Note: Descriptions are shown in the official language in which they were submitted.
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ADJUVANT AND CELL MATURATION AGENT
The invention relates to the use of CD38, or a portion or analogue thereof, as
an adjuvant or to cause maturation of dendritic cells in vitro; and to uses of
the
mature dendritic cells. The invention also relates to the natural ligand of
CD38 on
follicular and non-follicular dendritic cells and to a method of identifying
adjuvants.
Follicular dendritic cells (FDCs) and non-follicular dendritic cells (DCs)
present antigen to T cells and B cells. FDC networks are present in lymph
nodes and
play a critical role in the development of memory B cells in germinal centres.
DCs
are important in the development of primary T cell responses, particularly CD8
T cell
responses.
Although the role of CD38 has previously been a matter of much
investigation, the effect of CD38 on dendritic cells has not been
investigated. The
inventors have shown that administration of a CD38 construct increases the
size and
number of FDC networks in germinal centres. This would cause an increased
efficiency in antigen presentation to B cells and result in an improvement in
long
term B cell memory and recall responses.
The inventors have also shown that the CD38 construct causes maturation of
DCs. Since immatiu-e DCs are better than mature DCs in taking up antigen and
mature DCs are better at stimulating primary T cell responses, this property
of CD38
can be used when preparing DCs in vitro.. Immature DCs can be used to take up
antigen and then be treated with CD38 to change the phenotype to the mature
form.
Such mature DCs will be more efficient at stimulating primary T cell responses
(for
example in vitro or after administration to a patient).
Using the CD38 construct the inventors have also identified the natural ligand
of CD38 on FDC and DC.
The invention thus provides use of CD38, or a portion thereof or analogue
thereof which can inhibit the binding of CD38 to a non-follicular dendritic
cell (DC)
or a follicular dendritic cell (FDC) and which retains the ability of CD38 to
stimulate
a DC or FDC, for use in the manufacture of an adjuvant for use in
immunotherapy.
Such an adjuvant may be administered together with, or separately from, an
antigen
for immunisation. It may, for example, preferably be administered several days
after
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_7_
immunisation, e.g. 4-5 days, after immunisation.
The invention also provides a method of causing maturation of DCs
comprising contacting DCs ex vivo with CD38, or a CD38 portion or analogue of
the
invention. The invention additionally provides an ex vivo mature DC that has
been
made using the method.
The invention provides (i) the native ligand present on a FDC or DC which
binds CD38 and which is substantially free of FDC or DC cell membrane,
excluding
CD31: (ii) a protein which is at least 70% homologous to (i) and binds, CD38;
or (iii)
a fragment of (i) or (ii) which retains the ability to bind CD38. The
invention also
provides an inhibitor of the same CD38 ligand which specifically inhibits
activation
of the ligand by CD38.
The invention is illustrated by the accompanying drawings in which:
Figiu-e la shows the molecular weight of the CD38/IgGl construct in
reducing (on the right) and non-reducing (on the left) conditions.
Figure lb shows the molecular weight of the construct after N-
endoglucosidase treatment.
Figure 2 and Figure 3 show cytometry of DCs.
Figure 4 shows CD38 ligand.
Figure 4a shows MHC class I levels on DCs.
Figure 4b shows MHC class II levels on DCs.
Figure 4c shows B7.1 levels on DCs.
Figure 4d shows B7.2 levels on DCs.
Figure 5 shows anti-DNP antibody responses.
Figure 6 shows CTL assay results.
The team 'analogue' as used below also includes portions of CD38.
The CD38 used in the invention is generally a mammalian or avian CD38,
such as a human, primate or rodent CD38 (e.g. mouse or rat CD38). Thus the
CD38
can be any of the different species fomns, or any of the naturally occurnng
allelic
forms (including variants in an individual such as splice variants) which
occur
naturally in such animals. Generally such forms of CD38 will be able to bind
FDCs
and/or DCs and will be able to stimulate FDCs and/or DCs.
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The CD38 may be one which is soluble i~z vivo, e.g. a naturally occurring
soluble form. Such naturally-occurnng soluble forms have previously been
described (see, for example Funaro et al., Int. Immunol. (1996) 8. 1643-1650
and
Horenstein et al., Biochem. J. (1998) 330, 1129-1135.)
When CD38 (or an analogue with CD38 sequence) is administered to humans
according to the invention, it is preferably human CD38 (or preferably the
analogue
has human CD38 sequence). Human CD38 has previously been studied and
techniques for obtaining the extracellular domains of human CD38 in soluble
form
have also previously been described (see, for example, Daeglio et al., J.
Immunol.
(1996) 156, 727-734).
The analogue of CD38 can inhibit the binding of CD38 to a FDC or DC.
Therefore the amount of CD38 which can bind an FDC or DC in the presence of
the
analogue is decreased. This is because the analogue is able to bind CD38
ligand on
the FDC or DC in a specific manner, and therefore competes with the CD38 for
binding to CD38 ligand. The inhibition of binding can be determined using
known
binding assays, such as those discussed below. FDCs or DCs for use in such
assays
can be obtained by known methods, such as by sorting cells based on their
ability to
bind CD38.
Other binding characteristics of the analogue are generally also the same as
CD38, and thus typically the analogue binds to antibodies specific for CD38
(e.g.
specific for an extracellular portion or the ligand binding site of CD38), and
thus
inhibits binding of the CD38 to such an antibody.
The analogue retains the ability of CD38 to stimulate FDCs and/or DCs.
Thus typically the analogue is able to cause an increase in the FDC networks
and/or
maturation of DCs (the maturation generally determined by measuring the
upregulation of cell surface markers, such as MHC class I or II, or B7.2, on
the DCs).
In one embodiment the analogue has ADP-ribosyl transferase activity. It
typically has at least 10%, for example at least 20, 40, 60, 100, 150, 200% or
more of
the ADP-ribosyl transferase activity of CD38.
