Note: Descriptions are shown in the official language in which they were submitted.
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OPTIIVIIZATION OF IMMUNOMODULATORY PROPERTIES OF
GENETIC VACCINES
STATEMENT REGARDING GOVERNIYIENT SUPPORT
This invention was made with Government support under Grant No.
N65236-98-1-5401, awarded by the Defense Advanced Projects Agency. The
Government
has certain rights in this invention.
BACKGROUND OF THE INVENTION
Field of the Invention
I S This invention pertains to the field of modulation of immune responses
such
as those induced by genetic vaccines.
Background
Antigen processing and presentation is only one factor which determines the
effectiveness of vaccination, whether performed with genetic vaccines or more
classical
methods. Other molecules involved in determining vaccine effectiveness include
cytokines
(interleukins, interferons, chemokines, hematopoietic growth factors, tumor
necrosis factors
and transforming growth factors), which are small molecular weight proteins
that regulate
maturation, activation, proliferation and differentiation of the cells of the
imrriune system.
Characteristic features of cytokines are pleiotropy and redundancy; that is,
one cytokine
often has several functions and a given function is often mediated by more
than one
cytokine. In addition, several cytokines have additive or synergistic effects
with other
cytokines, and a number of cytokines also share receptor components.
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Due to the complexity of the cytokine networks, studies on the physiological
significance of a given cytokine have been difficult, although recent studies
using cytokine
gene-deficient mice have significantly improved our understanding on the
functions of
cytokines in vivo. In addition to soluble proteins, several membrane-bound
costimulatory
molecules play a fundamental role in the regulation of immune responses. These
molecules
include CD40, CD40 ligand, CD27, CD80, CD86 and CD 150 (SLAM), and they are
typically expressed on lymphoid cells aRer activation via antigen recognition
or through
cell-cell interactions.
T helper (TH) cells, key regulators of the immune system, are capable of
producing a large number of different cytokines, and based on their cytokine.
synthesis
pattern TH cells are divided into two subsets (Paul and Seder (1994) Cell76:
241-251). TH1
cells produce high levels of IL-2 and IFN-y and no or minimal levels of IL-4,
IL-5 and IL-
13. In contrast, TH2 cells produce high levels of IL-4, IL-5 and IL-13, and IL-
2 and IFN-y
production is minimal or absent. TH1 cells activate macrophages, dendritic
cells and augment
the cytolytic activity of CD8+ cytotoxic T lymphocytes and NK cells (Id.),
whereas TH2 cells
provide efficient help for B cells and they also mediate allergic responses
due to the capacity
of TH2 cells to induce IgE isotype switching and differentiation of B cells
into IgE secreting
cell (De Vries and Punnonen (1996) In Cytokine regulation of humoral immunity:
basic and
clinical aspects. Eds. Snapper, C.M., John Wiley & Sons, Ltd., West Sussex,
UK, p. 195-
215). The exact mechanisms that regulate the differentiation of T helper cells
are not fully
understood, but cytokines are believed to play a major role. IL-4 has been
shown to direct
TH2 differentiation, whereas IL-12 induces development of TH1 cells (Paul and
Seder,
supra.). In addition, it has been suggested that membrane bound costimulatory
molecules,
such as CD80, CD86 and CD150, can direct TH1 and/or TH2 development, and the
same
molecules that regulate TH cell differentiation also affect activation,
proliferation and
differentiation of B cells into Ig-secreting plasma cells (Cocks et al. (1995)
Nature 376:
260-263; Lenschow et al. (1996) Immunity 5: 285-293; Punnonen et al. (1993)
Proc. Nat'l.
Acad. Sci. USA. 90: 3730-3734; Punnonen et al. (1997) J. Exp. Med. 185: 993-
1004).
Studies in both man and mice have demonstrated that the cytokine synthesis
profile of T helper (TH) cells plays a crucial role in determining the outcome
of several viral,
bacterial and parasitic infections. High frequency of TH1 cells generally
protects from lethal
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infections, whereas dominant TH2 phenotype often results in disseminated,
chronic
infections. For example, TH 1 phenotype is observed in tuberculoid (resistant)
form of
leprosy and TH2 phenotype in lepromatous, multibacillary (susceptible) lesions
(Yamamura
et al. (1991) Science 254: 277-279). Similarly, late-stage HIV patients have
TH2-like
cytokine synthesis profiles, and TH1 phenotype has been proposed to protect
from AIDS
(Maggi et al. (1994} ,l. Exp. Med. 180: 489-495). Furthermore, the survival
from
meningococcal septicemia is genetically determined based on the capacity of
peripheral
blood leukocytes to produce TNF-a and IL-10. Individuals from families with
high
production of IL-10 have increased risk of fatal meningococcal disease,
whereas members of
families with high TNF-a production were more likely to survive the infection
(Westendorp
et al. (1997) Lancet 349: 170-173).
Cytokine treatments can dramatically influence TH1/TH2 cell differentiation
and macrophage activation, and thereby the outcome of infectious diseases. For
example,
BALB/c mice infected with Leishmania major generally develop a disseminated
fatal
disease with a TH2 phenotype, but when treated with anti-IL-4 mAbs or IL-12,
the frequency
of TH1 cells in the mice increases and they are able to counteract the
pathogen invasion
(Chatelain et al. (1992) J. Immunol. 148: 1182-1187). Similarly, IFN-y
protects mice from
lethal Herpes Simplex Virus (HSV) infection, and MCP-1 prevents lethal
infections by
Pseudomonas aeruginosa or Salmonella typhimurium. In addition, cytokine
treatments, such
as recombinant IL-2, have shown beneficial effects in human common variable
immunodeficiency (Cunningham-Rundles et al. (1994) N. Engl. J. Med. 331: 918-
921).
The administration of cytokines and other molecules to modulate immune
responses in a manner most appropriate for treating a particular disease can
provide a
significant tool for the treatment of disease. However, presently available
immunomodulator
treatments can have several disadvantages, such as insufficient specific
activity, induction of
immune responses against, the immunomodulator that is administered, and other
potential
problems. Thus, a need exists for immunomodulators that exhibit improved
properties
relative to those currently available. The present invention fulfills this and
other needs.
SUMMARY OF THE INVENTION
The present invention provides methods of obtaining a polynucleotide that
has a modulatory effect on an immune response that is induced by a genetic
vaccine, either
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directly (i.e., as an immunomodulatory polynucleotide) or indirectly (i.e.,
upon translation of
the polynucleotide to create an immunomodulatory polypeptide. The methods of
the
invention involve: creating a library of recombinant polynucleotides; and
screening the
library to identify at least one optimized recombinant polynucleotide that
exhibits, either by
itself or through the encoded polypeptide, an enhanced ability to modulate an
immune
response than a form of the nucleic acid from which the library was created.
Examples
include, for example, CpG-rich polynucleotide sequences, polynucleotide
sequences that
encode a costimulator (e.g., B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40),
CD150
(SLAM), or a cytokine. The screening step used in these methods can include,
for example,
introducing genetic vaccine vectors which comprise the library of recombinant
nucleic acids
into a cell, and identifying cells which exhibit an increased ability to
modulate an immune
response of interest or increased ability to express an immunomodulatory
molecule. For
example, a library of recombinant cytokine-encoding nucleic acids can be
screened by
testing the ability of cytokines encoded by the nucleic acids to activate
cells which contain a
receptor for the cytokine. The receptor for the cytokine can be native to the
cell, or can be
expressed from a heterologous nucleic acid that encodes the cytokine receptor.
For example,
the optimized costimulators can be tested to identify those for which the
cells or culture
medium are capable of inducing a predominantly TH2 immune response, or a
predominantly
TH1 immune response.
In some embodiments, the polynucleotide that has a modulatory effect on an
immune response is obtained by: (1) recombining at least first and second
forms of a nucleic
acid that is, or encodes a molecule that is, involved in modulating an immune
response,
wherein the first and second forms differ from each other in two or more
nucleotides, to
produce a library of recombinant polynucleotides; and (2) screening the
library to identify at
least one optimized recombinant polynucleotide that exhibits, either by itself
or through the
encoded polypeptide, an enhanced ability to modulate an immune response than a
form of
the nucleic acid from which the library was created. If additional
optimization is desired, the
method can further involve: (3) recombining at least one optimized recombinant
polynucleotide with a further form of the nucleic acid, which is the same or
different from
the first and second forms, to produce a further library of recombinant
polynucleotides; (4)
screening the further library to identify at least one further optimized
recombinant
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polynucleotide that exhibits an enhanced ability to modulate an immune
response than a
form of the nucleic acid from which the library was created.; and (5)
repeating {3) and (4),
as necessary, until the further optimized recombinant polynucleotide exhibits
an further
enhanced ability to modulate an immune response than a form of the nucleic
acid from
which the library was created.
In some embodiments of the invention, the library of recombinant
polynucleotides is screened by: expressing the recombinant polynucleotides so
that the
encoded peptides or polypeptides are produced as fusions with a protein
displayed on the
surface of a replicable genetic package; contacting the replicable genetic
packages with a
plurality of cells that display the receptor; and identifying cells that
exhibit a modulation of
an immune response mediated by the receptor.
The invention also provides methods for obtaining a polynucleotide that
encodes an accessory molecule that improves the transport or presentation of
antigens by a
cell. These methods involve creating a library of recombinant polynucleotides
by subjecting
to recombination nucleic acids that encode all or part of the accessory
molecule; and
screening the library to identify an optimized recombinant polynucleotide that
encodes a
recombinant accessory molecule that confers upon a cell an increased or
decreased ability to
transport or present an antigen on a surface of the cell compared to an
accessory molecule
encoded by the non-recombinant nucleic acids. In some embodiments, the
screening step
involves: introducing the library of recombinant polynucleotides into a
genetic vaccine
vector that encodes an antigen to form a library of vectors; introducing the
library of vectors
into mammalian cells; and identifying mammalian cells that exhibit increased
or decreased
immunogenicity to the antigen.
In some embodiments of the invention, the cytokine that is optimized is
interleukin-12 and the screening is performed by growing mammalian cells which
contain
the genetic vaccine vector in a culture medium, and detecting whether T cell
proliferation or
T cell differentiation is induced by contact with the culture medium. In
another embodiment,
the cytokine is interferon-a and the screening is performed by expressing the
recombinant
vector module as a fusion protein which is displayed on the surface of a
bacteriophage to
form a phage display library, and identifying phage library members which are
capable of
inhibiting proliferation of a B cell line. Another embodiment utilizes B7-1
(CD80) or B7-2
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(CD86) as the costimulator and the cell or culture medium is tested for
ability to modulate an
immune response.
The invention provides methods of using DNA shuffling to obtain optimized
recombinant vector modules that encode cytokines and other costimulators that
exhibit
reduced immunogenicity compared to a corresponding polypeptide encoded by a
non-
optimized vector module. The reduced immunogenicity can be detected by
introducing a
cytokine or costimulator encoded by the recombinant vector module into a
mammal and
determining whether an immune response is induced against the cytokine.
The invention also provides methods of obtaining optimized
immunomodulatory sequences that encode a cytokine antagonist. For example,
suitable
cytokine agonists include a soluble cytokine receptor and a transmembrane
cytokine receptor
having a defective signal sequence. Examples include DIL-l OR and AIL-4R, and
the like.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows an example of a cytotoxic T-cell inducing sequence (CTIS)
obtained from HBsAg polypeptide (PreS2 plus S regions).
Figure 2 shows a CTIS having heterologous epitopes attached to the
cytoplasmic portion.
Figure 3 shows the derivation of immunogenic agonistic sequences (IAS) as
described in Example 3. Specific killing (percent) is shown for an effector:
target (E:T) ratio
of five.
Figure 4 shows a method for preparing immunogenic agonist sequences
(IAS). Wild-type (WT) and mutated forms of nucleic acids encoding a
polypeptide of
interest are assembled and subj ected to DNA shuffling to obtain a nucleic
acid encoding a
poly-epitope region that contains potential agonist sequences.
Figure 5 shows a scheme for improving immunostimulatory sequences by
DNA shuffling.
Figure 6 is a diagram of a procedure by which recombinant libraries of human
IL-12 genes can be screened to identify shuffled IL-12 genes that encode
recombinant IL-12
having increased ability to induce T cell proliferation.
Figure 7 shows the results of a high-throughput functional assay for vectors
that encode variants of IL-12 obtained using the methods of the invention.
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Figure 8 shows the induction of T cell proliferation upon transfection of the
T
cells by individual vectors that encode IL-12 variants.
Figure 9 shows results of an experiment which demonstrates that a shuffled
IL-12 chimera obtained using the methods of the invention exhibits improved
ability to
activate human T cells.
Figure 10 shows a model of how T cell activation or anergy can be induced
by genetic vaccine vectors that encode different B7-1 (CD80) and/or B7-2
(CD86) variants.
Figure 11 shows a method for using DNA shuffling to obtain CD80/CD86
variants that have improved capacity to induce T cell activation or anergy.
Figure 12 shows results obtained in a screening assay for altered function of
B7.
Figure 13 provides experimental results which demonstrate that shuffled B7
chimeras provide potent T cell activation.
Figure 14 presents an alignment of the nucleotide sequences for human and
1 S mouse IL-10 receptor sequences.
Figure 15 shows an alignment of the nucleotide sequences of B7-1 (CD80)
genes from human, rhesus monkey, and rabbit.
DETAILED DESCRIPTION
Definitions
The term "cytokine" includes, for example, interleukins, interferons,
chemokines, hematopoietic growth factors, tumor necrosis factors and
transforming growth
factors. In general these are small molecular weight proteins that regulate
maturation,
activation, proliferation and differentiation of the cells of the immune
system.
The term "screening" describes, in general, a process that identifies optimal
immunomodulatory molecules. Several properties of the respective molecules can
be used
in selection and screening including, for example, ability to induce a desired
immune
response in a test system. Selection is a form of screening in which
identification and
physical separation are achieved simultaneously by expression of a selection
marker, which,
in some genetic circumstances, allows cells expressing the marker to survive
while other
cells d~ (or vice versa). Screening markers include, for example, luciferase,
beta-
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galactosidase and green fluorescent protein. Selection markers include drug
and toxin
resistance genes, and the like. Because of limitations in studying primary
immune responses
in vitro, in vivo studies are particularly useful screening methods. In these
studies, the
genetic vaccines that include immunomodulatory molecules are first introduced
to test
animals, and the immune responses are subsequently studied by analyzing
protective
immune responses or by studying the quality or strength of the induced immune
response
using lymphoid cells derived from the immunized animal. Although spontaneous
selection
can and does occur in the course of natural evolution, in the present methods
selection is
performed by man.
A "exogenous DNA segment", "heterologous sequence" or a "heterologous
nucleic acid", as used herein, is one that originates from a source foreign to
the particular
host cell, or, if from the same source, is modified from its original form.
Thus, a
heterologous gene in a host cell includes a gene that is endogenous to the
particular host cell,
but has been modified. Modification of a heterologous sequence in the
applications
described herein typically occurs through the use of DNA shuffling. Thus, the
terms refer to
a DNA segment which is foreign or heterologous to the cell, or homologous to
the cell but in
a position within the host cell nucleic acid in which the element is not
ordinarily found.
Exogenous DNA segments are expressed to yield exogenous polypeptides.
The term "gene" is used broadly to refer to any segment of DNA associated
with a biological function. Thus, genes include coding sequences and/or the
regulatory
sequences required for their expression. Genes also include nonexpressed DNA
segments
that, for example, form recognition sequences for other proteins. Genes can be
obtained from
a variety of sources, including cloning from a source of interest or
synthesizing from known
or predicted sequence information, and may include sequences designed to have
desired
parameters.
The term "isolated", when applied to a nucleic acid or protein, denotes that
the nucleic acid or protein is essentially free of other cellular components
with which it is
associated in the natural state. It is preferably in a homogeneous state
although it can be in
either a dry or aqueous solution. Purity and homogeneity are typically
determined using
analytical chemistry techniques such as polyacrylamide gel electrophoresis or
high
performance liquid chromatography. A protein which is the predominant species
present in a
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preparation is substantially purified. In particular, an isolated gene is
separated from open
reading frames which flank the gene and encode a protein other than the gene
of interest.
The term "purified" denotes that a nucleic acid or protein gives rise to
essentially one band
in an electrophoretic gel. Particularly, it means that the nucleic acid or
protein is at least
about 50% pure, more preferably at least about 85% pure, and most preferably
at least about
99% pure.
The term "naturally-occurring" is used to describe an object that can be found
in nature as distinct from being artificially produced by man. For example, a
polypeptide or
polynucleotide sequence that is present in an organism (including viruses,
bacteria, protozoa,
insects, plants or mammalian tissue) that can be isolated from a source in
nature and which
has not been intentionally modified by man in the laboratory is naturally-
occurring.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the
term encompasses nucleic acids containing known analogues of natural
nucleotides which
have similar binding properties as the reference nucleic acid and are
metabolized in a manner
similar to naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic
acid sequence also implicitly encompasses conservatively modified variants
thereof (e.g.
degenerate codon substitutions) and complementary sequences and as well as the
sequence
explicitly indicated. Specifically, degenerate codon substitutions may be
achieved by
generating sequences in which the third position of one or more selected (or
all) codons is
substituted with mixed-base andlor deoxyinosine residues (Batzer et al. (1991)
Nucleic Acid
Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; Cassol et
al. (1992) ;
Rossolini et al. (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is
used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
"Nucleic acid derived from a gene" refers to a nucleic acid for whose
synthesis the gene, or a subsequence thereof, has ultimately served as a
template. Thus, an
mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a
DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc.,
are all
derived from the gene and detection of such derived products is indicative of
the presence
and/or abundance of the original gene and/or gene transcript in a sample.
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A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is
operably linked to a coding sequence if it increases the transcription of the
coding sequence.
Operably linked means that the DNA sequences being linked are typically
contiguous and,
where necessary to join two protein coding regions, contiguous and in reading
frame.
However, since enhancers generally function when separated from the promoter
by several
kilobases and intronic sequences may be of variable lengths, some
polynucleotide elements
may be operably linked but not contiguous.
A specific binding affinity between two molecules, for example, a ligand and
10 a receptor, means a preferential binding of one molecule for another in a
mixture of
molecules. The binding of the molecules can be considered specific if the
binding affinity is
about 1 x 104 M -~ to about 1 x 106 M -' or greater.
The term "recombinant" when used with reference to a cell indicates that the
cell replicates a heterologous nucleic acid, or expresses a peptide or protein
encoded by a
1 S heterologous nucleic acid. Recombinant cells can contain genes that are
not found within
the native (non-recombinant) form of the cell. Recombinant cells can also
contain genes
found in the native form of the cell wherein the genes are modified and re-
introduced into
the cell by artificial means. The term also encompasses cells that contain a
nucleic acid
endogenous to the cell that has been modified without removing the nucleic
acid from the
cell; such modifications include those obtained by gene replacement, site-
specific mutation,
and related techniques.
