Note: Descriptions are shown in the official language in which they were submitted.
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
COMPOSITIONS AND METHODS FOR GENE-BASED
VACCINES TO PROVOKE T CELL RESPONSES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. ~ 119(e) to U.S.
Provisional Patent Application 60/078,725, filed March 20, 1998, the contents
of
which are hereby incorporated by reference into the present disclosure.
TECHNICAL FIELD
This invention is in the field of molecular immunology and medicine. In
particular, the present invention provides compositions and methods for
inducing
CD4 and CD8 T cell responses in a subject.
BACKGROUND OF THE INVENTION
The mammalian immune system is capable of generating responses to
foreign antigens, to self antigens present on cancerous cells, and to self
antigens
present on normal tissues. The immune system comprises two types of antigen-
specific cells: B cells and T cells. B cells synthesize both membrane-bound
and
secreted antibody. T cells can be characterized phenotypically by the manner
in
which they recognize antigen, by their cell surface markers, and by their
secreted
products. T cells express distinctive membrane molecules. Included among these
are the T cell antigen receptor (TCR), which appears on the cell surface in
association with CD3; and accessory molecules such as CDS, CD28 and CD45R.
Subpopulations of T cells can be distinguished by the presence of additional
membrane molecules. Thus, for example, T cells that express CD4 recognize
antigen associated with class II MHC molecules and generally function as
helper
cells whose roles include enhancement of antibody production by B cells, while
T
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
cells that express CD8 recognize antigen associated with class I MHC molecules
and generally function as cytotoxic cells.
Immune cells recognize discrete sites, known as epitopes or antigenic
determinants, on the antigen. Epitopes are regions of an immunogen or antigen
that bind to antigen-specific membrane-bound receptors on immune cells or to
their soluble counterparts, such as antibodies. Both membrane-bound antibody
on
the surface of a B lymphocyte and secreted antibody recognize soluble antigen.
Unlike B cells, which recognize soluble antigen, T cells recognize antigen
only
when the antigen is associated with self major histocompatibility complex
(MHC)
gene products on the surface of an antigen presenting cell. This antigen can
be
displayed together with MHC molecules on the surface of antigen-presenting
cells
or on virus-infected cells, cancer cells, and grafts.
Disease states can result from invasion by a pathogenic organisms,
including bacterial, viral, and protozoan pathogens, and subsequent
inefficient or
ineffective immune response to the invader. Disease states can also result
from
the activation of self reactive T lymphocytes, from the activation of T
lymphocytes that provoke allergic reactions, or from the activation of
autoreactive
T lymphocytes following certain bacterial and parasitic infections, which can
produce antigens that mimic human protein, rendering these protein
"autoantigens". These diseases include, but are not limited to, the autoimmune
diseases. autoimmune disorders that occur as a secondary event to infection
with
certain bacteria or parasites, T cell mediated allergies, and certain skin
diseases
such as psoriasis and vasculitis. Furthermore, undesired rejection of a
foreign
antigen can result in graft rejection or even infertility, and such rejection
may be
due to activation of specific T lymphocyte populations.
Foreign antigens include macromolecules associated with pathogens such
as bacteria, viruses. and protozoans; allergens; and allografts.
Self antigens, under normal physiological conditions, are usually non-
immunogenic. However, self antigens can also be immunogenic, as is the case
with autoimmune diseases. Autoimmune diseases affect approximately 5% of
adults in Europe and North America, often causing chronic debilitating
illnesses.
2
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
Steinman (1993) ScientifrcAmerican 269:107-114. Autoimmunity is
characterized by activation of auto-reactive clones of T or B cells,
generating
humoral or cell-mediated responses against self antigens.
Tumor cell antigens are also self antigens, and frequently do not elicit an
S immune response that results in elimination of the cancerous cells effective
to
control or eliminate the disease.
In addition, there are other, specific situations in which induction of an
immune response to a seif antigen is desirable. These include the induction of
an
immune response to certain antigens as a means of contraception.
The introduction into an animal of an antigen has been widely used for the
purposes of modulating the immune response, or lack thereof, to the antigen
for a
variety of purposes. These include vaccination against pathogens, induction of
an
immune response to a cancerous cell, reduction of an allergic response,
reduction
of an immune response to a self antigen that occurs as a result of an
autoimmune
disorder, reduction of allograft rejection, and induction of an immune
response to
a self antigen for the purpose of contraception.
A critical target of vaccines is the specialized professional antigen-
presenting cell ("APC"), the most immunologically powerful of which is the
bone
marrow-derived dendritic cell ("DC"). Presentation of antigenic peptides on
the
surface of APCs in the context of MHC class I molecules leads to a cellular
response while presentation of antigenic peptides by MHC class II molecules
provokes a humoral response. Arca et al. (1997) J. Immunol. 20(2):138-45,
examined the interactions of CD4+ and CD8+ T cells involved in the immune
response to a poorly immunogenic tumor transduced to secrete GM-CSF. The
authors report CD4+ and CD8+ cells are independently sensitized during tumor
growth and both have functional capacity as effector cells in adoptive
transfer.
Both CD4+ and CD8+ T cells were induced concurrently against a poorly
immunogenic tumor.
In general, all nucleated cells have the capacity to present endogenously
produced antigen (including, for example, self antigens as well as viral
antigens)
on MHC Class I molecules. Antigen-presenting cells, however, have the capacity
CA 02322659 2000-09-06
WO 99/47641 PCT/US99106030
to present endogenously produced antigen on MHC Class I molecules, and soluble
exogenous (i.e., extracellular) antigens on both MHC CIass I and MHC Class II
molecules, but they generally present soluble exogenous antigens on Class I
molecules relatively inefficiently. Much has been learned in recent years
about
S antigen presenting pathways for both endogenously produced antigen and
soluble
exogenous antigen. Extracellular antigens are internalized by antigen-
presenting
cells and are processed in endocytic vesicles, where they encounter and bind
to
MHC Class II molecules. In all nucleated cells, viral and cellular proteins
synthesized within the cell are hydrolyzed by proteasomes in the cytoplasm
into
peptides, some of which are transported by transporters associated with
antigen
processing (TAPs) into the endoplasmic reticulum, where they encounter and
bind
MHC Class I molecules. Raychaudhuri and Rock (1998) Nature 16:1025-1031;
and Lindauer et al. (1998) 3. Mol. Med. 76:32-47.
It would be beneficial to utilize both pathways, i.e, MHC Class I and Class
II presenting pathways, in the same antigen-presenting cell, to modulate a
humoral and a cellular immune response in a subject against a given antigen.
This
invention provides this and related benefits which have, until this invention,
remained unrealized.
DISCLOSURE OF THE INVENTION
This invention provides a polynucleotide encoding first antigen that is
processed and presented with an MHC Class I molecule on an antigen-presenting
cell (APC), and a second coding sequence encoding a second antigen that is
processed and presented with an MHC Class II molecule on the APC. The first
and second antigens may or may not have the same amino acid sequence. In some
embodiments, a polynucleotide of the invention may comprise: (a) a first
coding
sequence which comprises a nucleotide sequence encoding an antigen; and (b) a
second coding sequence which comprises a nucleotide sequence encoding an
antigen and a nucleotide sequence encoding an amino acid sequence that
promotes retention of the encoded antigen in the endoplasmic reticulum (ER).
4
CA 02322659 2000-09-06
WO 99/47641 PCT/US99106030
In other embodiments, a polynucieotide of the invention may comprise: (a)
a first coding sequence which comprises a nucleotide sequence encoding an
antigen; (b) a second coding sequence which comprises a nucleotide sequence
encoding an antigen and a nucleotide sequence encoding an amino acid sequence
that promotes retention of the encoded antigen in the endoplasmic reticulum
(ER);
and (c) a third coding sequence which comprises a nucleotide sequence encoding
an antigen and a nucleotide sequence encoding an amino acid sequence that
directs (or promotes retention of, or targets) the encoded antigen into a non-
endosomal MHC Class II pathway.
In some of these embodiments, the encoded antigen is a naturally
occurring antigen or fragment of a naturally occurring antigen. In other
embodiments, the antigen is a synthetic antigen. An encoded antigen can be
secreted or a cell-surface protein.
The invention further provides methods of increasing antigen presentation
on the surface of an APC, comprising introducing a polynucleotide of the
invention into the APC under conditions which favor expression of the
polynucleotide. The invention also provides APCs produced by these methods.
These APCs have enhanced presentation of antigen encoded by a polynucleotide
of the invention in both Class I and Class II MHC molecules.
Polynucleotides of the invention are useful in a variety of methods of
modulating an immune response to the antigen thereby encoded. In some
... . embodiments, the polynucleotides of the invention encode tumor antigens,
or
synthetic antigens corresponding to tumor antigens, and are thus useful as
vaccines against tumor cells expressing cell surface tumor antigen. In other
embodiments, the polynucleotides of the invention encode self antigens on
normal
(i.e., non-cancerous) tissues. In other embodiments, the polynucleotides of
the
invention encode foreign (non-self] antigens, such as those associated with
organisms such as pathogenic bacteria, viruses and protozoans. In still other
embodiments, the polynucleotides of the invention encode antigens present on
allografts which mediate their rejection.
5
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
The invention further provides polypeptides encoded by polynucleotides
of the invention, and compositions comprising the polypeptides. The
polypeptides are useful in immunomodulatory methods of the invention.
Compositions containing the polynucleotides are further provided by this
invention. The polynucleotides can be contained in gene delivery vehicles such
as
viral vectors and liposomes, and in host cells. These polynucleotides are
useful
diagnostically and therapeutically, as well as in methods of expressing the
polynucleotide for reproduction, expression and purification of the antigenic
products.
The polynucleotides can be used to modulate an immune response to an
antigen in a subject. Accordingly, the invention further provides methods for
modulating a cellular and/or a humoral immune response to the antigen in a
subject by administering to the subject an effective amount of the
polynucleotide.
The polynucleotide can be delivered as naked DNA or in a gene delivery
vehicle.
In one aspect, a host cell containing the polynucleotide is administered to
the
subject.
BRIEF DESCRIPTION OF THE FIGURE
The figure is a schematic of one embodiment of the polynucleotide of this
invention. The figure shows a single transcription cassette that encodes
intracellular and secreted forms of the same antigen. This polynucleotide can
be
. . .. . . .. . incorporated into a replication defective adenoviral vector
deleted in the El, E3
and/E4 regions. A suitable helper cell line is utilized to produce infectious
virions. Alternatively, a helper plasmid or helper virus can be co-transfected
with
the viral vector so that infectious, replication defective virions capable of
expressing the polynucleotide are produced.
MODES FOR CARRYING OUT THE INVENTION
Throughout this disclosure, various publications, patents and published
patent specifications are referenced by an identifying citation. The
disclosures of
these publications, patents and published patent specifications are hereby
6
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
incorporated by reference into the present disclosure to more fully describe
the
state of the art to which this invention pertains.
Definitions
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of immunology, molecular bioiogy,
microbiology, cell biology and recombinant DNA, which are within the skill of
the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A
LABORATORY MANUAL, 2"d edition (1989}; CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (1987) and updates); the series
METHODS IN ENZYMOLOGY (Academic Press. Inc.): PCR 2: A PRACTICAL
APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. ( 1995)}, Harlow
and Lane eds. (1989) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL
CULTURE (R.I. Freshney ed. (1987)).
As used herein, certain terms may have the following defined meanings.
As used in the specification and claims, the singular form "a", "an" and
"the" include plural references unless the context clearly dictates otherwise.
For
example, the term "a cell" includes a plurality of cells, including mixtures
thereof.
The terms "polynucleotide" and "nucleic acid molecule" are used
interchangeably to refer to polymeric forms of nucleotides of any length. The
polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or
their
.. . . analogs. Nucleotides may have any three-dimensional structure, and may
perform .
any function, known or unknown. The term "polynucleotide" includes, for
example, single-, double-stranded and triple helical molecules, a gene or gene
fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of
any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
A
nucleic acid molecule may also comprise modified nucleic acid molecules.
Hybridization refers to a reaction in which
one or more polynucleotides react to form a complex that is
stabilized via hydrogen bonding between the bases of the nucleotide residues.
7
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any other sequence-specific manner. The complex may comprise
two strands forming a duplex structure, three or more strands forming a
multi-stranded complex, a single self hybridizing strand, or any combination
of
these. A hybridization reaction may constitute a step in a more extensive
process,
such as the initiation of a PCR reaction, or the enzymatic cleavage of a
polynucleotide by a ribozyme.
Examples of stringent hybridization conditions include: incubation
temperatures of about 25°C to about 37°C; hybridization buffer
concentrations of
about 6 X SSC to about 10 X SSC; formamide concentrations of about 0% to
about 25%; and wash solutions of about 6 X SSC. Examples of moderate
hybridization conditions include: incubation temperatures of about 40°C
to about
50°C; buffer concentrations of about 9 X SSC to about 2 X SSC;
formamide
concentrations of about 30% to about 50%; and wash solutions of about 5 X SSC
to about 2 X SSC. Examples of high stringency conditions include: incubation
temperatures of about 55°C to about 68°C; buffer concentrations
of about 1 X
SSC to about 0.1 X SSC; formamide concentrations of about 55% to about 75%;
and wash solutions of about 1 X SSC, 0.1 X SSC, or deionized water. In
general,
hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or
more
washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is
0.15 M NaCI and 15 mM citrate buffer. It is understood that equivalents of SSC
. . . , using other buffer systems can be employed. _ , , . . .. .
A polynucleotide or polynucleotide region (or a polypeptide or
polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or
95%) of "sequence identity" to another sequence means that, when aligned, that
percentage of bases (or amino acids) are the same in comparing the two
sequences. This alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example those
described in Current Protocols in Molecular Biology (F.M. Ausubel et al.,
eds.,
1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default
parameters
are used for alignment. A preferred alignment program is BLAST, using default
8
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
parameters. In particular, preferred programs are BLASTN and BLASTP, using
the following default parameters: Genetic code = standard; filter = none;
strand =
both; cutoff= 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50
sequences; sort by = HIGH SCORE: Databases = non-redundant. GenBank +
EMBL + DDBJ + pDB + GenBank CDS translations + SwissProtein + SPupdate
+ PIR. Details of these programs can be found at the following Internet
address:
http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
As used herein, the term "classical MHC Class I processing pathway"
intends that a protein is processed within a proteasome, transported to the
ER,
where it associates with an MHC Class I molecule.
As used herein, the term "an endoplasmic reticulum retention sequence"
intends an amino acid sequence which, when in the same translation unit with
an
amino acid sequence of a peptide antigen, increases the probability that the
antigen will be located in the endoplasmic reticulum (ER) of a eukaryotic
cell,
compared with the same antigen not in the same translation unit with the amino
acid sequence. "Increases the probability that the antigen will be located in
the
ER" intends that a given antigen which is in the same translation unit with an
amino acid sequence of an amino acid sequence which promotes retention in the
ER will be found in the ER at levels at least about 2-fold, more preferably at
least
about 5-fold, even more preferably at least about 10-fold or higher that the
same
antigen which is in a translation unit lacking the amino acid sequence which
.promotes retention in the ER. Methods of determining,whether an antigen is in
."
the endoplasmic reticulum are known in the art and include, but are not
limited to,
a process involving disrupting the cell, fractionating the subcellular
components,
and determining in which subcellular compartment the protein is located by,
e.g.,
immunoassay.
As used herein, the term "classical MHC Class II processing pathway"
intends that a protein is taken up by an antigen-presenting cell by
endocytosis or
phagocytosis and the protein is processed in an endosome, before associating
with
MHC Class II molecules. This is also referred to herein as "processing by the
endosomal pathway". In the present invention, the terms "endosome", "endocytic
9
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
compartment", "endocytic vesicle", and "endosomal compartment' are used
interchangeably.
As used herein, the term "an amino acid sequence that promotes
processing of a protein by a non-endosomal MHC Class II pathway", is an amino
acid sequence which, when in the same translation unit with an amino acid
sequence of a peptide antigen, increases the probability that the antigen will
be
located in a subcellular compartment of a eukaryotic cell which is nat an
endosome, and where processing of the protein and subsequent association with
MHC CIass II molecules can occur, compared with the same antigen not in the
same translation unit with the amino acid sequence. "Increases the
probability"
intends that a given antigen which is in the same translation unit with an
amino
acid sequence of an amino acid sequence which promotes retention in a non-
endosomal subcellular compartment where processing and subsequence
association with MHC Class II molecules can occur, will be found in such a
subcellular compartment at levels at least about 2-fold, more preferably at
least
about 5-fold, even more preferably at least about 10-fold or higher that the
same
antigen which is in a translation unit lacking the amino acid sequence which
promotes retention in such a subcellular compartment. Non-endosomal
subcellular compartments where processing and subsequence association with
MHC Class II molecules can occur include, but are not limited to. lysosomes,
Golgi, and melanosomes. Methods to determine whether a protein is in a given
subcellular compartment are described above.