In one embodiment the analogue is a peptide or comprises 1. 2, 3 or more
peptides typically joined together by peptide or non-peptide linkers. The
analogue
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may comprise sequence from tcvo or more of the proteins discussed below either
(e.g.
fragments from the proteins) as a fusion protein or joined together by the
linker(s).
A peptide analogue or a peptide which is present in an analogue typically has
or comprises the same sequence as all or part of CD38, or a sequence which is
homologous to pant of or all of CD38.
The peptide may comprise a fragment of CD38 or a fragment of the
homologous sequence, typically with a length of at least 10, 20, 30, 50 or 100
amino
acids long. Such a fragment may be the extracellular portion or ligand binding
site of
CD38, or the equivalent sequence in the homologous sequence. In a preferred
embodiment the fragment is the portion of CD38 which is encoded by the
polynucleotide sequence which is amplified when SEQ ID NO:1 and 2 are used as
primers in a PCR reaction in which a murine CD38 gene is used as template. The
portion of human CD38 which is equivalent to such an amplified sequence (the
'equivalent' sequence is determined based on homology, e.g. using the PILEUP
program in the UWGCG package) is also preferred and may be obtained in similar
manner.
The peptide analogue or peptide which is present in the analogue may
comprise sequence from other proteins (typically naturally occurring
proteins). The
sequence may be from an antibody (e.g. IgG), such as a Fab, (Fab)= fragment or
Fc
fragment. Such an antibody may be one which can bind CD38 ligand or a cell
surface protein on a FDC or DC. In a preferred embodiment the peptide is a
fusion
protein which comprises an extracellular domain portion of CD38 and a non-CD38
sequence.
A preferred analogue comprises a fusion protein of the extracellular domain
of CD38 (such as the murine fragment discussed above encoded by the sequence
amplified by SEQ ID NO: l and SEQ ID N0:2, or the equivalent human fragment)
and the CH2CH3 (Fc) domains of a human IgGI .
The sequence may be one that causes the analogue to associate with FDCs or
DCs, such as the cell membranes of these cells or with proteins on the surface
of
these cells. Such sequence may be from a protein that binds CD38 ligand or
that
binds a cell surface protein of FDCs or DCs.
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Typically the peptide analogue or peptide present in the analogue comprises
1, 2, 3, 4 or more modifications, which may be natural post-translational
modifications or artificial modifications. The modifications are typically the
same as
the modifications (e.g. the glycosylation) present on natural CD38, typically
at the
same or equivalent positions. The modification may provide a chemical moiety
(typically by substitution of a hydrogen, e.g. the hydrogen of a C-H bond in
the
peptide), such as an amino, acetyl, hydroxy or halogen (e.g. fluorine) group
or
carbohydrate group. Typically the modification is present on the N or C
terminus of
the peptide.
The analogue (or the peptide of the analogue) may comprise one or more non-
natural amino acids, e.g. amino acids with a side chain different from natural
amino
acids. Generally, the non-natural amino acid will have an N terminus and/or a
C
terminus. The non-natural amino acid may be an L-amino acid.
The analogue typically has or comprises a part which has a shape, size,
flexibility or electronic configuration which is substantially similar to CD38
or any
of the fragments of CD38 discussed above. It is typically a derivative of CD38
or of
such a fragment.
The analogue may be soluble or insoluble in water. The analogue may be
capable of forming an oil-in-water or water-in-oil emulsion. The analogue may
be
capable of associating with a lipid membrane, such as a cell membrane.
The analogue is typically designed by computational means and then
synthesised using methods known in the art. Alternatively the analogue can be
selected from a library of compounds. The library may be a combinatorial
library or a
display library, such as a phage display library. Analogues are generally
selected
from the library based on their ability to mimic the binding characteristics
and/or the
ability to stimulate a FDC or DC. Thus they may be selected based on ability
to bind
a CD38 ligand (for example on a FDC or DC) or antibody which binds CD38.
Substances that provide CD38 or the analogue in vivo
As discussed above CD38 or an analogue can be used as an adjuvant in vivo.
In one embodiment a substance capable of providing CD38 or the analogue in
vivo
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can be administered. Such a substance is included in the term 'analogue'
herein.
The substance is typically a precursor of CD38 or the analogue and is capable
of being modified (e.g. hydrolysed) in vivo, typically in a cell, to provide
CD38 or
the analogue.
The substance may be polynucleotide capable of being expressed to provide
CD38, the analogue or the precursor. The polynucleotide is typically DNA or
RNA,
and is single or double stranded. The polynucleotide generally comprises
sequence
that encodes CD38, the analogue or the precursor. The coding sequence is
typically
operably linked to a control sequence (e.g. a promoter) capable of providing
for
expression of the polynucleotide. Thus typically the polynucleotide comprises
5' and
3' to the coding sequence sequences which aid expression, such as
transcription
and/or translation of the coding sequence.
The polynucleotide is typically capable of being expressed in the cells of any
of the animals mentioned herein. The polynucleotide may be present in a viral
or
cellular vector.
The invention provides CD38 or an analogue for use as an adjuvant, in
particular for stimulating a larger and/or longer lasting immune response
against an
antigen. The immune response may be an antibody response, or a CD4 or CD8 T
cell response. CD38 or the analogue are also provided for increasing the
length of
time of B cell memory: and/or for increasing the size and/or number of FDC
networks. In the case of an antibody response CD38 or the analogue are
provided to
increase the amount of specific antibodies and/or to increase the time for
which such
antibodies are produced. In the case of CD4 and CD8 cells the CD38 or analogue
are
provided to increase the numbers of such cells produced which are specific for
the
antigen.
The invention provides CD38 or an analogue for use in causing maturation of
DCs in vivo.