A "recombinant expression cassette" or simply an "expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically, with
nucleic.acid elements
that are capable of effecting expression of a structural gene in hosts
compatible with such
sequences. Expression cassettes include at /east promoters and optionally,
transcription
termination signals. Typically, the recombinant expression cassette includes a
nucleic acid
to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a
promoter.
Additional factors necessary or helpful in effecting expression may also be
used as described
herein. For example, an expression cassette can also include nucleotide
sequences that
encode a signal sequence that directs secretion of an expressed protein from
the host cell.
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Transcription termination signals, enhancers, and other nucleic acid sequences
that influence
gene expression, can also be included in an expression cassette.
A "recombinant polynucleotide" or a "recombinant polypeptide" is a non-
naturally occurring polynucleotide or polypeptide that includes nucleic acid
or amino acid
sequences, respectively, from more than one source nucleic acid or
polypeptide, which
source nucleic acid or polypeptide can be a naturally occurring nucleic acid
or polypeptide,
or can itself have been subjected to mutagenesis or other type of
modification. The source
polynucleotides or polypeptides from which the different nucleic acid or amino
acid
sequences are derived are sometimes homologous (i.e., have, or encode a
polypeptide that
encodes, the same or a similar structure and/or function), and are often from
different
isolates, serotypes, strains, species, of organism or from different disease
states, for example.
The terms "identical" or percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that are the
same, when compared and aligned for maximum correspondence, as measured using
one of
the following sequence comparison algorithms or by visual inspection.
The phrase "substantially identical," in the context of two nucleic acids or
polypeptides, refers to two or more sequences or subsequences that have at
least 60%,
preferably 80%, most preferably 90-95% nucleotide or amino acid residue
identity, when
compared and aligned for maximum correspondence, as measured using one of the
following
sequence comparison algorithms or by visual inspection. Preferably, the
substantial identity
exists over a region of the sequences that is at least about 50 residues in
length, more
preferably over a region of at least about 100 residues, and most preferably
the sequences are
substantially identical over at least about 150 residues. In some embodiments,
the sequences
are substantially identical over the entire length of the coding regions.
For sequence comparison, typically one sequence acts as a reference sequence
to which test sequences are compared. When using a sequence comparison
algorithm, test
and reference sequences are input into a computer, subsequence coordinates are
designated,
if necessary, and sequence algorithm program parameters are designated. The
sequence
comparison algorithm then calculates the percent sequence identity for the
test sequences)
relative to the reference sequence, based on the designated program
parameters.
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Optimal alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat'1. Acad. Sci. USA
85:2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575
Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et
al., infra).
One example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in
Altschul et al., J. Mol. Biol. 215:403-410 (1990}. Software for performing
BLAST analyses
is publicly available through the National Center for Biotechnology
Information
(http://www.ncbi.nlm.nih.gov~. This algorithm involves first identifying high
scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence, which
either match or satisfy some positive-valued threshold score T when aligned
with a word of
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et al., supra). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always > 0) and N (penalty
score for
mismatching residues; always < 0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved
value; the cumulative score goes to zero or below, due to the accumulation of
one or more
negative-scoring residue alignments; or the end of either sequence is reached.
The BLAST
algorithm parameters W, T, and X determine the sensitivity and speed of the
alignment. The
BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of
11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For
amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of
3, an
expectation (E} of 10, and the BLOSLTM62 scoring matrix (see Henikoff &
Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915).
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In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see, e.g., Karlin
& Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid to
the reference nucleic acid is less than about 0.1, more preferably less than
about 0.01, and
most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially identical
is that the two molecules hybridize to each other under stringent conditions.
The phrase
"hybridizing specifically to", refers to the binding, duplexing, or
hybridizing of a molecule
only to a particular nucleotide sequence under stringent conditions when that
sequence is
present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially"
refers to complementary hybridization between a probe nucleic acid and a
target nucleic acid
and embraces minor mismatches that can be accommodated by reducing the
stringency of
the hybridization media to achieve the desired detection of the target
polynucleotide
sequence.
"Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and
northern hybridizations are sequence dependent, and are different under
different
environmental parameters. Longer sequences hybridize specifically at higher
temperatures.
An extensive guide to the hybridization of nucleic acids is found in Tijssen
(1993)
Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic
Acid Probes part I chapter 2 "Overview of principles of hybridization and the
strategy of
nucleic acid probe assays", Elsevier, New York. Generally, highly stringent
hybridization
and wash conditions are selected to be about S° C lower than the
thermal melting point (Tm)
for the specific sequence at a defined ionic strength and pH. Typically, under
"stringent
conditions" a probe will hybridize to its target subsequence, but to no other
sequences.
The Tm is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent
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14
conditions are selected to be equal to the Tm for a particular probe. An
example of stringent
hybridization conditions for hybridization of complementary nucleic acids
which have more
than 100 complementary residues on a filter in a Southern or northern blot is
50%
formamide with 1 mg of heparin at 42°C, with the hybridization being
carried out overnight.
An example of highly stringent wash conditions is O.ISM NaCI at 72°C
for about 15
minutes. An example of stringent wash conditions is a 0.2x SSC wash at
65°C for 15
minutes (see, Sambrook, infra., for a description of SSC buffer). Often, a
high stringency
wash is preceded by a low stringency wash to remove background probe signal.
An example
medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx
SSC at 45°C
for 1 S minutes. An example low stringency wash for a duplex of, e.g., more
than 100
nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes
(e.g., about 10 to SO
nucleotides), stringent conditions typically involve salt concentrations of
less than about 1.0
M Na+ ion, typically about 0.01 to 1.0 M Na' ion concentration (or other
salts) at pH 7.U to
8.3, and the temperature is typically at least about 30°C. Stringent
conditions can also be
achieved with the addition of destabilizing agents such as formamide. In
general, a signal to
noise ratio of 2x (or higher) than that observed for an unrelated probe in the
particular
hybridization assay indicates detection of a specific hybridization. Nucleic
acids which do
not hybridize to each other under stringent conditions are still substantially
identical if the
polypeptides which they encode are substantially identical. This occurs, e.g.,
when a copy of
a nucleic acid is created using the maximum codon degeneracy permitted by the
genetic
code.
A further indication that two nucleic acid sequences or polypeptides are
substantially identical is that the polypeptide encoded by the first nucleic
acid is
immunologically cross reactive with, or specifically binds to, the polypeptide
encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially identical
to a second
polypeptide, for example, where the two peptides differ only by conservative
substitutions.
The phrase "specifically (or selectively) binds to an antibody" or
"specifically
(or selectively) immunoreactive with", when referring to a protein or peptide,
refers to a
binding reaction which is determinative of the presence of the protein, or an
epitope from the
protein, in the presence of a heterogeneous population of proteins and other
biologics. Thus,
under designated immunoassay conditions, the specified antibodies bind to a
particular
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protein and do not bind in a significant amount to other proteins present in
the sample. The
antibodies raised against a multivalent antigenic polypeptide will generally
bind to the
proteins from which one or more of the epitopes were obtained. Specific
binding to an
antibody under such conditions may require an antibody that is selected for
its specificity for
5 a particular protein. A variety of immunoassay formats may be used to select
antibodies
specifically immunoreactive with a particular protein. For example, solid-
phase ELISA
immunoassays, Western blots, or immunohistochemistry are routinely used to
select
monoclonal antibodies specifically immunoreactive with a protein. See Harlow
and Lane
(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New
York
10 "Harlow and Lane"), for a description of immunoassay formats and conditions
that can be
used to determine specific immunoreactivity. Typically a specific or selective
reaction will
be at least twice background signal or noise and more typically more than 10
to 100 times
background.
"Conservatively modified variations" of a particular polynucleotide sequence
15 refers to those polynucleotides that encode identical or essentially
identical amino acid
sequences, or where the polynucleotide does not encode an amino acid sequence,
to
essentially identical sequences. Because of the degeneracy of the genetic
code, a large
number of functionally identical nucleic acids encode any given polypeptide.
For instance,
the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid
arginine.
Thus, at every position where an arginine is specified by a codon, the codon
can be altered to
any of the corresponding codons described without altering the encoded
polypeptide. Such
nucleic acid variations are "silent variations," which are one species of
"conservatively
modified variations." Every polynucleotide sequence described herein which,
encodes a
polypeptide also describes every possible silent variation, except where
otherwise noted.
One of skill will recognize that each codon in a nucleic acid (except AUG,
which is
ordinarily the only codon for methionine) can be modified to yield a
fimctionally identical
molecule by standard techniques. Accordingly, each "silent variation" of a
nucleic acid
which encodes a polypeptide is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small percentage of
amino acids (typically less than 5%, more typically less than 1%) in an
encoded sequence are
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16
"conservatively modified variations" where the alterations result in the
substitution of an
amino acid with a chemically similar amino acid. Conservative substitution
tables providing
functionally similar amino acids are well known in the art. The following five
groups each
contain amino acids that are conservative substitutions for one another:
Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I);
Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
Sulfur-containing: Methionine (M), Cysteine (C);
Basic: Arginine (R), Lysine (K), Histidine (H);
Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (I~, Glutamine (Q).
See also, Creighton (1984) Proteins, W.H. Freeman and Company, for additional
groupings
of amino acids. In addition, individual substitutions, deletions or additions
which alter, add
or delete a single amino acid or a small percentage of amino acids in an
encoded sequence
are also "conservatively modified variations".
A "subsequence" refers to a sequence of nucleic acids or amino acids that
comprise a part of a longer sequence of nucleic acids or amino acids (e.g.,
polypeptide)
respectively.
Description of the Preferred Embodiments
The present invention provides methods for obtaining polynucleotide
sequences that, either directly or indirectly (i.e., through encoding a
polypeptide), can
modulate an immune response when present on a genetic vaccine vector. In
another
embodiment, the invention provides methods for optimizing the transport and
presentation of
antigens. The optimized immunomodulatory polynucleotides obtained using the
methods of
the invention are particularly suited for use in conjunction with vaccines,
including genetic
vaccines. One of the advantages of genetic vaccines is that one can
incorporate genes
encoding immunomodulatory molecules, such as cytokines, costimulatory
molecules, and
molecules that improve antigen transport and presentation into the genetic
vaccine vectors.
This provides opportunities to modulate immune responses that are induced
against the
antigens expressed by the genetic vaccines.
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A. Creation of Recombinant Libraries
The invention involves creating recombinant libraries of polynucleatides that
are then screened to identify those library members that exhibit a desired
property. The
recombinant libraries can be created using any of various methods.
The substrate nucleic acids used for the recombination can vary depending
upon the particular application. For example, where a polynucleotide that
encodes a
cytokine, chemokine, or other accessory molecule is to be optimized, different
forms of
nucleic acids that encode all or part of the cytokine, chemokine, or other
accessory molecule
are subjected to recombination. The methods require at least two variant forms
of a starting
substrate. The variant forms of candidate substrates can show substantial
sequence or
secondary structural similarity with each other, but they should also differ
in at least two
positions. The initial diversity between forms can be the result of natural
variation, e.g., the
different variant forms (homologs) are obtained from different individuals or
strains of an
organism (including geographic variants) or constitute related sequences from
the same
1 S organism (e.g., allelic variations). Alternatively, the initial diversity
can be induced, e.g., the
second variant form can be generated by error-prone transcription, such as an
error-prone
PCR or use of a polymerase which lacks proof reading activity (see Liao (1990)
Gene
88:107-111), of the first variant form, or, by replication of the first form
in a mutator strain
(mutator host cells are discussed in further detail below). The initial
diversity between
substrates is greatly augmented in subsequent steps of recursive sequence
recombination.
Often, improvements are achieved after one round of recombination and
selection. However, recursive sequence recombination can be employed to
achieve still
further improvements in a desired property. Sequence recombination can be
achieved in
many different formats and permutations of formats, as described in further
detail below.
These formats share some common principles. Recursive sequence recombination
entails
successive cycles of recombination to generate molecular diversity. That is,
one creates a
family of nucleic acid molecules showing some sequence identity to each other
but differing
in the presence of mutations. In any given cycle, recombination can occur in
vivo or in vitro,
intracellular or extracellular. Furthermore, diversity resulting from
recombination can be
augmented in any cycle by applying prior methods of mutagenesis (e.g., error-
prone PCR or
cassette mutagenesis) to either the substrates or products for recombination.
In some
instances, a new or improved property or characteristic can be achieved after
only a single
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18
cycle of in vivo or in vitro recombination, as when using different, variant
forms of the
sequence, as homologs from different individuals or strains of an organism, or
related
sequences from the same organism, as allelic variations.
In a presently preferred embodiment, the recombinant libraries are prepared
using DNA shuffling. The shuffling and screening or selection can be used to
"evolve"
individual genes, whole plasmids or viruses, multigene clusters, or even whole
genomes
(Stemmer (1995) BiolTechnology 13:549-553). Reiterative cycles of
recombination and
screening/selection can be performed to further evolve the nucleic acids of
interest. Such
techniques do not require the extensive analysis and computation required by
conventional
methods for polypeptide engineering. Shuffling allows the recombination of
large numbers
of mutations in a minimum number of selection cycles, in contrast to
traditional, pairwise
recombination events. Thus, the sequence recombination techniques described
herein
provide particular advantages in that they provide recombination between
mutations in any
or all of these, thereby providing a very fast way of exploring the manner in
which different
combinations of mutations can affect a desired result. In some instances,
however, structural
and/or functional information is available which, although not required for
sequence
recombination, provides opportunities for modification of the technique.
Exemplary formats and examples for sequence recombination, sometimes
referred to as DNA shuffling, evolution, or molecular breeding, have been
described by the
present inventors and co-workers in co-pending applications U.S. Patent
Application Serial
No. 08/198,431, filed February 17, 1994, Serial No. PCT1CTS95/02126, filed,
February 17,
1995, Serial No. 08/425,684, filed April 18, 1995, Serial No. 08/537,874,
filed October 30,
1995, Serial No. 08/564,955, filed November 30, 1995, Serial No. 08/621,859,
filed March
25, 1996, Serial No. 08/621,430, filed March 25, 1996, Serial No.
PCT/LTS96/05480, filed
April 18, 1996, Serial No. 08/650,400, filed May 20, 1996, Serial No.
08/675,502, filed July
3, 1996, Serial No. 08/721, 824, filed September 27, 1996, Serial No.
PCT/LJS97/17300,
f led September 26, 1997, and Serial No. PCT/LTS97/24239, filed December 17,
1997;
Stemmer, Science 270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995);
Stemmer,
BiolTechnology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci. U.S.A.
91:10747-10751
(1994); Stemmer, Nature 370:389-391 (1994); Crameri et al., Nature Medicine
2(1):1-3
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19
(1996); Crameri et al., Nature Biotechnology 14:315-319 (1996), each of which
is
incorporated by reference in its entirety for all purposes.
Other methods for obtaining recombinant polynucleotides and/or for
obtaining diversity in nucleic acids used as the substrates for shuffling
include, for example,
homologous recombination (PCT/US98/05223; Publ. No. W098/42727};
oligonucleotide-
directed mutagenesis (for review see, Smith, Ann. Rev. Genet. 19: 423-462
(1985); Botstein
and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J. 237: 1-7
(1986); Kunkel;
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic acids &
Molecular
Biology, Eckstein and Lilley, eds., Springer Verlag, Berlin (1987)). Included
among these
methods are oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl.
Acids Res. 10:
6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983), and Methods in
Enzymol.
154: 329-350 (1987)) phosphothioate-modified DNA mutagenesis (Taylor et al.,
Nucl. Acids
Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye
and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.
Acids Res. 16:
791-802 (1988); Sayers et al., Nucl. Acids Res. 16: 803-814 (1988)),
mutagenesis using
uracii-containing templates (Kunkel, Proc. Nat'l. Acad. Sci. USA 82: 488-492
(1985) and
Kunkel et al., Methods in Enzymol. 154: 367-382)); mutagenesis using gapped
duplex DNA
(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and Fritz,
Methods in
Enzymol. 154: 350-367 (1987); Kramer et al., Nucl. Acids Res. 16: 7207
(1988)); and Fritz et
al., Nucl. Acids Res. 16: 6987-6999 (1988)). Additional suitable methods
include point
mismatch repair (Kramer et al., Cell 38: 879-887 (1984)), mutagenesis using
repair-deficient
host strains (Carter et al., Nucl. Acids Res. 13: 4431-4443 (1985); Carter,
Methods in
Enzymol. 154: 382-403 (1987)), deletion mutagenesis (Eghtedarzadeh and
Henikoff, Nucl.
Acids Res. 14: 5115 (1986)), restriction-selection and restriction-
purification (Wells et al.,
Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986)), mutagenesis by total gene
synthesis
(Nambiar et al., Science 223: 1299-1301 {1984); Sakamar and Khorana, Nucl.
Acids Res. 14:
6361-6372 (1988); Wells et al., Gene 34: 315-323 {1985); and Grundstrom et
al., Nucl.
Acids Res. 13: 3305-3316 (1985). Kits for mutagenesis are commercially
available (e.g.,
Bio-Rad, Amersham International, Anglian Biotechnology).
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B. Screening Methods
A recombination cycle is usually followed by at least one cycle of screening
or selection for molecules having a desired property or characteristic. If a
recombination
cycle is performed in vitro, the products of recombination, i.e., recombinant
segments, are
sometimes introduced into cells before the screening step. Recombinant
segments can also
be linked to an appropriate vector or other regulatory sequences before
screening.
Alternatively, products of recombination generated in vitro are sometimes
packaged as
viruses before screening. If recombination is performed in vivo, recombination
products can
sometimes be screened in the cells in which recombination occurred. In other
applications,
10 recombinant segments are extracted from the cells, and optionally packaged
as viruses,
before screening.
The nature of screening or selection depends on what property or
characteristic is to be acquired or the property or characteristic for which
improvement is
sought, and many examples are discussed below. It is not usually necessary to
understand
15 the molecular basis by which particular products of recombination
(recombinant segments)
have acquired new or improved properties or characteristics relative to the
starting
substrates. For example, a genetic vaccine vector can have many component
sequences each
- having a different intended role (e.g., coding sequence, regulatory
sequences, targeting
sequences, stability-confernng sequences, immunomodulatory sequences,
sequences
20 affecting antigen presentation, and sequences affecting integration). Each
of these
component sequences can be varied and recombined simultaneously.
Screening/selection
can then be performed, for example, for recombinant segments that have
increased episomal
maintenance in a target cell without the need to attribute such improvement to
any of the
individual component sequences of the vector.
Depending on the particular screening protocol used for a desired property,
initial rounds) of screening can sometimes be performed in bacterial cells due
to high
transfection efficiencies and ease of culture. Later rounds, and other types
of screening
which are not amenable to screening in bacterial cells, are performed in
mammalian cells to
optimize recombinant segments for use in an environment close to that of their
intended use.
Final rounds of screening can be performed in the precise cell type of
intended use (e.g., a
human antigen-presenting cell). In some instances, this cell can be obtained
from a patient
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21
to be treated with a view, for example, to minimizing problems of
immunogenicity in this
patient.
