Title of Invention

MULTIMERIC COMPLEXES OF ANTIGENS AND ADJUVANTS

Abstract The present invention provides a product comprising: a first component which is a scaffold; a second component which is an adjuvant, preferably a polypeptide which is a ligand for CD21 or a cell surface molecule on B cells or T cells or follicular dendritic or other antigen presenting cells; and a third component which is an antigen.
Full Text MULTIMERIC COMPLEXES OF ANTIGENS AND ADJUVANTS
Introduction.
This invention relates to macromolecular assemblies, such as
fusion proteins, comprising an adjuvant and an antigen, which
assemblies provoke an enhanced immune response to the antigen
in comparison to the antigen alone.
Background of the Invention.
Adjuvants enhance the immune response to antigens and are
therefore useful in vaccines. However, there are only a
limited number of adjuvants approved for use in humans, and as
stronger adjuvants are known from research on animals, a clear
need exists for stronger immunological adjuvants which are
safe to use in man. For a recent review, see "Advances in
vaccine adjuvants" (Nature Biotechnology, 1999, Volume 17,
pages 1075-1081). A critical feature of any adjuvant for
widespread use in man is that it should be very safe,
particularly if it is to be used in routine prophylaxis in
very large numbers of healthy people.
The complement system consists of a set of serum proteins that
are important in the response of the immune system to foreign
antigens. The complement system becomes activated when its
primary components are cleaved and the products, alone or with
other proteins, activate additional complement proteins
resulting in a proteolytic cascade. Activation of the
complement system leads to a variety of responses including
increased vascular permeability, chemotaxis of phagocytic
cells, activation of inflammatory cells, opsonisation of
foreign particles, direct killing of cells and tissue damage.

Activation of the complement system may be triggered by
antigen-antibody complexes (the classical pathway) or a normal
slow activation may be amplified in the presence of cell walls
of invading organisms such as bacteria and viruses (the
alternative pathway). The complement system interacts with
the cellular immune system through a specific pathway
involving C3, a protein central to both classical and
alternative pathways. The proteolytic activation of C3 gives
rise to a large fragment (C3b) and exposes a chemically
reactive internal thiolester linkage which can react
covalently with external nucleophiles such as the cell surface
proteins of invading organisms or foreign cells. As a result,
the potential antigen is "tagged" with C3b and remains
attached to that protein as it undergoes further proteolysis
to iC3b and C3d,g. The latter fragments are, respectively,
ligands for the complement receptors CR3 and CR2; (CR2 is also
referred to as CD21). Thus the labelling of antigen by C3b
can result in a targeting mechanism for cells of the immune
system bearing these receptors.
That such targeting is important for augmentation of the
immune response is first shown by experiments in which mice
were depleted of circulating C3 and then challenged with an
antigen (sheep erythrocytes). Removal of C3 reduced the
antibody response to this antigen (M.B. Pepys, J. Exp. Med.,
140, 126-145, 1974). The role of C3 was confirmed by studies
in animals genetically deficient in either C3 or the upstream
components of the complement cascade which generate C3b, i.e.
C2 and C4 (J.M. Ahearn and D.T. Fearon, Adv. Immunol., 46,
183-219, 1989). More recently, it has been shown that linear
conjugation of a model antigen with more than two copies of
the murine C3d fragment sequence resulted in a very large
(1000-10000-fold) increase in antibody response in mice

compared with unmodified antigen controls (P.W. Dempsey et al,
Science, 271, 348-350, 1996; W096/17625, PCT/GB95/02851). The
increase could be produced without the use of conventional
adjuvants such as Freund's complete adjuvant, which is too
toxic to be used in humans. The mechanism of this remarkable
effect was demonstrated to be high-affinity binding of the
multivalent C3d construct to CR2 on B-cells, followed by co-
ligation of CR2 with another B-cell membrane protein, CD19 and
with membrane-bound immunoglobulin to generate a signal to the
B-cell nucleus.
However, it has proved difficult to produce large amounts of
homogenous recombinant proteins containing three copies of
C3d. The principal problems have been :
i) the genetic instability of the constructs containing
(three) repeated sequences and
ii) the folding (or solubilisation and refolding) of the
recombinant protein from inclusion bodies formed in
Escherichia coli.
One approach taken to minimise the genetic instability of
constructs containing repeated copies of the C3d gene is
described in WO99/35260 and WO01/77324. The technology
described in these applications is to use non-identical
sequences of DNA encoding repeats of C3d.
WO00/69907 and WO00/69886, the contents of which are
incorporated herein by reference, describe polypeptide
monomers capable of assembling into a multimeric form. The
monomers are derived from chaperone proteins, particularly
GroES or Cpn10 family members.
A multimerisation system using the complement 4 binding

protein (C4bp) is described in WO 91/114 61. Human C4b-binding
protein (C4BP) is a plasma glycoprotein of high molecular mass
(570 kDa) which has a spider like structure made of seven
identical alpha-chains and a single beta-chain. The C4bp alpha
chain has a C-terminal core region responsible for assembly of
the molecule into a multimer. According to the standard
model, the cysteine at position +4 98 of one C4bp monomer forms
a disulphide bond with the cysteine at position +510 of
another monomer. A minor form comprising only seven alpha-
chains has also been found in human plasma. The natural
function of this plasma glycoprotein is to inhibit the
classical pathway of complement activation.
WO 91/11461 proposes that the ability of the C4bp protein to
multimerise can be used to make fusion proteins comprising all
or part of C4bp and a biological protein of interest. The
fusion protein will form multimers which provides a platform
for the protein of interest, in which said protein has an
enhanced serum half-life and increased affinity or avidity for
its targets'. Fusion proteins of C4bp were targeted as the
focus of novel delivery and carrier systems for therapeutic
products in WO 91/11461.
Most of the alpha-chain of C4bp is composed of eight tandemly
arranged domains of approximately 60 amino acids in length
known as complement control protein (CCP) repeats. Inclusion
of one or more of these domains was preferred in the fusion
proteins described in WO 91/114 61, but it has since been
demonstrated that all CCPs can be deleted (leaving only the C-
terminal 57 amino acids) without preventing multimerisation
(Libyh M. T. et al., (1997) Blood, 90, 3978-3983). This C-
terminal region of C4bp is referred to as the C4bp core.