Generally in the method of causing maturation of a DC i» vitro 10' to 101
(e.g. 10~ to 109) DCs are present. Typically CD38 or the analogue are
contacted with
DCs at a concentration of 10-'- to 103 ~g/ml (e.g. 1 to 10 ~.g/ml). The DCs
are
typically contacted with CD38 or the analogue for from 1 hour to 7 days (e.g.
from 6
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hours to 3 days). Generally the DCs will be contacted in conditions which
support
them (i.e. which keep them alive) and typically allow the DCs to grow or
replicate.
In the method the DCs may also be contacted with other agents which bind
proteins on the surface of the DCs (e.g. MHC class I or II molecules, or CD~O)
and/or which stimulate the DCs. Suitable agents include antibodies to the
surface
proteins or the natural receptors of the surface proteins. Soluble (for
example
truncated) forms of the receptors may be used or cells may be used which
naturally
express the receptors on their surface.
The invention provides a vaccine which comprises CD38 or the analogue and
an antigenic component. Such a vaccine is generally capable of stimulating an
immune response to the antigenic component, such as any of the immune
responses
discussed herein. Thus the antigenic component v~~ill comprise antibody or T
cell
(e.g. CD4 or CD8) epitopes.
The vaccine may also comprise other adjuvants or delivery systems, such as
adjuvants which stimulate a CD8 T cell response. In a preferred embodiment the
antigenic component of the vaccine comprises a CD8 epitope.
The antigenic component is typically from a cancer cell (e.g. specific to a
cancer cell, such as a neo-antigen) or a pathogen. The cancer or pathogen are
typically ones which can be damaged or killed by an antibody or CD8 T cell
response. The pathogen may be an intracellular or extracellular pathogen (e.g.
a
bacterium or virus).
The invention also provides an ex vivo mature DC which has been made using
the method of the invention. As discussed below such a cell may be in an
isolated or
purified form. The cell may be in a composition which also comprises T cells
or B
cells. The cell may be in a composition of mononuclear cells (e.g. from
peripheral
blood). The mature DC may have been provided with antigen (such as any of the
antigens, or proteins comprising any of the antigens, discussed above which
are
present in the antigenic component) when it was immature. Such a mature DC
will
generally comprise the antigen inside the cell (e.g. in the class I or II
antigen
processing pathway) or on its surface bound to MHC molecules.
Thus in one embodiment an immature DC is contacted with any of the
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antigens or proteins discussed above (typically under conditions in which the
DC is
able to take in the antigen and protein and process it) and is then contacted
with
CD38 or the analogue to provide a mature DC of the invention. Such contacting
may
preferably be carried out simultaneously with provision of conditions such
that the
DCs correspond to DCs subject to T cell signalling. Such conditions may be
conditions which mimic T cell signalling, e.g. contacting additionally with
anti-
CD40 and anti-MHC class II as described in the examples. In another
embodiment,
the antigen is provided inside the DC (in the same manner as discussed above
with
regards to substances that provide CD38 or the analogue in vivo). Thus the DC
may
be contacted with a precursor of the antigen or a polynucleotide that is
capable of
being expressed to provide the antigen.
Mature DCs produced in accordance with the invention typically have higher
levels of MHC class I or B7.2 expression than an immature (e.g. splenic) DC,
typically at least 2, 4, 6, 10 or more fold higher.
The invention provides a DC of the invention for use in a method of treating
the human or animal body by therapy. In particular the DC is provided for use
in
stimulating a T cell response ire vivo. Typically the response is a CD8 T cell
response. In the case of the DC discussed above which was provided with
antigen
when immature the immune response is typically directed to the antigen used.
The invention also provides a vaccine comprising a DC of the invention.
The invention provides a method of stimulating T cells specific to an epitope
in vitro comprising contacting the T cells with a DC of the invention under
conditions in which the DC presents the epitope to the T cell. The method may
be
used to increase the numbers of such T cells. This may be for the purpose of
administering them to a patient or to increase their numbers so that they can
be
detected. The T cells are CD4 or CD8 T cells.
The invention provides the ligand of CD38 as presented by FDC or DC in
substantially isolated form (hereinafter referred to as CD38 ligand) and
homologues
of the ligand; and fragments thereof. Such homologues and fragments are
included
in the term 'ligand' herein. CD38 ligand may be obtained from a cell membrane
(e.g. a FDC or DC cell membrane).
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The ligands which are homologues or fragments are capable of binding
CD38, and thus can typically inhibit the binding of CD38 to CD38 ligand.
Preferred
ligands retain the ability to cause CD38 mediated activation of FDCs or DCs
when
present in the cell membrane of FDCs or DCs.
S The ligand is typically substantially free of FDC or DC cell membrane. The
ligand may be substantially free of cell membrane or of cellular components.
Such
cell membranes may be those of prokaryotes or eukaryotes, mammals (such as
humans, primates or rodents) or of the animal in which the particular CD38
naturally
occurs.
The ligand may be identified (for example in its naturally occurring form) on
a gel, such as under non-reducing conditions (see Example 2).
Ligands which are fragments preferably include the extracellular part of the
natural CD38 ligand or the CD38 binding site (or the equivalent sequence in a
homologue) .
1 S The ligand may or may not be able to bind CD38 or an analogue of the
invention. The ligand may be soluble in water. The ligand may be able to
associate
with lipids, such as cell membranes.
The ligand is typically at least 5, 10, 20, S0, 100 or more amino acids in
length. The ligand may be present in the form of a fusion protein which has
additional amino acid sequence N and/or C terminal to the ligand sequence.
Polynucleotides of the invention also include sequences that encode a ligand
of the invention. Such polynucleotides may also be DNA or RNA, and may be
single or double stranded.
Polynucleotides of the invention can be incorporated into a recombinant
2S replicable vector. The vector may be used to replicate the nucleic acid in
a
compatible host cell. Thus a polynucleotide of the invention may be made in a
process comprising introducing a polynucleotide of the invention into a
replicable
vector, introducing the vector into a compatible host cell, and growing the
host cell
under conditions which bring about replication of the vector. The vector may
be
recovered from the host cell. Suitable host cells are described below in
connection
with expression vectors.