The screening or selection step identifies a subpopulation of recombinant
segments that have evolved toward acquisition of a new or improved desired
property or
properties useful in genetic vaccination. Depending on the screen, the
recombinant segments
can be identified as components of cells, components of viruses or in free
form. More than
one round of screening or selection can be performed after each round of
recombination.
If further improvement in a property is desired, at least one and usually a
collection of recombinant segments surviving a first round of
screening/selection are subject
to a further round of recombination. These recombinant segments can be
recombined with
each other or with exogenous segments representing the original substrates or
further
variants thereof. Again, recombination can proceed in vitro or in vivo. If the
previous
screening step identifies desired recombinant segments as components of cells,
the
components can be subjected to further recombination in vivo, or can be
subjected to further
recombination in vitro, or can be isolated before performing a round of in
vitro
recombination. Conversely, if the previous screening step identifies desired
recombinant
segments in naked form or as components of viruses, these segments can be
introduced into
cells to perform a round of in vivo recombination. The second round of
recombination,
irrespective how performed, generates further recombinant segments which
encompass
additional diversity than is present in recombinant segments resulting from
previous rounds.
The second round of recombination can be followed by a further round of
screening/selection according to the principles discussed above for the first
round. The
stringency of screening/selection can be increased between rounds. Also,
the,nature of the
screen and the property being screened for can vary between rounds if
improvement in more
than one property is desired or if acquiring more than one new property is
desired.
Additional rounds of recombination and screening can then be performed until
the
recombinant segments have sufficiently evolved to acquire the desired new or
improved
property or function.
Various screening methods for particular applications are described herein. In
several instances, screening involves expressing the recombinant peptides or
polypeptides
encoded by the recombinant polynucleotides of the library as fusions with a
protein that is
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22
displayed on the surface of a replicable genetic package. For example, phage
display can be
used. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (
1990); Devlin et al.,
Science 249: 404-406 (1990), Scott & Smith, Science 249: 386-388 (1990);
Ladner et al., US
5,571,698. Other replicable genetic packages include, for example, bacteria,
eukaryotic
viruses, yeast, and spores.
The genetic packages most frequently used for display libraries are
bacteriophage, particularly filamentous phage, and especially phage MI3, Fd
and F1. Most
work has involved inserting libraries encoding polypeptides to be displayed
into either gIII
or gVIII of these phage forming a fusion protein. See, e.g., Dower, WO
91/19818; Devlin,
WO 91/18989; MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO
92/18619 (gene VIII). Such a fusion protein comprises a signal sequence,
usually but not
necessarily, from the phage coat protein, a polypeptide to be displayed and
either the gene III
or gene VIII protein or a fragment thereof. Exogenous coding sequences are
often inserted
at or near the N-terminus of gene III or gene VIII although other insertion
sites are possible.
Eukaryotic viruses can be used to display polypeptides in an analogous
manner. For example, display of human heregulin fused to gp70 of Moloney
murine
leukemia virus has been reported by Han et al., Proc. Natl. Acad. Sci. USA 92:
9747-9751
(1995). Spores can also be used as replicable genetic packages. In this case,
polypeptides
are displayed from the outer surface of the spore. For example, spores from B.
subtilis have
been reported to be suitable. Sequences of coat proteins of these spores are
provided by
Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cells can also be used as
replicable genetic
packages. Polypeptides to be displayed are inserted into a gene encoding a
cell protein that
is expressed on the cells surface. Bacterial cells including Salmonella
typhimurium, Bacillus
subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia,
Neisseria
gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and
especially
Escherichia coli are preferred. Details of outer surface proteins are
discussed by Ladner et
al., US 5,571,698 and references cited therein. For example, the lama protein
of E. coli is
suitable.
A basic concept of display methods that use phage or other replicable genetic
package is the establishment of a physical association between DNA encoding a
polypeptide
to be screened and the polypeptide. This physical association is provided by
the replicable
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23
genetic package, which displays a polypeptide as part of a capsid enclosing
the genome of
the phage or other package, wherein the polypeptide is encoded by the genome.
The
establishment of a physical association between polypeptides and their genetic
material
allows simultaneous mass screening of very large numbers of phage bearing
different
polypeptides. Phage displaying a polypeptide with affinity to a target, e.g.,
a receptor, bind
to the target and these phage are enriched by affinity screening to the
target. The identity of
polypeptides displayed from these phage can be determined from their
respective genomes.
Using these methods a polypeptide identified as having a binding affinity for
a desired target
can then be synthesized in bulk by conventional means, or the polynucleotide
that encodes
the peptide or polypeptide can be used as part of a genetic vaccine.
C. Evolution of Improved Immunomodulatory Sequences
Cytokines can dramatically influence macrophage activation and TH 1/TH2
cell differentiation, and thereby the outcome of infectious diseases. In
addition, recent
studies strongly suggest that DNA itself can act as adjuvant by activating the
cells of the
1 S immune system. Specifically, unmethylated CpG-rich DNA sequences were
shown to
enhance TH 1 cell differentiation, activate cytokine synthesis by monocytes
and induce
proliferation of B lymphocytes. The invention thus provides methods for
enhancing the
immunomodulatory properties of genetic vaccines (a) by evolving the
stimulatory properties
of DNA itself and (b) by evolving genes encoding cytokines and related
molecules that are
involved in immune system regulation. These genes are then used in genetic
vaccine vectors.
Of particular interest are IFN-a and IL-12, which skew immune responses
towards a T helper 1 (TH1) cell phenotype and, thereby, improve the host's
capacity to
counteract pathogen invasions. Also provided are methods of obtaining improved
immunomodulatory nucleic acids that are capable of inhibiting or enhancing
activation,
differentiation, or anergy of antigen-specific T cells. Because of the limited
infonmation
about the structures and mechanisms that regulate these events, molecular
breeding
techniques of the invention provide much faster solutions than rational
design.
The methods of the invention typically involve the use of DNA shuffling or
other methods to create a library of recombinant polynucleotides. The library
is then
screened to identify recombinant polynucleotides in the library, when included
in a genetic
vaccine vector or administered in conjunction with a genetic vaccine, are
capable of
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24
enhancing or otherwise altering an immune response induced by the vector. The
screening
step, in some embodiments, can involve introducing a genetic vaccine vector
that includes
the recombinant polynucleotides into mammalian cells and determining whether
the cells, or
culture medium obtained by growing the cells, is capable of modulating an
immune
response.
Optimized recombinant vector modules obtained through polynucleotide
recombination are useful not only as components of genetic vaccine vectors,
but also for
production of polypeptides, e.g., modified cytokines and the like, that can be
administered to
a mammal to enhance or shift an immune response. Polynucleotiiie sequences
obtained
using the DNA shuffling methods of the invention can be used as a component of
a genetic
vaccine, or can be used for production of cytokines and other immunomodulatory
polypeptides that are themselves used as therapeutic or prophylactic reagents.
If desired, the
sequence of the optimized immunomodulatory polypeptide-encoding
polynucleotides can be
determined and the deduced amino acid sequence used to produce polypeptides
using
1 S methods known to those of skill in the art.
1. Immunostimulatory DNA sequences
The invention provides methods of obtaining polynucleotides that are
immunostimulatory when introduced into a mammal. Oligonucleotides that contain
hexamers with a central CpG flanked by two 5' purines (GpA or ApA) and two 3'
pyrimidines (TpC or TpT) efficiently induce cytokine synthesis and B cell
proliferation
(Krieg et al. (1995) Nature 374: 546; Klinman et al. (1996) Proc. Nat'1. Acad.
Sci. USA 93:
2879; Pisetsky (1996) Immunity 5: 303-10) in vitro and act as adjuvants in
vivo. Genetic
vaccine vectors in which immunostimulatory sequence- (ISS) containing oligos
are inserted
have increased capacity to enhance antigen-specific antibody responses after
DNA
vaccination. The minimal length of an ISS oligonucleotide for functional
activity in vitro is
eight (Klinman et al., supra.}. Twenty-mers with three CG motifs were found to
be
significantly more efficient in inducing cytokine synthesis than a 15-mer with
two CG motifs
(Id.). GGGG tetrads have been suggested to be involved in binding of DNA to
cell surfaces
(macrophages express receptors. for example scavenger receptors, that bind
DNA) (Pisetsky
et al., supra.).
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According to the invention, a library is generated by subjecting to
recombination random DNA (e.g., fragments of human, murine, or other genomic
DNA),
oligonucleotides that contain known ISS, poly A, C, G or T sequences, or
combinations
thereof. The DNA, which includes at least first and second forms which differ
from each
5 other in two or more nucleotides, are recombined to produce a library of
recombinant
polynucleotides.
The library is then screened to identify those recombinant polynucleotides
that exhibit immunostimulatory properties. For example, the library can be
screened for
induction cytokine production in vitro upon introduction of the library into
an appropriate
10 cell type. A diagram of this procedure is shown in Figure 5. Among the
cytokines that can
be used as an indicator of immunostimulatory activity are, for example, IL-2,
IL-4, IL-5, IL-
6, IL-10, IL-12, IL-13, IL-15, and IFN-y. One can also test for changes in
ratios of IL-4/IF'N-
y, IL-4/IL-2, IL-5/IFN-y, IL-5/IL-2, IL-13/IFN-y, IL-13/IL-2. An alternative
screening
method is the determination of the ability to induce proliferation of cells
involved in immune
15 responses, such as B cells, T cells, monocytes/macrophages, total PBL, and
the like. Other
screens include detecting induction of APC activation based on changes in
expression levels
of surface antigens, such as B7-1 (CD80), B7-2 (CD86), MHC class I and II, and
CD14.
Other useful screens include identifying recombinant polynucleotides that
induce T cell proliferation. Because ISS sequences induce B cell activation,
and because of
20 several homologies between surface antigens expressed by T cells and B
cells,
polynucleotides can be obtained that have stimulatory activities on T cells.
Libraries of recombinant polynucleotides can also be screened for improved
CTL and antibody responses in vivo and for improved protection from infection,
cancer,
allergy or autoimmunity. Recombinant polynucleotides that exhibit the desired
property can
25 be recovered from the cell and, if further improvement is desired, the
shuffling and screening
can be repeated. Optimized ISS sequences can used as an adjuvant separately
from an actual
vaccine, or the DNA sequence of interest can be fused to a genetic vaccine
vector.
2. Cytokines, chemokines, and accessory molecules
The invention also provides methods for obtaining optimized cytokines,
cytokine antagonists, chemokines, and other accessory molecules that direct,
inhibit, or
enhance immune responses. For example, the methods of the invention can be
used to obtain
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genetic vaccines and other reagents (e.g., optimized cytokines, and the like)
that, when
administered to a mammal, improve or alter an immune response. These optimized
immunomodulators are useful for treating infectious diseases, as well as other
conditions
such as inflammatory disorders, in an antigen non-specific manner.
For example, the methods of the invention can be used to develop optimized
immunomodulatory molecules for treating allergies. The optimized
immunomodulatory
molecules can be used alone or in conjunction with antigen-specific genetic
vaccines to
prevent or treat allergy. Four basic mechanisms are available by which one can
achieve
specific immunotherapy of allergy. First, one can administer a reagent that
causes a decrease
in allergen-specific TH2 cells. Second, a reagent can be administered that
causes an increase
in allergen-specific TH1 cells. Third, one can direct an increase in
suppressive CD8+ T cells.
Finally, allergy can be treated by inducing anergy of allergen-specific T
cells. In this
Example, cytokines are optimized using the methods of the invention to obtain
reagents that
are effective in achieving one or more of these immunotherapeutic goals. The
methods of the
1 S invention are used to obtain anti-allergic cytokines that have one or more
properties such as
improved specific activity, improved secretion after introduction into target
cells, are
effective at a lower dose than natural cytokines, and fewer side effects.
Targets of particular
interest include interferon-a,/y, IL-10, IL-12, and antagonists of IL-4 and IL-
13.
The optimized immunomodulators, or optimized recombinant polynucleotides
that encode the immunomodulators, can be administered alone, or in combination
with other
accessory molecules. Inclusion of optimal concentrations of the appropriate
molecules can
enhance a desired immune response, and/or direct the induction or repression
of a particular
type of immune response. The polynucleotides that encode the optimized
molecules can be
included in a genetic vaccine vector, or the optimized molecules encoded by
the genes can
be administered as polypeptides.
In the methods of the invention, a library of recombinant polynucleotides that
encode immunomodulators is created by subjecting substrate nucleic acids to a
recombination protocol, such as DNA shuffling or other method known to those
of skill in
the art. The substrate nucleic acids are typically two or more forms of a
nucleic acid that
encodes an immunomodulator of interest.
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Cytokines are among the immunomodulators that can be improved using the
methods of the invention. Cytokine synthesis profiles play a crucial role in
the capacity of
the host to counteract viral, bacterial and parasitic infections, and
cytokines can dramatically
influence the efficacy of genetic vaccines and the outcome of infectious
diseases. Several
S cytokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-
12, IL-13, IL-I4, IL-15, IL-16, IL-17, IL-18, G-CSF, GM-CSF, IFN-a, IFN-y, TGF-
(3, TNF-
a, TNF-Vii, IL-20 (MDA-7), and flt-3 ligand have been shown stimulate immune
responses in
vitro or in vivo. Immune functions that can be enhanced using appropriate
cytokines
include, for example, B cell proliferation, Ig synthesis, Ig isotype
switching, T cell
proliferation and cytokine synthesis, differentiation of THI and TH2 cells,
activation and
proliferation of CTLs, activation and cytokine production by
monocytes/macrophages/
dendritic cells, and differentiation of dendritic cells from
monocytes/macrophages.
In some embodiments, the invention provides methods of obtaining optimized
immunomodulators that can direct an immune response towards a TH 1 or a TH2
response.
I S The ability to influence the direction of immune responses in this manner
is of great
importance in development of genetic vaccines. Altering the type of TH
response can
fundamentally change the outcome of an infectious disease. A high frequency of
TH1 cells
generally protects from lethal infections with intracellular pathogens,
whereas a dominant
TH2 phenotype often results in disseminated, chronic infections. For example,
in human, the
TH1 phenotype is present in the tuberculoid (resistant) form of leprosy, while
the TH2
phenotype is found in iepromatous, multibacillary (susceptible) lesions
(Yamamura et al.
(1991) Science 254: 277). Late-stage AIDS patients have the TH2 phenotype.
Studies in
family members indicate that survival from meningococcal septicemia depends on
the
cytokine synthesis profile of PBL, with high IL-10 synthesis being associated
with a high
risk of lethal outcome and high TNF-a being associated with a low risk.
Similar examples
are found in mice. For example, BALB/c mice are susceptible to Leishmania
major
infection; these mice develop a disseminated fatal disease with a TH2
phenotype. Treatment
with anti-IL-4 monoclonal antibodies or with IL-12 induces a TH1 response,
resulting in
healing. Anti-interferon-y monoclonal antibodies exacerbate the disease. For
some
applications, it is preferable to direct an immune response in the direction
of a TH2 response.
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For example, where increased mucosal immunity is desired, including protective
immunity,
enhancing the TH2 response can lead to increased antibody production,
particularly IgA.
T helper (TH) cells are probably the most important regulators of the immune
system. TH cells are divided into two subsets, based on their cytokine
synthesis pattern
(Mosmann and Coffman (1989) Adv. Immunol. 46: 111). TH1 cells produce high
levels of
the cytokines IL-2 and IFN-'y and no or minimal levels of II,-4, IL-5 and IL-
13. In contrast,
TH2 cells produce high levels of IL-4, IL-5 and IL-I3, and IL-2 and IFN-y
production is
minimal or absent. TH1 cells activate macrophages, dendritic cells and augment
the cytolytic
activity of CD8+ cytotoxic T lymphocytes and natural killer (NK) cells (Paul
and Seder
( 1994} Cell 76: 241 ), whereas TH2 cells provide efficient help for B cells
and also mediate
allergic responses due to the capacity of TH2 cells to induce IgE isotype
switching and
differentiation of B cells into IgE secreting cells (Punnonen et al. (1993)
Proc. Nat'1. Acad.
Sci. USA 90: 3730).
The screening methods for improved cytokines, chemokines, and other
accessory molecules are generally based on identification of modified
molecules that exhibit
improved specific activity on target cells that are sensitive to the
respective cytokine,
chemokine, or other accessory molecules. A library of recombinant cytokine,
chemokine, or
accessory molecule nucleic acids can be expressed on phage or as purified
protein and tested
using in vitro cell culture assays, for example. Importantly, when analyzing
the recombinant
nucleic acids as components of DNA vaccines, one can identify the most optimal
DNA
sequences (in addition to the functions of the protein products) in terms of
their
immunostimulatory properties, transfection efficiency, and their capacity to
improve the
stabilities of the vectors. The identified optimized recombinant nucleic acids
can then be
subjected to new rounds of shuffling and selection.
In one embodiment of the invention, cytokines are evolved that direct
differentiation of TH1 cells. Because of their capacities to skew immune
responses towards a
Tnl phenotype, the genes encoding interferon-a (IFN-a) and interleukin-12 (IL-
12) are
preferred substrates far recombination and selection in order to obtain
maximal specific
activity and capacity to act as adjuvants in genetic vaccinations. IFN-a is a
particularly
preferred target for optimization using the methods of the invention because
of its effects on
the immune system, tumor cells growth and viral replication. Due to these
activities, IFN-a
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29
was the first cytokine to be used in clinical practice. Today, IFN-a is used
for a wide variety
of applications, including several types of cancers and viral diseases. IFN-a
also efficiently
directs differentiation of human T cells into TH1 phenotype (Parronchi et al.
(1992) J.
Immunol. 149: 2977). However, it has not been thoroughly investigated in
vaccination
models, because, in contrast to human systems, it does not affect TH1
differentiation in mice.
The species difference was recently explained by data indicating that, like IL-
12, IFN-a
induces STAT4 activation in human cells but not in marine cells, and STAT4 has
been
shown to be required in IL-12 mediated TH1 differentiation (Thierfelder et al.
(1996) Nature
382: 171).
Family DNA shuffling is a preferred method for optimizing IFN-a, using as
substrates the mammalian IFN-a genes, which are 85% - 97% homologous. Greater
than
1 O26 distinct recombinants can be generated from the natural diversity in
these genes. To
allow rapid parallel analysis of recombinant interferons, one can employ high
throughput
methods for their expression and biological assay as fusion proteins on
bacteriophage.
Recombinants with improved potency and selectivity profiles are being
selectively bred for
improved activity. Variants which demonstrate improved binding to IFN-a
receptors can be
selected for further analysis using a screen for mutants with optimal capacity
to direct TH1
differentiation. More specifically, the capacities of IFN-a mutants to induce
IL-2 and IFN-y
production in in vitro human T lymphocyte cultures can be studied by cytokine-
specific
ELISA and cytoplasmic cytokine staining and flow cytometry.