The term "antigen" is well understood in the art and includes substances
which are immunogenic, i.e., immunogens, as well as substances which induce
immunological unresponsiveness, or anergy, i.e., anergens. An antigen may
comprise one or more antigenic determinants, or epitopes. In the description
of
the present invention, the terms "antigen", "antigenic determinant'', and
"epitope"
are used interchangeably. As used herein, the term "antigen" includes
naturally
occurring antigens, synthetic antigens, foreign antigens, self antigens,
modified
self antigens, and altered antigens.
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
A ''native" or "natural" antigen is a polypeptide, protein or a fragment
which contains an epitope, which has been isolated from a natural biological
source, and which can specifically bind to an antigen receptor, in particular
a T
cell antigen receptor (TCR), in a subject.
A synthetic antigen is said to "correspond" to a native epitope if the
peptide binds to the same TCR as the natural epitope.
The term "peptide", used interchangeably herein with "polypeptide" is
used in its broadest sense to refer to a compound of two or more subunit amino
acids, amino acid analogs, or peptidomimetics. The subunits may be linked by
peptide bonds. In another embodiment, the subunit may be linked by other
bonds,
e.g. ester. ether. etc. As used herein the term "amino acid" refers to either
natural
and/or unnatural or synthetic amino acids, including glycine and both the D or
L
optical isomers. and amino acid analogs and peptidomimetics. A peptide of
three
or more amino acids is commonly called an oligopeptide if the peptide chain is
short. If the peptide chain is long, the peptide is commonly called a
polypeptide
or a protein.
The term "sequence motif' refers to a pattern present in a group of
molecules (e.g., amino acids or nucleotides). A typical pattern may be
identified
by characteristic amino acid residues, such as hydrophobic, hydrophilic,
basic,
acidic. and the like.
A "signal sequence" is a short amino acid sequence that directs newly
synthesized secretory or membrane proteins to and through cellular membranes,
including, but not limited to, the endoplasmic reticulum, and an endosomal
compartment (or endosome). Signal sequences can be in the amino-terminal (N-
terminal), and/or the carboxy-terminal (C-terminal) portion of a polypeptide
and
are generally cleaved after the polypeptide has crossed the membrane.
A ''gene delivery vehicle" is defined as any molecule that can carry
inserted polynucleotides into a host cell. Examples of gene delivery vehicles
are
liposomes, biocompatible polymers, including natural polymers and synthetic
polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides;
artificial viral envelopes; metal particles; and bacteria, viruses, such as
11
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal
vectors and other recombination vehicles typically used in the art which have
been
described for expression in a variety of eukaryotic and prokaryotic hosts, and
may
be used for gene therapy as well as for simple protein expression.
The term "antigen-presenting matrix", as used herein, intends a molecule
or molecules which can present antigen in such a way that the antigen can be
bound by a T-cell antigen receptor on the surface of a T cell. An antigen-
presenting matrix can be on the surface of an antigen-presenting cell (APC),
on a
vesicle preparation of an APC, or can be in the form of a synthetic matrix on
a
solid support such as a bead or a plate. An example of a synthetic antigen-
presenting matrix is purified MHC class I molecules complexed to /32-
microglobulin, or purified MHC Class II molecules, or functional portions
thereof, attached to a solid support.
The term "antigen presenting cell", as used herein, intends any cell which
presents on its surface an antigen in association with a major
histocompatibility
complex molecule, or portion thereof, or, alternatively, one or more non-
classical
MHC molecules, or a portion thereof. Examples of suitable APCs are discussed
in detail below and include, but are not limited to, whole cells such as
macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen
presenting
cells. Methods of making hybrid APCs have been described. see, for example,
International Patent Application No. WO 98/46785; and WO 95/16775.
Dendritic cells (DCs) are potent antigen-presenting cells. It has been
shown that DCs provide all the signals required for T cell activation and
proliferation. These signals can be categorized into two types. The first
type,
which gives specificity to the immune response, is mediated through
interaction
between the T-cell receptor/CD3 ("TCR/CD3") complex and an antigenic peptide
presented by a major histocompatibility complex ("MHC") class I or II protein
on
the surface of APCs. This interaction is necessary, but not sufficient, for T
cell
activation to occur. In fact, without the second type of signals, the first
type of
signals can result in T cell anergy. The second type of signals, called co-
stimulatory signals, is neither antigen-specific nor MHC-restricted, and can
lead
12
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
to a full proliferation response of T cells and induction of T cell effector
functions
in the presence of the first type of signals. As used herein, "dendritic cell"
is to
include. but not be limited to a pulsed dendritic cell, a foster cell or a
dendritic cell
hybrid.
A "foster antigen presenting cell" is a genetically modified dendritic cell
that, from the restricted association of endogeneous peptides, will take up
and
present exogeneous antigen on the cell surface.
The terms "major histocompatibility complex" or "MHC" refers to a
complex of genes encoding cell-surface molecules that are required for antigen
presentation to T cells and for rapid graft rejection. In humans, the MHC
complex
is also known as the HLA complex. The proteins encoded by the MHC complex
are known as "MHC molecules" and are classified into class I and class II MHC
molecules. Class I MHC molecules include membrane heterodimeric proteins
made up of an a chain encoded in the MHC associated noncovalently with ~i2-
microglobulin. Class I MHC molecules are expressed by nearly all nucleated
cells and have been shown to function in antigen presentation to CD8+ T cells.
Class I molecules include HLA-A, -B, and -C in humans. Class II MHC
molecules also include membrane heterodimeric proteins consisting of
noncovalently associated I and ~ chains. Class II MHC are known to be present
on CD4+ T cells and, in humans, include HLA-DP, -DQ, and DR. The term
"MHC restriction" refers to a characteristic of T cells that permits them to
recognize antigen only after it is processed and the resulting antigenic
peptides are
displayed in association with either a class I or class lI MHC molecule.
Methods
of identifying and comparing MHC are well known in the art and are described
in
Allen et al. (1994) Human Imm. 40:25-32; Santamaria et al. (1993) Human Imm.
37:39-50 and HurIey et al. (1997) Tissue Antigens 50:401-415.
"Co-stimulatory molecules" are involved in the interaction between
receptor-ligand pairs expressed on the surface of antigen presenting cells and
T
cells. Research accumulated over the past several years has demonstrated
convincingly that resting T cells require at least two signals for induction
of
cytokine gene expression and proliferation (Schwartz, R.H. ( 1990) Science
13
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
248:1349-1356; Jenkins, M.K. (1992) Immunol. Today 13:69-73). One signal, the
one that confers specificity, can be produced by interaction of the TCR/CD3
complex with an appropriate MHC/peptide complex. The second signal is not
antigen specific and is termed the "co-stimulatory" signal. This signal was
originally defined as an activity provided by bone-marrow-derived accessory
cells
such as macrophages and dendritic cells, the so called "professional" APCs.
Several molecules have been shown to enhance co-stimulatory activity. These
are
heat stable antigen (HSA) (Liu, Y., et al. (1992) J. Exp. Med. 175:437-445),
chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas, M.F., et
al.
(1993) Cel174:257-268), intracellular adhesion molecule 1 (ICAM-1) (Van
Seventer, G.A. ( 1990) J. Immunol. 144:4579-4586), B7-l, and B7-2/B70
(Schwartz, R.H. ( 1992) Cell 71:1065-1068). These molecules each appear to
assist co-stimulation by interacting with their cognate ligands on the T
cells. Co-
stimulatory molecules mediate co-stimulatory signals) which are necessary,
under normal physiological conditions, to achieve full activation of naive T
cells.
One exemplary receptor-ligand pair is the B7 co-stimulatory molecule on the
surface of APCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et
al. ( 1993 ) Science 262: 909-911; Young et al . ( 1992) J. CI in. Invest. 90:
229;
Nabavi et al. (1992) Nature 360:266-268). Other important co-stimulatory
molecules are CD40, CD54, CD80, CD86. The term "co-stimulatory molecule"
encompasses any single molecule or combination of molecules which, when
acting together with a peptide/MHC complex bound by a TCR on the surface of a
T cell, provides a co-stimulatory effect which achieves activation of the T
cell that
binds the peptide. The term thus encompasses B7, or other co-stimulatory
molecules) on an antigen-presenting matrix such as an APC, fragments thereof
(alone. complexed with another molecule(s), or as part of a fusion protein)
which,
together with peptide/MHC complex, binds to a cognate ligand and results in
activation of the T cell when the TCR on the surface of the T cell
specifically
binds the peptide. Co-stimulatory molecules are commercially available from a
variety of sources, including, for example, Beckman Coulter. It is intended,
although not always explicitly stated, that molecules having similar
biological
14
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
activity as wild-type or purified co-stimulatory molecules (e.g.,
recombinantly
produced or muteins thereof) are intended to be used within the spirit and
scope of
the invention.
As used herein, "solid phase support'' or "solid support", used
interchangeably, is not limited to a specific type of support. Rather a large
number of supports are available and are known to one of ordinary skill in the
art.
Solid phase supports include silica gels, resins, derivatized plastic films,
glass
beads, cotton, plastic beads, alumina gels. As used herein, "solid support"
also
includes synthetic antigen-presenting matrices, cells, and liposomes. A
suitable
solid phase support may be selected on the basis of desired end use and
suitability
for various protocols. For example, for peptide synthesis, solid phase support
may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem
Inc., Peninsula Laboratories, etc.), POLYHIPE~ resin (obtained from Aminotech,
Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene
1 S resin grafted with polyethylene glycol (TentaGel~, Rapp Polymere,
Tubingen,
Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch,
California).
The term "genetically modified" means containing and/or expressing a
foreign gene or nucleic acid sequence which in turn, modifies the genotype or
phenotype of the cell or its progeny. In other words, it refers to any
addition,
deletion or disruption to a cell's endogenous nucleotides.
As used herein, the term "cytokine" refers to any one of the numerous
factors that exert a variety of effects on cells, for example, inducing growth
or
proliferation. Non-limiting examples of cytokines which may be used alone or
in
combination in the practice of the present invention include, interleukin-2
(IL-2),
stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6),
interleukin 12
(IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF),
interleukin-I alpha (IL-II ), interleukin-11 (IL-I I), MIP-lI , leukemia
inhibitory
factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The present
invention also includes culture conditions in which one or more cytokine is
specifically excluded from the medium. Cytokines are commerciaily available
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
from several vendors such as, for example, Genzyme (Framingham, MA),
Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D
Systems and Immunex (Seattle, WA). It is intended, although not always
explicitly stated, that molecules having similar biological activity as wild-
type or
purif ed cytokines (e.g., recombinantly produced or muteins thereof) are
intended
to be used within the spirit and scope of the invention.
"Vector" means a self replicating nucleic acid molecule that transfers an
inserted nucleic acid molecule into and/or between host cells. The term is
intended to include vectors that function primarily for insertion of a nucleic
acid
molecule into a cell, replication vectors that function primarily for the
replication
of nucleic acid and expression vectors that function for transcription and/or
translation of the DNA or RNA. Also intended are vectors that provide more
than
one of the above functions.
"PCR primers" refer to primers used in "polymerase chain reaction" or
"PCR," a method for amplifying a DNA base sequence using a heat-stable
polymerase such as Taq polymerase, and two oligonucleotide primers, one
complementary to the (+)-strand at one end of the sequence to be amplified and
the other complementary to the (- )-strand at the other end. Because the newly
synthesized DNA strands can subsequently serve as additional templates for the
same primer sequences, successive rounds of primer annealing, strand
elongation,
and dissociation produce exponential and highly specific amplification of the
desired sequence. (See, e.g., PCR 2: A PRACTICAL APPROACH, supra). PCR also
can be used to detect the existence of the defined sequence in a DNA sample.
"Host cell" is intended to include any individual cell or cell culture which
can be or have been recipients for vectors or the incorporation of exogenous
nucleic acid molecules, polynucleotides and/or proteins. It also is intended
to
include progeny of a single cell, and the progeny may not necessarily be
completely identical (in morphology or in genomic or total DNA complement) to
the original parent cell due to natural, accidental, or deliberate mutation.
The cells
may be procaryotic or eucaryotic, and include but are not limited to bacterial
cells,
yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or
human.
16
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
An "antibody" is an immunoglobulin molecule capable of binding an
antigen. As used herein, the term encompasses not only intact immunoglobulin
molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion
proteins,
humanized proteins and modifications of the immunoglobulin molecule that
comprise an antigen recognition site of the required specificity.
An "antibody complex" is the combination of antibody (as defined above)
and its binding partner or ligand.
The term "immunomodulatory agent", as used herein, is a molecule, a
macromolecular complex, or a cell that modulates an immune response and
encompasses an antigenic peptide of the invention alone or in any of a variety
of
formulations described herein; a polypeptide comprising an antigenic peptide
of
the invention; a polynucleotide encoding a peptide or polypeptide of the
invention; an antigenic peptide of the invention bound to a Class I or a Class
II
MHC molecule on an antigen-presenting matrix, including an APC and a
synthetic antigen-presenting matrix (in the presence or absence of co-
stimulatory
molecule(s)); an antigenic peptide of the invention covalently or non-
covalently
complexed to another molecules) or macromolecular structure; and an educated,
antigen-specific immune effector cell which is specific for a peptide of the
invention.
The term ''modulate an immune response'' includes inducing (increasing,
eliciting) an immune response; and reducing (suppressing) an immune response.
An immunomodulatory method (or protocol) is one that modulates an immune
response in a subject.
As used herein, the term "inducing an immune response in a subject" is a
term well understood in the art and intends that an increase of at least about
2-
fold, more preferably at least about 5-fold, more preferably at least about 10-
fold,
more preferably at least about 100-fold, even more preferably at least about
500-
fold, even more preferably at least about 1000-fold or more in an immune
response to an antigen (or epitope) can be detected (measured), after
introducing
the antigen (or epitope) into the subject, relative to the immune response (if
any)
before introduction of the antigen (or epitope) into the subject. An immune
17
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
response to an antigen (or epitope), includes, but is not limited to,
production of
an antigen-specific (or epitope-specific) antibody, and production of an
immune
cell expressing on its surface a molecule which specifically binds to an
antigen (or
epitope). Methods of determining whether an immune response to a given antigen
(or epitope) has been induced are well known in the art. For example, antigen-
specific antibody can be detected using any of a variety of immunoassays known
in the art, including, but not limited to, ELISA, wherein, for example,
binding of
an antibody in a sample to an immobilized antigen (or epitope) is detected
with a
detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig
antibody). Immune effector cells specific for the antigen can be detected any
of a
variety of assays known to those skilled in the art, including, but not
limited to,
FACS, or. in the case of CTLs, '~Cr-release assays, or 3H-thymidine uptake
assays.
The term "immune effector cells" refers to cells capable of binding an
antigen or which mediate an immune response. These cells include, but are not
limited to. T cells, B cells. monocytes, macrophages, NK cells and cytotoxic T
lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor,
inflammatory, or other infiltrates. Certain diseased tissue expresses specific
antigens and CTLs specific for these antigens have been identified. For
example,
approximately 80% of melanomas express the antigen known as gp100.
The term "immune effector molecule", as used herein, refers to molecules
capable of antigen-specific binding, and includes antibodies, T cell antigen
receptors, and MHC Class I and Class II molecules.
A "naive" immune effector cell is an immune effector cell that has never
been exposed to an antigen.
As used herein, the term ''educated, antigen-specific immune effector
cell", is an immune effector cell as defined above, which has encountered
antigen
and which is specific for that antigen. An educated, antigen-specific immune
effector cell may be activated upon binding antigen. "Activated" implies that
the
cell is no longer in Go phase, and begins to produce cytokines characteristic
of the
cell type. For example, activated CD4+ T cells secrete IL-2 and have a higher
18
CA 02322659 2000-09-06
WO 99/47641 PG"TNS99/06030
number of high affinity IL-2 receptors on their cell surfaces relative to
resting
CD4+ T cells.
A peptide or polypeptide of the invention may be preferentially recognized
by antigen-specific immune effector cells, such as B cells and T cells. In the
S context of T cells, the term "recognized" intends that a peptide or
polypeptide of
the invention, comprising one or more antigenic epitopes, is recognized, i.e.,
is
presented on the surface of an APC together with (i.e., bound to) an MHC
molecule in such a way that a T cell antigen receptor (TCR) on the surface of
an
antigen-specific T cell binds to the epitope wherein such binding results in
activation or anergy of the T cell. The term "preferentially recognized"
intends
that a polypeptide of the invention is substantially not recognized, as
defined
above. by a T cell specific for an unrelated antigen. Assays for determining
whether an epitope is recognized by an antigen-specific T cell are known in
the art
and are described herein.
The term "autogeneic", or "autologous", as used herein, indicates the
origin of a cell. Thus, a cell being administered to an individual (the
"recipient")
is autogeneic if the cell was derived from that individual (the "donor") or a
genetically identical individual. An autogeneic cell can also be a progeny of
an
autogeneic cell. The term also indicates that cells of different cell types
are
derived from the same donor or genetically identical donors. Thus, an effector
cell and an antigen presenting cell are said to be autogeneic if they were
derived
from the same donor or from an individual genetically identical to the donor,
or if
they are progeny of cells derived from the same donor or from an individual
genetically identical to the donor.