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In one embodiment the polynucleotide of the invention in a vector is operably
linked to a control sequence which is capable of providing for the expression
of the
coding sequence by the host cell.
The term "operably linked" refers to a juxtaposition wherein the components
described are in a relationship permitting them to function in their intended
manner.
A control sequence "operably linked" to a coding sequence is ligated in such a
way
that expression of the coding sequence is achieved under conditions compatible
with
the control sequences.
Such vectors may be transformed into a suitable host cell as described above
to provide for expression of a ligand of the invention. Thus, in a further
aspect the
invention provides a process for preparing a ligand of the invention. which
process
comprises cultivating a host cell transformed or transfected with an
expression vector
as described above under conditions to provide for expression of the ligand,
and
recovering the expressed ligand.
The vectors may be for example, plasmid, virus or phage vectors provided
with an origin of replication, optionally a promoter for the expression of the
said
polynucleotide and optionally a regulator of the promoter. The vectors may
contain
one or more selectable marker genes, for example an ampicillin resistance gene
in the
case of a bacterial plasmid or a neomycin resistance gene for a mammalian
vector.
Vectors may be used in vitro, for example for the production of RNA or used to
transfect or transform a host cell. The vector may also be adapted to be used
in vitro.
for example in a method of gene therapy.
A further embodiment of the invention provides host cells transformed or
transfected with the vectors for the replication and expression of
polynucleotides of
the invention. The cells will be chosen to be compatible with the said vector
and
may for example be bacterial, yeast, insect or mammalian.
Promoters and other expression regulation signals may be selected to be
compatible with the host cell for which the expression vector is designed. For
example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S.
pombe
nmtl and adh promoters. Mammalian promoters include the metallothionein
promoter which can be induced in response to heavy metals such as cadmium.
Most
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preferably, the expression vectors are possible for use in insect or mammalian
cells.
For use in insect cells, strong baculovirus promoters such as the polyhedron
promoter
are preferred. For expression in mammalian cells, strong viral promoters such
as the
SV40 large T antigen promoter, a CMV promoter or an adenovirus promoter may
also be used. All these promoters are readily available in the art.
Suitable cells include cells in which the abovementioned vectors may be
expressed. These include microbial cells such as bacteria (e.g. E. coli),
mammalian
cells such as CHO cells, COS7 cells, P388 cells, HepG2 cells, KB cells, EL4
cells or
HeLa cells, insect cells or yeast such as Saccharomyces. Baculovirus or
vaccinia
expression systems may be used.
The invention provides an antibody that binds CD38 ligand and inhibits the
binding of CD38 to CD38 ligand. The antibody may be monoclonal or polyclonal.
Such antibody may be produced in a process that comprises contacting CD38 with
a
population of B cells (in vivo or ex vivo), and then isolating antibody of the
invention
which is produced by such cells. As discussed below such antibody may be
collected
from the sera of animals to which CD38 ligand has been administered.
Alternatively
B cells (e.g. in the form of spleen) may be removed from such an animal,
immortalised; and selected based on their ability to produce antibody that
binds
CD38 ligand. Antibody may be obtained from such selected cells.
An adjuvant may be identified in a process comprising determining whether a
candidate substance binds specifically to CD38 ligand, specific binding
indicating
that the substance is an adjuvant. An adjuvant may be identified in a process
comprising determining whether a candidate substance is capable of causing
maturation of DCs, a substance which is capable of maturing DCs being an
adjuvant.
One aspect of the invention provides a method of identifying an adjuvant
comprising
contacting CD38 ligand with a candidate substance in the presence of CD38 or
an
analogue and determining whether said substance competes with CD38 or the
analogue for binding to CD38 ligand and whether said substance is capable of
maturing DCs. Adjuvants identified in these methods can be used in the in
vitro and
in vivo methods or vaccine discussed herein in the same manner as CD38 or the
analogue.
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The invention also provides an inhibitor of CD38 ligand which specifically
inhibits activation of CD38 ligand by CD38. Generally such an inhibitor binds
CD38 ligand. It may have any of the structural characteristics discussed above
in
relation to the analogue of CD38. Thus the inhibitor may comprise a peptide
with
homology to CD38.
The inhibitor can be identified, for example, in a process comprising
providing (contacting) a candidate substance to CD38 ligand in conditions in
which
in the absence of the candidate substance CD38 ligand would be activated, and
determining whether the candidate substance causes inhibition of the
activation of
CD381igand.
The invention provides the inhibitor for use in a method of treatment of the
human or animal body by therapy. In particular the inhibitor is provided for
use in a
method of immunotherapy. Such immunotherapy is generally immunosuppression,
such as by inhibition of CD38 mediated activity of FDC or DC. Thus the ligand
ma~~
be used to inhibit the maturation of DC or the activation of T cells by DC in
vivo.
The inhibitor may be used to inhibit the activity of FDC networks in vivo,
such as by
causing a decrease in the number of FDC networks. Thus the inhibitor may be
used
to decrease FDC mediated B cell activity, which would generally lead to a
decreased
amount of antibody being produced by the B cells and/or a decrease in the
length of
time of B cell memory.
Thus the inhibitor is provided for use in a method of treating a disease which
is caused by an immune response. The disease may be an autoimmune disease. In
one embodiment the disease is one in which the immune response is caused by a
foreign agent (such as a pathogen), but the immune response has a deleterious
effect
on the host.
The inhibitor may also be used to treat DCs or FDCs itz vitro in a process in
which the DC or FDC is contacted with the inhibitor, generally in conditions
which
support (i.e. keep alive) the DC or FDC.
The substances and cells of the invention
The discussion below includes substances provided by the invention,
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substances identified in the methods discussed herein, cells provided by the
invention
and cells which have been produced in the methods discussed herein.