Libyh et al., (1997), describe a protein multimerisation
system which is based on the C-terminal part of the alpha
chain of C4bp. The C-terminal part of the C4bp lacks lacks the
ability to inhibit the classical pathway of complement
activation, but is responsible for polymerisation of C4bp in
the cytoplasm of CHO cells producing C4bp. Libyh et al. were
able to induce spontaneous multimerisation of associated
antibody fragments to create homomultimers of scFv fragments
using the C4bp fragment. The C-terminal portion of C4bp used
was placed C-terminal to the scFv sequence, optionally spaced
by a MYC tag.
The use of C4bp is also described in Oudin et al. (2000,
Journal of Immunology, Vol. 164:1505) and Christiansen et al.
(2000, Journal of Virology, Vol. 74:4672). Self-assembling
multimeric soluble CD4-C4bp fusion protein have also been
demonstrated in Shinya et al (1999, Biomed & Pharmacother,
Vol. 53: 471) where the fusion proteins were expressed in the
human 2 93 cell line.
Summary of the Invention.
The present invention provides a product comprising:
a first component which is a scaffold;
a second component which is an adjuvant, preferably a
polypeptide which is a ligand for CD21 or a cell surface
molecule on B cells or T cells or follicular dendritic or
other antigen presenting cells; and
a third component which is an antigen.
The first component provides for assembly of multiple copies
of the second component in a multi-component product such that
the multiple copies of the second component are associated

with one or more copies of the antigen.
In a preferred aspect, the invention provides:
a first component which is a polypeptide scaffold;
a second component which is a polypeptide which is a
ligand for CD21 or a cell surface molecule on B cells or T
cells or on follicular dendritic or other antigen presenting
cells; and
a third component which is an antigen.
The first and second components may be in the form of a fusion
protein. When the third component is also a polypeptide, the
three components are present as a fusion protein.
Alternatively the third component is covalently linked to a
fusion of the first two components.
In some cases, where the first component is itself an antigen,
the first and third components may be the same molecule.
For the avoidance of doubt, the designation of "first",
"second" and "third" components does not imply or indicate a
specific linear order in the product of the three components.
The three components may be joined in any order.
Thus where all three components are polypeptides and the
product is made as a fusion protein, the N- to C- terminal
order of the three components may be in any permutation.
Further, as indicated below, in some cases the first component
may include loop regions which can be replaced by one or other
of the second and third components.
The product of the present invention provides for the
immunostimulatory second component to be formed into a multi-

component product, and to be expressed using recombinant DNA
technology without the need to use DNA sequences having tandem
repeat sequences.
The invention further provides nucleic acid encoding a fusion
protein of said first and second components and, where said
third component is a polypeptide, nucleic acid encoding all
three components. The invention also provides vectors
comprising said nucleic acids and host cells carrying said
vectors.
In another embodiment, the invention provides a method of
making a product comprising:
a first component which is a polypeptide scaffold;
a second component which is a polypeptide which is a
ligand for CD21 or a cell surface molecule on B cells or T
cells or follicular dendritic or other antigen presenting
cells; and
a third component which is a polypeptide antigen,
the method comprising expressing nucleic acid encoding the
three components in the form of a fusion protein, and
recovering the product.
In another embodiment, the invention provides a method of
making a product comprising:
a first component which is a polypeptide scaffold;
a second component which is a polypeptide which is a
ligand for CD21 or a cell surface molecule on B cells or T
cells or follicular dendritic or other antigen presenting
cells; and
a third component which is a non-polypeptide antigen,
the method comprising expressing nucleic acid encoding the
first and second components in the form of a fusion protein,

joining said fusion protein to the third component, and
recovering the product.
The methods of making the product may be performed in
eukaryotic or prokaryotic cells.
The invention also provides a method of inducing an immune
response to an antigen which method comprises administering to
a subject an effective amount of a product according to the
invention.
The invention also provides the use of a product of the
invention for a method of treatment of the human or animal
body, particularly a method of inducing an immune response.
The invention further provides a pharmaceutical composition
comprising a product of the invention in association with a
pharmaceutically acceptable carrier or diluent.
Description of the Drawings.
Figure 1 shows an alignment of C4bp core proteins.
Figure 2 shows the binding of the epitope-C3d-C4bp fusion
protein and of C3d7(l)to CR2 (also known as CD21) in
comparison to monomeric C3d and a linear trimeric version of
C3d, called C3d3.
Figure 3 is a cartoon representing the format of the CR2
binding assay.
Figure 4 shows the binding of C3d7(l) to CR2 CD21) in
comparison to monomeric C3d and a linear trimeric version of

C3d, called C3d3.
Figure 5 shows the binding of C3d7(l), (2) and (3) to CR2
(CD21) in comparison to monomeric C3d and a linear trimeric
version of C3d, called C3d3.
Figure 6 shows a flow cytometry analysis of C3d7(l), C3d7(2)
and C3d7(3) binding to Raji and Jurkat cells.
Detailed Description of the Invention.
Scaffold
This refers to any macromolecular assembly which is capable of
being a scaffold to which the second and third components may
be attached. It may be a protein or other polymeric molecule
(composed for example of sugars) or a prokaryotic or
eukaryotic cell wall or a virus. The cell walls, or viruses
or proteins may be incomplete, that is lacking components
normally present in the organism in which it is found; the
important feature for this invention is that the scaffold is
capable of uniting into a single assembly more than one
adjuvant molecule and more that one antigen molecule.
As is described in more detail herein, there are two main
classes of scaffold contemplated. The first is a complex
macromolecular product, including a virus or cell, onto which
multiple copies of the second and, where applicable, third
component are attached, either separately or as a fusion of
the second and third components. Alternatively, the scaffold
is present in a 1:1 ratio with the second component. When the
product is in the form of a fusion protein then the third
component is also present in a 1:1:1 ratio with the second and
first components.

Cell wall or viral scaffolds are known in the art for other
purposes. Surface display of proteins, whether on prokaryotic
(Samuelson et al., 2002, J. Biotechnol 96,129-154; Lang H.,
2001, Nat. Biotechnol.,19, 75-78) or eukaryotic cell walls
(Shusta E.V. et al., 1999, J. Mol. Biol. 292, 949-956) or
viruses, such as bacteriophages (Sidhu S.S., 2001, Biomol.
Eng.,18, 57-63) have been described. The distinctive feature
of this aspect of the invention is that the objects displayed
on the cell wall include more than one copy of an adjuvant
molecule simultaneously present with an antigen. The antigen
may be fused directly to the adjuvant (such as C3d), but need
not be.
Thus in one embodiment, the surface of a cell, such as a
bacterium, can serve as the scaffold. When the adjuvant is
fused genetically to a second component normally expressed on.
the surface of the bacterium, multiple copies of the adjuvant
are displayed on the surface of the bacterium. This has the
effect of eliciting an improved immune response against the
bacterium, when the bacterium infects a host. The antigen may
be the cell wall of the bacterium, or an antigen separately
but simultaneously expressed on the surface of the bacterium.
The infection may be deliberate, by the administration of the
modified bacterium to the host. The bacterium may be
administered either after being killed, or in a live but
attenuated form.
Similarly, eukaryotic cells may be used as the scaffold. In
such a case, the cell surface displaying more than one copy of
the adjuvant can be used to elicit an immune response to other
(normal or abnormal) cell surface components.
In contrast to the fusion proteins described in W096/17625,