IL-12 is perhaps the most potent cytokine that directs TH1 responses, and it
has also been shown to act as an adjuvant and enhance TH1 responses following
genetic
vaccinations (Kim et al. (1997) J. Immunol. 158: 816). IL-12 is both
structurally and
functionally a unique cytokine. It is the only heterodimeric cytokine known to
date,
composed of a 35 kD Iight chain (p35) and a 40 kD heavy chain (p40) (Kobayashi
et al
(1989) J. Bxp. Med. 170: 827; Stern et al. (1990} Proc. Nat'1. Acad. Sci. USA
87: 6808).
Recently Lieschke et al. ((1997) Nature Biotech. 15: 35) demonstrated that a
fusion between
p35 and p40 genes results in a single gene that has activity comparable to
that of the two
genes expressed separately. These data indicate that it is possible to shuffle
IL-12 genes as
one entity, which is beneficial in designing the shuffling protocol. Because
of its T cell
growth promoting activities, one can use normal human peripheral blood T cells
in the
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selection of the most active IL-12 genes, enabling direct selection of IL-I2
mutants with the
most potent activities on human T cells. IL-12 mutants can be expressed in CH4
cells, for
example, and the ability of the supernatants to induce T cell proliferation
determined (Figure
6). The concentrations of IL-12 in the supernatants can be normalized based on
a specific
S ELISA that detects a tag fused to the shuffled IL-12 molecules.
Incorporation of evolved IFN-a and/or IL-12 genes into genetic vaccine
vectors is expected to be safe. The safety of IFN-a has been demonstrated in
numerous
clinical studies and in everyday hospital practice. A Phase II trial of IL-12
in the treatment
of patients with renal cell cancer resulted in several unexpected adverse
effects (Tahara et al.
10 (1995) Human Gene Therapy 6: 1607). However, IL-12 gene as a component of
genetic
vaccines aims at high local expression levels, whereas the levels observed in
circulation are
minimal compared to those observed after systemic bolus injections. In
addition, some of
the adverse effects of systemic IL-12 treatments are likely to be related to
its unusually long
half life (up to 48 hours in monkeys). DNA shuffling may allow selection for a
shorter half
15 life, thereby reducing the toxicity even after high bolus doses.
In other cases, genetic vaccines that can induce TH2 responses are preferred,
especially when improved antibody production is desired. As an example, IL-4
has been
shown to direct differentiation of TH2 cells (which produce high levels of IL-
4, IL-S and IL-
13, and mediate allergic immune responses). Immune responses that are skewed
towards TH2
20 phenotype are preferred when genetic vaccines are used to immunize against
autoimmune
diseases prophylactically. TH 1 responses are also preferred when the vaccines
are used to
treat and modulate existing autoimmune responses, because autoreactive T cells
are
generally of THI phenotype (Liblau et al. (1995) Immunol. Today 16:34-38). .IL-
4 is also the
most potent cytokine in induction of IgE synthesis; IL-4 deficient mice are
unable to produce
25 IgE. Asthma and allergies are associated with an increased frequency of IL-
4 producing
cells, and are genetically linked to the locus encoding IL-4, which is on
chromosome 5 (in
close proximity to genes encoding IL-3, IL-5, IL-9, IL-13 and GM-CSF). IL-4,
which is
produced by activated T cells, basophils and mast cells, is a protein that has
153 amino acids
and two potential N-glycosylation sites. Human IL-4 is only approximately 50%
identical to
30 mouse IL-4, and IL-4 activity is species-specific. In human, IL-13 has
activities similar to
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31
those of IL-4, but IL-13 is less potent than IL-4 in inducing IgE synthesis.
IL-4 is the only
cytokine known to direct TH2 differentiation.
Improved IL-2 agonists are also useful in directing TH2 cell differentiation,
whereas improved IL-4 antagonists can direct TH 1 cell differentiation.
Improved IL-4
agonists and antagonists can be generated by shuffling of IL-4 or soluble IL-4
receptor. The
IL-4 receptor consists of an IL-4R a-chain (140 kD high-affinity binding unit)
and an IL-2R
y-chain (these cytokine receptors share a common y-chain). The IL-4R a-chain
is shared by
IL-4 and IL-13 receptor complexes. Both IL-4 and IL-13 induce phosphorylation
of the IL-
4R a-chain, but expression of IL-4R a-chain alone on transfectants is not
sufficient to
provide a functional IL-4R. Soluble IL-4 receptor currently in clinical trials
for the treatment
of allergies. Using the DNA shuffling methods of the invention, one can evolve
a soluble IL-
4 receptor that has improved affinity for IL-4. Such receptors are useful for
the treatment of
asthma and other TH2 cell mediated diseases, such as severe allergies. The
shuffling
reactions can take advantage of natural diversity present in cDNA libraries
from activated T
cells from human and other primates. In a typical embodiment, a shuffled IL-4R
a-chain
library is expressed on a phage, and mutants that bind to IL-4 with improved
affinity are
identified. The biological activity of the selected mutants is then assayed
using cell-based
assays.
IL-2 and IL-15 are also of particular interest for use in genetic vaccines. IL-
2
acts as a growth factor for activated B and T cells, and it also modulates the
functions of
NK-cells. IL-2 is predominantly produced by TH1-like T cell clones, and,
therefore, it is
considered mainly to function in delayed type hypersensitivity reactions.
However, IL-2 also
has potent, direct effects on proliferation and Ig-synthesis by B cells. The
complex
immunoregulatory properties of IL-2 axe reflected in the phenotype of IL-2
deficient mice,
which have high mortality at young age and multiple defects in their immune
functions
including spontaneous development of inflammatory bowel disease. IL-15 is a
more recently
identified cytokine produced by multiple cell types. IL-15 shares several, but
not all,
activities with IL-2. Both IL-2 and IL-15 induce B cell growth and
differentiation. However,
assuming that IL-15 production in IL-2 deficient mice is normal, it is clear
that IL-15 cannot
substitute for the function of IL-2 in vivo, since these mice have multiple
immunodeficiencies. IL-2 has been shown to synergistically enhance IL-10-
induced human
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32
Ig production in the presence of anti-CD40 mAbs, but it antagonized the
effects of IL-4. IL-
2 also enhances IL-4-dependent IgE synthesis by purified B cells. On the other
hand, IL-2
was shown to inhibit IL-4-dependent marine IgGl and IgE synthesis both in
vitro and in
vivo. Similarly, IL-2 inhibited IL-4-dependent human IgE synthesis by
unfractionated
human PBMC, but the effects were less significant than those of IFN-oc or IFN-
y. Due to
their capacities to activate both B and T cells, IL-2 and IL-1 S are useful in
vaccinations. In
fact, IL-2, as protein and as a component of genetic vaccines, has been shown
to improve the
efficacy of the vaccinations. Improving the specific activity and/or
expression
levels/kinetics of IL-2 and IL-15 through use of the DNA shuffling methods of
the invention
IO increases the advantageous effects compared to wild-type IL-2 and IL-15.
Another cytokine of particular interest for optimization and use in genetic
vaccines according to the methods of the invention is interleukin-6. IL-6 is a
monocyte-
derived cytokine that was originally described as a B cell differentiation
factor or B cell
stimulatory factor-2 because of its ability to enhance Ig levels secreted by
activated B cells.
IL-6 has also been shown to enhance IL-4-induced IgE synthesis. It has also
been suggested
that IL-6 is an obligatory factor for human IgE synthesis, because
neutralizing anti-IL-6
mAbs completely blocked IL-4-induced IgE synthesis. IL-6 deficient mice have
impaired
capacity to produce IgA. Because of its potent activities on the
differentiation of B cells, IL-
6 can enhance the levels of specific antibodies produced following
vaccination. It is
particularly useful as a component of DNA vaccines because high local
concentrations can
be achieved, thereby providing the most potent effects on the cells adjacent
to the transfected
cells expressing the immunogenic antigen. IL-6 with improved specific activity
and/or with
improved expression levels, obtained by DNA shuffling, will have more
beneficial effects
than the wild-type IL-6.
Interleukin-8 is another example of a cytokine that, when modified according
to the methods of the invention, is useful in genetic vaccines. IL-8 was
originally identified
as a monocyte-derived neutrophil chemotactic and activating factor.
Subsequently, IL-8 was
also shown to be chemotactic for T cells and to activate basophils resulting
in enhanced
histamine and leukotriene release from these cells. Furthermore, IL-8 inhibits
adhesion of
neutrophils to cytokine-activated endothelial cell monolayers, and it protects
these cells from
neutrophil-mediated damage. Therefore, endothelial cell derived IL-8 was
suggested to
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33
attenuate inflammatory events occurring in the proximity of blood vessel
walls. IL-8 also
modulates immunoglobulin production, and inhibits IL-4-induced IgG4 and IgE
synthesis by
both unfractionated human PBMC and purified B cells in vitro. This inhibitory
effect was
independent of IFN-a, IFN-~ or prostaglandin E2. In addition, IL-8 inhibited
spontaneous
IgE synthesis by PBMC derived from atopic patients. Due to its capacity to
attract
inflammatory cells, IL-8, like other chemotactic agents, is useful in
potentiating the
fimctional properties of vaccines, including DNA vaccines (acting as an
adjuvant). The
beneficial effects of IL-8 can be improved by using the DNA shuffling methods
of the
invention to obtain IL-8 with improved specific activity and/or with improved
expression in
target cells.
Interleukin-5, and antagonists thereof, can also be optimized using the
methods of the invention for use in genetic vaccines. IL-5 is primarily
produced by TH2-type
T cells and appears to play an important role in the pathogenesis of allergic
disorders
because of its ability to induce eosinophilia. IL-5 acts as an eosinophil
differentiation and
survival factor in both mouse and man. Blocking IL-5 activity by use of
neutralizing
monoclonal antibodies strongly inhibits pulmonary eosinophilia and
hyperactivity in mouse
models, and IL-5 deficient mice do not develop eosinophilia. These data also
suggest that
IL-5 antagonists may have therapeutic potential in the treatment of allergic
eosinophilia.
IL-5 has also been shown to enhance both proliferation of, and Ig synthesis
by, activated mouse and human B cells. However, other studies suggested that
IL-5 has no
effect on proliferation of human B cells, whereas it activated eosinophils. IL-
5 apparently is
not crucial for maturation or differentiation of conventional B cells, because
antibody
responses in IL-5 deficient mice are normal. However, these mice have a
developmental
defect in their CDS+ B cells indicating that IL-5 is required for normal
differentiation of this
B cell subset in mice. At suboptimal concentrations of IL-4, IL-5 was shown to
enhance IgE
synthesis by human B cells in vitro. Furthermore, a recent study suggested
that the effects of
IL-5 on human B cells depend on the mode of B cell stimulation. IL-5
significantly
enhanced IgM synthesis by B cells stimulated with Moraxella catarrhalis. In
addition, IL-5
synergized with suboptimal concentrations of IL-2, but had no effect on Ig
synthesis by
SAC-activated B cells. Activated human B cells also expressed IL-5 mRNA
suggesting that
IL-5 may also regulate B cell function, including IgE synthesis, by autocrine
mechanisms.
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34
The invention provides methods of evolving an IL-5 antagonist that
efficiently binds to and neutralizes IL-5 or its receptor. These antagonists
are useful as a
component of vaccines used for prophylaxis and treatment of allergies. Nucleic
acids
encoding IL-5, for example, from human and other mammalian species, are
shuffled and
screened for binding to immobilized IL-5R for the initial screening.
Polypeptides that
exhibit the desired effect in the initial screening assays can then be
screened for the highest
biological activity using assays such as inhibition of growth of IL-5
dependent cells lines
cultured in the presence of recombinant wild-type IL-5. Alternatively,
shuffled IL-5R a-
chains are screened for improved binding to IL-5.
Turnor necrosis factors (a and (3) and their receptors are also suitable
targets
for modification and use in genetic vaccines. TNF-a, which was originally
described as
cachectin because of its ability to cause necrosis of tumors, is a 17 kDa
protein that is
produced in low quantities by almost all cells in the human body following
activation. TNF-
a acts as an endogenous pyrogen and induces the synthesis of several
proinflammatory
cytokines, stimulates the production of acute phase proteins, and induces
proliferation of
fibroblasts. TNF-a plays a major role in the pathogenesis of endotoxin shock.
A
membrane-bound form of TNF-a (mTNF-a), which is involved in interactions
between B-
and T-cells, is rapidly upregulated within four hours of T cell activation.
mTNF-a plays a
role in the polyclonal B cell activation observed in patients infected with
HIV. Monoclonal
antibodies specific far mTNF-a or the p55 TNF-a receptor strongly inhibit IgE
synthesis
induced by activated CD4+ T cell clones or their membranes. Mice deficient for
p55 TNF-
aR are resistant to endotoxic shock, and soluble TNF-aR prevents autoimmune
diabetes
mellitus in NOD mice. Phase III trials using sTNF-aR in the treatment of
rheumatoid
arthritis are in progress, after promising results obtained in the phase II
trials.
The methods of the invention can be used to, for example, evolve a soluble
TNF-aR that has improved affinity, and thus is capable of acting as an
antagonist for TNF
activity. Nucleic acids that encode TNF-aR and exhibit sequence diversity,
such as the
natural diversity observed in cDNA libraries from activated T cells of human
and other
primates, are shuffled. The shuffled nucleic acids are expressed, e.g., on
phage, after which
mutants are selected that bind to TNF-a with improved affinity. If desired,
the improved
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mutants can be subjected to further assays using biological activity, and the
shuffled genes
can be subjected to one or more rounds of shuffling and screening.
Another target of interest for application of the methods of the invention is
interferon-y, and the evolution of antagonists of this cytokine. The receptor
for IFN-y
5 consists of a binding component glycoprotein of 90 kD, a 228 amino acid
extracellular
portion, a transmembrane region, and a 222 amino acid ;ntracellular region.
Glycosylation is
not required for functional activity. A single chain provides high affinity
binding (10-9 -10''°
M), but is not sufficient for signaling. Receptor components dimerize upon
ligand binding.
The mouse IFN-'y receptor is 53% identical to that of mouse at the amino acid
level. The
10 human and mouse receptors only bind human and mouse IFN-y, respectively.
Vaccinia,
cowpox and camelpox viruses have homologues of sIFN-yR, which have relatively
low
amino acid sequence similarity (~20%), but are capable of efficient
neutralization of IFN-y
in vitro. These homologues bind human, bovine, rat (but not mouse) IFN-'y, and
may have in
vivo activity as IFN-y antagonists. All eight cysteines are conserved in
human, mouse,
15 myxoma and Shope fibroma virus (6 in vaccinia virus) IFN-~yR polypeptides,
indicating
similar 3-D structures. An extracellular portion of mIFN-yR with a kD of 100-
300 pM has
been expressed in insect cells. Treatment of NZB/W mice (a mouse model of
human SLE)
with msIFN-y receptor (100 mg/three times a week i.p.) inhibits the onset of
glomerulonephritis. All mice treated with sIFN-y or anti-IFN-y mAbs were alive
4 weeks
20 after the treatment was discontinued, compared with 50% in a placebo group,
and 78% of
IFN-y-treated mice died.
The methods of the invention can be used to evolve soluble IFN-yR receptor
polypeptides with improved affinity, and to evolve IFN-y with improved
specific activity and
improved capacity to activate cellular immune responses. In each case nucleic
acids
25 encoding the respective polypeptide, and which exhibit sequence diversity
(e.g., that
observed in cDNA libraries from activated T cells from human and other
primates), are
subjected to recombination and screened to identify those recombinant nucleic
acids that
encode a polypeptide having improved activity. In the case of shuffled IFN-yR,
the library
of shuffled nucleic acids can be expressed on phage, which are screened to
identify mutants
30 that bind to IFN-Y with improved affinity. In the case of IFN-y, the
shuffled library is
analyzed for improved specific activity and improved activation of the immune
system, for
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36
example, by using activation of monocytes/macrophages as an assay. The evolved
IFN-y
molecules can improve the efficacy of vaccinations (e.g. when used as
adjuvants).
Diseases that can be treated using high-affinity sIFN-yR polypeptides
obtained using the methods of the invention include, for example, multiple
sclerosis,
systemic lupus erythematosus (SLE), organ rejection after treatment, and graft
versus host
disease. Multiple sclerosis, for example, is characterized by increased
expression of IFN-y
in the brain of the patients, and increased production of IFN-y by patients' T
cells in vitro.
IFN-y treatment has been shown to significantly exacerbate the disease (in
contrast to EAE
in mice).
Transforming growth factor (TGF)-(3 is another cytokine that can be
optimized for use in genetic vaccines using the methods of the invention. TGF-
(3 has growth
regulatory activities on essentially all cell types, and it has also been
shown to have complex
modulatory effects on the cells of the immune system. TGF-~i inhibits
proliferation of both
B and T cells, and it also suppresses development of and differentiation of
cytotoxic T cells
and NK cells. TGF-(3 has been shown to direct IgA switching in both marine and
human B
cells. It was also shown to induce germline a transcription in marine and
human B cells,
supporting the conclusion that TGF-~3 can specifically induce IgA switching.
Due to its capacity to direct IgA switching, TGF-~i is useful as a component
of DNA vaccines which aim at inducing potent mucosal immunity, e.g. vaccines
for
diarrhea. Also, because of its potent anti-proliferative effects TGF-(3 is
useful as a
component of therapeutical cancer vaccines. TGF-(3 with improved specific
activity and/or
with improved expression levels/kinetics will have increased beneficial
effects compared to
the wild-type TGF-~.
Cytokines that can be optimized using the methods of the invention also
include granulocyte colony stimulating factor (G-CSF) and
granulocyte/macrophage colony
stimulating factor (GM-CSF). These cytokines induce differentiation of bone
marrow stem
cell into granulocytes/macrophages. Administration of G-CSF and GM-CSF
significantly
improve recovery from bone marrow (BM) transplantation and radiotherapy,
reducing
infections and time the patients have to spend in hospitals. GM-CSF enhances
antibody
production following DNA vaccination. G-CSF is a 175 amino acid protein, while
GM-CSF
has 127 amino acids. Human G-CSF is 73% identical at the amino acid level to
marine G-
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37
CSF and the two proteins show species cross-reactivity. G-CSF has a
homodimeric receptor
(dimeric with kD of 200 pM, monomeric ~ 2-4 nM), and the receptor for GM-CSF
is a
three subunit complex. Cell lines transfected with cDNA encoding G-CSF R
proliferate in
response to G-CSF. Cell lines dependent of GM-CSF available (such as TF-1). G-
CSF is
nontoxic and is presently working very well as a drug. However, the treatment
is expensive,
and more potent G-CSF might reduce the cost for patients and to the health
care. Treatments
with these cytokines are typically short-lasting and the patients are likely
to never need the
same treatment again reducing likelihood of problems with immunogenicity.
The methods of the invention are useful for evolving G-CSF and/or GM-CSF
which have improved specific activity, as well as other polypeptides that have
G-CSF and/or
GM-CSF activity. G-CSF and/or GM-CSF nucleic acids having sequence diversity,
e.g.,
those obtained from cDNA libraries from diverse species, are shuffled to
create a library of
shuffled G-CSF and/or GM-CSF genes. These libraries can be screened by, for
example,
picking colonies, transfecting the plasmids into a suitable host cell (e.g.,
CHO cells), and
assaying the supernatants using receptor-positive cell lines. Alternatively,
phage display or
related techniques can be used, again using receptor-positive cell lines. Yet
another
screening method involves transfecting the shuffled genes into G-CSF/GM-CSF-
dependent
cell lines. The cells are grown one cell per well and/or at very low density
in Large flasks,
and the cells that grow fastest are selected. Shuffled genes from these cells
are isolated; if
desired, these genes can be used for additional rounds of shuffling and
selection.