The CD38, analogues, CD38 ligands, antibodies, polynucleotides (e.g. that
encode analogues or ligands) or inhibitors (of CD38 ligand) discussed herein
may be
in substantially purified form. They may be in substantially isolated form, in
which
case they will generally comprise at least 70%, e.g. at least 80, 90, 95, 97
or 99% of
the peptide, polynucleotide or dry mass in the preparation. These substances
may be
substantially free of cells or of cellular components (such as cell
membranes).
The DCs of the invention or the T cells produced in the method of stimulating
T cells may be in substantially purified form. They may be in substantially
isolated
fomn, in which case they will generally comprise at least 70%, e.g. at least
80, 90, 95,
97 or 99% of the cells or dry mass in the preparation.
Any of the above substances or cells may be in the form of a pharmaceutical
composition which comprises the substance or cell and a pharmaceutically
acceptable carrier or diluent. Suitable carriers and diluents include isotonic
saline
solutions, for example phosphate-buffered saline.
CD38, the analogue or CD38 ligand may be in a soluble form or be
associated with lipid, for example a lipid membrane, such as a vesicle or cell
membrane. Thus these substances may be present on the surface of a cell, such
as a
cell on which CD38 or CD 38 ligand is or is not naturally expressed. The cell
may
be a T cell, B cell, macrophage or NK cell which may be intact or lysed. The
CD38,
analogue or CD38 ligand may be present in the a preparation made from such a
cell,
such as an extract (e.g. a partially purified extract) from such a cell.
Any of the above substances may be in any of the above forms when present
in the vaccines of the invention or when used in the in vitro or in vivo
methods of the
invention.
Methods of producing antibodies
The antibodies mentioned herein may be produced by raising antibody in a
host animal. Such antibodies will be specific to CD38 or to the products
mentioned
above which bind antibodies. CD38 or the products are referred to as the
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'immunogen' below. Methods of producing monoclonal and polyclonal antibodies
are well-known. A method for producing a polyclonal antibody comprises
immunising a suitable host animal, for example an experimental animal, with
the
immunogen and isolating immunoglobulins from the serum. The animal may
therefore be inoculated with the immunogen, blood subsequently removed from
the
animal and the IgG fraction purified. A method for producing a monoclonal
antibody comprises immortalising cells which produce the desired antibody.
Hybridoma cells may be produced by fusing spleen cells from an inoculated
experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256.
495-
497).
An immortalized cell producing the desired antibody may be selected by a
conventional procedure. The hybridomas may be grown in culture or injected
intraperitoneally for formation of ascites fluid or into the blood stream of
an
allogenic host or immunocompromised host. Human antibody may be prepared by in
vitro immunisation of human lymphocytes, followed by transformation of the
lymphocytes with Epstein-Barr virus.
For the production of both monoclonal and polyclonal antibodies, the
experimental animal is suitably a goat, rabbit, rat or mouse. If desired. the
immunogen may be administered as a conjugate in which the immunogen is
coupled,
for example via a side chain of one of the amino acid residues, to a suitable
earner.
The carrier molecule is typically a physiologically acceptable carrier. The
antibody
obtained may be isolated and. if desired, purified.
Homologous sequences
Peptides which have a homologous sequence to a given (original) peptide are
discussed herein (for example peptide analogues or peptides) present in an
analogue
which are homologous to CD38, or homologues of CD38 ligand). The discussion
below describes how such homologues may be related to the original peptide.
The homologous sequence is typically at least 70% homologous to the
original peptide, preferably at least 80 or 90% and more preferably at least
95%, 97%
or 99% homologous thereto, for example over a region of at least 20,
preferably at
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least 30, for instance at least 40. 60 or 100 or more contiguous amino acids.
Methods
of measuring protein homology are well known in the art and it will be
understood
by those of skill in the art that in the present context, homology is
calculated on the
basis of amino acid identity (sometimes referred to as "hard homology").
Homology can be measured using known methods. For example the
UWGCG Package provides the BESTFIT program which can be used to calculate
homology (for example used on its default settings) (Devereux et al (1984)
Nucleic
Acids Research I2, p387-395). The PILEUP and BLAST algorithms can be used to
calculate homology or line up sequences (typically on their default settings),
for
example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul,
S, F
- et al ( 1990) J Mol Biol 215:403-I 0.
Software for performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (http://www.ncbi.nlm.nih. ov/).
This algorithm involves first identifying high scoring sequence pair (HSPs) by
identifying short words of length W in the query sequence that 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 neighbourhood word score
threshold
(Altschul et al, supra). These initial neighbourhood word hits act as seeds
for
initiating searches to find HSPs containing them. The word hits are extended
in both
directions along each sequence for as far as the cumulative alignment score
can be
increased. Extensions for 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 BLAST program uses as defaults a word length (V~ of 1 l,
the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5,
N=4,
and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity
between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad.
Sci.
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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 sequence is considered similar to another
sequence
if the smallest sum probability in comparison of the first sequence to the
second
sequence is less than about 1, preferably less than about 0.1, more preferably
less
than about 0.01, and most preferably less than about 0.001.
The homologous sequence typically differs from the original sequence by
substitution, insertion or deletion. Generally from l, 2, 3, 4 or more
substitutions.
deletions or insertions, for example over a region of at least 10, preferably
at least 20.
for instance at least 30, 40, 60 or 100 or more contiguous amino acids in the
analogue. Thus the homologous sequence may differ from the original sequence
by
at least 2, 5, 10, 20, 30 or more substitutions, deletions or insertions.