(PCT/GB95/02851), there need not be a covalent linkage either
at all between the antigen and adjuvant, or the covalent
linkage may only be indirect, being mediated by the scaffold.
Furthermore, W096/17625 teaches that the fusion of a single
copy of the C3d protein to an antigen decreases the immune
response to that antigen. In this invention, in direct
contrast, either the display of multiple single (monomeric)
copies of the adjuvant, or the fusion of a single copy of the
antigen to a single copy of the adjuvant, which are then fused
to a scaffold, results in an increased immune response to the
antigen.
The antigen may be the cell wall itself or a second protein or
glycoprotein. In the case of organisms where the protective
antigen is the capsule, as in the case of pneumococci, the
display of more than one copy of the adjuvant will improve the
immune response to the capsular antigens.
In the case of viruses, the antigen may be the virus itself,
which thus acts simultaneously as antigen and scaffold. An
example is provided of the hepatitis B virus surface antigen.
Methods for preparing a recombinant HBsAg vaccine are
described in United States Patent 4,769,238. Although this
recombinant HBsAg is a very successful vaccine, there remain a
substantial number of vaccine recipients who are "poor
responders". The addition of a new adjuvant to this existing
vaccine will enable the vaccination of such poor responders,
and the post-infection vaccination of chronic carriers of this
virus. One method envisaged of adding the adjuvant to this
vaccine is the genetic fusion of the coding sequence of the an
adjuvant protein, such as and preferably the human C3d
protein, to the C-terminus of the gene encoding the 226 amino
acid residue protein that is the S protein of the hepatitis B

virus. The coding sequence for the adjuvant can be added,
optimally with codons preferred for high-level expression in
yeast, in-frame to the S protein coding sequence present in
the plasmids described in the United States Patent 4,769,238
referred to above. The sequence of the S protein may be
modified to include variant sequences, known as "escape
mutants" (Cooreman M.P. et al., 2001, J. Biomed. Sci. 8, 237r-
24 7) or antigens not normally found in the hepatitis B vaccine
(Fomsgaard A. et al. 1998, Scand. J. Immunol., 47, 289-295).
As described in that article, the modified vaccine containing
the C3d adjuvant can be administered as DNA in order to obtain
an immune response.
Thus in another embodiment, the polypeptide scaffold may be
itself an antigen. Thus the surface antigen of hepatitis B
virus, which assembles into oligomeric structures, can
simultaneously be the first and third component of the
invention. As first remarked on in 1956 (FHC Crick, JD
Watson, Nature, 177, 473) the finite nucleic acid content of
viruses severely restricts the number of amino acids that
viruses can encode. As a consequence, the protein coat can
not be constructed from a very large number of different
protein molecules. Instead it must be constructed from a
number of identical small sub-units arranged in a regular
manner. Thus most viruses will be capable of simultaneously
being both the first and third component of the invention.
A polypeptide scaffold is a protein, or part thereof, whose
function is to determine the structure of the protein itself,
or of a group of associated proteins or other molecules.
Polypeptide scaffolds therefore have a defined three-
dimensional structure when assembled, and have the capacity to
support molecules or polypeptides - in or on the said

structure. Advantageously, a scaffold has the ability to
assume a variety of viable geometries, in relation to the
three-dimensional structure of the scaffold and/or the
insertion site of the polypeptides.
In another embodiment, the scaffold may serve as the adjuvant,
i.e. the first and second components will be the same. The
scaffold which is an adjuvant may be a C4bp core protein or a
fragment of the C4bp alpha chain, described in further detail
herein.
In one embodiment, the scaffold is a cochaperonin Cpn10/Hsp10
scaffold. Cpn10 is a widespread component of the Cpn60/Cpnl0
chaperonin system. Examples of Cpn10 include human
mitochondrial Cpn10, bacterial GroES and bacteriophage T4
Gp31. Further members of the Cpn10 family will be known to
those skilled in the art.
The invention moreover comprises the use of derivatives of
naturally-occurring scaffolds. Derivatives of scaffolds
(including scaffolds of the Cpn10 and 60 families) comprise
mutants thereof, which may contain amino acid deletions,
additions or substitutions (especially replacement of Cys
residues in Gp31), hybrids formed by fusion of different
members of the Cpn10 or Cpn60 families and/or circular
permutated protein scaffolds, subject to the maintenance of
the "oligomerisation" property described herein.
Polypeptide scaffolds assemble to form a multimeric product.
In the context of the present invention, the multimeric
product may have any shape and may comprise any number of
individual scaffold units.

Preferably, the mutimeric product comprises between 2 and 20
scaffold units, advantageously between 5 and 15 units, and
ideally about 10 units. The scaffold of Cpn10 family members
comprises seven protein units, in the shape of a seven-
membered ring or annulus. Advantageously, therefore, the
multimeric product is a seven-membered ring.
It is known that Cpn10 subunits possess a "mobile loop" within
their structure. The mobile loop is positioned between amino
acids 15 and 34, preferably between amino acids 16 to 33, of
the sequence of Escherichia coli GroES, and equivalent
positions on other members of the Cpn10 family. The mobile
loop of T4 Gp31 is located between residues 22 to 45,
advantageously 23 to 44. The polypeptide sequence of the
second or third component may be inserted by replacing all or
part of the mobile loop of a Cpn10 family polypeptide.
Where the polypeptide scaffold is a Cpn10 family polypeptide,
the second or third component polypeptide may moreover be
incorporated at the N or C terminus thereof, (which terminus
may be the natural or a modified N or C terminus) or in
positions which are equivalent to the roof beta hairpin of
Cpn10 family peptides. This position is located between
positions 54 and 67, advantageously 55 to 66, and preferably
59 to 61 of bacteriophage T4 Gp31, or between positions 43 to
63, preferably 44 to 62, advantageously 56 to 57 of E. coli
GroES.
In another embodiment, the polypeptide scaffold may be a C4bp
protein or part thereof retaining the C4bp core protein
region.
Human C4 binding protein (hC4bp) is a molecule possessing many