Similarly, the term "allogeneic", as used herein, indicates the origin of a
cell. Thus, a cell being administered to individual (the "recipient") is
allogeneic if
the cell was derived from an individual not genetically identical to the
recipient;
in particular, the term relates to non-identity in expressed MHC molecules. An
allogeneic cell can also be a progeny of an allogeneic cell. The term also
indicates that cells of different cell types are derived from genetically non-
identical donors, or if they are progeny of cells derived from genetically non-
19
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
identical donors. For example, an APC is said to be allogeneic to an effector
cell
if they are derived from genetically non-identical donors.
As used herein, the term "a disease or condition related to a population of
CD4+ or CD8+ T cells" is one which can be related to a population of CD4+ or
S CD8+ T cells, such that these cells are primarily responsible for the
pathogenesis
of the disease; it is also one in which the presence of CD4+ or CD8+ T cells
is an
indicia of a disease state; it is also one in which the presence of a
population CD4+
or CD8+ T cells is not the primary cause of the disease, but which plays a key
role
in the pathogenesis of the disease; it is also one in which a population of
CD4+ or
CD8+ T cells mediates an undesired rejection of a foreign antigen. Examples of
a
condition related to a population of CD4+ or CD8+T cells include. but are not
limited to, autoimmune disorders, graft rejection, immunoregulatory disorders,
and anaphylactic disorders.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor
cells", "cancer" and "cancer cells", (used interchangeably) refer to cells
which
exhibit relatively autonomous growth, so that they exhibit an aberrant growth
phenotype characterized by a significant loss of control of cell proliferation
(i.e.,
de-regulated cell division). Neoplastic cells can be malignant or benign.
"Suppressing" tumor growth indicates a growth state that is curtailed when
compared to growth without contact with educated. antigen-specific immune
effector cells described herein. Tumor cell growth can be assessed by any
means
known in the art, .including, but not limited to, measuring tumor size,
determining
whether tumor cells are proliferating using a 3H-thymidine incorporation
assay, or
counting tumor cells. "Suppressing" tumor cell growth means any or all of the
following states: slowing, delaying, and stopping tumor growth, as well as
tumor
shrinkage.
The term "culturing'' refers to the in vitro propagation of cells or
organisms on or in media of various kinds. It is understood that the
descendants
of a cell grown in culture may not be completely identical (either
morphologically, genetically, or phenotypically) to the parent cell. By
"expanded" is meant any proliferation or division of cells.
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
A "subject" is a vertebrate, preferably a mammal, more preferably a
human. Mammals include, but are not limited to, marines, simians. humans, farm
animals, sport animals, and pets.
As used herein, "expression" refers to the process by which
polynucleotides are transcribed into mRNA and translated into peptides,
polypeptides, or proteins. If the polynucleotide is derived from genomic DNA,
expression may include splicing of the mRNA, if an appropriate eukaryotic host
is
selected. Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation sequences for
ribosome binding. For example, a bacterial expression vector includes a
promoter
such as the lac promoter and for transcription initiation the Shine-Dalgarno
sequence and the start codon AUG (Sambrook et al. (1989) supra ). Similarly,
an
eukaryotic expression vector includes a heterologous or homologous promoter
for
RNA polymerase II, a downstream polyadenylation signal, the start codon AUG,
and a termination codon for detachment of the ribosome. Such vectors can be
obtained commercially or assembled by the sequences described in methods well
known in the art, for example, the methods described below for constructing
vectors in general.
An "isolated" or "purified" population of cells, nucleic acids, peptides or
proteins that is individually substantially free of cells and materials with
which it
is associated in nature. By substantially free or substantially purified is
meant at
. list SD% of the composition contains the population.of interest,
preferably.at
least 70%, more preferably at least 80%, and even more preferably at least 90%
free of molecules which associate with the molecule of interest in nature.
A "composition" is intended to mean a combination of active agent and
another compound or composition, inert (for example, a detectable agent or
label)
or active, such as an adjuvant.
A "pharmaceutical composition" is intended to include the combination of
an active agent with a carrier, inert or active, making the composition
suitable for
diagnostic or therapeutic use in vitro, in vivo or ex vivv.
21
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
As used herein, the term "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers. such as a phosphate
buffered saline solution, water, and emulsions, such as an oil/water or
water/oil
emulsion, and various types of wetting agents. The compositions also can
include
stabilizers and preservatives. For examples of Garners, stabilizers and
adjuvants,
see Martin, REMINGTON'S PHARM. Scl., 15th Ed. (Mack Publ. Co., Easton (1975)).
As used herein, the term "comprising" is intended to mean that the
compositions and methods include the recited elements, but not excluding
others.
"Consisting essentially of when used to define compositions and methods, shall
mean excluding other elements of any essential significance to the
combination.
Thus, a composition consisting essentially of the elements as defined herein
would not exclude trace contaminants from the isolation and purification
method
and pharmaceutically acceptable carriers, such as phosphate buffered saline,
preservatives, and the like. "Consisting of" shall mean excluding more than
trace
I 5 elements of other ingredients and substantial method steps for
administering the
compositions of this invention. Embodiments defined by each of these
transition
terms are within the scope of this invention.
An "effective amount" is an amount sufficient to effect beneficial or
desired results. An effective amount can be administered in one or more
administrations, applications or dosages.
This invention provides improved vaccines and, methods of using the
vaccines to modulate cellular and humoral immune responses to an antigen or
antigens in a subject. An antigen, for the purposes of the present invention,
is a
protein antigen. The term "protein antigen'' is used in its broadest sense and
includes minimal epitopes, chimeric molecules, synthetic antigens, in addition
to
isolated full length proteins. The epitopes can also be derived altered
antigens.
While the embodiments described below are directed to tumor antigens. it
should
be understood, although not explicitely stated, that any protein or
polypeptide
which induces an immune response is intended to be within the scope of this
invention. Such antigens include, but are not limited to tumor antigens, viral
22
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
antigens, bacterial antigens, and self antigens. The antigen of this vaccine
may be
autologous or heterologous (i.e., allogeneic) or a homolog from a isolated
species,
e.g., a murine antigen administered to a human patient.
Polynucleotides of the invention
The poiynucleotides of this invention comprise at least two coding
sequences. The first sequence encodes a first antigen that is processed and
presented with an MHC Class I molecule on an antigen-presenting cell (APC).
The second coding sequence codes for second antigen that is processed and
presented with an MHC Class II molecule on the APC. In some embodiments, the
first and second antigens have the same amino acid sequence. In an alternative
embodiment, the polynucleotides encode antigens comprising epitopes that are
recognized by the same T cell receptor. In a further embodiment. encoded
antigens comprise different epitopes from the same native protein. In a
further
embodiment, the first and second antigens share at least about SO% amino acid
sequence identity with one another, as determined using alignment programs
using default parameters, or are encoded by polynucleotides that hybridize to
each
other under stringent conditions.
A polynucleotide of the invention may comprise:
(a) a first coding sequence which comprises a nucleotide sequence
encoding an antigen; and
(b) a second coding sequence which comprises a nucleotide
sequence encoding an antigen and a nucleotide sequence encoding an amino acid
sequence that promotes retention of the encoded antigen in the endoplasmic
reticulum (ER).
The polynucleotide can be constructed such that the amino acid sequence
that promotes retention of the encoded antigen in the ER is appended to the N-
terminus or the C-terminus of the encoded antigen. Alternatively, the sequence
that promotes retention of the encoded antigen in the ER can occur internally,
i.e.,
between the N-terminus and C-terminus of the encoded antigen.
23
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
A number of amino acid sequences that promote retention in the ER are
known in the art. For example, the amino acid sequence Lys-Asp-Glu-Leu
(KDEL), when appended to the carboxy-terminus of a protein. promotes retention
of that protein in the ER. Pelham ( 1990) Trends in Biochemical Sciences
15:483-
486. Others have reported that the carboxy-terminal peptide His-Asp-Glu-Phe
(HDEF), promotes retention (targets) the appended protein to the ER. In
addition
to the "KDEL" and "HDEL" sequences, other sequences such as "DDEL",
"ADEL", "SDEL", "RDEL", "KEEL", "QEDL", "HIEL", "HTEL" and "KQDL"
are known as signals for staying in endoplasmic reticulum, and polypeptides
having these sequences are generally retained in the ER. Pelham (1990) TIBS,
15:483. Accordingly, in some embodiments, the amino acid sequence that
promotes retention of the encoded antigen in the ER is selected from the group
consisting of KDEL, HDEL, DDEL, ADEL, SDEL, RDEL, KEEL, QEDL,
HIEL, HTEL, and KQDL
In some embodiments, the amino acid sequence that promotes retention of
the encoded antigen in the endoplasmic reticulum (ER) is KDEL. In these
embodiments, when the antigen encoded by the coding sequence which comprises
a nucleotide sequence encoding an antigen and a nucleotide sequence encoding
KDEL is transcribed and translated, the translation unit comprises the antigen
having KDEL as the carboxy-terminal amino acids.
In other embodiments, the amino acid sequence that promotes retention of
the encoded antigen in the endoplasmic reticulum (ER) is HDEF. In these
embodiments, when the antigen encoded by the coding sequence which comprises
a nucleotide sequence encoding an antigen and a nucleotide sequence encoding
HDEF is transcribed and translated, the translation unit comprises the antigen
having HDEF as the carboxy-terminal amino acids.
In other embodiments, a polynucleotide of the invention comprises:
(a) a first coding sequence which comprises a nucleotide sequence
encoding an antigen;
(b) a second coding sequence which comprises a nucleotide sequence
encoding an antigen and a nucleotide sequence encoding an amino acid sequence
24
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
that promotes retention of the encoded antigen in the endoplasmic reticulum
(ER);
and
(c) a third coding sequence which comprises a nucleotide sequence
encoding an antigen and a nucleotide sequence encoding an amino acid sequence
that directs (or promotes retention of, or targets) the encoded antigen into a
non-
endosomal MHC Class II pathway.
The polynucleotide can be constructed such that the amino acid sequence
that directs the encoded antigen into a non-endosomal MHC Class II pathway is
appended to the N-terminus or the C-terminus of the encoded antigen.
Alternatively, the sequence that directs the encoded antigen into a non-
endosomal
MHC Class II pathway can occur internally, i.e., between the N-terminus and C-
terminus of the encoded antigen.
A non-endosomal MHC Class II pathway includes, but is not limited to, a
lysosome, the Golgi, and a melanosome. Amino acid sequence that direct
proteins into a non-endosomal MHC Class II pathway are known in the art.
Amino acid sequences that direct (or promote retention of, or target) an
antigen to
a melanosome (Xu et al. (1998) J. Invest. Dermatol. 110:324-331; and Jimbow et
al. ( 1997) Pigment Cell Res. 10:206-213 ); a lysosome (Kornfeld et al. (
1987)
FASEB J. 1:462-468; and Hasilik et al. (1992) Experientia 48:130-151); and to
the Golgi (Nilsson and Warren ( 1994) Curr. Opinion Cell Biol. 6:517-521 )
have
been reported in the literature and can be used in the present invention.
In some embodiments, the encoded antigen is a naturally occurring antigen. -- -
or fragment of a naturally occurring antigen. In other embodiments. the
antigen is
a synthetic antigen. The antigen can be "self' or foreign, and can be derived
from
any organism. The antigen may be autologous or heterologous (i.e., allogeneic
or
a homolog from a isolated species, e.g., a murine antigen administered to a
human
patient). Each coding sequence is operatively linked to operational and
regulatory
sequences which control transcription and translation of the coding sequences.
These operational and regulatory sequences are well known to those skilled in
the
art.
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
The polynucleotides of this invention comprise coding regions which
encode, in one embodiment, previously characterized tumor-associated antigens
such as gp100 (Kawakami et al. (1997) Intern. Rev. Immunol. 14:173-192),
MUC-I (Henderson et al. ( 1996) Cancer Res. 56:3763-3770), MART-1
S (Kawakami et al. (1994) Proc. Natl. Acad. Sci. 91:3515-3519; Kawakami et al.
(1997) Intern. Rev. Immunol. 14:173-192; Ribas et al. (1997) Cancer Res.
57:2865-2869), HER-2/neu (U.S. Patent No. 5,550,214), MAGE
(PCT/US92/04354) HPV16, 18E6 and E7 (Ressing et al. (1996) Cancer Res.
56(1):582-588; Restifo (1996) Current Opinion in Immunol. 8:658-663; Stern
(1996) Adv. Cancer Res. 69:175-211; Tindle et al. (1995) Clin. Exp. Immunol.
101:265-271; van Driel et al. (1996) Annals of Medicine 28:471-477} CEA (U.S.
Patent No. 5,274,087), PSA (Lundwall, A. ( 1989) Biochem. Biophys. Research
Communications 161:1151-59), prostate membrane specific antigen (PSMA)
(Israeli et al. (1993) Cancer Research 53:227-30), tyrosinase (U.S. Patent
Nos.
5,530,096 and 4,898,814; Brichard et al. (1993) J. Exp. Med. 178:489-49);
tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2), NY-ESO-1 (Chen et al.
(1997) Proc. Natl. Acad. Sci. U.S.A. 94:1914-18), or the GA733 antigen (U.S.
Patent No. 5,185,254).
In some embodiments, an antigen encoded by a polynucleotide of the
invention is a secreted protein, or an antigen that has been recombinantly
altered
to be secreted from the cell.
The secreted form of the antigen can.be synthesized by.splicing a signal
sequence onto the sequence encoding the wild-type antigen or by removing the
sequences coding for the cytoplasmic and transmembrane portions of the antigen
and/or by removing the sequences that 1 ) direct the encoded protein to an
intracellular compartment such as the cytoplasm, nucleus or mitochondia, or 2}
cause the encoded protein to be inserted into cellular membranes (i.e., a
transmembrane region), or 3) cause the encoded protein to be retained within
cellular compartments such as a hydrophobic anchoring sequence or endoplasmic
reticulum retention signal.
26
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
More specifically, a secreted antigen can be generated by fusing a
secretory signal sequence to the wild-type antigen using standard recombinant
DNA methodology familiar to one of skill in the art. The secretory signal
sequence would typically be positioned at the N-terminus of the desired
protein
but can be placed at any position suitable to allow secretion of the antigen.
By
way of illustration, the figure shows a transmembrane cassette comprising DNA
sequences encoding a wild-type antigen fused in frame at the 5' end with a DNA
sequence encoding a secretory signal sequence such as an endoplasmic reticulum
targeting sequence (ERTS). An internal ribosome entry sequence (IRES) is
inserted between the wild type gene and the modified gene to ensure that both
genes are translated from the bi-cistronic mRNA produced by transcription of
the
cassette as directed by the CMV promoter. Suitable secretory signal sequences
include signal sequences or derivatives of signal sequences of known secretory
proteins. A variety of secretory proteins have been identified. They include,
but
I 5 are not limited to, certain growth factors such as f broblast growth
factors 4-6,
epidermal growth factor, and lymphokines such as interleukins 2-6.
Alternatively, a secreted antigen can be generated by appending a
secretory signal sequence onto the antigen and optionally removing sequences
that
1 ) direct the encoded protein to an intracellular compartment such as the
cytoplasm, nucleus or mitochondia, or 2) cause the encoded protein to be
inserted
into cellular membranes (i.e., a transmembrane region), or 3) cause the
encoded
pr4tein to_be, retained within cellular compartments such as a hydrophobic
anchoring sequence or endoplasmic reticulum retention signal. The modified
antigen lacks the sequences necessary for membrane anchorage, but retains the
sequences required for entry into the rough endoplasmic reticulum/golgi
complex
and eventual secretion from the cell.
In other embodiments, an antigen encoded by a polynucleotide of the
invention is a cell-surface protein. In some of these embodiments, the
polynucleotide encoding an antigen further comprises a sequence which encodes
an amino acid sequence which causes the encoded protein to be inserted into
cellular membranes (i.e., a transmembrane region).
27
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
With regard to nucleic acid sequences of the present invention, "isolated"
means: an RNA or DNA polymer, portion of genomic nucleic acid, cDNA, or
synthetic nucleic acid which, by virtue of its origin or manipulation: (i) is
not
associated with all of a nucleic acid with which it is associated in nature
(e.g. is
present in a host cell as a portion of an expression vector); or (ii) is
linked to a
nucleic acid or other chemical moiety other than that to which it is linked in
nature; or (iii) does not occur in nature. By "isolated" it is further meant a
nucleic
acid sequence: (i) amplified in vitro by, for example, polymerise chain
reaction
(PCR); (ii) synthesized by, for example, chemical synthesis; (iii)
recombinantly
produced by cloning; or (iv) purified, as by cleavage and gel separation.
The nucleic acid sequences of the present invention may be characterized,
isolated, synthesized and purified using no more than ordinary skill. See
Sambrook et al., ( 1989) supra.
The polynucleotides of the present invention also can serve as primers for
the detection of genes or gene transcripts that are expressed in APC, for
example,
to confirm transduction of the polynucleotides into host cells. In this
context,
amplification means any method employing a primer-dependent polymerise
capable of replicating a target sequence with reasonable fidelity.