Ciliary neurotrophic factor (CNTF) is another suitable target for application
of the methods of the invention. CNTF has 200 amino acids which exhibit 80%
sequence
identity between rat and rabbit CNTF polypeptides. CNTF has IL-6-like
inflammatory
effects, and induces synthesis of acute phase proteins. CNTF is a cytosolic
protein which
belongs to the IL-6/IL-11/LIF/oncostatin M -family, and becomes biologically
active only
after becoming available either by cellular lesion or by an unknown release
mechanism.
CNTF is expressed by myelinating Schwann cells, astrocytes and sciatic nerves.
Structurally, CNTF is a dimeric protein, with a novel anti-parallel
arrangement of the
subunits. Each subunit adopts a double crossover four-helix bundle fold, in
which two
helices contribute to the dimer interface. Lys-155 mutants lose activity, and
some Glu-153
mutants have 5-10 higher biological activity. The receptor for CNTF consists
of a specific
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CNTF receptor chain, gp130, and a LIF-(3 receptor. The CNTFR a-chain lacks a .
transmembrane domain portion, instead being GPI-anchored. At high
concentration, CNTF
can mediate CNTFR-independent responses. Soluble CNTFR binds CNTF and
thereafter
can bind to LIFR and induce signaling through gp130. CNTF enhances survival of
several
types of neurons, and protects neurons in an animal model of Huntington
disease (in contrast
to NGF, neurotrophic factor, and neurotrophin-3). CNTF receptor knockout mice
have
severe motor neuron deficits at birth, and CNTF knockout mice exhibit such
deficits
postnatally. CNTF also reduces obesity in mouse models. Decreased expression
of CNTF is
sometimes observed in psychiatric patients. Phase I studies in patients with
ALS (annual
incidence 1/100 000, S% familiar cases, 90% die within 6 years) found
significant side
effects after doses higher than 5 mg/kg/day subcutaneously (including
anorexia, weight loss,
reactivation of herpes simplex virus (HSV1), cough, increased oral
secretions). Antibodies
against CNTF were detected in almost all patients, thus illustrating the need
for alternative
CNTF with different immunological properties.
The recombination and screening methods of the invention can be used to
obtain modified CNTF polypeptides that exhibit decreased immunogenicity in
vivo; higher
specific activity is also obtainable using the methods. Shuffling is conducted
using nucleic
acids encoding CNTF. In a preferred embodiment, an IL-6/LIF/(CNTF) hybrid is
obtained
by shuffling using an excess of oligonucleotides that encode to the receptor
binding sites of
CNTF. Phage display can then be used to test for lack of binding to the IL-
6/LIF receptor.
This initial screen is followed by a test for high affinity binding to the
CNTF receptor, and, if
desired, functional assays using CNTF responsive cell lines. The shuffled CNTF
polypeptides can be tested to identify those that exhibit reduced
immunogenicity upon
administration to a mammal.
Another way in which the recombination and screening methods of the
invention can be used to optimize CNTF is to improve secretion of the
polypeptide. When a
CNTF cDNA is operably linked to a leader sequence of hNGF, only 35-40 percent
of the
total CNTF produced is secreted.
Target diseases for treatment with optimized CNTF, using either the shuffled
gene in an expression vector as in DNA vaccines, or a purified protein,
include obesity,
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amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), diabetic
neuropathy, stroke, and
brain surgery.
Polynucleotides that encode chemokines can also be optimized using the
methods of the invention and included in a genetic vaccine vector. At least
three classes of
chemokines are known, based on structure: C chemokines (such as Iymphotactin),
C-C
chemokines (such as MCP-1, MCP-2, MCP-3, MCP-4, MIP-la, MIP-lb, RANTES), C-X-C
chemokines (such as IL-8, SDF-1, ELR, Mig, IP10) (Premack and Schall (1996)
Nature
Med. 2: 1174). Chemokines can attract other cells that mediate immune and
inflammatory
functions, thereby potentiating the immune response. Cells that are attracted
by different
types of chemokines include, for example, lymphocytes, monocytes and
neutrophils.
Generally, C-X-C chemokines are chemoattractants for neutrophils but not for
monocytes,
C-C chemokines attract monocytes and lymphocytes but not neutrophils, C
chemokine
attracts lymphocytes.
Genetic vaccine vectors can also include optimized recombinant
polynucleotides that encode surface-bound accessory molecules, such as those
that are
involved in modulation and potentiation of immune responses. These molecules,
which
include, for example, B7-1 (CD80), B7-2 (CD86), CD40, ligand for CD40, CTLA-4,
CD28,
and CD150 (SLAM), can be subjected to DNA shuffling to obtain variants have
altered
and/or improved activities.
Optimized recombinant polynucleotides that encode CD1 molecules are also
useful in a genetic vaccine vector for certain applications. CD1 are
nonpolymorphic
molecules that are structurally and functionally related to MHC molecules.
Importantly,
CD1 ,has MHC-like activities, and it can function as an antigen presenting
molecule (Porcelli
(I995) Adv. Immunol. 59: 1). CDl is highly expressed on dendritic cells, which
are very
efficient antigen presenting cells. Simultaneous transfection of target cells
with DNA
vaccine vectors encoding CD1 and an antigen of interest is likely to boost the
immune
response. Because CD1 cells, in contrast to MHC molecules, exhibit limited
allelic diversity
in an outbred population (Porcelli, supra.), large populations of individuals
with different
genetic backgrounds can be vaccinated with one CD1 allele. The functional
properties of
CD1 molecules can be improved by the DNA shuffling methods of the invention.
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Optimized recombinant TAP genes and/or gene products can also be included
in a genetic vaccine vector. TAP genes and their optimization for various
purposes are
discussed in more detail below. Moreover, heat shock proteins (HSP), such as
HSP70, can
also be evolved for improved presentation and processing of antigens. HSP70
has been
5 shown to act as adjuvant for induction of CD8+ T yell activation and it
enhances
immunogenicity of specific antigenic peptides (Blachere et al. (1997) J. Exp.
Med.
186:131 S-22). When HSP70 is encoded by a genetic vaccine vector, it is likely
to enhance
presentation and processing of antigenic peptides and thereby improve the
efficacy of the
genetic vaccines. DNA shuffling can be used to further improve the properties,
including
10 adjuvant activity, of heat shock proteins, such as HSP70.
Recombinantly produced cytokine, chemokine, and accessory molecule
polypeptides, as well as antagonists of these molecules, can be used to
influence the type of
immune response to a given stimulus. However, the administration of
polypeptides
sometimes has shortcomings, including short half life, high expense, difficult
to store (must
15 be stored at 4°C), and a requirement for large volumes. Also, bolus
injections can
sometimes cause side effects. Administration of polynucleotides that encode
the
recombinant cytokines or other molecules overcomes most or all of these
problems. DNA,
for example, can be prepared in high purity, is stable, temperature resistant,
noninfectious,
easy to manufacture. In addition, polynucleotide-mediated administration of
cytokines can
20 provide long-lasting, consistent expression, and administration of
polynucleotides in general
is regarded as being safe.
The functions of cytokines, chemokines and accessory molecules are
redundant and pleiotropic, and therefore can be difficult to determine which
cytokines or
cytokine combinations are the most potent in inducing and enhancing antigen
specific
25 immune responses following vaccination. Furthermore, the most useful
combination of
cytokines and accessory molecules is typically different depending on the type
of immune
response that is desired following vaccination. As an example, IL-4 has been
shown to
direct differentiation of TH2 cells (which produce high levels of IL-4, IL-5
and IL-13, and
mediate allergic immune responses), whereas IFN-y and IL-12 direct
differentiation of TH1
30 cells (which produce high levels of IL-2 and IFN-'y), and mediate delayed
type immune
responses. Moreover, the most useful combination of cytokines and accessory
molecules is
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41
also likely to depend on the antigen used in the vaccination. The invention
provides a
solution to this problem of obtaining an optimized genetic vaccine cocktail.
Different
combinations of cytokines, chemokines and accessory molecules are assembled
into vectors
using the methods described herein. These vectors are then screened for their
capacity to
induce immune responses in vivo and in vitro.
Large libraries of vectors, generated by gene shuffling and combinatorial
molecular biology, are screened for maximal capacity to direct immune
responses towards,
for example, a TH1 or TH2 phenotype, as desired. A library of different
vectors can be
generated by assembling different evolved promoters, (evolved) cytokines,
(evolved)
cytokine antagonists, (evolved) chemokines, (evolved) accessory molecules and
immunostimulatory sequences, each of which can be prepared using methods
described
herein. DNA sequences and compounds that facilitate the transfection and
expression can be
included. If the pathogens) is known, specific DNA sequences encoding
immunogenic
antigens from the pathogen can be incorporated into these vectors providing
protective
immunity against the pathogens) (as in genetic vaccines).
Initial screening is preferably carried out in vitro. For example, the library
can be introduced into cells which are tested for ability to induce
differentiation of T cells
capable of producing cytokines that are indicative of the type of immune
response desired.
For a TH 1 response, for example, the library is screened to identify
recombinant
polynucleotides that are capable of inducing T cells to produce IL-2 and IFN-
y, while
screening for induction of T cell production of IL-4, IL-5, and IL-13 is
performed to identify
recombinant polynucleotides that favor a T~2 response.
Screening can also be conducted in vivo, using animal models., For example,
vectors produced using the methods of the invention can be tested for ability
to protect
against a lethal infection. Another screening method involves injection
ofLeishmania major
parasites into footpads of BALB/c mice (nonhealer). Pools of plasmids are
injected i.v., i.p.
or into footpads of these mice and the size of the footpad swelling is
followed. Yet another
in vivo screening method involves detection of IgE levels after infection with
Nippostrongylus brasiliensis. High levels indicate a TH2 response, while low
levels of IgE
indicate a TH 1 response.
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Successful results in animal models are easy to verify in humans. In vitro
screening can be conducted to test for human TH1 or TH2 phenotype, or for
other desired
immune response. Vectors can also be tested for ability to induce protection
against
infection in humans.
Because the principles of immune functions are similar in a wide variety of
infections, immunostimulating DNA vaccine vectors may not only be useful in
the treatment
of a number of infectious diseases but also in prevention of the infections,
when the vectors
are delivered to the sites of the entry of the pathogen (e.g., the lung or
gut).
3. Agonists or antagonists of cellular receptors
The invention also provides methods for obtaining optimized recombinant
polynucleotides that encode a peptide or polypeptide that can interact with a
cellular receptor
that is involved in mediating an immune response. The optimized recombinant
palynucleotides can act as an agonist or an antagonist of the receptor.
Cytokine antagonists can be used as components of genetic vaccine cocktails.
Blocking immunosuppressive cytokines, rather than adding single
proinflammatory
cytokines, is likely to potentiate the immune response in a more general
manner, because
several pathways are potentiated at the same time_ By appropriate choice of
antagonist, one
can tailor the immune response induced by a genetic vaccine in order to obtain
the response
that is most effective in achieving the desired effect. Antagonists against
any cytokine can be
used as appropriate; particular cytokines of interest for blocking include,
for example, IL-4,
IL-13, IL-10, and the like.
The invention provides methods of obtaining cytokine antagonists that exhibit
greater effectiveness in blocking the action of the respective cytokine.
Polynucleotides that
encode improved cytokine antagonists can be obtained by using gene shuffling
to generate a
recombinant library of polynucleotides which are then screened to identify
those that encode
an improved antagonist. As substrates for the DNA shuffling, one can use, for
example,
polynucleotides that encode receptors for the respective cytokine. At least
two forms of the
substrate will be present in the recombination reaction, with each form
differing from the
other in at least one nucleotide position. In a preferred embodiment, the
different forms of
the polynucleotide are homologous cytokine receptor genes from different
organisms. The
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43
resulting library of recombinant polynucleotides is then screened to identify
those that
encode cytokine antagonists with the desired affinity and biological activity.
As one example of the type of effect that one can achieve by including a
cytokine antagonist in a genetic vaccine cocktail, as well as how the effect
can be improved
using the DNA shuffling methods of the invention, IL-10 is discussed. The same
rationale
can be applied to obtaining and using antagonists of other cytokines.
Interleukin-10 (IL-10)
is perhaps the most potent anti-inflammatory cytokine known to date. IL-10
inhibits a
number of pathways that potentiate inflammatory responses. The biological
activities of IL-
include inhibition of MHC class II expression on monocytes, inhibition of
production of
10 IL-l, IL-6, IL-12, TNF-a by monocytes/macrophages, and inhibition of
proliferation and IL-
2 production by T lymphocytes. The significance of IL-10 as a regulatory
molecule of
immune and inflammatory responses was clearly demonstrated in IL-10 deficient
mice.
These mice are growth-retarded, anemic and spontaneously develop an
inflammatory bowel
disease (Kahn et al. (1993) Cell 75: 263). In addition, both innate and
acquired immunity to
Listeria monocytogenes were shown to be elevated in IL-10 deficient mice (Dai
et al, (1997)
J. Immunol. 158: 2259). It has also been suggested that genetic differences in
the levels of
IL-10 production may affect the risk of patients to die from complications
meningococcal
infection. Families with high IL-10 production had 20-fold increased risk of
fatal outcome
of meningococcal disease (Westendorp et al. (1997) Lancet 349: 170).
IL-10 has been shown to activate normal and malignant B cells in vitro, but it
does not appear to be a major growth promoting cytokine for normal B cells in
vivo, because
IL-10 deficient mice have normal levels of B lymphocytes and Ig in their
circulation. In
fact, there is evidence that IL-10 can indirectly downregulate B cell function
through
inhibition of the accessory cell function of monocytes. However, IL-10 appears
to play a
role in the growth and expansion of malignant B cells. Anti-IL-10 monoclonal
antibodies
and IL-10 antisense oligonucleotides have been shown to inhibit transformation
of B cells by
EBV in vitro. In addition, B cell lymphomas are associated with EBV and most
EBV+
lymphomas produce high levels of IL-10, which is derived both from the human
gene and
the homologue of IL-10 encoded by EBV. AIDS-related B cell lymphomas also
secrete high
levels of IL-10. Furthermore, patients with detectable serum IL-10 at the time
of diagnosis
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44
of intermediateJhigh-grade non-Hodgkin's lymphoma have short survival, further
suggesting
a role for IL-10 in the pathogenesis of B cell malignancies.
Antagonizing IL-10 in vivo can be beneficial in several infectious and
malignant diseases, and in vaccination. The effect of blocking of IL-10 is an
enhancement of
immune responses that is independent of the specificity of the response. This
is useful in
vaccinations and in the treatment of serious infectious diseases. Moreover, an
IL-10
antagonist is useful in the treatment of B cell malignancies which exhibit
overproduction of
IL-10 and viral IL-10, and it may also be useful in boosting general anti-
tumor immune
response in cancer patients. Combining an IL-10 antagonist with gene therapy
vectors may
be useful in gene therapy of tumor cells in order to obtain maximal immune
response against
the tumor cells. If shuffling of IL-10 results in IL-10 with improved specific
activity, this
IL-10 molecule would have potential in the treatment of autoimmune diseases
and
inflammatory bowel diseases. IL-10 with improved specific activity may also be
useful as a
component of gene therapy vectors in reducing the immune response against
vectors which
are recognized by memory cells and it may also reduce the immunogenicity of
these vectors.
An antagonist of IL-10 has been made by generating a soluble form of IL-10
receptor (sIL-IOR; Tan et al. (1995) J. Biol. Chem. 270: 12906). However, sIL-
lOR binds
IL-10 with Kd of 560 pM, whereas the wild-type, surface-bound receptor has
affinity of 35-
200 pM. Consequently, 150-fold molar excess of sIL-lOR is required for half
maximal
inhibition of biological function of IL-10. Moreover, affinity of viral IL-10
(IL-10
homologue encoded by Epstein-Barr virus) to sIL-lOR is more than 1000 fold
less than that
of hIL-10, and in some situations, such as when treating EBV-associated B cell
malignancies, it may be beneficial if one can also block the function of viral
IL-10. Taken
together, this soluble form of IL-lOR is unlikely to be effective in
antagonizing IL-10 in
vivo.
To obtain an IL-10 antagonist that has sufficient affinity and antagonistic
activity to function in vivo, DNA shuffling can be performed using
polynucleotides that
encode IL-10 receptor. IL-10 receptor with higher than normal affinity will
function as an
IL-10 antagonist, because it strongly reduces the amount of IL-10 available
for binding to
functional, wild-type IL-l OR. In a preferred embodiment, IL-l OR is shuffled
using
homologous cDNAs encoding IL-lOR derived from human and other mammalian
species.
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An alignment of human and mouse IL-10 receptor sequences is shown in Figure 14
to
illustrate the feasibility of family DNA shuffling when evolving IL-10
receptors with
improved affinity. A phage library of IL-10 receptor recombinants can be
screened for
improved binding of shuffled IL-lOR to human or viral IL-10. Wild-type IL-10
and/or viral
5 IL-10 are added at increasing concentrations to demand for higher affinity.
Phage bound to
IL-10 can be recovered using anti-IL-10 monoclonal antibodies. If desired, the
shuffling can
be repeated one or more times, after which the evolved soluble IL-l OR is
analyzed in
functional assays for its capacity to neutralize the biological activities of
IL-10/viral IL-10.
More specifically, evolved soluble IL-lOR is studied for its capacity to block
the inhibitory
10 effects of IL-10 on cytokine synthesis and MHC class II expression by
monocytes,
proliferation by T cells, and for its capacity to inhibit the enhancing
effects of IL-10 on
proliferation of B cells activated by anti-CD40 monoclonal antibodies.
An IL-10 antagonist can also be generated by evolving IL-10 to obtain
variants that bind to IL-lOR with higher than wild-type affinity, but without
receptor
15 activation. The advantage of this approach is that one can evolve an IL-10
molecule with
improved specific activity using the same methods. In a preferred embodiment,
IL-10 is
shuffled using homologous cDNAs encoding IL-10 derived from human and other
mammalian species. In addition, a gene encoding viral IL-10 can be included in
the
shuffling. A library of IL-10 recombinants is screened for improved binding to
human IL-10
20 receptor. Library members bound to IL-lOR can be recovered by anti-IL-lOR
monoclonal
antibodies. This screening protocol is likely to result in IL-10 molecules
with both
antagonistic and agonistic activities. Because initial screen demands for
higher affinity, a
proportion of the agonists are likely to have improved specific activity when
compared to
wild-type human IL-10. The functional properties of the mutant IL-10 molecules
are
25 determined in biological assays similar to those described above for
ultrahigh-affinity IL-10
receptors (cytokine synthesis and MHC class II expression by monocytes,
proliferation of B
and T cells). An antagonistic IL-4 mutant has been previously generated
illustrating the
general feasibility of the approach (Kruse et al. ( 1992) EMBO J. 11: 3237-
3244). One
amino acid mutation in IL-4 resulted in a molecule that efficiently binds to
IL-4R a-chain
30 but has minimal IL-4-like agonistic activity.