attractive characteristics as a delivery vehicle for bioactive
molecules. Human C4bp is involved in the human complement
system - a group of immune system proteins whose functions
include lysing invading cells, activating phagocytic cells and
facilitating the clearance of foreign substances from the
system. It regulates the activity of proteins in this system,
particularly C4 protein. Structurally, hC4bp is a flexible,
disulfide-bonded molecule expected to have long serum half-
life and the ability to target bioactive molecules to the
lymph nodes. The serum form of hC4bp has a molecular weight of
about 590 kD. On reducing SDS gels, hC4bp produces a strong
band at about 70 kD, indicating a disulfide-bonded multimeric
protein.
A cDNA encoding the C4bp monomer has been cloned and
characterized [L.P. Chung et al., (1985) "Molecular Cloning
and Characterization of the cDNA Coding for C4b-Binding
Protein of the Classical Pathway of the Human Complement
System", Biochem. J., 230, 133-141 ]. Chung et al. refers to
hC4bp as a polypeptide of 54 9 amino acids. The polypeptide
predicted from the DNA sequence has a molecular weight of
about 61.5 kD, rather than 70 kD as actually measured on
reducing SDS gels. The difference in molecular weight
apparently is due to glycosylation of the serum form of the
polypeptide. The first 4 91 amino acids from the N-terminus of
the Chung et al. sequence are divisible into eight domains
called short consensus repeat regions (SCRs) of about sixty
amino acids each. These regions are designated, from N-
terminus to C-terminus, SCR8 to SCR1. The SCR domains are
defined as follows: SCR8 - +1 to +61; SCR7 - +62 to +123; SCR6
- +124 to +187; SCR5 - +188 to +247; SCR4 - +248 to +313; SCR3
- +314 to +374; SCR2 - +375 to +432; SCRl - +433 to +491.
These domains, which share significant sequence homology, each

contain four similarly situated cysteine residues. These
cysteine residues form intra-domain disulfide bonds in a
regular pattern [J. Janatova et al.r (1989) "Disulfide Bonds
Are Localized Within the Short Consensus Repeat Units of
Complement Regulatory Proteins: C4b-Binding Protein",
Biochemistry, 28, 4754-47 61 ]. Within each SCR domain, the
first cysteine residue bonds with the third and the second
cysteine residue bonds with the fourth, forming a double-loop
amino acid sequence. Thus, the SCRs are connected like beads
on a string. This pattern of intra-domain disulfide bonding is
responsible for the conformational flexibility of the C4bp
monomer. In addition to the eight SCR domains, hC4bp also has
a 57 amino acid sequence at the C-terminus, the C4bp core,
which bears no homology to the other regions of the protein.
This region is responsible for assembly of the molecule into a
multimer.
Thus the polypeptide scaffold may be a C4bp core and
optionally one or more SCRs fused to the core.
In a particularly preferred embodiment, the polypeptide
scaffold is the core protein of C4bp alpha chain.
A polypeptide scaffold may additionally comprise N- or C-
terminal extensions such as flexible linkers such as (Glym-
Ser)n (where m and n -are from 1 to 4) . These are used in the
art to attach protein domains (particularly antibody V
domains) to each other. Thus the first component may be
linked to the second and/or third component by such a linker.
It is preferred that the first component is at the C-terminal
of the product, when the core protein of C4bp alpha chain is
the scaffold.

Core protein of C4bp alpha, chain.
This is referred to herein as the "C4bp core protein" or "core
protein", or "C4bp scaffold". The terms are used
interchangeably. This protein may be a mammalian C4bp core
protein or a fragment thereof capable of forming multimers, or
a synthetic variant thereof capable of forming multimers.
The sequences of a number of mammalian C4bp proteins are
available in the art. These include human C4bp core protein
(SEQ ID N0:1). There are a number of homologues of human C4bp
core protein available in the art. There are two types of
homologue: orthologues and paralogues. Orthologues are defined
as homologous genes in different organisms, i.e. the genes
share a common ancestor coincident with the speciation event
that generated them. Paralogues are defined as homologous
genes in the same organism derived from a gene, chromosome or
genome duplication, i.e. the common ancestor of the genes
occurred since the last speciation event.
For example, a search of GenBank indicates mammalian C4bp core
homologue proteins in species including rabbit, rat, mouse and
bovine origin (SEQ ID NO:2-5 respectively). Paralogues have
been identified in pig (ApoR), guinea pig (AM67) and mouse
(ZP3); shown as SEQ ID NO:6-8 respectively.
An alignment of SEQ ID N0s:l-8 is shown as Figure 1. It can
be seen that all eight sequences have a high degree of
similarity, though with a greater degree of variation at the
C-terminal end. Further C4bp core proteins may be identified
by searching databases of DNA or protein sequences, using
commonly available search programs such as BLAST.
Where a C4bp protein from a desired mammalian source is not

available in a database, it may be obtained using routine
cloning methodology well established in the art. In essence,
such techniques comprise using nucleic acid encoding one of
the available C4bp core proteins as a probe to recover and to
determine the sequence of the C4bp core proteins from other
species of interest. A wide variety of techniques are
available for this, for example PCR amplification and cloning
of the gene using a suitable source of mRNA (e.g. from an
embryo or an actively dividing differentiated or tumour cell),
or by methods comprising obtaining a cDNA library from the
mammal, e.g. a cDNA library from one of the above-mentioned
sources, probing said library with a known C4bp nucleic acid
under conditions of medium to high stringency (for example
0.03M sodium chloride and 0.03M sodium citrate at from about
50°C to about 60°C), and recovering a cDNA encoding all or
part of the C4bp protein of that mammal. Where a partial cDNA
is obtained, the full length coding sequence may be determined
by primer extension techniques.
A fragment of a C4bp core protein capable of forming multimers
may comprise at least 47 amino acids, preferably at least 50
amino acids. The ability of the fragment to form multimers
may be tested by expressing the fragment in a prokaryotic host
cell according to the invention, and recovering the C4bp
fragment under conditions which result in multimerisation of
the full 57 amino acid C4bp core, and determining whether the
fragment also forms multimers. Desirably a fragment of C4bp
core comprises at least residues 6-52 of SEQ ID NO:l or the
corresponding residues of its homologues.
The human C4bp core protein of SEQ ID NO:l corresponds to
amino acids +493 to +549 of full length C4bp protein sequence.
A fragment of this known in the art to form multimers