Amplification
may be carried out by natural or recombinant DNA-polymerises such as T7 DNA
polymerise, Klenow fragment of E.coli DNA polymerise, and reverse
transcriptase. A preferred length of the primer is the same as that identified
for
probes, above.
The invention further provides the isolated polynucleotide operatively
linked to a promoter of RNA transcription, as well as other regulatory
sequences
for replication and/or transient or stable expression of the DNA or RNA. As
used
herein, the term "operatively linked" means positioned in such a manner that
the
promoter will direct transcription of RNA off the DNA molecule. Examples of
such promoters are SP6, T4 and T7. In certain embodiments, cell-specific
promoters are used for cell-specific expression of the inserted
polynucleotide.
Vectors which contain a promoter or a promoter/enhancer, with termination
codons and selectable marker sequences, as well is a cloning site into which
an
28
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
inserted piece of DNA can be operatively linked to that promoter are well
known
in the art and commercially available. For general methodology and cloning
strategies, see GENE EXPRESSION TECHNOLOGY (Goeddel ed., Academic Press,
Inc. ( 1991 )) and references cited therein and VECTORS: ESSENTIAL DATA SERIES
(Gacesa and Ramji, eds., John Wiley & Sons, N.Y. ( 1994)), which contains
maps,
functional properties, commercial suppliers and a reference to GenEMBL
accession numbers for various suitable vectors. Preferably, these vectors are
capable of transcribing RNA in vitro or in vivo.
Methods of increasing presentation of peptide on the surface of an APC
The present invention provides methods to enhance, or increase,
presentation of a peptide on the surface of an antigen-presenting cell.
comprising
introducing into the cell a polynucleotide of the invention. Since
polynucleotides
of the invention encoding an antigen that is processed and presented with an
MHC Class I molecule on an antigen-presenting cell (APC) and an antigen that
is
processed and presented with an MHC Class II molecule on the APC, one
observes increased presentation of peptide antigens to CD4+ and to CD8+ T
cells.
The invention further provides an APC produced by the method of the
present invention. These APCs exhibit enhanced presentation of a peptide
antigen
by both Class I and Class II MHC molecules. These levels of antigen loading on
the APC surface was not achieved by prior art methods, which enhance either
Class I or Class II presentation, but not both. Accordingly, these methods,
and the
APCs produced thereby, are navel and non-obvious over the prior art.
Whether presentation of peptide on the surface of an APC is enhanced or
increased can be determined by comparing the CD4+ and CD8+ T cell response to
the encoded peptide presented on the surface of an APC into which has been
introduced a polynucleotide of the invention under conditions which favor
expression (i.e., transcription and translation), to the CD4+ and CD8+ T cell
response to the peptide when the peptide expressed on the surface of a control
APC, i.e, an APC presenting the peptide encoded by a polynucleotide lacking
either the nucleotide sequences encoding an amino acid sequence that promotes
29
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
retention in the ER, the nucleotide sequences encoding an amino acid sequence
that directs the encoded antigen into a non-endosomal MHC Class II pathway, or
both. The methods of the present invention
Various methods are known to evaluate T cell activation. see, for
example. Gong et al. (1997) Gene Therapy 4:1023-1028. CD8+ T cell activation
can be detected by any known method, including but not limited to, tritiated
thymidine incorporation (indicative of DNA synthesis), and examination of the
population for growth or proliferation, e.g., by identification of colonies.
Alternatively, the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-
diphenyl tetrazolium bromide) may be added. Mossman (1983) J. Immunol.
Methods 65:55-63; Niks and Otto ( 19901 J. Immunol. Methods 130:140-1 S 1.
Succinate dehydrogenase, found in mitochondria of viable cells, converts the
MTT to formazan blue. Thus, concentrated blue color would indicate
metabolically active cells. In yet another embodiment, incorporation of
radiolabel, e.g., tritiated thymidine, may be assayed to indicate
proliferation of
cells. Similarly, protein synthesis may be shown by incorporation of 35S-
methionine. Cytotoxicity and cell killing assays, such as the classical
chromium
release assay, may be employed to evaluate epitope-specific CTL activation. To
detect activation of CD4+ T cells, any of a variety of methods can be used,
including, but not limited to. measuring cytokine production; and
proliferation, for
example, by tritiated thymidine incorporation
Production of polynucleotides of the invention
Any known method of producing, replicating, and expressing the isolated
polynucleotides of the invention can be used. Vectors and methods for in vitro
and in vivo transduction are briefly described below and are well known in the
art.
The incorporation and expression of the exogenous nucleic acid can be
confirmed
using RT-PCR, Northern and Southern blotting analysis. Sambrook et al. (1989)
supra.
The polynucleotides of this invention can be replicated using PCR. PCR
technoiogy is the subject matter of United States Patent Nos. 4,683,195,
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
4,800,159, 4,754,065, and 4,683,202 and described in PCR: THE POLYMERASE
CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston ( 1994)) and
references cited therein.
Alternatively, one of skill in the art can use the sequences provided herein
and a commercial DNA synthesizer to replicate the DNA. Accordingly, this
invention also provides a process for obtaining the polynucleotides of this
invention by providing the linear sequence of the polynucleotide. appropriate
primer molecules, chemicals such as enzymes and instructions for their
replication
and chemically replicating or linking the nucleotides in the proper
orientation to
obtain the polynucleotides. In a separate embodiment, these polynucleotides
are
further isolated. Still further, one of skill in the art can insert the
polynucleotide
into a suitable replication vector and insert the vector into a suitable host
cell
(procaryotic or eucaryotic) for replication and amplification. The DNA so
amplified can be isolated from the cell by methods well known to those of
skill in
the art. A process for obtaining polynucleotides by this method is further
provided herein as well as the polynucleotides so obtained.
RNA can be obtained by first inserting a DNA polynucleotide into a
suitable host cell. The DNA can be inserted by any appropriate method, e.g.,
by
the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or
vector)
or by electroporation. When the cell replicates and the DNA is transcribed
into
RNA; the RNA can then be isolated using methods well known to those of skill
in
the art, for example, as set forth in Sambrook et al. (1989) Supra. For
instance,
mRNA can be isolated using various lytic enzymes or chemical solutions
according to the procedures set forth in Sambrook et al. ( 1989) Supra or
extracted
by nucleic-acid-binding resins following the accompanying instructions
provided
by manufactures.
Gene delivery vehicles comprising polynucleotides of the invention
The present invention also provides delivery vehicles suitable for delivery
of a polynucleotide of the invention into cells (whether in vivo, ex vivo, or
in
vitro). A polynucleotide of the invention can be contained within a cloning or
31
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
expression vector. These vectors (especially expression vectors) can in turn
be
manipulated to assume any of a number of forms which may, for example,
facilitate delivery to and/or entry into a cell.
Expression vectors containing these nucleic acids are useful to obtain host
vector systems to produce proteins and polypeptides. It is implied that these
expression vectors must be replicable in the host organisms either as episomes
or
as an integral part of the chromosomal DNA. Suitable expression vectors
include
plasmids, viral vectors, including adenoviruses, adeno-associated viruses,
retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for
introducing genes into tissues in vivo because of their high levels of
expression
and efficient transformation of cells both in vitro and in vivo. When a
nucleic acid
is inserted into a suitable host cell, e.g., a procaryotic or a eucaryotic
cell and the
host cell replicates, the protein can be recombinantly produced. Suitable host
cells will depend on the vector and can include mammalian cells, animal cells,
human cells, simian cells, insect cells, yeast cells, and bacterial cells
constructed
using well known methods. See Sambrook, et al. (1989) Supra. In addition to
the
use of viral vector for insertion of exogenous nucleic acid into cells, the
nucleic
acid can be inserted into the host cell by methods well known in the art such
as
transformation for bacterial cells; transfection using calcium phosphate
precipitation for mammalian cells; or DEAE-dextran; electroporation; or
microinjection. See Sambrook et al. ( 1989) Supra for this methodology. Thus,
this invention also provides a host cell, e.g. a mammalian cell, an animal
cell (rat
or mouse), a human cell, or a procaryotic cell such as a bacterial cell,
containing a
polynucleotide encoding a protein or polypeptide or antibody.
When the vectors are used for gene therapy in vivo or ex vivo, a
pharmaceutically acceptable vector is preferred, such as a replication-
incompetent
retroviral or adenoviral vector. Pharmaceutically acceptable vectors
containing
the nucleic acids of this invention can be further modified for transient or
stable
expression of the inserted polynucleotide. As used herein, the term
"pharmaceutically acceptable vector" includes, but is not limited to, a vector
or
delivery vehicle having the ability to selectively target and introduce the
nucleic
32
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
acid into dividing cells. An example of such a vector is a "replication-
incompetent" vector defined by its inability to produce viral proteins,
precluding
spread of the vector in the infected host cell. An example of a replication-
incompetent retroviral vector is LNL6. Miller et al. ( 1989) BioTechniques
7:980-
990. The methodology of using replication-incompetent retroviruses for
retroviral-mediated gene transfer of gene markers is well established. Correll
et
al. ( 1989) PNAS USA 86:8912; Bordignon ( 1989) PNAS USA 86:8912-52; Culver
( 1991 ) PNAS USA 88:3155; and Rill ( 1991 ) Blood 79( 10):2694-700.
In general, genetic modifications of cells employed in the present
invention are accomplished by introducing a vector containing a poIynucleotide
comprising sequences encoding a synthetic antigenic peptide of the invention.
A
variety of different gene transfer vectors, including viral as well as non-
viral
systems can be used.
A wide variety of non-viral vehicles for delivery of a polynucleotide of the
invention are known in the art and are encompassed in the present invention. A
polynucleotide of the invention can be delivered to a cell as naked DNA. WO
97/40163. Alternatively, a polynucleotide of the invention can be delivered to
a
cell associated in a variety of ways with a variety of substances (forms of
delivery) including, but not limited to cationic lipids; biocompatible
polymers,
including natural polymers and synthetic polymers; lipoproteins; polypeptides;
polysaccharides; lipopolysaccharides; artificial viral envelopes; metal
particles;
and bacteria. A delivery vehicle may take the form of a microparticle.
Mixtures
or conjugates of these various substances can also be used as delivery
vehicles. A
polynucleotide of the invention can be associated with these various forms of
delivery non-covalently or covalently.
Included in the non-viral vector category are prokaryotic plasmids and
eukaryotic plasmids. Non-viral vectors (i.e., cloning and expression vectors)
having cloned therein a polynucleotide(s) of the invention can be used for
expression of recombinant polypeptides as well as a source of polynucleotide
of
the invention. Cloning vectors can be used to obtain replicate copies of the
polynucleotides they contain, or as a means of storing the polynucleotides in
a
33
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
depository for future recovery. Expression vectors (and host cells containing
these expression vectors) can be used to obtain polypeptides produced from the
polynucleotides they contain. They may also be used where it is desirable to
express polypeptides, encoded by an operably linked polynucleotide, in an
individual, such as for eliciting an immune response via the polypeptide(s)
encoded in the expression vector(s). Suitable cloning and expression vectors
include any known in the art, e.g., those for use in bacterial, mammalian,
yeast
and insect expression systems. Specific vectors and suitable host cells are
known
in the art and need not be described in detail herein. For example, see Gacesa
and
Ramji, Vectors, John Wiley & Sons (1994).
Cloning and expression vectors typically contain a selectable marker (for
example, a gene encoding a protein necessary for the survival or growth of a
host
cell transformed with the vector), although such a marker gene can be carried
on
another polynucleotide sequence co-introduced into the host cell. Only those
host
cells into which a selectable gene has been introduced will survive and/or
grow
under selective conditions. Typical selection genes encode proteins) that (a)
confer resistance to antibiotics or other toxins substances, e.g., ampicillin,
neomycyin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c)
supply critical nutrients not available from complex media. The choice of the
proper marker gene will depend on the host cell, and appropriate genes for
different hosts are known in the art. Cloning and expression vectors also
typically
contain a replication system recognized by the host.
Suitable cloning vectors may be constructed according to standard
techniques, or may be selected from a large number of cloning vectors
available in
the art. While the cloning vector selected may vary according to the host cell
intended to be used, useful cloning vectors will generally have the ability to
self
replicate, may possess a single target for a particular restriction
endonuclease,
and/or may carry genes for a marker that can be used in selecting clones
containing the vector. Suitable examples include plasmids and bacterial
viruses,
e.g., pUCl8, pUCl9, Bluescript (e.g., pBS SK+) and its derivatives, mpl8,
mpl9,
pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as
34
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
pSA3 and pAT28. These and many other cloning vectors are available from
commercial vendors such as BioRad, Strategene, and Invitrogen.
Expression vectors generally are replicable polynucleotide constructs that
contain a polynucleotide encoding a polypeptide of interest. The
polynucleotide
encoding the polypeptide of interest is operably linked to suitable
transcriptional
controlling elements, such as promoters, enhancers arid terminators. For
expression (i.e., translation), one or more translational controlling elements
are
also usually required, such as ribosome binding sites, translation initiation
sites,
and stop codons. A polynucleotide sequence encoding a signal peptide can also
be included to allow a polypeptide, encoded by an operably linked
polynucleotide,
to cross and/or lodge in cell membranes or be secreted from the cell. A number
of
expression vectors suitable for expression in eukaryotic cells including
yeast,
avian, and mammalian cells are known in the art. Examples of mammalian
expression vectors contain both prokaryotic sequence to facilitate the
propagation
of the vector in bacteria, and one or more eukaryotic transcription units that
are
expressed in eukaryotic cells. Examples of mammalian expression vectors
suitable for transfection of eukaryotic cells include the pcDNAI/amp,
pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pRSVneo, and pHyg derived
vectors. Alternatively, derivatives of viruses such as the bovine papilloma
virus
(BPV-1 ), or Epstein-Barr virus (pHEB, pREP derived vectors) can be used for
expression in mammalian cells. Examples of expression vectors for yeast
systems, include YEP24, YIPS, YEP51, YEP52, YES2 and YRP17, which are
cloning and expression vehicles useful for introduction of constructs into S.
cerevisiae. Broach et al. (1983) Experimental Manipulation of Gene Expression,
ed. M. Inouye, Academic Press. p. 83. Baculovirus expression vectors for
expression in insect cells include pVL-derived vectors (such as pVL 1392,
pVL1393 and pVL941), pAcUW-derived vectors and pBlueBac-derived vectors.
Viral vectors include, but are not limited to, DNA viral vectors such as
those based on adenoviruses, herpes simplex virus, poxviruses such as vaccinia
virus, and parvoviruses, including adeno-associated virus; and RNA viral
vectors,
including, but not limited to, the retroviral vectors. Retroviral vectors
include
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
marine leukemia virus, and lentiviruses such as human immunodeficiency virus.
Naldini et al. ( 1996) Science 272:263-267.
Replication-defective retroviral vectors harboring a polynucleotide of the
invention as part of the retroviral genome can be used. Such vectors have been
described in detail. (Miller et al. ( 1990) Mol. Cell Biol. 10:4239; Kolberg,
R.
( 1992) J. NIH Res. 4:43; Cornetta et al. ( 1991 ) Hum. Gene Ther 2:21 S).
Adenovirus and adeno-associated virus vectors useful in the genetic
modifications of this invention may be produced according to methods already
taught in the art. (See, e.g., Karlsson et al. (1986) EMBO 5:2377; Carter
(1992)
Current Opinion in Biotechnology 3:533-539; Muzcyzka ( 1992) Current Top.
Microbiol. Immunol. 158:97-129; GENE TARGETING: A PRACTICAL APPROACH
( I 992) ed. A. L. Joyner, Oxford University Press, NY). Several different
approaches are feasible.
Additional references describing viral vectors which could be used in the
methods of the present invention include the following: Horwitz, M.S.,
Adenoviridae and Their Replication, in Fields, B., et al. (eds.) viROLOGY,
Vol. 2,
Raven Press New York, pp. 1679-1721, 1990); Graham, F. et al., pp. 109-128 in
METHODS IN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION
PROTOCOLS, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller et al.
(1995) FASEBJournal 9:190-199, Schreier (1994) PharmaceuticaActa Helvetiae
68:145-159; Schneider and French (1993) Circulation 88:1937-1942; Curiel et
al.
(1992) Human Gene Therapy 3:147-154; Graham et al., WO 95/00655 (5 January
1995); Falck-Pedersen WO 95/16772 (22 June 1995); Denefle et al. WO 95/23867
(8 September 1995); Haddada et al. WO 94/26914 (24 November 1994);
Perricaudet et al. WO 95/02697 (26 January 1995); Zhang et al. WO 95/25071 (12
October 1995).
The efficiency of transduction of DCs or other APCs can be assessed by
immunofluorescence using fluorescent antibodies specific for the tumor antigen
being expressed (Kim et al. ( 1997) J. Immunother. 20:276-286). Alternatively,
the antibodies can be conjugated to an enryme (e.g. HRP) giving rise to a
colored
36
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
product upon reaction with the substrate. The actual amount of antigenic
polypeptides being expressed by the APCs can be evaluated by ELISA.