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46
Another example of an IL-10 antagonist is IL-20/mda-7, which is a 206
amino acid secreted protein. This protein was originally characterized as mda-
7, which is a
melanoma cell-derived negative regulator of tumor cell growth (Jiang et al.
(1995)
Oncogene 11: 2477; (1996) Proc. Nat'1. Acad. Sci. USA 93: 9160). IL-20/mda-7
is
structurally related to IL-10, and it antagonizes several functions of IL-10
(Abstract of the
13th European Immunology Meeting, Amsterdam, 22-25 June 1997). In contrast to
IL-10,
IL-20/mda-7 enhances expression of CD80 (B7-1) and CD86 (B7-2) on human
monocytes
and it upregulates production of TNF-a and IL-6. IL-20/mda-7 also enhances
production of
IFN-y by PHA-activated PBMC. The invention provides methods of improving
genetic
vaccines by incorporation of IL-20/mda-7 genes into the genetic vaccine
vectors. The
methods of the invention can be used to obtain IL-20/mda-7 variants that
exhibit improved
ability to antagonize IL-10 activity.
When a cytokine antagonist is used as a component of DNA vaccine or gene
therapy vectors, maximal local effect is desirable. Therefore, in addition to
a soluble form of
a cytokine antagonist, a transmembrane form of the antagonist can be
generated. The
soluble form can be given in purified polypeptide form to patients by, for
example,
intravenous injection. Alternatively, a polynucleotide encoding the cytokine
antagonist can
be used as a component as a component of a genetic vaccine or a gene therapy
vector. In
this case, either or both of the soluble and transmembrane forms can be used.
Where both
soluble and transmembrane forms of the antagonist are encoded by the same
vector, the
target cells express both forms, resulting in maximal inhibition of cytokine
function on the
target cell surface and in their immediate vicinity.
The peptides or polypeptides obtained using these methods can substitute for
the natural ligands of the receptors, such as cytokines or other costimulatory
molecules in
their ability to exert an effect on the immune system via the receptor. A
potential
disadvantage of administering cytokines or other costimulatory molecules
themselves is that
an autoimmune reaction could be induced against the natural molecule, either
due to
breaking tolerance (if using a natural cytokine or other molecule) or by
inducing cross-
reactive immunity (humoral or cellular) when using related but distinct
molecules. Through
using the methods of the invention, one can obtain agonists or antagonists
that avoid these
potential drawbacks. For example, one can use relatively small peptides as
agonists that can
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47
mimic the activity of the natural immunomodulator, or antagonize the activity,
without
inducing cross-reactive immunity to the natural molecule. In a presently
preferred
embodiment, the optimized agonist or antagonist obtained using the methods of
the
invention is about SO amino acids or length or less, more preferably about 30
amino acids or
less, and most preferably is about 20 amino acids in length, or less. The
agonist or antagonist
peptide is preferably at least about 4 amino acids in length, and more
preferably at least
about 8 amino acids in length. Polynucleotides that flank the coding sequence
of the mimetic
peptide can also be optimized using the methods of the invention in order to
optimize the
expression, conformation, or activity of the mimetic peptide.
The optimized agonist or antagonist peptides or polypeptides are obtained by
generating a library of recombinant polynucleotides and screening the library
to identify
those that encode a peptide or polypeptide that exhibits an enhanced ability
to modulate an
immune response. The library can be produced using methods such as DNA
shuffling or
other methods described herein or otherwise known to those of skill in the
art. Screening is
conveniently conducted by expressing the peptides encoded by the library
members on the
surface of a population of replicable genetic packages and identifying those
members that
bind to a target of interest, e.g., a receptor.
The optimized recombinant polynucleotides that are obtained using the
methods of the invention can be used in several ways. For example, the
polynucleotide can
be placed in a genetic vaccine vector, under the control of appropriate
expression control
sequences, so that the mimetic peptide is expressed upon introduction of the
vector into a
mammal. If desired, the polynucleotide can be placed in the vector embedded in
the coding
sequence of the surface protein (e.g., geneIII or geneVIII) in order to
preserve the
conformation of the mimetic. Alternatively, the mimetic-encoding
polynucleotide can be
inserted directly into the antigen-encoding sequence of the genetic vaccine to
form a coding
sequence for a "mimotope-on-antigen" structure. The polynucleotide that
encodes the
mimotope-on-antigen structure can be used within a genetic vaccine, or can be
used to
express a protein that is itself administered as a vaccine. As one example of
this type of
application, a coding sequence of a mimetic peptide is introduced into a
polynucleotide that
encodes the "M-loop" of the hepatitis B surface antigen (HBsAg) protein. The M-
loop is a
six amino acid peptide sequence bounded by cysteine residues, which is found
at amino
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48
acids I39-147 (numbering within the S protein sequence). The M-loop in the
natural HBsAg
protein is recognized by the monoclonal antibody RFHB7 (Chen et al., Proc.
Nat'l. Acad.
Sci. USA, 93: 1997-2001 (1996)). According to Chen et al., the M-Ioop forms an
epitope of
the HBsAg that is non-overlapping and separate from at least four other HBsAg
epitopes.
Because of the probable Cys-Cys disulfide bond in this hydrophilic part of the
protein, amino acids 139-147 are likely in a cyclic conformation. This
structure is therefore
similar to that found in the regions of the filamentous phage proteins pIII
and pVIII where
mimotope sequences are placed. Therefore, one can insert a mimotope obtained
using the
methods of the invention into this region of the HBsAg amino acid sequence.
The chemokine receptor CCR6 is an example of a suitable target for a peptide
mimetic obtained using the methods. The CCR6 receptor is a 7-transmembrane
domain
protein (Dieu et al., Biochem. Biophys. Res. Comm. 236: 212-2I7 (1997) and J.
Biol. Chem.
272: 14893-14898 (1997)) that is involved in the chemoattraction of immature
dendritic
cells, which are found in the blood and migrate to sites of antigen uptake
(Dieu et al., J. Exp.
Med. 188: 373-386 (1998)). CCR6 binds the chemokine MIP-3a, so a mimetic
peptide that
is capable of activating CCR6 can provide a further chemoattractant function
to a given
antigen and thus promote uptake by dendritic cells after immunization with the
antigen
antigen-mimetic fusion or a DNA vector that expresses the antigen.
Another application of this method of the invention is to obtain molecules
that can act as an agonist for the macrophage scavenger receptor (MSR; see,
Wloch et al.,
Hum. Gene Ther. 9: 1439-1447 (1998)). The MSR is involved in mediating the
effects of
various immunomodulators. Among these are bacterial DNA, including the
plasmids used in
DNA vaccination, and oligonucleotides, which are often potent
immunostimulators.
Oligonucleotides of certain chemical structure (e.g., phosphothio-
oligonucleotides) are
2~ particularly potent, while bacterial or plasmid DNA must be used in
relatively large
quantities to produce an effect. Also mediated by the MSR is the ability of
oligonucleotides
that contain dG residues to stimulate B cells and enhance the activity of
immunostimulatory
CpG motifs, and of lipopolysaccharides to activate macrophages. Some of these
activities
are toxic. Each of these immunomodulators, along with a variety of polyanionic
ligands,
binds to the MSR. The methods of the invention can be used to obtain mimetics
of one or
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49
more of these immunomodulators that bind to the MSR with high affinity but are
devoid of
toxic properties. Such mimetic peptides are useful as immunostimulators or
adjuvants.
The MSR is a trimeric integral membrane glycoprotein. The three
extracellular C-terminal cysteine-rich regions are connected to the
transmembrane domain
by a f brous region that is composed of an a-helical coil and a collagen-like
triple helix (see,
Kodama et al., Nature 343: 531-535 (1990)). Therefore, screening of the
library of
recombinant polynucleotides can be accomplished by expressing the
extracellular receptor
structure and artificially attaching it to plastic surfaces. The libraries can
be expressed, e.g.,
by phage display, and screened to identify those that bind to the receptors
with high affinity.
The optimized recombinant polynucleotides identified by this method can be
incorporated
into antigen-encoding sequences to evaluate their modulatory effect on the
immune
response.
4. Costimulatory molecules capable of inhibiting or enhancing activation,
or
Also provided are methods of obtaining optimized recombinant
polynucleotides that, when expressed, are capable of inhibiting or enhancing
the activation,
differentiation, or anergy of antigen-specific T cells. T cell activation is
initiated when T
cells recognize their specific antigenic peptides in the context of MHC
molecules on the
plasma membrane of antigen presenting cells (APC), such as monocytes,
dendritic cells
(DC), Langerhans cells or B cells. Activation of CD4+ T cells requires
recognition by the T
cell receptor (TCR) of an antigenic peptide in the context of MHC class II
molecules,
whereas CD8+ T cells recognize peptides in the context of MHC class I
molecules.
Importantly, however, recognition of the antigenic peptides is not sufficient
for induction of
T cell proliferation and cytokine synthesis. An additional costimulatory
signal, "the second
signal", is required. The costimulatory signal is mediated via CD28, which
binds to its
ligands B7-1 (CD80) or B7-2 (CD86), typically expressed on the antigen
presenting cells. In
the absence of the costimulatory signal, no T cell activation occurs, or T
cells are rendered
anergic. In addition to CD28, CTLA-4 (CD152) also functions as a ligand for B7-
1 and B7-
2. However, in contrast to CD28, CTLA-4 mediates a negative regulatory signal
to T cells
and/or to induce anergy and tolerance (Walunas et al. (1994) Immunity 1: 405;
Karandikar et
al. ( 1996) ,I. Exp. Med. 184: 783).
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B7-l and B7-2 have been shown to be able to regulate several immunological
responses, and they have been implicated to be of importance in the immune
regulation in
vaccinations, allergy, autoimmunity and cancer. Gene therapy and genetic
vaccine vectors
expressing B7-1 and/or B7-2 have also been shown to have therapeutic potential
in the
S treatment of the above mentioned diseases and in improving the efficacy of
genetic vaccines.
Figure 10 illustrates interaction of APC and CD4+ T cells, but the same
principle is true with CD8+ T cells, with the exception that the T cells
recognize the
antigenic peptides in the context of MHC class I molecules. Both B7-1 and B7-2
bind to
CD28 and CTLA-4, even though the sequence similarities between these four
molecules are
10 very limited (20-30%). It is desirable to obtain mutations in B7-1 and B7-2
that only
influence binding to one ligand but not to the other, or improve activity
through one ligand
while decreasing the activity through the other. Moreover, because the
affinities of B7
molecules to their ligands appear to be relatively low, it would also be
desirable to f nd
mutations that improve/alter the activities of the molecules. However,
rational design does
15 not enable predictions of useful mutations because of the complexity of the
molecules.
The invention provides methods of overcoming these difficulties, enabling
one to generate and identify functionally different B7 molecules with altered
relative
capacities to induce T cell activation, differentiation, cytokine production,
anergy and/or
tolerance. Through use of the methods of the invention, one can find mutations
in B7-1 and
20 B7-2 that only influence binding to one ligand but not to the other, or
that improve activity
through one ligand while decreasing the activity through the other. DNA
shuffling is likely
to be the most powerful method in discovering new B7 variants with altered
relative binding
capacities to CD28 and CTLA-4. B7 variants which act through CD28 with
improved
activity (and with decreased activity through CTLA-4) are expected to have
improved
25 capacity to induce activation of T cells. In contrast, B7 variants which
bind and act through
CTLA-4 with improved activity (and with decreased activity through CD28) are
expected to
be potent negative regulators of T cell functions and to induce tolerance and
anergy.
DNA shuffling or other recombination method is used to generate B7 (e.g.,
B7-1/CD80 and B7-2/CD86) variants which have altered relative capacity to act
through
30 CD28 and CTLA-4 when compared to wild-type B7 molecules. In a preferred
embodiment,
the different forms of substrate used in the recombination reaction are B7
cDNAs from
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51
various species. Such cDNAs can be obtained by methods known to those of skill
in the art,
including RT-PCR.
Typically, genes encoding these variant B7 molecules are incorporated into
genetic vaccine vectors encoding an antigen, so that one the vectors can be
used to modify
antigen-specific T cell responses. Vectors that harbor B7 genes that
efficiently act through
CD28 are useful in inducing, for example, protective immune responses, whereas
vectors
that harbor genes encoding B7 genes that efficiently act through CTLA-4 are
useful in
inducing, for example, tolerance and anergy of allergen- or autoantigen-
specific T cells. In
some situations, such as in tumor cells or cells inducing autoimmune
reactions, the antigen
may already be present on the surface of the target cell, and the variant B7
molecules may be
transfected in the absence of additional exogenous antigen gene. Figure 11
illustrates a
screening protocol that one can use to identify B7-1 (CD80) and/or B7-2 (CD86)
variants
that have increased capacity to induce T cell activation or anergy, and the
application of this
strategy is described in more detail in Example 1.
Several approaches for screening of the variants can be taken. For example,
one can use a flow cytometry-based selection systems. The library of B7-1 and
B7-2
molecules is transfected into cells that normally do not express these
molecules (e.g., COS-7
cells or any cell line from a different species with limited or no cross-
reactivity with man
regarding B7 ligand binding). An internal marker gene can be incorporated in
order to
analyze the copy number per cell. Soluble CTLA-4 and CD28 molecules can be
generated
to for use in the flow cytometry experiments. Typically, these will be fused
with the Fc
portion of IgG molecule to improve the stability of the molecules and to
enable easy staining
by labeled anti-IgG mAbs, as described by van der Merwe et al. (J. Exp. Med.
185: 393,
1997). The cells transfected with the library of B7 molecules are then stained
with the
soluble CTLA-4 and CD28 molecules. Cells demonstrating increased or decreased
CTLA-
4/CD28 binding ratio will be sorted. The plasmids are then recovered and the
shuffled B7
variant-encoding sequences identified. These selected B7 variants can then be
subjected to
new rounds of shuffling and selection, and/or they can be further analyzed
using functional
assays as described below.
The B7 variants can also be directly selected based on their functional
properties. For in vivo studies, the B7 molecules can also be evolved to
function on mouse
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52
cells. Bacterial colonies with plasmids with mutant B7 molecules are picked
and the
plasmids are isolated. These plasmids are then transfected into antigen
presenting cells, such
as dendritic cells, and the capacities of these mutants to activate T cells is
analyzed. One of
the advantages of this approach is that no assumptions on the binding
affinities or
specificities to the known ligands are made, and possibly new activities
through yet to be
identified ligands can be found. In addition to dendritic cells, other cells
that are relatively
easy to transfect (e.g., U937 or COS-7) can be used in the screening, provided
that the "first
T cell signal" is induced by, for example, anti-CD3 monoclonal antibodies. T
cell activation
can be analyzed by methods known to those of skill in the art, including, for
example,
measuring proliferation, cytokine production, CTL activity or expression of
activation
antigens such as IL-2 receptor, CD69 or HLA-DR molecules. Usage of antigen-
specific T
cell clones, such as T cells specific for house dust mite antigen Der p I,
will allow analysis of
antigen-specific T cell activation (Yssel et al. (1992) J. Immunol. 148: 738-
745). Mutants
are identified that can.enhance or inhibit T cell proliferation or enhance or
inhibit CTL
responses. Similarly variants that have altered capacity to induce cytokine
production or
expression of activation antigens as measured by, for example, cytokine-
specific ELISAs or
flow cytometry can be identif ed.
The B7 variants are useful in modulating immune responses in autoimmune
diseases, allergy, cancer, infectious disease and vaccination. B7 variants
which act through
CD28 with improved activity (and with decreased activity through CTLA-4) will
have
improved capacity to induce activation of T cells. In contrast, B7 variants
which bind and
act through CTLA-4 with improved activity (and with decreased activity through
CD28) will
be potent negative regulators of T cell functions and to induce tolerance and
anergy. Thus,
by incorporating genes encoding these variant B7 molecules into genetic
vaccine vectors
encoding an antigen, it is possible to modify antigen-specific T cell
responses. Vectors that
harbor B7 genes that efficiently act through CD28 are useful in inducing, for
example,
protective immune responses, whereas vectors that harbor genes encoding B7
genes that
efficiently act through CTLA-4 are useful in inducing, for example, tolerance
and anergy of
allergen- or autoantigen-specific T cells. In some situations, such as in
tumor cells or cells
inducing autoimmune reactions, the antigen may already be present on the
surface of the
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53
target cell, and the variant B7 molecules may be transfected in the absence of
additional
exogenous antigen gene.
The methods of the invention are also useful for obtaining B7 variants that
have increased effectiveness in directing either TH1 or TH2 cell
differentiation. Differential
roles have been observed for B7-1 and B7-2 molecules in the regulation of T
helper (TH) cell
differentiation (Freeman et al. (1995) Immunity 2: 523; Kuchroo et al. (1995)
Cell 80: 707).
TH cell differentiation can be measured by analyzing the cytokine production
profiles
induced by each particular variant. High levels of IL-4, IL-5 and /or IL-13
are an indication
of efficient T~2 cell differentiation whereas high levels of IFN-y or IL-2
production can be
used as a marker of THl cell differentiation. B7 variants with altered
capacity to induce TH 1
or TH2 cell differentiation are useful, for example, in the treatment of
allergic, malignant,
autoimmune and infectious diseases and in vaccination.
Also provided by the invention are methods of obtaining B7 variants that
have enhanced capacity to induce IL-10 production by antigen-specific T cells.
Elevated
production of IL-10 is a characteristic of regulatory T cells, which can
suppress proliferation
of antigen-specific CD4+ T cells (Groux et al. (1997) Nature 389: 737). DNA
shuffling is
performed as described above, after which recombinant nucleic acids encoding
B7 variants
having enhanced capability of inducing IL-10 can be identified by, for
example, ELISA or
flow cytometry using intracytoplasmic cytokine staining. The variants that
induce high
levels of IL-10 production are useful in the treatment of allergic and
autoimmune diseases.
D. Optimization of Transport and Presentation of Antigens
The invention also provides methods of obtaining genetic vaccines and
accessory molecules that can improve the transport and presentation of
antigenic peptides. A
library of recombinant polynucleotides is created and screened to identify
those that encode
molecules that have improved properties compared to the wild-type
counterparts. The
polynucleotides themselves can be used in genetic vaccines, or the gene
products of the
polynucleotides can be utilized for therapeutic or prophylactic applications.