corresponds to amino acids +498 to +549 of C4bp core protein.
Variants of C4bp core and fragments capable of forming
multimers, which variants likewise retain the ability to form
multimers (which may be determined as described above for
fragments) may also be used. The variant will preferably have
at least 70%, more preferably at least 80%, even more
preferably at least 90%, for example at least 95% or most
preferably at least 98% sequence identity to a wild type
mammalian C4bp core or a multimer-forming fragment thereof.
In one aspect, the C4bp core will be a core which includes the
two cysteine residues which appear at positions 6 and 18 of
SEQ ID Nos:l-3 and 5-8. Desirably, the variant will retain
the relative spacing between these two residues.
The above-specified degree of identity will be to any one of
SEQ ID NOs::l-8 or a multimer-forming fragment thereof.
Most preferably the specified degree of identity will be to
SEQ ID N0:1 or a multimer-forming fragment thereof.
The degree of sequence identity may be determined by the
algorithm GAP, part of the "Wisconsin package" of algorithms
widely used in the art and available from Accelrys (formerly
Genetics Computer Group, Madison, WI) . GAP uses the Needleman
and Wunsch algorithm to align two complete sequences in a way
that maximises the number of matches and minimises the number
of gaps. GAP is useful for alignment of short closely related
sequences of similar length, and thus is suitable for
determining if a sequence meets the identity levels mentioned
above. GAP may be used with default parameters.
Synthetic variants of a mammalian C4bp core protein include

those with one or more amino acid substitutions, deletions or
insertions or additions to the C- or N-termini. Substitutions
are particularly envisaged. Substitutions include
conservative substitutions. Examples of conservative
substitutions include those set out in the following table,
where amino acids on the same block in the second column and
preferably in the same line in the third column may be
substituted for each other:

Examples of fragments and variants of the C4bp core protein
which may be made and tested for their ability to form
multimers thus include SEQ ID NOs: 9 to 16, shown in Table 1
below:

A=SEQ ID NO:; B= sequence, C= % identity, calculated by
reference to a fragment of SEQ ID N0:1 of the same length.
where deletions of the sequence are made, apart from N- or C-

terminal truncations, these will preferably be limited to no
more than one, two or three deletions which may be contiguous
or non-contiguous.
Where insertions are made, or N- or C-terminal extensions to
the core protein sequence, these will also be desirably
limited in number so that the size of the core protein does
not exceed the length of the wild type sequence by more than
20, preferably by more than 15, more preferably no more than
10, amino acids. Thus in the case of SEQ ID NO:l, the core
protein, when modified by insertion or elongation, will
desirably be no more than 77 amino acids in length.
Second component.
The product, of the invention will comprise the scaffold as
described above linked to the second component either directly
or indirectly and the third component.
The second component may be any ligand for CD21 or CD19, as
described in US-A-6,238,670, and WO99./35260, the contents of
which are hereby incorporated by reference. The second
component may also be a ligand for a cell surface molecule on
B cells or T cells or follicular dendritic or other antigen
presenting cells.
Preferably, the second component is C3d, particularly human
C3d.
The nucleotide sequence and predicted amino acid sequence of
mouse C3d are disclosed in Domdey et al. (1982) Proc. Natl.
Acad. Sci. USA 79: 7619-7623 and Fey et al. (1983) Ann. N.Y.
Acad. Sci. 421: 307-312). The nucleotide sequence and
predicted amino acid sequence for human C3d are disclosed in

de Bruijn and Fey (1985) Proc. Natl. Acad. Scl. USA 82:708-
712. Nucleic acid encoding C3d from other species may be
isolated using the human or mouse sequence information to
prepare one or more probes for use in standard hybridisation
methods. When C3d is to be employed in the invention and
administered to a subject, the C3d may be matched to the
species to be immunised (e.g. mouse C3d to be used in mouse,
human C3d in human and so on) . Furthermore, the codons chosen
may also be optimised for the species to be immunised, for
example using codons that are efficiently translated in
mammalian hosts.
Where the second component is linked by a peptide linker to
the first and/or third component, the linker may be a flexible
linker as described above.
In a preferred embodiment, the second component is N-terminal
to the first component, and C-terminal to the antigen (where
the antigen is a polypeptide) when the scaffold is the C4bp
core protein. Where the antigen is not a polypeptide, the
antigen may be covalently linked to either of the first or
second components.
Antigen.
Antigens may be any product of prophylactic value; they might
be useful for vaccination. The invention allows rapid
progress from nucleotide sequences to the production of
recombinant antigens attached to an adjuvant in a polyvalent
form.
Bacterial immunogens, parasitic immunogens and viral
immunogens are useful as polypeptide moieties to create
multimeric or hetero-multimeric C4bp fusion proteins useful as

vaccines.
Bacterial sources of these immunogens include those
responsible for bacterial pneumonia, Pneumocystis pneumonia,
meningitis, cholera, tetanus, tuberculosis and leprosy.
Parasitic sources include malarial parasites, such as
Plasmodium.
Viral sources include poxviruses, e.g., cowpox virus and orf
virus; herpes viruses, e.g., herpes simplex virus type 1 and
2, B-virus, varicellazoster virus, cytomegalovirus, and
Epstein-Barr virus; adenoviruses, e.g., mastadenovirus;
papovaviruses, e.g., papillomaviruses such as HPV16, and
polyomaviruses such as BK and JC virus; parvoviruses, e.g.,
adeno-associated virus; reoviruses, e.g., reoviruses 1, 2 and
3; orbiviruses, e.g., Colorado tick fever; rotaviruses, e.g.,
human rotaviruses; alphaviruses, e.g., Eastern encephalitis
virus and. Venezuelan encephalitis virus; rubiviruses, e.g.,
rubella; flaviviruses, e.g., yellow fever virus, Dengue fever
viruses, Japanese encephalitis virus, Tick-borne encephalitis
virus and hepatitis C virus; coronaviruses, e.g., human
coronaviruses; paramyxoviruses, e.g., parainfluenza 1, 2, 3
and 4 and mumps; morbilliviruses, e.g., measles virus;
pneumovirus, e.g., respiratory syncytial virus;
vesiculoviruses, e.g., vesicular stomatitis virus;
lyssaviruses, e.g., rabies virus; orthomyxoviruses, e.g.,
influenza A and B; bunyaviruses e.g., LaCrosse virus;
phleboviruses, e.g., Rift Valley fever virus; nairoviruses,
e.g., Congo hemorrhagic fever virus; hepadnaviridae, e.g.,
hepatitis B; arenaviruses, e.g., 1cm virus, Lasso virus and
Junin virus; retroviruses, e.g., HTLV I, HTLV II, HIV-1 and
HIV-2; enteroviruses, e.g., polio virus 1,- 2 and 3, coxsackie