In vivo transduction of DCs, or other APCs, can be accomplished by
administration of a viral vectors comprising a polynucleotide of the invention
via
different routes including intravenous, intramuscular, intranasal,
intraperitoneal or
cutaneous delivery. One method which can be used is cutaneous delivery of Ad
vector at multiple sites using a total dose of approximately 1 x 10 ~ °-
1 x 10 ~ 2 i.u.
Levels of in vivo transduction can be roughly assessed by co-staining with
antibodies directed against APC markers) and the peptide epitope being
expressed. The staining procedure can be carried out on biopsy samples from
the
site of administration or on cells from draining lymph nodes or other organs
where APCs (in particular DCs) may have migrated. Condon et al. ( 1996) Nature
Med. 2:1122-1128; Wan et al. (1997) Human Gene Therapy 8:1355-1363. The
amount of antigen being expressed at the site of injection or in other organs
where
transduced APCs may have migrated can be evaluated by ELISA on tissue
homogenates.
APCs can also be transduced in vitro%x vivo by non-viral gene delivery
methods such as electroporation, calcium phosphate precipitation or cationic
lipid/plasmid DNA complexes. Arthur et al. ( 1997) Cancer Gene Therapy 4:17-
25. Transduced APCs can subsequently be administered to the host via an
intravenous. subcutaneous, intranasal, intramuscular or intraperitoneal route
of
delivery.
In vivo transduction of DCs, or other APCs, can potentially be
accomplished by administration of cationic lipid/plasmid DNA complexes
delivered via the intravenous, intramuscular, intranasal, intraperitoneal or
cutaneous route of administration. Gene gun delivery or injection of naked
plasmid DNA into the skin also leads to transduction of DCs. Condon et al.
( 1996) Nature Med. 2:1122-1128; Raz et al. ( 1994) PNAS 91:9519-9523.
Intramuscular delivery of plasmid DNA may also be used for immunization.
Rosato et al. (1997) Human Gene Therapy 8:1451-1458.
37
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
The transduction efficiency and levels of transgene expression can be
assessed as described above for viral vectors.
Host cells comprising polynucleotides of the invention
The present invention further provides host cells comprising
polynucleotides of the invention. Host cells containing the polynucleotides of
this
invention are useful for the recombinant replication of the polynucleotides
and for
the recombinant production of peptides of the invention. In addition, host
cells
comprising a polynucleotide of the invention can be used to induce an immune
response in a subject in the methods described herein.
Host cells which are suitable for recombinant replication of the
polynucleotides of the invention, and for the recombinant production of
peptides
of the invention can be prokaryotic or eukaryotic. Host systems are known in
the
art and need not be described in detail herein. Prokaryotic hosts include
bacterial
I S cells, for example E. coli, B. subtilis, and mycobacteria. Among
eukaryotic hosts
are yeast, insect, avian, plant, C. elegans (or nematode) and mammalian cells.
These cells are cultured in conventional nutrient media modified as
appropriate
for inducing promoters, selecting transformants, or amplifying the genes
encoding
the desired sequences.
When the host cells are antigen presenting cells, they can be used to
expand a population of immune effector cells such as tumor infiltrating
lymphocytes which in turn are useful in adoptive immunotherapies. Antigen
presenting cells are described in more detail below.
In some of these embodiments, isolated host cells are APCs. APCs
include, but are not limited to, dendritic cells (DCs), monocytes/macrophages,
B
lymphocytes or other cell types) expressing the necessary MHC/co-stimulatory
molecules.
In some embodiments, the immune effector cells and/or the APCs are
genetically modified. Using standard gene transfer, genes coding for co-
stimulatory molecules and/or stimulatory cytokines can be inserted prior to,
concurrent to or subsequent to expansion of the immune effector cells.
38
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
In one embodiment. the host cell containing the genes) coding for the
cytokine and/or co-stimulatory molecule is a professional antigen-presenting
cell
such as a dendritic cell which includes, but is not limited to, a pulsed
dendritic
cell, a dendritic cell hybrid or an antigen-presenting foster cell.
Antigen presenting cells
APCs suitable for use in the present invention are capable of presenting
exogenous peptide or protein or endogenous antigen to T cells in association
with
an antigen-presenting molecule, such as an MHC molecule. APCs include, but
are not limited to, macrophages, dendritic cells, CD40-activated B cells,
antigen-
specific B cells, tumor cells, virus-infected cells, and genetically modified
cells.
APCs can obtained from a variety of sources, including but not limited to,
peripheral blood mononuclear cells (PBMC), whole blood or fractions thereof
containing mixed populations, spleen cells, bone marrow cells, tumor
infiltrating
lymphocytes, cells obtained by leukapheresis, lymph nodes, e.g., lymph nodes
draining from a tumor. Suitable donors include an immunized donor, a non-
immunized (naive) donor, treated or untreated donors. A "treated" donor is one
that has been exposed to one or more biological modifiers. An "untreated"
donor
has not been exposed to one or more biological modifiers. APC's can also be
treated in vitro with one or more biological modifiers.
The APCs are generally alive but can also be irradiated. mitomycin C
treated, attenuated, or chemically fixed. Further, the APCs need not be whole
cells. Instead, vesicle preparations of APCs can be used.
APCs can be genetically modified, i.e., transfected with a recombinant
polynucleotide construct such that they express a polypeptide or an RNA
molecule which they would not normally express or would normally express at
lower Levels. Examples of polynucleotides include, but are not limited to,
those
which encode an MHC molecule; a co-stimulatory molecule such as B7; and a
peptide or poIypeptide of the invention.
Cells which do not normally function in vivo in mammals as APCs can be
modified in such a way that they function as APCs. A wide variety of cells can
function as APCs when appropriately modified. Examples of such cells are
insect
39
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
cells, for example Drosophila or Spodoptera; and foster cells, such as the
human
cell line T2. For example, expression vectors which direct the synthesis of
one or
more antigen-presenting polypeptides, such as MHC molecules, optionally also
accessory molecules such as B7, can be introduced into these cells to effect
the
expression on the surface of these cells antigen presentation molecules and,
optionally, accessory molecules or functional portions thereof. Alternatively,
antigen-presenting polypeptides and accessory molecules which can insert
themselves into the cell membrane can be used. For example, glycosyl-
phosphotidylinositol (GPI)-modified polypeptides can insert themselves into
the
membranes of cells. Hirose et al. (1995) Methods Enzymol. 250:582-614; and
Huang et al. ( 1994) Immunity I :607-613. Accessory molecules include. but are
not limited to, co-stimulatory antibodies such as antibodies specific for
CD28,
CD80, or CD86; costimulatory molecules, including, but not limited to, B7.1
and
B7.2; adhesion molecules such as ICAM-1 and LFA-3; and survival molecules
such as Fas ligand and CD70. See, for example, PCT Publication No. WO
97/46256.
Foster antigen presenting cells are particularly useful as APCs. Foster
APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which
contains a mutation in its antigen processing pathway that restricts the
association
of endogenous peptides with cell surface MHC class I molecules. Zweerink et
al.
( 1993) J. Immunol. 150:1763-1771. This is due to a large homozygous deletion
in
the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and
LMP2, which are required for antigen presentation to MHC class 1-restricted
CD8+ CTLs. In effect, only "empty" MHC class I molecules are presented on the
surface of these cells. Exogenous peptide added to the culture medium binds to
these MHC molecules provided that the peptide contains the allele-specific
binding motif. These T2 cells are referred to herein as "foster" APCs. They
can
be used in conjunction with this invention to present antigen(s).
Transduction of T2 cells with specific recombinant MHC alleles allows for
redirection of the MHC restriction profile. Libraries tailored to the
recombinant
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
allele will be preferentially presented by them because the anchor residues
will
prevent efficient binding to the endogenous allele.
High level expression of MHC molecules makes the APC more visible to
the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful
transcriptional promoter (e.g., the CMV promoter) results in a more reactive
APC
(most likely due to a higher concentration of reactive MHC-peptide complexes
on
the cell surface}.
Methods for determining whether an antigen-presenting cell is capable of
presenting antigen to an immune effector cell in such a manner as to effect
activation of the immune effector cell, are known in the art and include, for
example. 3H-thymidine uptake by effector cells, cytokine production by
effector
cells. and cytolytic 5' Cr-release assays.
In some embodiments, an antigenic peptide of the invention is presented
on an antigen-presenting cell in a Class I or Class II MHC molecule such that
the
peptide is bound by a TCR on a CD4+ or CD8+ T cell, but the antigen-presenting
cell lacks one or more co-stimulatory molecules required for activation of the
T
cell. These antigen-presenting cells induce T cell anergy (unresponsiveness),
and
are useful in methods described herein for reducing or suppressing an immune
response. Methods for determining whether an antigen-presenting cell is
capable
of presenting antigen to an immune effector cell, in such a manner as to
effect T
cell anergy, are known in the art.
The following is a brief description of two fundamental approaches for the
isolation of APC. These approaches involve ( 1 ) isolating bone marrow
precursor
cells (CD34+) from blood and stimulating them to differentiate into APC; or
(2)
collecting the precommitted APCs from peripheral blood. In the first approach,
the patient must be treated with cytokines such as GM-CSF to boost the number
of circulating CD34+ stem cells in the peripheral blood.
The second approach for isolating APCs is to collect the relatively large
numbers of precommitted APCs already circulating in the blood. Previous
techniques for isolating committed APCs from human peripheral blood have
involved combinations of physical procedures such as metrizamide gradients and
41
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
adherence/nonadherence steps (Freudenthal et al. ( 1990) PNAS 87:7698-7702);
Percoll gradient separations (Mehta-Damani et al. ( 1994) J. Immunol. 153:996-
1003); and fluorescence activated cell sorting techniques (Thomas et al.
(1993) J.
Immunol. 151:6840-52).
One technique for separating large numbers of cells from one another is
known as countercurrent centrifugal elutriation (CCE). In this technique,
cells are
subject to simultaneous centrifugation and a washout stream of buffer which is
constantly increasing in flow rate. The constantly increasing countercurrent
flow
of buffer leads to fractional cell separations that are largely based on cell
size.
In one aspect of the invention, the APC are precommitted or mature
dendritic cells which can be isolated from the white blood cell fraction of a
mammal, such as a marine, simian or a human (See, e.g., WO 96/23060). The
white blood cell fraction can be from the peripheral blood of the mammal. This
method includes the following steps: (a) providing a white blood cell fraction
obtained from a mammalian source by methods known in the art such as
leukopheresis; (b) separating the white blood cell fraction of step (a) into
four or
more subfractions by countercurrent centrifugal elutriation, (c) stimulating
conversion of monocytes in one or more fractions from step (b) to dendritic
cells
by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF
and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c),
and (e)
collecting the enriched fraction of step (d), preferably at about 4°C.
One way to
identify the dendritic cell-enriched fraction is by fluorescence-activated
cell
sorting. The white blood cell fraction can be treated with calcium ionophore
in
the presence of other cytokines, such as recombinant (rh) rhIL-12, rhGM-CSF,
or
rhIL-4. The cells of the white blood cell fraction can be washed in buffer and
suspended in Ca++/Mg~ free media prior to the separating step. The white blood
cell fraction can be obtained by leukopheresis. The dendritic cells can be
identified by the presence of at least one of the following markers: HLA-DR,
HLA-DQ, or B7. 2, and the simultaneous absence of the following markers: CD3,
CDI4, CD16, 56, 57, and CD 19, 20. Monoclonal antibodies specific to these
cell
surface markers are commercially available.
42
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
More specifically, the method requires collecting an enriched collection of
white cells and platelets from leukopheresis that is then further fractionated
by
countercurrent centrifugal elutriation (CCE). Abrahamsen et al. ( 1991 ) J.
Clin.
Apheresis. 6:48-53. Cell samples are placed in a special elutriation rotor.
The
rotor is then spun at a constant speed of, for example, 3000 rpm. Once the
rotor
has reached the desired speed, pressurized air is used to control the flow
rate of
cells. Cells in the elutriator are subjected to simultaneous centrifugation
and a
washout stream of buffer which is constantly increasing in flow rate. This
results
in fractional cell separations based largely but not exclusively on
differences in
cell size.
Quality control of APC and more specifically DC collection and
confirmation of their successful activation in culture is dependent upon a
simultaneous mufti-color FACS analysis technique which monitors both
monocytes and the dendritic cell subpopulation as well as possible contaminant
T
lymphocytes. It is based upon the fact that DCs do not express the following
markers: CD3 (T cell); CD14 (monocyte); CD16, 56, 57 (NK/LAK cells); CD19,
(B cells). At the same time, DCs do express large quantities of HLA-DR,
significant HLA-DQ and B7.2 (but little or no B7.1 ) at the time they are
circulating in the blood (in addition they express Leu M7 and M9, myeloid
20 markers which are also expressed by monocytes and neutrophils).
Once collected, the DC rich/monocyte APC fractions (usually 150 through
190) can be pooled and cryopreserved for future use, or immediately placed in
short term culture.
Alternatively, others have reported that a method for upregulating
(activating) dendritic cells and converting monocytes to an activated
dendritic cell
phenotype. This method involves the addition of calcium ionophore to the
culture
media convert monocytes into activated dendritic cells. Adding the calcium
ionophore A23187, for example, at the beginning of a 24-48 hour culture period
resulted in uniform activation and dendritic cell phenotypic conversion of the
pooled "monocyte plus DC" fractions: characteristically, the activated
population
43
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
becomes uniformly CD14 (Leu M3} negative, and upregulates HLA-DR, HLA-
DQ, ICAM-1. B7.1, and B7.2.
Specific combinations) of cytokines have been used successfully to
amplify (or partially substitute) for the activation/conversion achieved with
calcium ionophore: these cytokines include but are not limited to purified or
recombinant human ("rh") rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when
given alone is inadequate for optimal upregulation.
Foster Antigen Presenting Cells
A foster antigen presenting cell is a genetically modified dendritic cell
that, from the restricted association of endogeneous peptides, will take up
and
present exogeneous antigen on the cell surface. In one embodiment, the human
cell line 174xCEM.T2, referred to as T2, contains a mutation in its antigen
processing pathway that restricts the association of endogenous peptides with
cell
surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763-
1771 ) and therefore can be used for the manufacture of a foster antigen
presenting
cell. (The cell line is available from the ATCC.) This is due to a large
homozygous deletion in the MHC class II region encompassing the genes TAP1,
TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC
class 1-restricted CD8+ CTLs. In effect, only "empty" MHC class I molecules
are
presented on the surface of these cells. Exogenous peptide added to the
culture
medium binds to these MHC molecules provided that the peptide contains the
allele-specific binding motif. These T2 cells are referred to herein as
"foster"
APCs. They can be used in conjunction with this invention to present the
antigens.
Transduction of T2 cells with specific recombinant MHC alleles allows for
redirection of the MHC restriction profile. Libraries tailored to the
recombinant
allele will be preferentially presented by them because the anchor residues
will
prevent efficient binding to the endogenous allele.
Transduction of non-professional APCs with allogeneic MHC alleles aids
greatly in the immunogenicity of the recombinant cell line (Leong et al.
(1994)
Inter. J. Cancer 59:212-216; Ostrand-Rosenberg et al. ( 1991 ) Inter. J.
Cancer
44
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
Suppl. 6:61-68). Immunogenicity was reported to be proportional to the level
of
expression of the MHC proteins. T2 cells are ideal APCs.
High level expression of MHC molecules makes the APC more visible to
the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful
transcriptional promoter (e.g., the CMV promoter) results in a more reactive
APC
(most likely due to a higher concentration of reactive MHC-peptide complexes
on
the cell surface). Note that since only one type of MHC allele will be able to
interact with a given library, the presence of or expression level of the
endogenous
allele will not compromise specificity if the library is designed to bind to
the
newly transduced cells.
Hybrid cells also will present antigens) and therefore, are useful in the
methods of this invention.
Preparation of Hybrid Cells Utilizing Dendritic Cells
Hybrid cells typically retain the phenotypic characteristics of the APCs.
Thus. hybrids made with dendritic cells will express the same MHC class II
proteins and other cell surface markers. Moreover, the hybrids will express
those
antigens expressed on the cells from which they are derived. The procedure for
making these hybrids is described in WO 96/30030 and Gong et al. (1997) Nature
Medicine 3(5):558-561.
A population of APCs are collected and isolated. Preferably, the ratio of
APCs:antigen-expressing cells is between about 1:100 and about 1000:1. For
example, in one aspect, the fraction enriched for antigen-expressing cells is
then
fused to APCs, preferably dendritic cells. Fusion between the APCs and antigen-
expressing cells can be carried out with any suitable method, for example
using
polyethylene glycol (PEG) or Sendia virus. In a preferred embodiment. the
hybrid
cells are created using the procedure described by Gong et al. ( 1997) Nat.
Med.
3(5):558-561.
Typically, unfused cells will die off after a few days in culture, therefore,
the fused cells can be separated from the parent cells simply by allowing the
culture to grow for several days. In this embodiment, the hybrid cells both
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
survive more and, additionally, are only lightly adherent to tissue culture
surfaces.