The substitutions are preferably 'conservative'. These are defined according
to the following Table. Amino acids in the same block in the second column and
preferably in the same line in the third column may be substituted for each
other:
ALIPHATIC Non-polar G A P
ILV
Polar - uncharged C S T M
NQ
Polar- - charged D E
KR
AROMATIC H F W Y
A polynucleotide sequence encoding the homologous peptide typically
hybridises with a polynucleotide encoding the original peptide. It typically
hybridises at a level significantly above background. The signal level
generated by
the interaction is typically at least 10 fold, preferably at least 100 fold,
as intense as
'background' hybridisation. The intensity of interaction may be measured, for
example, by radiolabelling the probe, e.g. with ~ZP. Selective hybridisation
is
typically achieved using conditions of medium to high stringency (for example
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0.03M sodium chloride and 0.003M sodium citrate at from about 50°C to
about
60°C).
Bi'ndin~
The binding between any two substances mentioned herein is typically a
specific binding. The binding may be reversible or non-reversible binding.
Determination of binding, for example in the method of identifying an
adjuvant, can be done by using any known binding assay. Typically whether or
not a
first substance binds to a second substance is done by determining whether the
second substance is able to inhibit the binding of a substance known to bind
the first
substance (for example a specific antibody).
Binding may be determined by measuring a characteristic of any of the
substances that changes upon binding, such as spectroscopic changes.
Binding between may be detemined in a 'band shift' system, in which the
retardation of a substance on a gel can be used to detect when ii is bound to
another
substance. The binding may be determined in a competitive binding method.
Administration
The substances and cells of the invention (in pazrticular CD38, analogues,
DCs, vaccines. T cells produced in the method of stimulating T cells,
adjuvants
identified in the method of identifying an adjuvant and inhibitors CD38 ligand
of the
invention) are refewed to as the 'substances' below. The substances may be
administered to a human or animal in need of treatment. The condition of the
human
or animal can thus be improved.
The invention provides the substances for use in a method of treating the
human or animal body by therapy. Thus the substances are provided for use in
immunotherapy. The substances are provided for use in the manufacture of an
adjuvant (or an immunosuppressant in the case of an inhibitor of CD38 ligand)
for
use in immunotherapy. Thus the invention provides a method of treating a
disease
comprising administering a substance of the invention.
The substances may be combined with a pharmaceutically acceptable carrier
or diluent to produce a pharmaceutical composition. Suitable carriers and
diluents
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include isotonic saline solutions, for example phosphate-buffered saline. The
composition may be formulated for parenteral, intramuscular, intravenous,
subcutaneous, or transdermal administration.
The dose at which the substance of the invention is administered to a patient
will depend upon a variety of factors such as the age, weight and general
condition of
the patient, the condition that is being treated and the particular substance
that is
being administered. A suitable dose may however be from 0.1 to 100 mg/kg body
weight such as 1 to 40 mg/kg body weight.
Substances which are polynucleotides can be administered directly as a naked
nucleic acid construct. Uptake of naked nucleic acid constructs by mammalian
cells
is enhanced by several known transfection techniques for example those
including
the use of transfection agents. Example of these agents include cationic
agents (for
example calcium phosphate and DEAE-dextran) and lipofectants (for example
lipofectamT'~~ and transfectamTM ). Typically, nucleic acid constructs are
mixed
with the transfection agent to produce a composition.
When the polynucleotide is delivered to cells by a viral vector, the amount of
virus administered is in the range of from 106 to 101° pfu, preferably
from 10' to 109
pfu, more preferably about lOs pfu for adenoviral vectors. When injected,
typically
1-? ml of virus in a pharmaceutically acceptable suitable carrier or diluent
is
administered. When the polynucleotide of the invention is administered as a
naked
nucleic acid, the amount of nucleic acid administered is typically in the
range of from
1 pg to 10 mg. The polynucleotide may be administered in a cellular vector.
In the case where the substance is a cell (or the substance is a
polynucleotide
in a cellular vector) then typically 103 to 10'2 cells are administered, such
as 106 to
10~ cells.
The following Examples illustrate the invention:
Example 1
Materials and Methods
Construction of soluble mouse CD38 Human IgGl Fc Chimeric protein
The primers used to amplify the extracellular domain of mouse CD38 were 5'
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(AGG CCG CGC TCA CTC CTG GTG GTG GTG TGG (SEQ ID N0:1)) and 3'
(TAC TCA CGT ATT AAG TCT ACA CGA TGG GTG CTC (SEQ ID N0:2)). A
cDNA library of splenic cells was used as template for PCR amplification. Each
primer contained restriction sites that allowed subcloning into a vector that
contained
the sequence encoding the CH2CH3 (Fc) domains of human IgGl . The resulting
plasmid was transfected into J558L cells. Soluble mouse construct (CD38~y1)
was
purified from culture supernants using Protein G columns.
In vivo administration of soluble CD38yl
Mice were immunized with SO~g of soluble DNP-KL,H (Calbiochem-
_ Novobiochem, USA). Groups of 4 mice were injected i.v. with either CD38~y1
or
human IgG (Binding Site, Birmingham, UK) at 100gg/mouse/day for 4 days
starting
at the day of immunization.
Effect on FDC networks using tissue sections
Spleen and lymph nodes were collected from mice after 14 days and frozen
for cryostat sections. Sections were fixed with either 2% paraformaldehyde or
cold
acetone. The sections were then stained with either FDC-Ml (FDC and tingible
body macrophages), PNA (germinal centres), B220, CD3 or Ml 15.4 (Anti-class
II).
And anti-Rat-Ig labelled with peroxidase. The number of FDC networks /x20
field,
stained by FDC-M 1, was counted in spleen sections of 4 treated and 4
untreated
mice. Between 1 l and 15 fields were counted per section/mouse and a t test
used to
determine statistical significance of differences in mean number of networks
per
fteld.
Distribution of the li~and for CD38
Mice were given SRBC and at the time of immunisation and after l, 2, 5. 7
and 9 days the spleen and lymph nodes were collected. Tissue sections of
spleen and
lymph node from naive and mice immunised with CRBC were labelled with the
construct and anti-human IgG-Fc-peroxidase.