viruses, echoviruses, human enteroviruses, hepatitis A virus,
hepatitis E virus, and Norwalk-virus; rhinoviruses e.g., human
rhinovirus; and filoviridae, e.g., Marburg (disease) virus and
Ebola virus.
Antigens from these bacterial, viral and parasitic sources may
be used in the production of multimeric proteins useful as
vaccines. The multimers may comprise a mixture of monomers
carrying different antigens.
Immunogens to human proteins for research or therapeutic
purposes may be made. These have many applications not only
in vaccination but also in research. For example, the
generation of human gene sequence data by the human genome
project has made the generation of antisera reactive to new
polypeptides a pressing requirement. The same requirement
applies to prokaryotic, such as bacterial, and other
eukaryotic, including fungal, gene products.
Non-polypeptide immunogens may be, for example, carbohydrates
or nucleic acids. The polysaccharide coats of Neisseria
species or of Streptococcus pneumoniae species are examples of
carbohydrates which may be used for the purposes of the
invention.
The antigen may be any size conventional in the art for
vaccines, ranging from small polypeptides to larger proteins.
Due to the nature of the present invention, antigens of up to
100 kDa, and more preferably up to 50 kDa, such as up to 30
kDa in size are preferred.
Where a non-polypeptide immunogen is part of the product of
the invention, the immunogen may be covalently attached to the

first and second components of the product using routine
synthetic methods. Generally, the immunogen may be attached
to either the N- or C-terminal of a fusion protein comprising
the first and second components, or to an amino acid side
chain group (for example the epsilon-amino group of lysine),
or a combination thereof. More than one immunogen per fusion
protein may be added. To facilitate the coupling, a cysteine
residue may be added to the fusion protein, for example as the
C-terminus.
The present invention has many advantages in the generation of
an immune response. For example, the use of multimers can
permit the presentation of a number of antigens,
simultaneously, to the immune system. This allows the
preparation of polyvalent vaccines, capable of raising an
immune response to more than one epitope, which may be present
on a single organism or a number of different organisms. Thus,
vaccines formed according to the invention may be used for
simultaneous vaccination against more than one disease, or to
target simultaneously a plurality of epitopes on a given
pathogen. The epitopes may be present in a single monomer
units or on different monomer units which are combined to
provide a heteromultimer.
Human C4bp core fusion proteins or human Cpn10 fusion proteins
in particular are useful in the context of immunisations,
because the core protein and human Cpn10 are not only present
normally in the serum or plasma of the recipient of the
immunisation, but also because they do not themselves evoke an
immune response. C4bp proteins are known in a number of
mammalian species, and the appropriate homologues for
mammalian species may be found by those skilled in the art
using standard gene cloning techniques.

Nucleic Acids.
Products of the invention may be produced by expression of a
fusion protein of at least the first and second components in
a prokaryotic or eukaryotic host cell, using a nucleic acid
construct encoding the protein. Where the third component is
a polypeptide, the expression of all three components from a
nucleic acid sequence can be used to produce a product of the
invention.
Thus the invention provides a nucleic acid construct,
generally DNA or RNA, which encodes a product of the
invention.
The construct will generally be in the form of a replicable
vector, in which sequence encoding the protein is operably
linked to a promoter suitable for expression of the protein in
a desired host cell.
The vectors may be provided with an origin of replication and
optionally a regulator of the promoter. The vectors may
contain one; or more selectable marker genes. There are a wide
variety of prokaryotic and eukaryotic expression vectors known
as such in the art, and the present invention may utilise any
vector according to the individual preferences of those of
skill in the art.
A wide variety of prokaryotic host cells can be used in the
method of the present invention. These hosts may include
strains of Escherichia, Pseudomonas, Bacillus, Lactobacillus,
Thermophilus, Salmonella, Enterobacteriacae or Streptomyces.
For example, if E. coli from the genera Escherichia is used in
the method of the invention, preferred strains of this
bacterium to use would include BL21(DE3) and their derivatives

including C41(DE3), C43(DE3) or C0214(DE3), as described and
made available in WO98/02559.
Even more preferably, derivatives of these strains lacking the
prophage DE3 may be used when the promoter is not the T7
promoter.
Prokaryotic vectors includes vectors bacterial plasmids, e.g.,
plasmids derived from E. coli including ColEI, pCRl, pBR322,
pMB9 and their derivatives, wider host range plasmids, e.g.,
RP4; phage DNAs, e.g., the numerous derivatives of phage A,
e.g., NM989, and other DNA phages, e.g., M13 and filamentous
single stranded DNA phages. These and other vectors may be
manipulated using standard recombinant DNA methodology to
introduce a nucleic acid of the invention operably linked to a
promoter.
The promoter may be an inducible promoter. Suitable promoters
include the T7 promoter, the tac promoter, the trp promoter,
the lambda promoters PL or PR and others well known to those
skilled in the art.
A wide variety of eukaryotic host cells may also be used,
including for example yeast, insect and mammalian cells.
Mammalian cells include CHO and mouse cells, African green
monkey cells, such as COS-1, and human cells.
Many eukaryotic vectors suitable for expression of proteins
are known. These vectors may be designed to be chromosomally
incorporated into a eukaryotic cell genome or to be maintained
extrachromosomally, or to be maintained only transiently in
eukaryotic cells. The nucleic acid may be operably linked to
a suitable promoter, such as a strong viral promoter including

a CMV promoter, and SV40 T-antigen promoter or a retroviral
LTR.
To obtain a product of the invention, host cells carrying a
vector of the invention may be cultured under conditions
suitable for expression of the protein, and the protein
recovered from the cells of the culture medium.
Compositions
Products according to the invention may be prepared in the
form of a pharmaceutical composition. The product will be
present with one or more pharmaceutically acceptable carriers
or diluents. The composition will be prepared according to
the intended use and route of administration of the product.
Thus the invention provides a composition comprising a product
of the invention in multimeric form together with one or more
pharmaceutically acceptable carriers or diluents, and the use
of such a composition in methods of immunotherapy for
treatment or prophylaxis of a human or animal subject.
Pharmaceutically acceptable carriers or diluents include those
used in formulations suitable for oral, rectal, nasal, topical
(including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous,
intradermal, intrathecal and epidural) administration. The
formulations may conveniently be presented in unit dosage form
and may be prepared by any of the methods well known in the
art of pharmacy.
Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc, a fusion
protein of the invention optional pharmaceutical adjuvants in
a carrier, such as, for example, water, saline aqueous

dextrose, glycerol, ethanol, and the like, to thereby form a
solution or suspension. If desired, the composition to be
administered may also auxiliary substances such as pH
buffering agents and the like. Actual methods of preparing
such dosage forms are known, or will be apparent, to those
skilled in this art; for example, see Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton,
Pennsylvania, 19th Edition, 1995.
The composition or formulation to be administered will, in any
event, contain a quantity of the active compound(s) in an
amount effective to alleviate the symptoms of the subject
being treated. Dosage forms or compositions containing active
ingredient in the range of 0.25 to 95% with the balance made
up from non-toxic carrier may be prepared.
Parenteral administration is generally characterized by
injection, either subcutaneously, intramuscularly or
intravenously. Injectables can be prepared in conventional
forms, either as liquid solutions or suspensions, solid forms
suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol or the
like. A more recently devised approach for parenteral
administration employs the implantation of a slow-release or
sustained-release system, such that a constant level of dosage
is maintained. See, e.g., US Patent No. 3,710,795.
Doses of the product will be dependent upon the nature of the
antigen and may be determined according to current practice
for administration of that antigen in conventional vaccine
formulations.

