desired volume.
Cytokines. In order to promote CTL generation, human recombinant
cytokines, IL-12 (R&D Systems, Minneapolis. MN), IL-7 (Intermedico, Markham,
Ontario)
were diluted in serum-free AIM-V media (Life Technologies) just prior to use.
Genercal Procedttresrfor Loacfrng APCs with Liposonae-encapsulated peptide.
Human peripheral blood lymphocytes (PBLs) were purified from heparinized blood
by
centrifugation in Ficoll-Hypaque (Pharmacia. Uppsala, Sweden). The Ficoll-
blood interface
layer obtained by centrifugation was collected and washed twice with RPMI
before use.
Briefly, to 2-10x106 PBLs in 0.9 mL AIM-V media, one dose of liposome
containing peptide formulation was added and the PBLs were incubated overnight
at 37°C
with CO, supplemented incubator. After incubation, the PBLs were treated with
mitomycin C
or y-irradiated (3000 rads) followed by washing v;~ith AIM-V media.
Cvtotoxic T Ivmphocvte assaus. For the CTL assay, three (HLA.A2+) normal
donors' PBLs were used. The T-cells were Grown for five weeks in bulk cultures
as described
above. At the end of two weeks, live T-cells were harvested from flasks and
counted. The
targets were mutant T2 cells. Houbiers et al., Eur. J. Immunol 23:2072-2077
(1993); Stauss
et al., Proc. Natl. Acad. Sci. U.S.A. 89:7871-787 (1992). The telomerase
peptide-mediated
upregulation of HLA.A2 expression on T2 cells was examined using the HLA-A2-
associated
peptides ILAKFLHWL (BPl-187), RLVDDFLLV (BP1-190), and ELLRSFFYV (BP1-191)
using known methods. Townsend et al., Nature 346:476 (1989). T2 cells were
loaded
overnight at 37°C in 7% CO, with various the telomerase synthetic
peptides at 200 pM in
presence of 8 yg exogenous (32 microglobulin. Houbiers et al., supra; Stauss
et al., supra.
The peptide-loaded T2 target cells were loaded with''Cr (using NaCrO~) for 90
minutes and
washed. CTL assays were performed as pre~~iously described. Agrawal et al., J.
Immunol.
156:2089 (1996). Percent specific killing was calculated as: experimental
release -
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spontaneous release/maximum release - spontaneous release x 100. The effector
versus target
ratios used were 50:1, 25:1, 10:1 and S:l . Each group was set up in four
replicate and mean
percent specific killing was calculated.
Cell Sz~r~'ace Immuno~la~orescence Staining. For detection of cell surface
antigens, the peptide-fed T2 cells were washed once in cold PBS containing I%
BSA
followed by addition of 1 pg of anti-A2 monoclonal antibody, .MA2.1 or a
control antibody
and incubated for 45 minutes on ice. Cells were then washed and a secondary
antibody, goat
anti-mouse IgG (H+L)-FITC labeled (Southern Biotech) was added for 30 minutes
on ice.
B. Results
Using the foregoing methods, the cytotoxic activity of T-cells stimulated with
autologous APCs pulsed with liposomal telomerase peptide was determined. The
source of
T-cells was PBLs from three HLA.A2' donors. Target T2 cells (HLA.A2+) were
loaded with
the telomerase peptide indicated above.
The negative control was T2 cells, and the positive control was the 10-mer flu
peptide FLPSDYFPSV, which strongly upreQulates HLA.A2 expression on T2 cells.
These
data confirm that the present methods can be used to generate specific,
biologically relevant
T-cell responses to telomerase, such as cytotoxicity.
Figure 1 shows FACS analysis of cells treated according to the foregoing
methods, panel A shows the negative control. Panel B, is the positive control,
showing a
227% increase in median channel intensity of A2 expression as compared to the
negative
control. Panel C, is the experimental with BPl-187, and shows a 396% increase
in median
channel intensity of A2 expression as compared to the negative control. Panels
D and E show
the results with BPl-190 and BPl-191, respectively yielding 0% and 6%
increases over the
negative control.
Table I shows specific killing of targets by telomerase antigen-primed (BP1-
187) cytotoxic T-cells. The negative control was SIINFEKL.
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These data indicate that, contrary to expectations, an immune response can be
generated against telomerase, a self antigen.
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Table I: Telomerase Peptide-Specific Killing
TARGET EFFECTOR:T.aRGET RATIO PERCENT KILLING
Unloaded X0:1 35.7
Unloaded 25:1 23.8
Unloaded 10:1 13.1
Unloaded 5:1 5.0
Telomerase Peptide-Loaded50:1 56.7
Telomerase Peptide-Loaded25:1 33.5
Telomerase Peptide-Loaded10:1 21.1
Telomerase Peptide-Loaded5:1 15.5
Control Peptide-Loaded X0:1 32.3
Control Peptide-Loaded 25:1 22.1
Control Peptide-Loaded 10:1 11.3
Control Peptide-Loaded ~:1 7.6
*******
The foregoing detailed description and examples are presented for illustrative
purposes only and are not meant to be limiting. Further embodiments of the
invention will be
ready apparent to the skilled worker in view of this disclosure.
?5