Sections were also double labelled with alkaline phosphatase labelled class II
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antibody or F480. Some sections were treated with an rat anti-mouse Fc
antibody
known to block Fc binding and then labelled with the construct. but these
sections
could not be double labelled.
Flow cvtometry
Purified DC (Wykes et al (1998), J.Immunol., 161, 1313-1319) were labelled
with the construct and biotin labelled anti-human Ig and Streptavidin-FITC.
Isolation of immature splenic DC
Spleens from 8-10 week old C57BL/6 mice were digested in collagenase D
and DNAase (Boehringer Mannheim, UK), RBC were lysed and cells incubated with
KT3 (anti-CD3), anti-CD4 (TYS.191.1), anti-~ (B cells), 3D6 (marginal zone
metalophillic macrophages) and biotinylated anti-IgI and anti-IgG2a antibodies
(Binding Site Ltd., UK) for lh at 4°C. Labelled cells were depleted by
rosetting with
anti-rat and anti-mouse Ig-coated SRBCs and layering over histopaque (Sigma,
USA). Rosetting has the added advantage of depleting macrophages via their Fc
receptors. Contaminating cells were depleted using MACS system (Miltenyi
Biotec.
Germany). The final DC-enriched population was examined by flow cytometry and
immunocytochemistry and contained >95% DC based on morphology and expression
of MHC Class II, CDl lc (very weak expression) and CD 40 (very weak
expression.
with less than 1 % T cells, B cells or macrophages. Dendritic cells were also
produced using the adherence method described by Steinman and Cohn (1974) J.
Exp. Med., 139, p380, which was known to produce mature DC. These adherent
splenic DC were further treated to remove contaminating T, B cells and
macrophages.
Immunoprecipitaion and western Blotting
The ligand for CD38 was immunoprecipitated from DC pulsed with 35S-
methionine radiolabel for 4 hours. The ligand was then immunoprecipitated and
run
on SDS-PAGE gels as described in Wykes et al (1998) Euro. J. Immunol. 28,
548).
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Maturation of DC
Immature DC were treated with either 30~giml Human IgGI (Binding Site
Ltd., UK) or CD38~y1. Other treatments included anti-CD40 (FGK-45) and anti-
Class
II (M115.4), or CD38~r1, anti-CD40 and anti-Class II, CD38~y1 and anti-CD40 or
LPS.
All antibodies were used at 10~g/ml and the LPS at SOg/ml. The cells were
cultured
for 18 hours and labelled with antibodies to detect Class I (28.8.65), class
II
(Cadaxlane, Canada) B7.1 and B7.2.
Example 2
Results
Characterisation of the construct
As indicated above, a chimeric protein of murine CD38 and the Fc portion of
human IgGl (CD38~~1) was created and purified on protein G columns. The
construct
occurs predominantly as a dimer but some monomeric protein is seen under non-
reducing conditions (Figure 1 a). Under reducing conditions, a single band
with an
apparent molecular weight of S~kD is observed. The protein was shown to be
glycosylated using N-endoglucosidase, with a reduction of approximately SkD
(Figure 1 b).
Localization of ligand for CD38 in mice using Immunocvtochemistry
CD38~y1 was used to localise the ligand for CD38 in the spleen and lymph
node. Human IgGI was used to detect any binding due to the Fc portion of Ig.
Mice
were given SRBC i.v. via the tail vein and the spleen and LN collected at day
0, 2, 5,
7, and 9. The sections were treated with the construct or human IgGI and
immuncytochemistry carried out.
While the human IgG bound occasional cells in the red pulp, the CD38~y1
bound to different cell types. Five to ten percent of single cells in the red
pulp of the
spleen expressed the ligand and MHC class II. The ligand was also weakly
expressed by dendritic cells in the T cell area of the spleen. Due to weak
detection of
the ligand in tissue sections, flowcytometry of purified DC (Figure 2) was
used to
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demonstrate the ligand (Figure 3 and 4). The ligand was also found on networks
of
cells in the secondary follicles by days 3-5 following immunisation. The
networks
were FDC that co-expressed FDC-M l , a FDC specific marker. Antibody to Fc
receptor was used to treat tissue sections prior to binding of the construct
to rule out
Fc mediated binding. We did not detect any ligand on T cells or B cells in the
appropriate areas (flowcytometry did not show any evidence of binding to T
cells and
B cells either). There was some very weak staining of the vascular
endothelium.
The absence of any binding of the construct to cells expressing CD38 rules out
homotypic adhesion.
There have been studies that suggested that human CD38 mediates adhesion
of lymphoid cells to the endothelium via homotypic adhesion and that CD31 was
possibly the receptor for CD38. However, in those studies western blotting was
used
and no direct binding between CD38 and the CD31 was shown.
Characterisation of the lisand for CD38
Fresh DC were lysed and run on a 3-15% gradient gel under non-reducing
conditions. A western blot of the gel was then stained with the construct and
anti-
human Fc-HRP antibody (Figure 4). Bands of about 130kD, 65 and a doublet at
33kD were seen. A weak band of about 50 kD was also seen, especially in fresh
immature DC. An in-elevant cell type or control antibody were not included in
these
experiments. 35S-methionine,~cystine labelling also detected these bands.
CD38 has a role in Germinal centre development in vivo
To identify a function for CD38, groups of mice were given soluble DNP-
KLH either with human IgGl or the CD38~y1 every day for 4 days. The spleens
were
collected after 14 days and the sections stained with various markers.
Staining with
antibodies to class II, B220 or CD3 did not show any change to the
architecture or
distribution of cells in lymphoid tissue. However, treatment of mice with
CD38~1
had markedly increased the numbers of FDC networks in GC, stained by the
antibody FDC-M1 by at least 2.8 fold (t test, p=<0.001). The apparent size of
the
germinal centres were also several fold larger as seen by PNA and FDC-M 1
staining
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and could account for the apparent increase in number of GC.