1. Proteasomes
The class I peptides presented on major histocompatibility complex
molecules are generated by cellular proteasomes. Interferon-gamma can
stimulate antigen
presentation, and part of the mechanism of action of interferon may be due to
induction of
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54
the proteasome beta-subunits LMP2 and LMP7, which replace the homologous beta-
subunits
Y (delta) and X (epsilon). Such a replacement changes the peptide cleavage
specificity of the
proteasome and can enhance class I epitope immunogenicity. The Y (delta) and X
(epsilon)
subunits, as well as other recently discovered proteasome subunits such as the
MECL-1
homologue MC14, are characteristic of cells which are not specialized in
antigen
presentation. Thus, the incorporation into cells by DNA transfer of LMP2,
LMP7, MECL-1
and/or other epitope presentation-specific and potentially interferon-
inducible subunits can
enhance epitope presentation. It is likely that the peptides generated by the
proteasome
containing the interferon-inducible subunits are transported to the
endoplasmic reticulum by
the TAP molecules.
The invention provides methods of obtaining proteasomes that exhibit
increased or decreased ability to specifically process MHC class I epitopes.
According to the
methods, DNA shuffling is used to obtain evolved proteins that can either have
new
specificities which might enhance the immunogenicity of some proteins and/or
enhance the
activity of the subunits once they are bound to the proteasome. Because the
transition from a
non-specific proteasome to a class I epitope-specific proteasome can pass
through several
states (in which some but not all of the interferon-inducible subunits are
associated with the
proteasome), many different proteolytic specificities can potentially be
achieved. Evolving
the specific LMP-like subunits can therefore create new proteasome
compositions which
have enhanced functionality for the presentation of epitopes.
The methods involve performing DNA shuffling using as substrates two or
more forms of polynucleotides which encode proteasome components, where the
forms of
polynucleotides differ in at least one nucleotide. Shuffling is performed as
described herein,
using polynucleotides that encode any one or more of the various proteasome
components,
including, for example, LMP2, LMP7, MECL-1 and other individual proteasome
components that are specifically involved in class I epitope presentation.
Examples of
suitable substrates are described in, e.g., Stohwasser et al. (1997) Eur. ,l.
Immunol. 27: 1182-
1187 and Gaczynska et al. (1996) J. Biol. Chem. 271: 17275-17280. In a
preferred
embodiment, family shuffling is used, in which the different substrates are
proteasome
component-encoding polynucleotides from different species.
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After the recombination reaction is completed, the resulting library of
recombinant polynucleotides is screened to identify those which encode
proteasome
components having the desired effect on class I epitope production. For
example, the
recombinant polynucleotides can be introduced into a genetic vaccine vector
which also
5 encodes a particular antigen of interest. The library of vectors can then be
introduced into
mammalian cells which are then screened to identify cells which exhibit
increased antigen-
specific immunogenicity. Methods of analyzing proteasome activity are
described in, for
example, Groettrup et al. (1997) Proc. Nat'l. Acad. Sci. USA 94: 8970-897 and
Groettrup et
al. ( 1997) Eur. J. Immunol. 26: 863-869.
10 Alternatively, one can use the methods of the invention to evolve proteins
which bind strongly to the proteasome but have decreased or no activity, thus
antagonizing
the proteasome activity and diminishing a cells ability to present class I
molecules. Such
molecules can be applied to gene therapy protocols in which it is desirable to
lower the
immunogenicity of exogenous proteins expressed in the cells as a result of the
gene therapy,
15 and which would otherwise be processed for class I presentation allowing
the cell to be
recognized by the immune system. Such high-affinity low-activity LMP-like
subunits will
demonstrate immunosuppressive effects which are also of use in other
therapeutic protocols
where cells expressing a non-self protein need to be protected from an immune
response.
The specificity of the proteasome and the TAP molecules (discussed below)
20 may have co-evolved naturally. Thus it may be important that the two
pathways of the class I
processing system be functionally matched. A further aspect of the invention
involves
performing DNA shuffling simultaneously on the two gene families followed by
random
combinations of the two in order to discover appropriate matched proteolytic
and transport
specificities.
25 2. Antigen Transport
The invention provides methods of improving transport of antigenic peptides
from the cytosolic compartment to the endoplasmic reticulum and thereby to the
cell surface
in the context of MHC class I molecules. Enhanced expression of antigenic
peptides results
in enhanced immune response, particularly in improved activation of CD8+
cytotoxic
30 lymphocytes. This is useful in the development of DNA vaccines and in gene
therapy.
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In one embodiment, the invention involves evolving TAP-genes (transporters
associated with antigen processing) to obtain genes that exhibit improved
antigen
presentation. TAP genes are members of ATP-binding cassette family of membrane
translocators. These proteins transport antigenic peptides to MHC class I
molecules and are
involved in the expression and stability of MHC class I molecules on the cell
surface. Two
TAP genes, TAP1 and TAP2, have been cloned to date (Powis et al. (1996) Proc.
Nat'1.
Acad Sci. USA 89: 1463-1467; Koopman et al. (1997) Curr. Opin. Immunol. 9: 80-
88;
Monaco (I995) J. Leukocyte Biol. 57: 543-57). TAP1 and TAP2 form a heterodimer
and
these genes are required for transport of peptides into the endoplasmic
reticulum, where they
bind to MHC class I molecules. The essential role of TAP gene products in
presentation of
antigenic peptides was demonstrated in mice with disrupted TAP genes. TAP1-
deficient
mice have drastically reduced levels of surface expression of MHC class I, and
positive
selection of CD8+ T cells in the thymus is strongly reduced. Therefore, the
number of CD8+
T lymphocytes in the periphery of TAP-deficient mice is extremely low.
Transfection of
TAP genes back into these cells restores the level of MHC class I expression.
TAP genes are a good target for gene shuffling because of natural
polymorphism and because these genes of several mammalian species have been
cloned and
sequenced, including human (Beck et al. (1992) J. Mol. Biol. 228: 433-441;
Genbank
Accession No. Y13582; Powis et al., supra.), gorilla TAP1 (Laud et al. (1996)
Human
Immunol. 50: 91-102), mouse (Reiser et al. (1988) Proc. Nat'l. Acad. Sci. USA
85: 2255-
2259; Marusina et al. (1997) J. Immunol. 158: 5251-5256, TAP1: Genbank
Accession Nos.
U60018, U60019, U60020, U60021, U60022, and L76468-L67470; TAP2: Genbank
Accession Nos. U60087, U60088, U6089, U60090, U60091 and U60092), hamster
(TAP1,
Genbank Accession Nos. AF001154 and AF001157; TAP2, Genbank Accession Nos.
AF001156 and AF001155). Furthermore, it has been shown that point mutations in
TAP
genes may result in altered peptide specificity and peptide presentation.
Also, functional
differences in TAP genes derived from different species have been observed.
For example,
human TAP and rat TAP containing the rTAP2a allele are rather promiscuous,
whereas
mouse TAP is restrictive and select against peptides with C-terminal small
polar/hydrophobic or positively charged amino acids. The basis for this
selectivity is
unknown.
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57
The methods of the invention involve performing DNA shuffling of TAP 1
and TAP2 genes using as substrates at least two forms of TAP 1 and/or TAP2
polynucleotide
sequences which differ in at least one nucleotide position. In a preferred
embodiment, TAP
sequences derived from several mammalian species are used as the substrates
for shuffling.
Natural polymorphism of the genes can provide additional diversity of
substrate. If desired,
optimized TAP genes obtained from one round of shuffling and screening can be
subjected
to additional shuffling/screening rounds to obtain further optimized TAP-
encoding
polynucleotides.
To identify optimized TAP-encoding polynucleotides from a library of
recombinant TAP genes, the genes can be expressed on the same plasmid as a
target antigen
of interest. If this step is limiting the extent of antigen presentation, then
enhanced
presentation to CD8+ CTL will result. Mutants of TAPs may act selectively to
increase
expression of a particular antigen peptide fragment for which levels of
expression are
otherwise limiting, or to cause transport of a peptide that would normally
never be
transferred into the RER and made available to bind to MHC Class I.
When used in the context of gene therapy vectors in cancer treatment,
evolved TAP genes provide a means to enhance expression of MHC class I
molecules on
tumor cells and obtain efficient presentation of antigenic tumor-specific
peptides. Thus,
vectors that contain the evolved TAP genes can induce potent immune responses
against the
malignant cells. Shuffled TAP genes can be transfected into malignant cell
lines that express
low levels of MHC class I molecules using retroviral vectors or
electroporation.
Transfection efficiency can be monitored using marker genes, such as green
fluorescent
protein, encoded by the same vector as the TAP genes. Cells expressing equal
levels of
green fluorescent protein but the highest levels of MHC class I molecules, as
a marker of
efficient TAP genes, are then sorted using flow cytometry, and the evolved TAP
genes are
then recovered from these cells by, for example, PCR or by recovering the
entire vectors.
These sequences can then subjected into new rounds of shuffling, selection and
recovery, if
further optimization is desired.
Molecular evolution of TAP genes can be combined with simultaneous
evolution of the desired antigen. Simultaneous evolution of the desired
antigen can further
improve the efficacy of presentation of antigenic peptides following DNA
vaccination. The
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antigen can be evolved, using gene shuffling, to contain structures that allow
optimal
presentation of desired antigenic peptides when optimal TAP genes are
expressed. TAP
genes that are optimal for presentation of antigenic peptides of one given
antigen may be
different from TAP genes that are optimal for presentation of antigenic
peptide of another
antigen. Gene shuffling technique is ideal, and perhaps the only, method to
solve this type
of problems. Efficient presentation of desired antigenic peptides can be
analyzed using
specific cytotoxic T lymphocytes, for example, by measuring the cytokine
production or
CTL activity of the T lymphocytes using methods known to those of skill in the
art.
3. Cytotoxic T-Cell Inducing Sequences And Immunogenic
Seauences
Certain proteins are better able than others to carry MHC class I epitopes
because they are more readily used by the cellular machinery involved in the
necessary
processing for class I epitope presentation. The invention provides methods of
identifying
expressed polypeptides that are particularly efficient in traversing the
various biosynthetic
and degradative steps leading to class I epitope presentation and the use of
these
polypeptides to enhance presentation of CTL epitopes from other proteins.
In one embodiment, the invention provides Cytotoxic T-cell Inducing
Sequences (CTIS), which can be used to carry heterologous class I epitopes for
the purpose
of vaccinating against the pathogen from which the heterologous epitopes are
derived. One
example of a CTIS is obtained from the hepatitis B surface antigen (HBsAg),
which has
been shown to be an effective carrier for its own CTL epitopes when delivered
as a protein
under certain conditions. DNA immunization with plasmids expressing the HBsAg
also
induces high levels of CTL activity. The invention provides a shorter,
truncated fragment of
the HBsAg polypeptide which functions very efficiently in inducing CTL
activity, and
attains CTL induction levels that are higher than with the HBsAg protein or
with the
plasmids encoding the full-length HBsAg polypeptide. Synthesis of a CTIS
derived from
HBsAg is described in Example 3; and a diagram of a CTIS is shown in Figure 1.
The ER localization of the truncated polypeptide may be important in
achieving suitable proteolytic liberation of the peptides) containing the CTL
epitopes (see
Cresswell & Hughes (1997) Curr. Biol. 7: 8552-RS55; Craiu et al. (1997) Proc.
Nat'1. Acad.
Sci. USA 94: 10850-10855). The preS2 region and the transmembrane region
provide T-
helper epitopes which may be important for the induction of a strong cytotoxic
immune
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59
response. Because the truncated CTIS polypeptide has a simple structure (see
Figure 1), it is
possible to attach one or more heterologous class I epitope sequences to the C-
terminal end
of the polypeptide without having to maintain any specific protein
conformation. Such
sequences are then available to the class I epitope processing mechanisms. The
size of the
S polypeptide is not subject to the normal constraints of the native HBsAg
structure. Therefore
the length of the heterologous sequence and thus the number of included CTL
epitopes is
flexible. This is shown schematically in Figure 2. The ability to include a
long sequence
containing either multiple and distinct class I sequences, or alternatively
different variations
of a single CTL sequence, allows DNA shuffling methodology to be applied.
The invention also provides methods of obtaining Immunogenic Agonist
Sequences (IAS) which induce CTLs capable of specific lysis of cells
expressing the natural
epitope sequence. In some cases, the reactivity is greater than if the CTL
response is induced
by the natural epitope (see, Example 3 and Figure 3). Such IA.S-induced CTL
may be drawn
from a T-cell repertoire different from that induced by the natural sequence.
In this way,
poor responsiveness to a given epitope can be overcome by recruiting T cells
from a larger
pool. In order to discover such IAS, the amino acid at each position of a CTL-
inducing
peptide (excluding perhaps the positions of the so-called anchor residues) can
be varied over
the range of the 19 amino acids not normally present at the position. DNA
shuffling
methodology can be used to scan a large range of sequence possibilities.
A synthetic gene segment containing multiple copies of the original epitope
sequence can be prepared such that each copy possesses a small number of
nucleotide
changes. The gene segment can be shuffled to create a diverse range of CTL
epitope
sequences, some of which should function as IAS. This process is illustrated
in Figure 4.
In practice, oligonucleotides are typically constructed in accordance with the
above design and polymerized enzymatically to form the synthetic gene segment
of the
concatenated epitopes. Restriction sites can be incorporated into a fraction
of the
oligonucleotides to allow for cleavage and selection of given size ranges of
the concatenated
epitopes, most of which will have different sequences and thus will be
potential IAS. The
epitope-containing gene segment can be joined by appropriate cloning methods
to a CTIS,
such as that of HBsAg. The resulting plasmid constructions can be used for DNA-
based
immunization and CTL induction.
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E. Genetic Vaccine Pharmaceutical Compositions and Methods of
Administration
The improved immunomodulatory polynucleotides and polypeptides of the
invention are useful for treating and/or preventing the various diseases and
conditions with
which the respective antigens are associated. For example, genetic vaccines
that employ the
reagents obtained according to the methods of the invention are useful in both
prophylaxis
and therapy of infectious diseases, including those caused by any bacterial,
fungal, viral, or
other pathogens of mammals. The reagents obtained using the invention can also
be used for
treatment of autoimmune diseases including, for example, rheumatoid arthritis,
SLE,
10 diabetes mellitus, myasthenia gravis, reactive arthritis, ankylosing
spondylitis, and multiple
sclerosis. These and other inflammatory conditions, including IBD, psoriasis,
pancreatitis,
and various immunodeficiencies, can be treated using genetic vaccines that
include vectors
and other components obtained using the methods of the invention. Genetic
vaccine vectors
and other reagents obtained using the methods of the invention can be used to
treat allergies
15 and asthma. Moreover, the use of genetic vaccines have great promise for
the treatment of
cancer and prevention of metastasis. By inducing an immune response against
cancerous
cells, the body's immune system can be enlisted to reduce or eliminate cancer.
In presently preferred embodiments, the reagents obtained using the invention
are used in conjunction with a genetic vaccine vector. The choice of vector
and components
20 can also be optimized for the particular purpose of treating allergy or
other conditions. For
example, an antigen associated with treating a particular condition can be
optimized using
recombination and selection methods analogous to those described herein. Such
methods,
and antigens appropriate for various conditions, are described in copending,
commonly
assigned US Patent Application Serial No. , entitled "Antigen Library
25 Immunization," which was filed on February 10, 1999 as TTC Attorney Docket
No. 18097-
028710US. The polynucleotide that encodes the recombinant antigenic
polypeptide can be
placed under the control of a promoter, e.g., a high activity or tissue-
specific promoter. The
promoter used to express the antigenic polypeptide can itself be optimized
using
recombination and selection methods analogous to those described herein, as
described in
30 International Application No. PCT/US97/17300 (International Publication No.
WO
98/13487). The reagents obtained using the methods of the invention can also
be used in
conjunction with multicomponent genetic vaccines, which are capable of
tailoring an
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61
immune response as is most appropriate to achieve a desired effect (see, e.g.,
copending,
commonly assigned US Patent Application Serial No.
entitled "Genetic
Vaccine Vector Engineering," filed on February 10, 1999 as TTC Attorney Docket
No.
18097-030100US). It is sometimes advantageous to employ a genetic vaccine that
is targeted
for a particular target cell type (e.g., an antigen presenting cell or an
antigen processing cell);
suitable targeting methods are described in copending, commonly assigned US
patent
application Serial No. , entitled "Targeting of Genetic Vaccine Vectors,"
filed on
February 10, 1999 as TTC Attorney Docket No. 18097-030200US.
Genetic vaccine vectors that include the optimized recombinant
polynucleotides obtained as described herein can be delivered to a mammal
(including
humans) to induce a therapeutic or prophylactic immune response. Vaccine
delivery vehicles
can be delivered in vivo by administration to an individual patient, typically
by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal,
intracranial,
anal, vaginal, oral, buccal route or they can be inhaled) or they can be
administered by
topical application. Alternatively, vectors can be delivered to cells ex vivo,
such as cells
explanted from an individual patient (e.g., lymphocytes, bone marrow
aspirates, tissue
biopsy) or universal donor hematopoietic stem cells, followed by
reimplantation of the cells
into a patient, usually after selection for cells which have incorporated the
vector.
A large number of delivery methods are well known to those of skill in the
art. Such methods include, for example liposome-based gene delivery (Debs and
Zhu (1993)
WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691;
Rose
U.S. Pat No. 5,279,833; Brigham (1991} WO 91/06309; and Felgner et al. (1987)
Proc. Natl.
Acad. Sci. USA 84: 7413-7414), as well as use of viral vectors (e.g.,
adenoviral (see, e.g.,
Berns et al. (1995) Ann. NYAcad. Sci. 772: 95-104; Ali et al. (1994) Gene
Ther. 1: 367-384;
and Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 ( Pt 3): 297-306
for review),
papillomaviral, retroviral (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)
2731-2739;
Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al.,
(1990) Virol.
176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al., J.
Virol. 65:2220-2224
(1991); Wong-Staal et al., PCT/CTS94/05700, and Rosenburg and Fauci (1993) in
Fundamentallmmunology, Third Edition Paul (ed) Raven Press, Ltd., New York and
the
references therein, and Yu et al., Gene Therapy (1994) supra.), and adeno-
associated viral
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62
vectors (see, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S.
Patent No.
4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy
5:793-
801; Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for an
overview of AAV
vectors; see also, Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985)
Mol. Cell.
Biol. 5(I 1):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-
2081; Hermonat and
Muzyczka (1984) Proc. Natl. Acad Sci. USA, 81:6466-6470; McLaughlin et al.
(1988) and
Samulski et al. (1989) J. Virol., 63:03822-3828), and the like.
"Naked" DNA and/or RNA that comprises a genetic vaccine can be
introduced directly into a tissue, such as muscle. See, e.g., USPN 5,580,859.
Other methods
such as "biolistic" or particle-mediated transformation (see, e.g., Sanford et
al., USPN
4,945,050; USPN 5,036,006) are also suitable for introduction of genetic
vaccines into cells
of a mammal according to the invention. These methods are useful not only for
in vivo
introduction of DNA into a mammal, but also for ex vivo modification of cells
for
reintroduction into a mammal. As for other methods of delivering genetic
vaccines, if
necessary, vaccine administration is repeated in order to maintain the desired
level of
immunomodulation.