The parent cells are strongly adherent to the containers. Therefore, after
about 5
to 10 days in culture. the hybrid cells can be gently dislodged and
transferred to
new containers, while the unfused cells remained attached.
Alternatively, it has been shown that fused cells lack functional
hypoxanthine-guaninephosphoribosyl transferase ("HGPRT") enzyme and are,
therefore, resistant to treatment with the compound HAT. Accordingly, to
select
these cells HAT can be added to the culture media. However, unlike
conventional
HAT selection, hybrid cell cultures should not be exposed to the compound for
more than I2 days. The APCs as described above can be assayed for antigens)
expression as described below.
In addition to their use as vaccines, they are useful to expand immune
effector cell population that can be used in adoptive immunotherapy.
Presentation of Antigen to the APC
The following briefly describes various methods for expression or
presentation of antigen on APCs.
Paglia et al. (1996) J. Exp. Med. 183:317-322 has shown that APC
incubated with whole protein in vitro were recognized by MHC class I-
restricted
CTLs, and that immunization of animals with these APCs led to the development
of antigen-specific CTLs in vivo. Hsu et al. (1996) Nature Medicine 2(1):52-
58;
Celluzzi et al. (1996) J. Exp. Med. 183:283-287; Mayordomo et al. (1995)
Nature
Medicine 1(I2):1297-1302; Bakker et al. (1995) Cancer Research 55:5330-5334;
and Mayordomo et al. (1997) Stem Cells 15:94-103, reported on the successful
use of peptide-pulsed dendritic cells in cancer immunotherapy.
In addition, several different techniques have been described which lead to
the expression of antigen in the cytosol of APCs, such as DCs. These include (
1 )
the introduction into the APCs of RNA isolated from tumor cells, (2) infection
of
APCs with recombinant vectors to induce endogenous expression of antigen, and
(3) introduction of tumor antigen into the DC cytosol using liposomes. (See
46
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
Boczkowski et al. (1996) J. Exp. Med. 184:465-472; Rouse et al. (1994) J.
Virol.
68:5685-5689; and Nair et al. ( 1992) J. Exp. Med. 175:609-612).
Genetically modified dendritic cells and their use in immunotherapy have
been described. Brossart et al. ( 1997) J. Immunol. 3270-3276 and Wan et al.
(1997) Human Gene Therapy 8:1355-1363; Gong et al. (1997) Gene Therapy
4:1023-1028; Kim et al.(1997) J. Immunotherapy 20(4):276-286; and Song et al.
( 1997) J. Exp. Med. 186(8):1247-1256, reported that adenovirus containing
antigenic peptides can be used was used to transduce DCs which are then
administered to animals to induce an immune response. Condon et al. (1996)
Nature Medicine 2(10):1122-1128 showed that cutaneous genetic immunization
with naked DNA resulted in a potent, antigen-specific, cytotoxic T-lymphocyte-
mediated protective immunity. Arthur et al. ( 1997) Cancer Gene Therapy
4(1):17-25 compare and analyze the gene transfer methods in human dendritic
cells.
However, none of the above report on the use of APCs to present cell-
surface and secreted forms of the antigen to induce an immune response as
claimed herein.
Insertion of the genes) into the APCs requires the making of appropriate
gene delivery vehicles and methods for efficient transduction. The following
are
useful for in vitro and in vivo transduction with the genes) of interest.
Immune Effector Cells
The present invention makes use of the above-described APCs to stimulate
production of an enriched population of antigen-specific immune effector
cells.
Accordingly, the present invention provides a population of cells enriched in
educated. antigen-specific immune effector cells, specific for an antigen
encoded
by a polynucleotide of the invention. In some embodiments, the antigen
corresponds to an antigen on the surface of tumor cells and the educated,
antigen-
specific immune effector cells of the invention suppress growth of the tumor
cells.
When APCs are used, the antigen-specific immune effector cells are expanded at
the expense of the APCs, which die in the culture. The process by which nave
47
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
immune effector cells become educated by other cells is described essentially
in
Coulie (1997) Molec. Med. Today 3:261-268.
The APCs prepared as described above are mixed with naive immune
effector cells. Preferably, the cells may be cultured in the presence of a
cytokine,
for example IL2. Because dendritic cells secrete potent immunostimulatory
cytokines, such as IL-12, it may not be necessary to add supplemental
cytokines
during the first and successive rounds of expansion. In any event, the culture
conditions are such that the antigen-specific immune effector cells expand (i.
e.
proliferate) at a much higher rate than the APCs. Multiple infusions of APCs
and
optional cytokines can be performed to further expand the population of
antigen-
specific cells.
In one embodiment, the immune effector cells are T cells. In a separate
embodiment, the immune effector cells can be genetically modified by
transduction with a transgene coding for example, IL-2, IL-11 or IL-13.
Methods
I 5 for introducing transgenes in vitro, ex vivo and in vivo are well known in
the art.
See Sambrook, et al. (1989) Supra.
An effector cell population suitable for use in the methods of the present
invention can be autogeneic or allogeneic, preferably autogeneic. When
effector
cells are allogeneic, preferably the cells are depleted of alloreactive cells
before
use. This can be accomplished by any known means, including, for example, by
mixing the allogeneic effector cells and a recipient cell population and
incubating
them for a suitable time, then depleting CD69+ cells, or inactivating
alloreactive
cells, or inducing anergy in the alloreactive cell population.
Hybrid immune effector cells can also be used. Immune effector cell
hybrids are known in the art and have been described in various publications.
See, for example, International Patent Application Nos. WO 98/46785; and WO
95/16775.
The effector cell population can comprise unseparated cells, i.e., a mixed
population. for example, a PBMC population, whole blood, and the like. The
effector cell population can be manipulated by positive selection based on
expression of cell surface markers, negative selection based on expression of
cell
48
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
surface markers, stimulation with one or more antigens in vitro or in vivo,
treatment with one or more biological modifiers in vitro or in vivo,
subtractive
stimulation with one or more antigens or biological modif ers, or a
combination of
any or all of these.
Effector cells can obtained from a variety of sources, including but not
limited to, PBMC, whole blood or fractions thereof containing mixed
populations,
spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells
obtained by
leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a
tumor. Suitable donors include an immunized donor, a non-immunized (naive)
donor, treated or untreated donors. A "treated" donor is one that has been
exposed to one or more biological modifiers. An ''untreated" donor has not
been
exposed to one or more biological modifiers.
Methods of extracting and culturing effector cells are well known. For
example, effector cells can be obtained by leukapheresis, mechanical apheresis
using a continuous flow cell separator. For example, lymphocytes and monocytes
can be isolated from the huffy coat by any known method, including, but not
limited to, separation over Ficoll-HypaqueTM gradient, separation over a
Percoll
gradient. or elutriation. The concentration of Ficoll-HypaqueTM can be
adjusted to
obtain the desired population, for example, a population enriched in T cells.
Other methods based on affinity are known and can be used. These include, for
example. fluorescence-activated cell sorting (FACS), cell adhesion, magnetic
bead separation, and the like. Affinity-based methods may utilize antibodies,
or
portions thereof, which are specific for cell-surface markers and which are
available from a variety of commercial sources, including, the American Type
Culture Collection (Manassas, MD). Affinity-based methods can alternatively
utilize ligands or ligand analogs, of cell surface receptors.
The effector cell population can be subjected to one or more separation
protocols based on the expression of cell surface markers. For example, the
cells
can be subjected to positive selection on the basis of expression of one or
more
cell surface polypeptides, including, but not limited to, ''cluster of
differentiation"
cell surface markers such as CD2, CD3, CD4, CDB, TCR, CD45, CD45R0,
49
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
CD45RA, CD 11 b, CD26, CD27, CD28, CD29, CD30, CD31, CD40L; other
markers associated with lymphocyte activation, such as the lymphocyte
activation
gene 3 product (LAG3), signaling lymphocyte activation molecule (SLAM),
T1/ST2; chemokine receptors such as CCR3, CCR4, CXCR3, CCRS; homing
receptors such as CD62L, CD44, CLA, CD146, a4~37, aE~37; activation markers
such as CD25, CD69 and OX40; and lipoglycans presented by CD1. The effector
cell population can be subjected to negative selection for depletion of non-T
cells
and/or particular T cell subsets. Negative selection can be performed on the
basis
of cell surface expression of a variety of molecules, including, but not
limited to,
B cell markers such as CD19, and CD20; monocyte marker CD14; the NK cell
marker CD56.
An effector cell population can be manipulated by exposure, in vivo or in
vitro, to one or more biological modifiers. Suitable biological modifiers
include,
but are not limited to, cytokines such as IL-2, IL-4, IL-10, TNF-a, IL-12, IFN-
y;
non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such
as
phorbol myristate acetate (PMA), concanavalin-A, and ionomycin; antibodies
specific for cell surface markers, such as anti-CD2, anti-CD3, anti-IL2
receptor,
anti-CD28; chemokines, including, for example, lymphotactin. The biological
modifiers can be native factors obtained from natural sources, factors
produced by
recombinant DNA technology, chemically synthesized polypeptides or other
molecules, or any derivative having the functional activity of the native
factor. If
~~ ~ - more than~one biological modifier is used; the exposure can-be
simultaneous or
sequential.
The present invention provides compositions comprising immune effector
cells, which may be T cells, enriched in antigen-specific cells, specific for
an
antigen encoded by a polynucleotide of the invention. By "enriched" is meant
that a cell population is at least about 50-fold, more preferably at least
about 500-
fold, and even more preferably at least about 5000-fold or more enriched from
an
original naive cell population. The proportion of the enriched cell population
which comprises antigen-specific cells can vary substantially, from less than
10%
up to 100% antigen-specific cells. If the cell population comprises at least
50%,
CA 02322659 2000-09-06
WO 99/47641 PGTNS99/06030
preferably at least 70%. more preferably at least 80%, and even more
preferably at
least 90%, antigen-specific immune effector cells, specific for a peptide of
the
invention, then the population is said to be "substantially pure". The
percentage
which are antigen-specific can readily be determined, for example, by a 3H-
thymidine uptake assay in which the effector cell population (for example, a T-
cell population) is challenged by an antigen-presenting cell presenting an
antigen
encoded by a polynucleotide of the invention.
Expansion of Immune Effector Cells
The present invention makes use of the above-described APCs to stimulate
production of an enriched population of antigen-specific immune effector
cells.
The antigen-specific immune effector cells are expanded at the expense of the
APCs, which die in the culture. The process by which naive immune effector
cells become educated by other cells is described essentially in Coulie (
1997)
Molec. Med. Today 3:261-268; Hwu et al. (1993) J. Immunol. 150:4104-4115;
and Rosenburg,et al. (I985) N. Eng. J. Med. 313(23):1485-1492.
In one embodiment, the antigen-specific immune effector cell population
comprises both CD4+ and CD8+ T cells.
In one aspect, the cytotoxic T cells (i.e., CD8+ T cells) are polyclonal T
cells isolated from a site of cytotoxic T cell infiltration from a subject.
Alternatively, such cells may be isolated from a site of cytotoxic T cell
infiltration
,. . .: from .two or more subjects or human patients;. in which the subjects
share an MHC
halotype. In another embodiment, the CTLs may be two or more cytotoxic T cell
lines. In yet another embodiment, the CTLs may be any combination of the
foregoing.
In a further aspect of the invention, the site of cytotoxic T cell
infiltration
is a tumor. The tumors from which cells or cell lines are obtained can be the
same
type of tumor in different individuals with a shared MHC halotype or different
types of tumors from different subjects who share an MHC haplotype.
51
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
Alternatively, CTL infiltrates can be from sites of viral infection.
autoimmune inflammation, transplantation rejection, an like sites of
inflammation
or lymphocyte/leukocyte infiltration.
The APCs prepared as described above are mixed with naive immune
effector cells. Preferably, the cells may be cultured in the presence of a
cytokine,
for example IL2. Because dendritic cells secrete potent immunostimulatory
cytokines. such as IL 12, it may not be necessary to add supplemental
cytokines
during the first and successive rounds of expansion. In any event, the culture
conditions are such that the antigen-specific immune effector cells expand
(i.e.
proliferate) at a much higher rate than the APCs. Multiple infusions of hybrid
cells and optional cytokines can be performed to further expand the population
of
antigen-specific cells.
In one embodiment, the immune effector cells are T cells and are specific
for tumor-specific antigens which are presented by the APCs.
Polypeptide Antigens of the Invention
The invention further provides peptide antigens encoded by the
polynucleotides of the invention. Peptide antigens encoded by polynucleotides
of
the invention can be produced by any known method. Isolated peptides of the
present invention can be synthesized using an appropriate solid state
synthetic
procedure. Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San
... .. Francisco..Calif..(19,68)..A preferred method is the Merrifield
process. Merrifield,
Recent Progress in Hormone Res., 23:451 ( 1967). The antigenic activity of
these
peptides may conveniently be tested using, for example, the assays as
described
herein.
Once an isolated peptide of the invention is obtained, it may be purified by
standard methods including chromatography (e.g., ion exchange, affinity, and
sizing column chromatography), centrifugation, differential solubility, or by
any
other standard technique for protein purification. For immunoaffmity
chromatography, an epitope may be isolated by binding it to an affinity column
52
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
comprising antibodies that were raised against that peptide. or a related
peptide of
the invention, and were affixed to a stationary support.
Alternatively, affinity tags such as hexa-His (Invitrogen), Maltose binding
domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991)
Methods Enzymol. 194:508-509), and glutathione-S-transferase can be attached
to
the peptides of the invention to allow easy purification by passage over an
appropriate affinity column. A DNA aff nity column using DNA containing a
sequence encoding the peptides of the invention could be used in purification.
Isolated peptides can also be physically characterized using such
techniques as proteolysis, nuclear magnetic resonance, and x-ray
crystallography.
Also included within the scope of the invention are antigenic peptides that
are differentially modified during or after translation, e.g., by
phosphorylation,
glycosylation, crosslinking, acylation, proteolytic cleavage, linkage to an
antibody
molecule, membrane molecule or other ligand, (Ferguson,et al., ( 1988) Ann.
Rev.
Biochem.57:285-320).
Assaying Antigen Specificity
The immune effector cells described herein are selected for their ability to
modulate an immune response. In one aspect, the immune effector cells are
selected both for their ability to actively lyse the cells expressing the
specific
antigen and for their ability to increase a humoral response to the antigen.
.... . .Cytolytic activity of.the.cells can be measured in various ways,
including, butpot. . .
limited to, tritiated thymidine incorporation (indicative of DNA synthesis),
and
examination of the population for growth or proliferation, e.g., by
identification of
colonies. (See, e.g., WO 94/21287). In another embodiment, the tetrazolium
salt
MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-diphenyl tetrazolium bromide) may be
added (Mossman (1983) J. Immunol. Methods 65:55-63: Niks and Otto (1990) J.
Immunol. Methods 130:140-151). Succinate dehydrogenase, found in
mitochondria of viable cells, converts the MTT to fonmazan blue. Thus,
concentrated blue: color would indicate metabolically active cells. Similarly,
protein synthesis may be shown by incorporation of 3'S-methionine. In still
53
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
another embodiment, cytotoxicity and cell killing assays, such as the
classical
chromium release assay, may be employed to evaluate epitope-specific CTL
activation. Other suitable assays will be known to those of skill in the art.
Stimulation of a humoral response to an antigen can be measured by specific
antibody production, using any known method, including ELISA and RIA.
Compositions of the invention
This invention also provides compositions containing any of the above-
mentioned peptides, polypeptides, polynucleotides, host cells, antigen-
presenting
cells, immune effector cells, vectors, antibodies and fragments thereof, and
an
acceptable solid or liquid carrier. When the compositions are used
pharmaceutically, they are combined with a "pharmaceutically acceptable
carrier"
for diagnostic and therapeutic use. These compositions also can be used for
the
preparation of medicaments for the diagnostic and immunomodulatory methods of
the invention.
Methods using the polynucleotides, antigen presenting cells, and immune
effector
cells of the invention
The present invention provides diagnostic and immunomodulatory
methods using polynucleotides, and host cells (including APCs and educated
immune effector cells), i.e., immunomodulatory agents, of the invention.
,. . . ,. ... , ~ . ...
Diagnostic methods
The present invention provides diagnostic methods using the
polynucleotides and APCs of the invention. The methods can be used to detect
the presence of an antigen-specific CD4+ and/or CD8+ T cell which binds an
antigen encoded by a polynucleotide of the invention.
The diagnostic methods of the invention include: ( 1 ) assays to predict the
efficacy of an antigen encoded by a polynucleotide of the invention; (2)
assays to
determine the precursor frequency (i.e., the presence and number of) of immune
effector cells specific for an antigen encoded by a polynucleotide of the
invention
54
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
and/or its natural counterpart: and (3) assays to determine the efficacy of an
antigen encoded by a polynucleotide of the invention once it has been used in
an
immunomodulatory method of the invention.