DNA vaccines
In another aspect, the invention provides a eukaryotic
expression vector comprising a nucleic acid sequence encoding
a recombinant fusion protein comprising the three component
product of the invention for use in the treatment of the human
or animal body.
Such treatment would achieve its therapeutic effect by
introduction of a nucleic acid sequence encoding an antigen
for the purposes of raising an immune response. Delivery of
nucleic acids can be achieved using a plasmid vector (in
"naked" or formulated form) or a recombinant expression
vector. To illustrate how the invention may be performed with
plasmid vectors, the publication of Green T.D, et al., 2001,
in Vaccine 20, 242-248 serves as an example. These authors
showed that using a DNA vaccine expressing a fusion of the
measles hemagglutinin protein and three copies of C3d,
enhanced titers of neutralizing antibody were obtained. In
the present invention, the second and third copies of C3d
would be replaced with the sequence encoding the C4bp alpha
chain core, resulting in an oligomeric antigen-adjuvant fusion
protein. This plasmid would be smaller in size (because the
core coding sequence is much shorter than that encoding two
copies of C3d) and more stable because of the absence of
repeated sequences.
Various viral vectors which can be utilized for gene delivery
include adenovirus, herpes virus, vaccinia or an RNA virus
such as a retrovirus. The retroviral vector may be a
derivative of a murine or avian retrovirus. Examples of
retroviral vectors in which a single foreign gene can be
inserted include, but are not limited to: Moloney murine
leukaemia virus (MoMuLV), Harvey murine sarcoma virus

(HaMuSV), murine mammary tumour virus (MuMTV), and Rous
Sarcoma Virus (RSV) . When the subject is a human, a vector
such as the gibbon ape leukaemia virus (GaLV) can be utilized.
The vector will include a transcriptional regulatory sequence,
particularly a promoter region sufficient to direct the
initiation of RNA synthesis. Suitable eukaryotic promoters
include the promoter of the mouse metallothionein I gene
(Hamer et al., 1982, J. Molec. Appl. Genet. 1: 273 )/ the TK
promoter of Herpes virus (McKnight, 1982, Cell 31: 355 ); the
SV40 early promoter (Benoist et al., 1981, Nature 290: 304 );
the Rous sarcoma virus promoter (Gorman et al., 1982, Proc.
Natl. Acad. Sci. USA 79: 6777); and the cytomegalovirus
promoter (Foecking et al., 1980, Gene 45: 101 ).
Administration of vectors of this aspect of the invention to a
subject, either as a plasmid vector or as part of a viral
vector can be affected by many different routes. Plasmid DNA
can be "naked" or formulated with cationic and neutral lipids
(liposomes) or microencapsulated for either direct or indirect
delivery. The DNA sequences can also be contained within a
viral (e.g., adenoviral, retroviral, herpesvius, pox virus)
vector, which can be used for either direct or indirect
delivery. Delivery routes include but are not limited to
intramuscular, intradermal (Sato, Y. et al., 1996, Science
273: 352-354), intravenous, intra-arterial, intrathecal,
intrahepatic, inhalation, intravaginal instillation (Bagarazzi
et al., 1997, J Med. Primatol. 26:27), intrarectal,
intratumour or intraperitoneal.
Thus the invention includes a vector as described herein as a
pharmaceutical composition useful for allowing transfection of
some cells with the DNA vector such that a therapeutic

polypeptide will be expressed and have a therapeutic effect,
namely to induce an immune response to an antigen. The
pharmaceutical compositions according to the invention are
prepared by bringing the construct according to the present
invention into a form suitable for administration to a subject
using solvents, carriers, delivery systems, excipients, and
additives or auxiliaries. Frequently used solvents include
sterile water and saline (buffered or not). One carrier
includes gold particles, which are delivered biolistically
(i.e., under gas pressure). Other frequently used carriers or
delivery systems include cationic liposomes, cochleates and
microcapsules, which may be given as a liquid solution,
enclosed within a delivery capsule or incorporated into food.
An alternative formulation for the administration of gene
delivery vectors involves liposomes. Liposome encapsulation
provides an alternative formulation for the administration of
polynucleotides and expression vectors. Liposomes are
microscopic vesicles that consist of one or more lipid
bilayers surrounding aqueous compartments. See, generally,
Bakker-Woudenberg et al, 1993, Eur. J. Clin. Microbiol.
Infect. Dis. 12 (Suppl. 1): S61, and Kim, 1993, Drugs 46: 618.
Liposomes are similar in composition to cellular membranes and
as a result, liposomes can be administered safely and are
biodegradable. Depending on the method of preparation,
liposomes may be unilamellar or multilamellar, and liposomes
can vary in size with diameters ranging from 0.02 uM to
greater than 10 uM. See, for example, Machy et al., 1987,
LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey) , and
Ostro et al., 1989, American J. Hosp. Phann. 46: 1576.
Expression vectors can be encapsulated within liposomes using
standard techniques. A variety of different liposome

compositions and methods for synthesis are known to those of
skill in the art. See, for example, US-A-4,844,904, US-A-
5,000,959, US-A-4,863,740, US-A-5,589,466, US-A-5,580,859, and
US-A-4,975,282, all of which are hereby incorporated by
reference.
In general, the dosage of administered liposome-encapsulated
vectors will vary depending upon such factors as the patient's
age, weight, height, sex, general medical condition and
previous medical history. Dose ranges for particular
formulations can be determined by using a suitable animal
model.
Cell culturing.
Plasmids encoding fusion proteins in accordance with the
invention may be introduced into the host cells using
conventional transformation techniques, and the cells cultured
under conditions to facilitate the production of the fusion
protein. Where an inducible promoter is used, the cells may
initially be cultured in the absence of the inducer, which may
then be added once the cells are growing at a higher density
in order to maximise recovery of protein.
Cell culture conditions are widely known in the art and may be
used in accordance with procedures known as such.
In a particular aspect, when the first component is a C4bp
core protein, the fusion of at least the first two components,
and where applicable, all three components, may be expressed
in a prokaryotic expression system. To date, fusion proteins
based on C4bp core protein have been expressed in eukaryotic
cells. The yields of fusion protein from eukaryotic cells has
rarely reached 2 micrograms per millilitre of culture