Genetic vaccine vectors (e.g., adenoviruses, liposomes, papillomaviruses,
retroviruses, etc.) can be administered directly to the mammal for
transduction of cells in
vivo. The genetic vaccines obtained using the methods of the invention can be
formulated as
pharmaceutical compositions for administration in any suitable manner,
including parenteral
(e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical,
oral, rectal,
intrathecal, buccal (e.g., sublingual), or local administration, such as by
aerosol or
transdermally, for prophylactic and/or therapeutic treatment. Pretreatment of
skin, for
example, by use of hair-removing agents, may be useful in transdermal
delivery. Suitable
methods of administering such packaged nucleic acids are available and well
known to those
of skill in the art, and, although more than one route can be used to
administer a particular
composition, a particular route can often provide a more immediate and more
effective
reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method used to
administer the
composition. Accordingly, there is a wide variety of suitable formulations of
pharmaceutical
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63
compositions of the present invention. A variety of aqueous carriers can be
used, e.g.,
buffered saline and the like. These solutions are sterile and generally free
of undesirable
matter. These compositions may be sterilized by conventional, well known
sterilization
techniques. The compositions may contain pharmaceutically acceptable auxiliary
substances
as required to approximate physiological conditions such as pH adjusting and
buffering
agents, toxicity adjusting agents and the like, for example, sodium acetate,
sodium chloride,
potassium chloride, calcium chloride, sodium lactate and the like. The
concentration of
genetic vaccine vector in these formulations can vary widely, and will be
selected primarily
based on fluid volumes, viscosities, body weight and the like in accordance
with the
particular mode of administration selected and the patient's needs.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the packaged nucleic acid suspended
in diluents,
such as water, saline or PEG 400; (b) capsules, sachets or tablets, each
containing a
predetermined amount of the active ingredient, as liquids, solids, granules or
gelatin; (c)
1 S suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet
forms can include
one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato
starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and other
excipients,
colorants, fillers, binders, diluents, buffering agents, moistening agents,
preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible
carriers.
Lozenge forms can comprise the active ingredient in a flavor, usually sucrose
and acacia or
tragacanth, as well as pastilles comprising the active ingredient in an inert
base, such as
gelatin and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in
addition to the active ingredient, carriers known in the art. It is recognized
that the genetic
vaccines, when administered orally, must be protected from digestion. This is
typically
accomplished either by complexing the vaccine vector with a composition to
render it
resistant to acidic and enzymatic hydrolysis or by packaging the vector in an
appropriately
resistant corner such as a liposome. Means of protecting vectors from
digestion are well
known in the art. The pharmaceutical compositions can be encapsulated, e.g.,
in liposomes,
or in a formulation that provides for slow release of the active ingredient.
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64
The packaged nucleic acids, alone or in combination with other suitable
components, can be made into aerosol formulations (e.g., they can be
"nebulized") to be
administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Suitable formulations for rectal administration include, for example,
suppositories, which consist of the packaged nucleic acid with a suppository
base. Suitable
suppository bases include natural or synthetic triglycerides or paraffin
hydrocarbons. In
addition, it is also possible to use gelatin rectal capsules which consist of
a combination of
the packaged nucleic acid with a base, including, for example, liquid
triglycerides,
polyethylene glycols, and paraffin hydrocarbons.
Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers,
and preservatives. In the practice of this invention, compositions can be
administered, for
example, by intravenous infusion, orally, topically, intraperitoneally,
intravesically or
intrathecally. Parenteral administration and intravenous administration are
the preferred
methods of administration. The formulations of packaged nucleic acid can be
presented in
unit-dose or mufti-dose sealed containers, such as ampoules and vials.
Injection solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described. Cells transduced by
the packaged
nucleic acid can also be administered intravenously or parenterally.
The dose administered to a patient, in the context of the present invention
should be sufficient to effect a beneficial therapeutic response in the
patient over time. The
dose will be determined by the efficacy of the particular vector employed and
the condition
of the patient, as well as the body weight or vascular surface area of the
patient to be treated.
The size of the dose also will be determined by the existence, nature, and
extent of any
adverse side-effects that accompany the administration of a particular vector,
or transduced
cell type in a particular patient.
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In determining the effective amount of the vector to be administered in the
treatment or prophylaxis of an infection or other condition, the physician
evaluates vector
toxicities, progression of the disease, and the production of anti-vector
antibodies, if any. In
general, the dose equivalent of a naked nucleic acid from a vector is from
about 1 p,g to 1 mg
5 for a typical 70 kilogram patient, and doses of vectors used to deliver the
nucleic acid are
calculated to yield an equivalent amount of therapeutic nucleic acid.
Administration can be
accomplished via single or divided doses.
In therapeutic applications, compositions are administered to a patient
suffering from a disease (e.g., an infectious disease or autoimmune disorder)
in an amount
10 sufficient to cure or at least partially arrest the disease and its
complications. An amount
adequate to accomplish this is defined as a "therapeutically effective dose."
Amounts
effective for this use will depend upon the severity of the disease and the
general state of the
patient's health. Single or multiple administrations of the compositions may
be administered
depending on the dosage and frequency as required and tolerated by the
patient. In any
15 event, the composition should provide a sufficient quantity of the proteins
of this invention
to effectively treat the patient.
In prophylactic applications, compositions are administered to a human or
other mammal to induce an immune response that can help protect against the
establishment
of an infectious disease or other condition.
20 The toxicity and therapeutic efficacy of the genetic vaccine vectors
provided
by the invention are determined using standard pharmaceutical procedures in
cell cultures or
experimental animals. One can determine the LDso (the dose lethal to 50% of
the
population) and the EDSO (the dose therapeutically effective in 50% of the
population) using
procedures presented herein and those otherwise known to those of skill in the
art.
25 A typical pharmaceutical composition for intravenous administration would
be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg
per patient
per day may be used, particularly when the drug is administered to a secluded
site and not
into the blood stream, such as into a body cavity or into a lumen of an organ.
Substantially
higher dosages are possible in topical administration. Actual methods for
preparing
30 parenterally administrable compositions will be known or apparent to those
skilled in the art
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and are described in more detail in such publications as Remington's
Pharmaceutical
Science, ISth ed., Mack Publishing Company, Easton, Pennsylvania (1980).
The multivalent antigenic polypeptides of the invention, and genetic vaccines
that express the polypeptides, can be packaged in packs, dispenser devices,
and kits for
administering genetic vaccines to a mammal. For example, packs or dispenser
devices that
contain one or more unit dosage forms are provided. Typically, instructions
for
administration of the compounds will be provided with the packaging, along
with a suitable
indication on the label that the compound is suitable for treatment of an
indicated condition.
For example, the label may state that the active compound within the packaging
is useful for
treating a particular infectious disease, autoinunune disorder, tumor, or for
preventing or
treating other diseases or conditions that are mediated by, or potentially
susceptible to, a
mammalian immune response.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present
I S invention.
Example 1
Altered ligand specificity of B7-I (CD80) and/or B7-2 (CD86) by DNA shuffling
This Example describes the use of the DNA shuffling methods of the
invention to obtain B7-1 and B7-2 polypeptides that have altered biological
activities.
DNA shuffling
DNA shuffling is used to generate a library of B7 (B7-1/CD80 and B7-
2/CD86) variants that have altered relative capacity to act through CD28 and
CTLA-4 when
compared to wild-type B7 molecules. Typically, B7 cDNAs from various species
are
generated by RT-PCR, and these sequences are shuffled using family DNA
shuffling.
Alignments of human, rhesus monkey and rabbit B7-1 nucleotide sequences are
shown in
Figure 1 S, demonstrating that family DNA shuffling is a feasible approach
when evolving
B7 molecules.
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Screening of B7 variants
The library is then screened to identify those variants that are useful in
modulating immune responses in autoimmune diseases, allergy, cancer,
infectious disease
and vaccination. Any of several approaches for screening of the variants can
be used:
A. Flow cytometry-based selection system.
The library of B7-l and B7-2 molecules is transfected into cells that normally
do not express these molecules (e.g., COS-7 cells or any cell line from
different species with
limited or no cross-reactivity with man regarding B7 ligand binding). An
internal marker
gene can be incorporated in order to analyze the copy number per cell.
Soluble CTLA-4 and CD28 molecules are generated to facilitate the flow
cytometry experiments. Typically, these soluble polypeptides are fused with
the Fc portion
of IgG molecule to improve the stability of the molecules and to enable easy
staining by
labeled anti-IgG monoclonal antibodies, as described by van der Merwe et al.
((1997) J. Exp.
Med. 185: 393). The cells transfected with the library of B7 molecules are
then stained with
the soluble CTLA-4 and CD28 molecules. Cells demonstrating increased or
decreased
CTLA-4/CD28 binding ratio are sorted. The plasmids are then recovered and the
shuffled
sequences identified. These selected B7 variants can then be subjected to new
rounds of
shuffling and selection, and can be further analyzed using functional assays
as described
below.
B. Selection based on functional properties.
Bacterial colonies that contain plasmids that include mutant B7 molecules are
picked and the plasmids are isolated. These plasmids are then transfected into
antigen
presenting cells, such as dendritic cells, and the capacities of these mutants
to. activate T cells
is analyzed. One of the advantages of this approach is that no assumptions are
made as to
the binding affinities or specificities to the known ligands, and possibly new
activities
through yet to be identified ligands can be found.
T cell activation can be analyzed by measuring proliferation, cytokine
production, CTL activity or expression of activation antigens such as IL-2
receptor, CD69 or
HLA-DR molecules. Usage of antigen-specific T cell clones, such as T cells
specific for
house dust mite antigen Der p I, allows analysis of antigen-specific T cell
activation.
Mutants are identified that can enhance or inhibit T cell proliferation or
enhance or inhibit
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CTL responses. Similarly variants that have altered capacity to induce
cytokine production
or expression of activation antigens as measured by, for example, cytokine-
specific ELISAs
or flow cytometry can be selected. Results obtained using a proliferation-
based assay is
shown are shown in Figure 13.
C. Ability to direct either THI or Tx2 cell differentiation.
Because differential roles for B7-1 and B7-2 molecules in the regulation of T
helper cell differentiation have been identified (Freeman et al. (1995)
Immunity 2: 523;
Kuchroo et al. (1995) Cell 80: 707), one can screen for B7 variants that are
the most
effective in directing either TH1 or TH2 cell differentiation. TH cell
differentiation can be
measured by analyzing the cytokine production profiles induced by each
particular variant.
High levels of IL-4, IL-5 and /or IL-13 are an indication of efficient TH2
cell differentiation
whereas high levels of IFN-y or IL-2 production can be used as a marker of TH1
cell
differentiation. B7 variants that altered capacity to induce TH1 or TH2 cell
differentiation are
likely to be useful in the treatment of allergic, malignant, autoimmune and
infectious
diseases and in vaccination.
D. Enhanced IL-10 production.
Elevated production of IL-10 is a characteristic of regulatory T cells, which
can suppress proliferation of antigen-specific CD4+ T cells (Groux et al.
(1997) Nature 389:
737). Therefore, B7 variants can be screened to identify those that have
enhanced capacity
to induce IL-10 production by antigen-specific T cells. IL-10 production can
be measured,
for example, by ELISA or flow cytometry using intracytoplasmic cytokine
stainings. The
variants that induce high levels of IL-10 production are useful in the
treatment of allergic
and autoimmune diseases.
Example 2
Evolution Of Cytokines Far Improved Specific Activity
And/Or Improved Expression Levels
This example describes a method to evolve a cytokine for improved specific
activity and/or improved expression levels when the genetic vaccine is
transfected into
mammalian cells. IL-12 is the most potent cytokine directing TH1 responses,
and it
improves the efficacy of genetic vaccinations. Evolved IL-12 molecules are
useful as
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components of genetic vaccines. IL-12 is a heterodimeric cytokine composed of
a 35 kD
light chain (p35) and a 40 kD heavy chain (p40) (Kobayashi et al. (1989) J.
Exp. Med. 170:
827; Stern et al. (1990) Proc. Nat'1. Acad. Sci. USA 87: 6808). Recently
Lieschke et al.
(Nature Biotechnol. (1997) 15: 35) demonstrated that a fusion between p35 and
p40 genes
results in a single gene that has activity comparable to that of the two genes
expressed
separately. Accordingly, an IL-12 gene is shuffled as one entity that encodes
both subunits,
which is beneficial in designing the shuffling protocol. The subunits of IL-12
can also be
expressed separately in the same expression vector, or the subunits can be
expressed
separately and screened using cotransfections of the two vectors, providing
additional
shuffling strategies.
IL-12 plays several roles in the regulation of allergic responses. For
example,
IL-12 induces TH1 cell differentiation and downregulates the TH2 response. IL-
12 inhibits
IgE synthesis both in vivo and in vitro, and also induces IFN-y production.
Accordingly, it is
desirable to obtain an optimized IL-12 that better able to carry out these
functions upon
administration to a mammal.
Cytokine genes, including IL-12 genes, from humans and nonhuman primates
are generally 93-99% homologous (Villinger et al. (1995) J. Immunol. 155:3946-
3954),
providing a good starting point for family shuffling. A library of shuffled IL-
12 genes was
obtained by shuffling p35 and p40 subunits derived from human, rhesus monkey,
cat, dog,
cow, pig, and goat, and incorporated into vectors and the supernatants of
these transfectants
are analyzed for biological activity as shown in Figure 6. Because of its T
cell growth
promoting activities, it is possible to use normal human peripheral blood T
cells in the
selection of the most active IL-12 genes, enabling directly to select IL-I2
mutants with the
most potent activities on human T cells.
As shown in Figure 7, a functional screening assay has been successfully
established. In this assay, COS-7 cells were first transfected with vectors
encoding IL-12
subunits. Forty-eight hours after transfection, the capacity of these culture
supernatants to
induce proliferation of activated human peripheral blood T cells was studied.
Figure 8
indicates the consistency of the level of T cell proliferation induced in this
assay, indicating
that the assay can be used to distinguish the activities between supernatants
that have
different capacities to induce T cell activation. In other words, the assay
provides means to
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screen for improved IL-12-Iike activities in culture supernatants of
transfected cells. A
vector with an optimized IL-12-encoding polynucleotide was tested for ability
to induce
human T cell activation. Results, shown in Figure 9, show that the shuffled IL-
12 has an
significantly increased ability to induce T cell activation compared to wild-
type IL-12.
5 Figure 6 illustrates a general strategy for screening of evolved cytokine
genes.
The specific example is given for IL-12 but similar approach applies to all
cytokines when
using cell types sensitive for each cytokine. For example, GM-CSF can be
evolved by the
same approach by using the GM-CSF sensitive cell line TF-1 in the screening.
In addition,
although in this example the vectors are transfected into CHO cells, any
mammalian cell that
10 can be transfected in vitro can be used as host cells. In addition to CHO
cell, other good host
cells include cell lines WI-26, COS-1, COS-7, 293, U937 and freshly isolated
human antigen
presenting cells, such as monocytes, B cells and dendritic cells.
Example 3
T-cell Inducing Sequences Derived from hepatitis B Surface Antigen and
This Example describes the preparation of a polypeptide sequence capable of
efficient presentation of T cell epitopes and a strategy for the application
of DNA shuffling
to discover strongly immunogenic agonistic T cell epitopes.
The HBsAg polypeptide (PreS2 plus S regions) was truncated by the
introduction of a stop codon at amino acid position 103 (counting from the
beginning of the
PreS2 initiator methionine), transforming a cysteine codon TGT into the Stop
codon TGA.
The amino acid sequence of the truncated protein was therefore:
MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNME
NITSGFLGPLLVLQAGFFLLTRILTIPQSLDS W WTSLNFLGGTTV
where the standard single-letter code for amino acids is used. The methionine
residues at the
start of the PreS2 and S regions are underlined, and the mouse La-restricted
CTL epitope is
double-underlined; the asterisk (*) represents the artificially introduced
Stop codon.
A likely structure for this truncated polypeptide is shown in Figure 1. During
protein biosynthesis, the N-terminal region of the HBsAg polypeptide is
transported through
the membrane of the endoplasmic reticulum (ER). The first part of the S region
is a
transmembrane structure which locks the polypeptide into the ER. The remaining
C-terminal
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region of the polypeptide is located in the cytoplasmic compartment where it
can be accessed
by the epitope processing mechanism of the cell. This structure forms what is
referred to as
the Cytotoxic T-cell Inducing Sequence (CTIS).
A CTIS is preferably used in conjunction with an immunogenic agonistic
sequence (IAS). As an example of an IAS, each position of a 12 amino acid Ld-
restricted
class I epitope from HBsAg was replaced with an alanine-encoding codon in the
DNA
sequence of the epitope. The results demonstrate that, in some cases, the
reactivity is greater
than if the CTL response is induced by the natural epitope (Figure 3).
Example 4
Treatment of Obesity, Anorexia, and Cachexia using
Optimized Immunomodulatory Molecules
Optimized immunomodulatory molecules that are obtained using the methods
of the invention find use in a wide variety of applications, in addition to
use in vaccination.
For example, there is increasing evidence that certain forms of obesity are
associated with
dysfunction of the immune system, and that molecules which regulate immune
responses,
e.g. cytokines, can induce or i'bit obesity. The invention provides methods of
optimizing
immune regulatory molecules for the treatment of obesity, anorexia and
cachexia.
Leptin and ciliary neurotrophic factor (CNTF) are examples of cytokines that
have been shown to play a role in the development of obesity. Congenital
leptin deficiency
results in severe early-onset obesity in human (Montague et al. (1997) Nature
387: 903-908),
and CNTF has been shown to correct obesity and diabetes associated with leptin
deficiency
and resistance (Gloaguen et al. (1997) Proc. Nat'1. Acad Sci. USA 94: 6456-
6461).
Antagonists of CNTF and/or leptin may be useful in the treatment of anorexia
and/or
cachexia.
The methods of the invention are used to generate leptin and/or CNTF
molecules that have improved specific activity. The methods are also useful
for obtaining
improved cytokines that exhibit reduced immunogenicity in vivo; immunogenicity
is a
particular concern for CNTF, because the wild-type CNTF is highly antigenic,
which results
in the production of high levels of anti-CNTF antibodies when administered to
a human.
Improved cytokine molecules prepared using the methods of the invention are
administered
as polypeptides, or the shuffled nucleic acids that encode improved leptin
and/or CNTF
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polypeptides are used in genetic vaccine vectors. The invention also provides
methods of
generating vectors that induce production of increased levels of leptin and/or
CNTF.
The methods of the invention can also be used to obtain reagents that are
useful for the treatment of anorexia, cachexia, and related disorders. In this
embodiment,
antagonists of leptin and/or CNTF are evolved using the DNA shuffling methods.
For
example, a leptin receptor can be evolved to obtain a soluble form that has an
enhanced
affinity for leptin. The receptor for leptin in mice is found in the
hypothalamus (Mercer et
al. (1996) FEBSLett. 387: 1 I3), a region known to be involved in maintenance
of energy
balance, and in the choroid plexus and leptomeninges, which form part of the
blood/brain
barrier.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publications, patents,
and patent
applications cited herein are hereby incorporated by-reference for all
purposes.