As B cells express CD38, we suggest that when B cells interact with CD38L
on FDC in follicles, this interaction stimulates an expansion of the FDC
network.
CD38L is the first molecule known to modulate FDC function which has
implications for germinal centre and antibody memory development. Germinal
centres are essential for the development of antibody memory and expansion and
enhancement of this environment should in theory increase the numbers of
memory
B cells that develop. Furthermore, it has also been shown that antibody memory
is
dependent on FDC retaining unprocessed antigen on their surface via
antibodies.
Thus a larger FDC network would also improve recall (memory) responses.
CD38~~ 1 improves antibody memory
As a follow on from the previous observation that CD38gl improved the size
and numbers of germinal centres, we immunised 2 groups of Balb/c mice with
75~.g
DNP-KLH and 5d later gave one group 75~g CD38~~1 and the remaining mice 75~g
human IgGl as a negative control. After 10 weeks, lymphois cells were purified
from the spleen of these mice and transferred to SCID mice of a B6 background,
i.v.
along with 10~.g DNP-KLH. These SCID mice were also given spleen cells from
Balb/c KLH primed mice to provide optimal T cell help. The mice were bled
after 8
and 14d (data not shown) and tested for IgH~ haplotype anti-DNP responses.
Balb/c
immunoglobulin is of IgH' haplotype and the B6 background is IgH~. Although
SCID mice produce very little or no immunoglobulin, detection reagents
specific for
the IgH~ haplotype confirmed that the immunoglobulin detected was of donor
origin.
The response after 8 days indicated memory responses since primary IgG
responses
take longer to develop.
The titres of IgM anti-DNP were low 8d after the transfer of memory cells in
all mice. However, 5/5 mice given control antibody produced IgGI anti-DNP
antibody (Figure 5a) but only 1/5 mice produced a low titre IgG2a anti-DNP
antibody (Figure 5b). In contrast, following treatment with CD38~y1 under
identical
conditions, 5/5 mice produced IgG2a and IgGl anti-DNP antibody (Figure 5a and
b).
The titres of IgG2a anti-DNP were 3-fold lower than IgGl anti-DNP but 10-fold
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higher than the single mouse that produced IgG2a anti-DNP.
CD38 has a role in DC maturation
To identify a function for CD38 ligand on DCs, fresh ex vivo immature DCs
were purified (without adherence) and treated with CD38~y1 alone or in
combination
with other antibodies. Human IgGl was used to control for non-specific effects
of Fc
binding, although this was unlikely since cells expressing Fc receptors were
remo~~ed
using rosetting. LPS was used to show maximal maturation of DCs. Mature
splenic
DCs prepared by the adherence technique were used to compare maturity of
treated
cells. LPS treatment of fresh DCs increased expression of class I, class II
and B7.2
to levels comparable with mature DCs. However, B7.1 expression was not
significantly affected.
Treatment of the DC with human ~~l showed minimal changes in expression or
all markers whereas treatment with anti-Class II and anti-CD40 (to mimic T
cell
signalling) induced a 28% increase in the numbers of cells expressing higher
levels
of class I. This treatment did not increase expression of any other marker.
However.
treatment with CD38yl increased Class I expression modestly and in combination
with anti-CD40, or anti-CD40 and Class II, increased class I expression to
levels
comparable to LPS treatment (Figure 4a). While the percentage of cells
expressing
"higher" class I levels was lower than mature splenic DC, the mean
fluorescence
intensity was higher. MHC class II levels on all treated cells were not
markedly
different from the mature splenic DC (Figure 4b). Moreover, B7.1 levels were
reduced by all treatments especially CD38~y1 in combination with anti-CD40 and
class II when compared to human ~yl and mature DC (Figure 4c). In contrast,
the
construct combination improved B7.2 expression to levels equal to LPS
treatment
and better than mature splenic DC (Figure 4d).
The implications for these observations are that these treated DC may have a
role in CTL development. Firstly, the phenotype of LPS treated DC was
comparable
to mature splenic DC. Furthermore, mature splenic DC have been shown to prime
CTL in vivo, suggesting that LPS- treated DC may also be capable of priming
CTL.
Since LPS induces IL,-12 production by DC driving a TH1 cytokine profile
essential
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for CTL responses this may indeed be the case. Secondly, class I and B7.2
expression are essential to prime CTL and these were found on LPS and mature
DC
but not on immature DC which do not prime CTL. Thus, signalling of class II,
CD40
and CD38L will mature DC, as determined by their in change in phenotypic
expression, to a form known to prime CTL. In fact the higher B7.2 levels on
these
cells may suggest a pre-disposition to priming CTL.
The clinical implications are two-fold. Firstly, DC are required to prime CTL
but a problem exists in that only immature DC will endocytose antigen.
However,
these DC will not prime CTL. Conversely, mature DC cannot take up antigen but
can prime CTL. Therefore, in a clinical application immature DC, which are
easier
to obtain, could be pulsed with antigen ex vivo and then treated to mature
them to a
fotzn which would prime CTL.
CD38vl treated DC improve cell-~ecific cytotoxicity
Since studies have shown that the expression of class I and B7.2 were
essential for the development of cytotoxic T cells, and our CD38~y1 based
treatment
improved expression of these molecules, we tested these cells in vivo. Fresh
DC
were pulsed with P815 tumour lysate and treated with either human IgGl control
antibody or CD38~1/anti-Class II/anti-CD40 antibodies for 20 hours, washed and
given to groups of 3 naive DBA/2 mice. After 1 week, this process was
repeated.
After another week, the spleens were collected and cultured with irradiated
P815
cells. After the third week, cell counts of spleen cells from CD38~y1 treated
DC were
3-fold higher than control treated mice. The absolute numbers of CD4, CD8 and
B
cells were increased (data not shown). The numbers of NK cells were also
apparently increased. CTL assays showed that CTL and non-CTL mediated killing
was also improved compared to untreated DC (Figure 6).