Diagnostic methods of the invention are generally carned out under
suitable conditions and for a sufficient time to allow specific binding to
occur
between an antigen encoded by a polynucleotide of the invention, usually
presented by an APC of the invention, and an immune effector molecule, such as
a TCR, on the surface of an immune effector cell, such as a CD4+ or CD8+ T
cell.
"Suitable conditions" and "sufficient time" are generally conditions and times
suitable for specific binding. Suitable conditions occur between about
4°C and
about 40°C, preferably between about 4°C and about 37°C,
in a buffered solution,
and within a pH range of between S and 9. A variety of buffered solutions are
known in the art, can be used in the diagnostic methods of this invention, and
include, but are not limited to, phosphate-buffered saline. Sufficient time
for
binding and response will generally be between about 1 second and about
24 hours after exposure of the sample to the antigen encoded by a
polynucleotide
of the invention.
In some embodiments, the invention provides diagnostic ssays to predict
the efficacy of an antigen encoded by a polynucleotide of the invention,
presented
by an APC of the invention. In some of these embodiments, defined T cell
epitopes are used to clinically characterize tumors and viral pathogens in
order to
.. . _ ,. determine, in advance, the predicted efficacy of an in vivo vaccine
trial. This can
be achieved by a simple proliferation assay of a patient's peripheral blood
mononuclear cells using defined T cell epitopes as stimulators. Peptides which
elicit a response are viable vaccine candidates for that patient.
In other embodiments, assays are provided to determine the precursor
frequency (i.e., the presence and number of) of resting (naive) immune
effector
cells specific for an antigen encoded by a polynucleotide of the invention and
which therefore have the potential to become activated. In these embodiments,
an
antigen-presenting cell bearing on its surface an antigen encoded by a
polynucleotide of the invention is used to detect the presence of immune
effector
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
cells in a biological sample which bind specifically to the natural epitope. A
functional assay is used to determine (and quantitate) the antigen-specific
immune
effector cells. As an illustrative example, PBMCs are isolated from a subject
with
a tumor. A sample of these PBMCs is cultured together for a suitable time with
the tumor cells from the same subject. A second sample of these PBMCs is
cultured together for a suitable time with surrogate APCs pulsed with an
antigen
encoded by a polynucleotide of the invention which corresponds to a natural
epitope(s) expressed on the surface of the tumor. Both tumor cells and
surrogate
APCs are loaded with''Cr. By comparing the amount of 5'Cr release from the
tumor cell and the antigen-pulsed surrogate APC, one can determine the
precursor
frequency of immune effector cells which are specific for tumor and the
precursor
frequency of immune effector cells which are specific for an antigen encoded
by a
polynucleotide. Functional assays include, but are not limited to, immune
effector
cell proliferation, cytokine production, specific lysis of an APC.
In other embodiments, the efficacy of an immunomodulatory method,
including immunomodulatory methods of the invention, in modulating an immune
response to an antigen encoded by a polynucleotide of the invention and/or its
natural counterpart, can be tested using diagnostic assays of the invention.
These
diagostic assays are also useful to assess or monitor the efficacy of an
immunotherapeutic agent. In some of these embodiments, the method allows
detection of immune effector cells, which may be activated CD4+ or CD8+ T
cells,
. . which. have become. activated or anergized as a result of exposure to an.
antigen
encoded by a polynucleotide of the invention. A sample containing cells from a
subject can be tested for the presence of CD4+ or CD8+ T cells which have
become activated or anergized as a result of binding to an antigen encoded by
a
polynucleotide of the invention. In some embodiments, the method comprises the
steps of: (a) contacting an immobilized APC presenting an antigen encoded by a
polynucleotide of the invention on its surface bound to Class I and Class II
MHC
molecules with a biological sample under suitable conditions and for a time
sufficient to allow binding of an immune effector cell which bears on its
surface
an antigen receptor specific for the peptide, thereby immobilizing the antigen-
56
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
specific immune effector cell; and (b) contacting the immobilized immune
effector cell with a detestably labeled molecule, such as an antibody, which
specifically binds the immune effector cell. In other embodiments, the method
comprises the steps of (a) contacting an immobilized antigen-presenting matrix
which presents an antigen encoded by a polynucleotide of the invention on its
surface bound to a Class I or Class II MHC molecule with a biological sample
under suitable conditions and for a time sufficient to allow binding of an
immune
effector cell which bears on its surface an antigen receptor specific for the
peptide,
thereby immobilizing the antigen-specific immune effector cell; and (b)
performing a functional assay on the immobilized immune effector cell. Once
the
immune effector cell is bound to the immobilized APC presenting on its surface
an antigen encoded by a polynucleotide of the invention, it can be labeled on
the
basis of characteristic cell surface molecules, including, but not limited to,
CD4,
CDB, and cell surface markers specific for activated T cells. A variety of
cell
surface markers specific to populations of immune effector cells are known to
those skilled in the art and have been described in numerous publications.
see, for
example, The Leukocyte Antigen Facts Book, Barclay et al., eds., 1995,
Academic
Press. Antibodies to these markers are commercially available from. inter
alia,
Beckman Coulter. The immobilized immune effector cell can also be
characterized by presence of mRNA and/or proteins in the cytosol which are
characteristic of a given T cell type in a given activated or anergic state. A
,. ~ .. ,. characteristic mRNA can be detected by any known.~means, including,
but not
limited to, a polymerase chain reaction. A detestably labeled antibody to a
cell
surface marker can be contacted with the immobilized immune effector cell
under
suitable conditions and for a time sufficient to allow specific binding. If
necessary or desired, the labeled cells can be physically removed from unbound
label or excess unbound label can be inactivated. The requirements of an
antibody specific for a cell surface marker on an immune effector cell are
that the
antibody bind specifically and that the antibody not interfere with binding
between a TCR and the immobilized synthetic antigenic peptide epitope.
57
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
Labels which may be employed are known to those skilled in the art and
include, but are not limited to, traditional labeling materials such as
fluorophores,
radioactive isotopes, chromophores, and magnetic particles. Enzyme labels
include, but are not limited to, luciferase; a green fluorescent protein
(GFP), for
example, a GFP from Aequorea victoria, or any of a variety of GFP known in the
art; (3-galactosidase, chloramphenicol acetyl transferase. See, for example,
Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, and
periodic updates). Any assay which detects the label, either by directly or
indirectly, is suitable for use in the present invention. Assays include
colorimetric, fluorimetric, or luminescent assays, radioimmunoassays or other
immunological assays.
Immunomodulatory methods
The invention provides methods of modulating an immune response in an
individual to an antigen encoded by a polynucleotide of the invention, and
thus to
the corresponding natural epitope. Immunomodulatory methods of the invention
include methods that result in induction or increase, as well as methods that
result
in suppression or reduction, of an immune response in a subject, and comprise
administering to the subject an effective amount of a polynucleotide, or an
APC,
or an immune effector cell, of the invention in formulations and/or under
conditions that result in the desired effect on an immune response (or lack
thereof)
M. ~ . . ao :the peptide antigen. Immunomodulatory methods of the
invention.include ..
vaccine methods, adoptive immunotherapy, and methods to induce T cell
unresponsiveness, or anergy.
Various methods are known to evaluate T celi activation. CTL activation
can be detected by any known method, including but not limited to, tritiated
thymidine incorporation (indicative of DNA synthesis), and examination of the
population for growth or proliferation, e.g., by identification of colonies.
Alternatively, the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-
diphenyl tetrazolium bromide) may be added. Mossman (1983) J. Immunol.
Methods 65:55-63; Niks and Otto (1990) J. Immunol. Methods 130:140-151.
58
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
Succinate dehydrogenase. found in mitochondria of viable cells, converts the
MTT to formazan blue. Thus, concentrated blue color would indicate
metabolically active cells. In yet another embodiment, incorporation of
radiolabeh e.g., tritiated thymidine, may be assayed to indicate proliferation
of
cells. Similarly, protein synthesis may be shown by incorporation of 3'S-
methionine. In still another embodiment, cytotoxicity and cell killing assays,
such
as the classical chromium release assay, may be employed to evaluate epitope-
specific CTL activation. To detect activation of CD4+ T cells, any of a
variety of
methods can be used, including, but not limited to, measuring cytokine
production; and proliferation, for example, by tritiated thymidine
incorporation
Release of 5' Cr from labeled target cells is a standard assay which can be
used to assess the number of peptide-specific CTLs in a biological sample.
Tumor cells, or APCs of the invention, are radiolabeled as targets with about
200
p,Ci of Na2 ''Cr04 for 60 minutes at 37° C, followed by washing. T
cells and
target cells (~1 x 104/well) are then combined at various effector-to-target
ratios
in 96-well, U-bottom plates. The plates are centrifuged at 100 x g for 5
minutes
to initiate cell contact, and are incubated for 4-16 hours at 37°C with
5% C02.
Release of''Cr is determined in the supernatant, and compared with targets
incubated in the absence of T cells (negative control) or with 0.1% TRITONTM X-
100 (positive control). See. e.g., Mishell and Shiigi, eds. Selected Methods
in
Cellular Immunology (1980) W.H. Freeman and Co.
. . , . . . . . , . The: formulation of an APC of the invention will vary;
depending on the
desired result. In general, peptides presented on an APC by Class I and Class
II
MHC molecules, together with the appropriate co-stimulatory molecules, will
result in induction of an immune response to the peptide. An anergic (or
unresponsive) state may be induced in T lymphocytes by presentation of an
antigen by an APC of the invention which contains appropriate MHC molecules
on its surface, but which lacks the appropriate co-stimulatory molecules.
Polynucleotides of the invention can be administered in a gene delivery
vehicle or by inserting into a host cell which in turn recombinantly
transcribes,
translates and processed the encoded polypeptide. Isolated host cells
containing a
59
CA 02322659 2000-09-06
WO 99/47641 PGT/US99/06030
polynucleotide of the invention in a pharmaceutically acceptable earner can be
combined with appropriate and effective amount of an adjuvant, cytokine or co-
stimulatory molecule for an effective vaccine regimen. In some embodiments,
the
host cell is an APC, such as a dendritic cell. The host cell can be further
modified
S by inserting of a polynucleotide coding for an effective amount of either or
both
of a cytokine a co-stimulatory molecule.
The methods of this invention can be further modified by co-administering
an effective amount of a cytokine or co-stimulatory molecule to the subject.
The agents provided herein as effective for their intended purpose can be
administered to subjects having a disease to be treated with an
immunomodulatory method of the invention or to individuals susceptible to or
at
risk of developing such a disease. When the agent is administered to a subject
such as a mouse, a rat or a human patient, the agent can be added to a
pharmaceutically acceptable carrier and systemically or topically administered
to
the subject. Therapeutic amounts can be empirically determined and will vary
with the pathology or condition being treated, the subject being treated and
the
efficacy and toxicity of the therapy.
The amount of a polynucleotide or APC or immune effector cell of the
invention will vary depending, in part, on its intended effect, and is
ultimately at
the discretion of the medical or veterinary practitioner. The factors to be
considered include the condition being treated, the route of adminitstration,
and
. . ~ . . , ... ., nature of the Formulation, the mammal's body weight,
surface area, age, and
general condition and the particular peptide to be administered. Cells can be
administered once, followed by monitoring of the clinical response, such as
diminution of disease symptoms or tumor mass. Administration may be repeated
on a monthly basis, for example, or as appropriate. Those skilled in the art
will
appreciate that an appropriate administrative regimen would be at the
discretion of
the physician or veterinary practitioner.
Administration in vivo can be effected in one dose, continuously or
intermittently throughout the course of treatment. Methods of determining the
most effective means and dosage of administration are well known to those of
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
skill in the art and will vary with the composition used for therapy, the
purpose of
the therapy, the target cell being treated, and the subject being treated.
Single or
multiple administrations can be carned out with the dose level and pattern
being
selected by the treating physician. Suitable dosage formulations and methods
of
administering the agents can be found below.
The agents and compositions of the present invention can be used in the
manufacture of medicaments and for the treatment of humans and other animals
by administration in accordance with conventional procedures, such as an
active
ingredient in pharmaceutical compositions.
More particularly, an agent of the present invention also referred to herein
as the active ingredient, may be administered for therapy by any suitable
route
including nasal, topical (including transdermal, aerosol, buccal and
sublingual),
parenteral (including subcutaneous, intramuscular, intravenous and
intradermal)
and pulmonary. It will also be appreciated that the preferred route will vary
with
the condition and age of the recipient, and the disease or condition being
treated.
Vaccines for cancer treatment and prevention
In one embodiment, immunomodulatory methods of the present invention
comprise vaccines for cancer treatment. Cancer cells contain many new antigens
potentially recognizable by the immune system. Given the speed with which
epitopes can be identified, custom anticancer vaccines can be generated for
affected individuals by isolating TILs from patients with solid tumors,
. , , . determining their MHC restriction, and assaying these CTLs against the
. . .
appropriate library for reactive epitopes. These vaccines will be both
treatments
for affected individuals as well as preventive therapy against recurrence (or
establishment of the disease in patients which present with a familial genetic
predisposition to it). Inoculation of individuals who have never had the
cancer is
expected to be quite successful as preventive therapy, even though a tumor
antigen-specific CTL response has not yet been elicited, because in most cases
high affinity peptides seem to be immunogenic suggesting that holes in the
functional T cell repertoire. if they exist, may be relatively rare. Sette et
al.
(1994) J. Immunol., 153:5586-5592. In mice, vaccination with appropriate
61
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
epitopes not only eliminates established tumors but also protects against
tumor re-
establishment after inoculation with otherwise lethal doses of tumor cells.
Bystryn et al. (1993) Supra.
Vaccines, for diseases caused by pathogenic organisms
Polynucleotides and APCs of the present invention are also useful in
methods to induce (or increase, or enhance) an immune response to a pathogenic
organism. These include pathogenic viruses, bacteria, and protozoans.
Viral infections are ideal candidates for immunotherapy. Immunological
responses to viral pathogens are sometimes ineffective as in the case of the
lentiviruses such as HIV which causes AIDS. The high rates of spontaneous
mutation make these viruses elusive to the immune system. However, a
saturating
profile of CTL epitopes presented on infected cells will identify shared
antigens
among different serotypes in essential genes that are largely intolerant to
mutation
which would allow the design of more effective vaccines.
Adoptive Immunotherapy Methods
The expanded populations of antigen-specific immune effector cells and
APCs of the present invention find use in adoptive immunotherapy regimes and
as
vaccines.
Adoptive immunotherapy methods involve, in one aspect, administering to
a subject a substantially pure population of educated, antigen-specific immune
effector cells made by culturing naive immune effector cells with APCs as
.. . ~ . described above. In some embodiments, the APCs are dendritic cells.
..
In one embodiment, the adoptive immunotherapy methods described
herein are autologous. In this case, the APCs are made using parental cells
isolated from a single subject. The expanded population also employs T cells
isolated from that subject. Finally, the expanded population of antigen-
specific
cells is administered to the same patient.
In a further embodiment, APCs or immune effector cells are administered
with an effective amount of a stimulatory cytokine, such as IL-2 or a co-
stimulatory molecule.
62
CA 02322659 2000-09-06
WO 99/47641 PCTNS99/06030
Methods of inducing T cell anergy
Synthetic antigenic peptide epitopes of the present invention are useful in
methods to induce T cell unresponsiveness, or anergy. Disorders which can be
treated using these methods include autoimmune disorders, allergies, and
allograft
rejection.
Autoimmune disorders are diseases in which the body's immune system
responds against self tissues. They include most forms of arthritis,
ulcerative
colitis, and multiple sclerosis. Polynucleotides of the invention encoding
antigens
corresponding to endogenous elements that are recognized as foreign can be
used
in the development of treatments using gene therapy or other approaches. For
example, synthetic CTL epitopes, which can act as "suicide substrates" for
CTLs
that mediate autoimmunity, can be designed as described above. That is to say,
peptides which have a high affinity for the MHC allele but fail to activate
the
TCR could effectively mask the cellular immune response against cells
presenting
1 S the antigen in question. In support of this approach, it is believed that
the long
latency period of the HIV virus is due to an antiviral immune response and a
mechanism by which the virus finally evades the immune system is by generating
epitopes that occupy the MHC molecules but do not stimulate a TCR lytic
response, inducing specific T cell anergy. Klenerman et al. (1995) Eur. J.
Immunol.. 25:1927-I 93 I .
In vitro stimulation of T cells through the complex of T cell-antigen
. ~. receptor and CD3 alone in the absence of other signals, induces T cell
anergy or
paralysis. T cell activation as measured by interleukin-2 production and
proliferation in vitro requires both antigenic and co-stimulatory signals
engendered by cell to cell interactions among antigen-specific T cells and
antigen
presenting cells. Various interactions of these CD2 proteins on the T-cell
surface
with CD58 (LFA-3) proteins and antigen-presenting cells, those of CD1 la/CD18
(LFA-I) proteins with CD54 (ICAM-1) proteins and those of CDS proteins with
CD72 proteins can impart such a co-stimulatory signal in vitro. Cytokines
derived from antigen-presenting cells (e.g., interleukin-1 and interleukin-6)
can
also provide co-stimulatory signals that result in T-cell activation in vitro.
The
63
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
delivery of both antigenic and co-stimulatory signals leads to stable
transcription
of the interleukin-2 gene and other pivotal T cell-activation genes. The
foregoing
co-stimulatory signals depend on protein kinase C and calcium. Potent antigen
presenting cells express CD80 (B7 and BB I ) and other related surface
proteins
and many T cells express B7 binding proteins, namely CD28 and CTLA-4
proteins. Binding of CD80 by CD28 and CDLA-4 stimulates a T cell co-
stimulatory pathway that is independent of protein kinase C and calcium
leading
to vigorous T cell proliferation. The stimulation of B cells also depends on
the
interaction between the specific antigen and the cell-surface immunoglobulin.
T cell derived cytokines (e.g., interleukins l and 4), physical contact
between
T cells and B cells through specific pairs of receptors and co-receptors, or
both,
provide the signal or signals essential for B cell stimulation.
Conventional routes of administration are used. A T-cell stimulating or
anergy producing amount (or therapeutically effective amount as described
above)
of an immunotherapeutic antigen-superantigen polymer according to the
invention
is contacted with the target cells. By "T-cell anergy effective amount" is
intended
an amount which is effective in producing a statistically significant
inhibition of a
cellular activity mediated by a TCR. This may be assessed in vitro using T-
cell
activation tests. Typically, T-cell anergy or activation is assayed by
tritiated
thymidine incorporation in response to specific antigen.
One way in which T cell anergy can be induced is to present to a T cell an
APC which presents an antigens in MHC Class I anti Class II molecules, but
which
lack co-stimulatory molecules necessary to activate a T cell. For example, a
cell
other than a normal antigen presenting cell (APC), which has been transfected
with MHC antigen to which a selected T cell clone is restricted, can be used.
Resting T cells are provided with an appropriate peptide recognized by the
resting
T cells in the context of the MHC transfected into a cellular host other than
an
APC. The MHC is expressed as a result of introduction into a mammalian cell
other than an antigen presenting cell of genes constitutively expressing the a
and ~i
chains of the MHC class II, or an MHC Class I molecule together with invariant
chain. Importantly, these cells do not provide other proteins, either cell
surface
64
CA 02322659 2000-09-06
WO 99/47641 PCT1US99/06030
proteins or secreted proteins, associated with antigen presenting cells, which
together with the MHC and peptide result in co-stimulatory signals.
To determine whether anergy has been induced, the T cells to be tested can
be cultured together with an APC which presents an antigen encoded by a
polynucleotide of the invention in MHC Class I and Class II molecules together
with co-stimulatory molecules necessary to activate the T cell. The cultures
are
incubated for about 48 hours, then pulsed with tritiated thymidine and
incorporation measured about 18 hours later. The absence of incorporation
above
control levels, where the T-cells are presented with antigen presenting cells
which
do not stimulate the T cells, either due to using an MHC to which the T cells
are
not restricted or using a peptide to which the T cells are not sensitive, is
indicative
of an absence of activation. One may use other conventional assays to
determine
the extent of activation, such as assaying for IL-2, -3, or -4, cell surface
proteins
associated with activation, e.g. CD71 or other convenient techniques. Another
method is to determine the expression of a protein which is expressed on
quiescent T cells, but not on anergic T cells. U.S. Patent No. 5,747,299.
Adoptive Immunotherapy
The cells and compositions of this invention are useful as cancer vaccines
and in adoptive immunotherapy. The ability of autologous antigen-pulsed
dendritic cells to induce a clinically relevant immune response has previously
been reported. Hsu et al. (1996) Nature Med. 2(1):52-58. Using this clinical
study as a guide, it is possible to administer an effective amount of the APCs
as
described herein to a subject to induce an anti-tumor immune response. After
isolation and purification of DCs (day 0) purified pulsed dendritic cells were
administered by subcutaneous injection on days 2, 28 and 56 and then 5 to 6
months later. At day 16, patients received subcutaneous injections with either
keyhole limpet hemocyanin or idiotype protein in saline at a site separate
from
intraveneous injection of the pulsed DCs.
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
The expanded populations of antigen-specif c immune effector cells of the
present invention also find use in adoptive immunotherapy regimes and as
vaccines.
Adoptive immunotherapy methods involve, in one aspect, administering to
S a subject a substantially pure population of educated, antigen-specific
immune
effector cells made by culturing naive immune effector cells with APCs as
described above. Preferably, the APCs are dendritic cells.
In one embodiment, the adoptive immunotherapy methods described
herein are autologous. In this case, the APCs are made using parental cells
isolated from a single subject. The expanded population also employs T cells
isolated from that subject. Finally, the expanded population of antigen-
specific
cells is administered to the same patient.
In another embodiment, the adoptive immunotherapy methods are
allogeneic. Here, cells from two or more patients are used to generate the
APCs,
and stimulate production of the immune effector cells. For instance, cells
from
other healthy or diseased subjects can be used to generate antigen-specific
cells in
instances where it is not possible to obtain autologous T cells and/or
dendritic
cells from the subject providing the biopsy. The expanded population can be
administered to any one of the subjects from whom cells were isolated, or to
another subject entirely.
In a further embodiment, APCs or immune effector cells are administered
with an effective amount of a stimulatory cytokine, such as IL-2 or a co-
stimulatory molecule.
The agents identified herein as effective for their intended purpose can be
administered to subjects having tumors or individuals susceptible to or at
risk of a
tumor. When the agent is administered to a subject such as a mouse, a rat or a
human patient, the agent can be added to a pharmaceutically acceptable carrier
and systemically or topically administered to the subject. To determine
patients
that can be beneficially treated, a tumor regression can be assayed.
Therapeutic
amounts can be empirically determined and will vary with the pathology being
treated, the subject being treated and the efficacy and toxicity of the
therapy.
66
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
When delivered to an animal, the method is useful to further conf rm efficacy
of
the agent.
Administration in vivo can be effecte3 in one dose, continuously or
intermittently throughout the course of treatment. Methods of determining the
most effective means and dosage of administration are well known to those of
skill in the art and will vary with the composition used for therapy, the
purpose of
the therapy, the target cell being treated, and the subject being treated.
Single or
multiple administrations can be carried out with the dose level and pattern
being
selected by the treating physician. Suitable dosage formulations and methods
of
administering the agents can be found below.
Also within the scope of this invention is an epitope or wild-type antigenic
peptide corresponding to a yet unidentified protein. A common strategy in the
search for tumor antigens is to isolate tumor-specific T-cells and attempt to
identify the antigens recognized by these cells. In patients with cancer,
specific
CTLs have been derived from lymphocytic infiltrates present~at the tumor site.
Weidmann, et al., supra. These TILs are unique cell population that can be
traced
back to sites of disease when they are labeled with indium and adoptively
transferred. Alternatively, large libraries of putative antigens can be
produced and
tested. Using the "phage method" (Scott and Smith (1990) Science 249:386-390;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6387-6382; Devlin et al. (1990)
Science 249:404-406), very large libraries can be constructed. Another
approach
uses primarily chemical methods, of which the Geysen method (Geysen et al.
(1986) Mol. Immunol. 23:709-715; Geysen et al. (1987) J. Immunol. Method
102:259-274) and the method of Fodor et al. ( 1991 ) Science 251:767-773, are
examples. Furka et al. (1988) 14th Inter. Cong. Bio. Vol. 5, Abst. FR:013;
Furka
(1991) Inter. J. Peptide Protein Res. 37:487-493), Houghton (U.S. Patent No.
4,683,211, issued December 1986) and Rutter et al. (U.S. Patent No. 5,010,175,
issued April 23, 1991 ) describe methods to produce a mixture of peptides that
can
be tested as agonists or antagonists.
In a further aspect of this invention, Solid-PHase Epitope REcovery
("SPHERE", described in PCT WO 97/35035) can be used to identify tumor
67
CA 02322659 2000-09-06
WO 99/47641 PGT/US99/06030
antigens. Briefly, SPHERE can be used to identify antigens by creating a
library
of molecules, preferably peptides, and attaching one type of molecule to a
solid
support via a releasable linker. At least a portion of the molecules bound to
each
support can be released and it can be determined if the antigen-specific
immune
effector cells recognized the peptide.
Thus, this invention also provides a screen to identify novel wild-type
antigens that can be further modified and used to induce a cellular and a
humoral
immune response in the subject. The antigens and their biological activity in
vitro and in vivo are positive controls. Using the methods described herein,
the
biological activity of the isolated antigen and its secreted form can be
compared to
their biological activity.
Furthermore, the invention provides a method for cloning the cDNA and
genomic DNA encoding such a protein by generating degenerate oligonucleotides
probes or primers based on the sequence of the epitope. Compositions
comprising
the nucleic acid and a carrier, such as a pharmaceutically acceptable carrier,
a
solid support or a detectable label, are further provided by this method as
well as
methods for detecting the sequences in a sample using methods such as Northern
analysis, Southern analysis and PCR.
Further provided by this invention are therapeutic and diagnostic
oligopeptide sequences determined according to the foregoing methods.
Compositions comprising the oligopeptide sequence and a carrier, such as a
pharmaceutically acceptable Garner, a solid support or a detectable label, are
further provided by this method as well as methods for detecting the
oligopeptide
sequence in a sample using methods such as Western analysis and ELISA.
Harlow and Lane ( 1989), supra.
The following examples are provided to illustrate, but not limit, the
invention.
EXAMPLES
68
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
Identiftcation of Tumor Associated Antigens
Any conventional method, e.g., expression cloning methodology as
described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. 91:351-19, can be
used to identify a novel tumor-associated antigen. Briefly, in this method, a
library of cDNAs corresponding to mRNAs derived from tumor cells is cloned
into an expression vector and introduced into target cells which are
subsequently
incubated with cytotoxic T cells. One identifies pools of cDNAs that are able
to
stimulate the CTL and through a process of sequential dilution and re-testing
of
less complex pools of cDNAs one is able to derive unique cDNA sequences that
are able to stimulate the CTL and thus encode the cognate tumor antigen.
SAGE (U.S. Patent No. 5,695,937) and SPHERE (described in PCT WO
97/35035), also can be used to identify putative antigens for use in the
subject
invention.
SAGE analysis can be employed to identify the antigens recognized by
1 S expanded immune effector cells such as CTLs, by identifying nucleotide
sequences expressed in the antigen-expressing cells. Briefly, SAGE analysis
begins with providing complementary deoxyribonucleic acid (cDNA) from ( 1 )
the
antigen-expressing population and (2) cells not expressing that antigen. Both
cDNAs can be linked to primer sites. Sequence tags are then created, for
example, using the appropriate primers to amplify the DNA. By measuring the
differences in these tag sets between the two cell types, sequences which are
aberrantly expressed in the antigen-expressing cell population can be
identified.
Another method which may be used to identify antigenic epitopes is Solid
PHase Epitope REcovery ("SPHERE") which is described in PCT WO 97/35035.
Briefly, roughly speaking, peptide libraries are loaded onto beads and
inserted
into 96-well plates. The plates with 1000 beads per well will accommodate 106
beads; ten 96-well plates with 100 beads per well will accommodate 105 beads.
In
order to minimize both the number of CTL cells required per screen and the
amount of manual manipulations, the eluted peptides can be further pooled to
yield wells with any desired complexity. For example, based on experiments
with
soluble libraries, it should be possible to screen 10' peptides in 96-well
plates
69
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
(10,000 peptides per well) with as few as 2 X lOb CTL cells. After cleaving a
percentage of the peptides from the beads, incubating them with gamma-
irradiated
APCs or foster APCs, and the cloned CTL line(s), positive wells are determined
by 3H-thymidine incorporation. Alternatively, as pointed out above. cytokine
production or cytolytic ' ~ Cr-release assays may be used (Coutic et al. ( I
992) Int.
J. Cancer. 50:289-291 ). Beads from each positive well are then separated and
assayed individually, utilizing an additional percentage of the peptide from
each
bead. Positive individual beads will then be decoded, identifying the reactive-
amino acid sequence. Analysis of all positives will give a partial profile of
conservatively substituted epitopes which stimulate the CTL clone tested. At
this
point, the peptide can be resynthesized and retested. Also, a second library
(of
minimal complexity) can be synthesized with representations of all
conservative
substitutions in order to enumerate the complete spectrum of derivatives
tolerated
by a particular CTL. By screening multiple CTLs (of the same MHC restriction)
simultaneously, the search for crossreacting epitopes is greatly facilitated.
Alternatively, muteins of the antigen as well as allogeneic and antigens
from a different species, of previously characterized antigens are useful in
the
subject invention. MART-1 and gp100 are melanocyte differentiation antigens
specifically recognized by HLA-A2 restricted tumor-infiltrating lymphocytes
(TILs) derived from patients with melanoma, and appear to be involved in tumor
regression (Kawakami et al. ( 1994) Proc. Natl. Acad. Sci. U.S.A. 91:6458-62;
Kawakami et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:91:3515-9). Recently,
the mouse homologue of human MART-1 has been isolated. The full-length open
-ending frame of the mouse MART-I consists of 342 bp, encoding a protein of
113 amino acid residues with a predicted molecular weight of ~l 3 kDa.
Alignment of human and marine MART-1 amino acid sequences showed 68.6%
identity.
In another embodiment, the described method for the identification of
CD8+ MHC Class I restricted CTL epitopes can be applied to the identification
of
CD4+ MHC Class II restricted helper T-cell (Th) epitopes. In this case, MHC
Class II allele-specific libraries are synthesized such that haplotype-
specific
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
anchor residues are represented at the appropriate positions. MHC Class II
agretopic motifs have been identified for the common alleles (Rammensee (1995)
Curr. Opin. Immunol. 7:85-96; Altuvia et al. (1994) Mol. Immunol. 24:375-379;
Reay et al. ( 1994) J. Immunol. 152:3946-3957; Verreck et al. ( 1994) Eur. J.
Immunol. 24:375-379; Sinigaglia and Hammer ( 1994) Curr. Opin. Immunol.
6:52-56: Rotzschke and Falk ( 1994) Curr. Opin. Immunol. 6:45-51 ). The
overall
length of the peptides will be 12-20 amino acid residues, and previously
described
methods may be employed to limit library complexity. The screening process is
identical to that described for MHC Class I-associated epitopes except that B
lymphoblastoid cell lines (B-LCL) are used for antigen presentation rather
than
T2 cells. In a preferred aspect, previously characterized B-LCLs that are
defective in antigen processing (Mellins et al. ( I 991 ) J. Exp. Med.
174:1607-
1615); thus allowing specific presentation of exogenously added antigen, are
employed. The libraries are screened for reactivity with isolated CD4+ MHC
Class II allele-specific Th cells. Reactivity may be measured by 3H-thymidine
incorporation according to the method of Mellins, et al. supra., or by any of
the
methods previously described for MHC Class I-associated epitope screening.
In vitro confirmation of the immunogenicity of a putative antigen of this
invention can be confirmed using the method described below which assays for
the production of CTLs. However, weakly immunogeneic antigens also can be
used for the making of compositions and in the methods of this invention.
Using these sequences and recombinant expression techniques
summarized below, polypeptides and proteins can be made for presentation on
the
APC of this invention.
Tumor Protection in Animal Models
The transcription cassette shown in the Figure is contained within an
adenoviral vector such that expression of the genes leads to the production of
intracellular and secreted forms of the same protein. The adenoviral vector
can be
employed either in vivo for direct immunization as described in Zhai et al. (
1996)
J. Immunol. 156:700-10 or ex vivo wherein antigen presenting cells (preferably
71
CA 02322659 2000-09-06
WO 99/47641 PCT/US99/06030
dendritic cells) derived from a host are transduced with the adenoviral vector
and
the genetically modified APCs are subsequently infused back into the host as
described in Ribas et al. (1997) Cancer Research 57:2865-9. For in vivo
immunization, C57BL/6 mice are injected subcutaneously at two sites (1.SE9 IU
per site) with the adenoviral vector encoding the wild type and secreted forms
of
the same antigen. Two weeks later, animals are challenged with a lethal dose
(2 x 10'x) of marine B 16F 10 melanoma tumor cells by subcutaenous injection.
Animals are scored for survival and tumor measurements are taken. For ex vivo
dendritic cell therapy, bone marrow cells are harvested from C57BL/6 mice and
cultured in vitro with GM-CSF and IL4 to derive dendritic cells. The DCs are
infected at an MOI of S00 with the adenoviral vector overnight, and the next
day
the DCs (S x 105) are washed and infused into recipient C57BL/6 mice via tail
vein injection. Two weeks later, animals are challenged with a lethal dose of
marine B 16F 10 melanoma tumor cells via subcutaneous injection as above and
tumor size and survival are recorded as a function of time.
It is to be understood that while the invention has been described in
conjunction with the above embodiments, that the foregoing description and the
following examples are intended to illustrate and not limit the scope of the
invention. For example, any of the above-noted compositions and/or methods can
be combined with known therapies or compositions. Other aspects, advantages
and modifications within the scope of the invention will be apparent to those
skilled in the art to which the invention pertains.
72