supernatant (Oudin et al, ibid) and this could be achieved
only after rounds of gene amplification. This level is too
low for the economic production of large quantities of many
fusion protein for therapeutic use.
Although WO91/00567 suggests that prokaryotic host cells may
be used in the production of C4bp-based proteins, there is no
experimental demonstration of any such production. A number
of considerations however, would suggest that the use of
prokaryotic systems would be disadvantageous. In particular,
many eukaryotic proteins lose some or all of their active
folded structure when expressed in cells such as E. coli.
Other eukaryotic proteins denature or are completely inactive
when expressed in prokaryotic cells.
C4bp is a secreted protein in mammals, and these are known in
the art to be particularly difficult to produce in a correctly
folded form in prokaryotes. Proteins with disulphide bridges
are particularly problematic, as are those that require
oligomerisation. Disulphide bonds are not normally produced in
the reducing environment of the bacterial cytoplasm, and when
they can form, they can stabilise misfolded or aggregated
forms of the protein.
Usually, recombinant proteins expressed in prokaryotes are
aggregated inside inclusion bodies within the host prokaryotic
cell. These are discrete particles or globules separate from
the rest of the cell which contain the expressed proteins
usually in an agglomerated or inactive form. The presence of
the expressed protein in the inclusion bodies makes it
difficult to recover the protein in active soluble form as the
necessary refolding techniques are techniques are inefficient
and costly. Proteins purified from inclusion bodies have to be

laboriously manipulated, denatured and refolded to obtain
active functional proteins at relatively poor yields.
With regard to expressing C4bp core fusion proteins in
prokaryotic cells, other considerations have also to be taken
into account. Firstly, each core monomer retains two cysteine
residues, and according to the model of C4bp multimers
accepted in the art, these cysteines are required to form
inter-molecular disulphide bonds during the assembly of
multimers. The reducing environment of the prokaryotic
cytosol such as the bacterial cytosol would be expected to
prevent the formation of C4bp core multimers by reducing these
disulphide bonds.
Secondly, multimers are assembled during passage through the
eukaryotic secretion apparatus, which is known to assist
protein folding in ways not available in prokaryotes (e.g. the
presence of protein disulphide isomerase and unique
chaperones). Thirdly, even under conditions where relatively
small yields were obtained in eukaryotic cells (micrograms per
millilitre), this secretory pathway is unable to produce
homogenous protein.
Further, the inventors have also found that proteins fused to
the C4bp core produced in the prokaryotic expression systems
retain their functional activity. The present invention
therefore provides a method for obtaining a recombinant fusion
protein comprising a scaffold of a C-terminal core protein of
C4bp alpha chain and a second component, and optionally a
third component, said recombinant fusion protein being capable
of forming multimers in soluble form in the cytosol of a
prokaryotic host cell, the method including the steps of
(i) providing a prokaryotic host cell carrying a nucleic

acid encoding said recombinant protein operably linked to
a promoter functional in said prokaryotic cell;
(ii) culturing the host cell under conditions wherein
said recombinant protein is expressed; and
(iii) recovering the recombinant protein wherein said
protein is recovered in multimeric form.
We have found that the yield of protein in cell cultures of
the invention can be relatively high, for example greater than
2 mg/1 of culture, such as greater than 5 mg/1 of culture,
preferably greater than 10 mg/1 of culture, such as greater
than 20 mg/1 culture, and even more preferably greater than
100 mg/1 culture.
C4bp core fusion proteins of the invention comprise a C4bp
core protein sequence fused, at the N- or C-terminus, to one
of the other components of the invention. In a preferred
arrangement, the order of components from the N- to C-terminal
of a fusion protein is N-third component - second component -
first component-C.
We have found that proteins falling within the above
definition can be expressed in and recovered from bacterial
expression systems in multimeric form without the need for
scaffold refolding. We have expressed proteins which include
C4bp core and which are capable of carrying an antigen and a
second component which have a monomer weight up to about 30
kDa. The invention may thus be used to express proteins in
this size range, and more generally for proteins up to about
100 kDa, more preferably about 50 kDa.
The fact that this system allows production of soluble protein
in E. coli enables using it to produce, as folded soluble

proteins, domains or fragments of proteins that would not fold
when expressed on their own due to a lack of constraint on
their C-terminal and /or N-terminal ends. Engineering a
specific cleavage site enables production of the free domain
of interest. Similarly constraining the N-terminal and/or C-
terminal end of a peptide of interest could be beneficial
during refolding processes. Furthermore, as the oligo-
merisation structure is very resistant to denaturation and to
disassembly, it would be stable during denaturation of the
inserted protein. Therefore, during refolding, for an equal
amount of protein of interest, the actual concentration of
free protein would be diminished by a factor equal to the
oligomerisation number. Oligomerisation may also be
beneficial for purification purposes as many methods in
protein technology are not optimised to work with proteins and
specifically peptides of very low molecular weight.
Recovery of protein from culture.
Once the cells have been grown to allow for production of the
protein, the protein may be recovered from the cells. Because
we have found that surprisingly, the protein remains soluble,
the cells will usually be spun down and lysed by sonication,
for example, which keeps the protein fraction soluble and
allows this fraction to remain in the supernatant following a
further higher speed (e.g. 15,000 rpm for 1 hour)
centrifugation.
The fusion protein in the supernatant protein fraction may be
purified further by any suitable combination of standard
protein chromatography techniques. We have used ion-exchange
chromatography followed by gel filtration chromatography.
Other chromatographic techniques, such as affinity
chromatography, may also be used.

In one embodiment, we have found that heating the supernatant
sample either after centrifugation of the lysate, or after any
of the other purification steps will assist recovery of the
protein. The sample may be heated to about 70 - 80 °C for a
period of about 10 to 30 minutes, though this embodiment is
not preferred when the second component is C3d.
Depending on the intended uses of the protein, the protein may
be subjected to further purification steps, for example
dialysis, or to concentration steps, for example freeze
drying.
The invention is illustrated by the following examples.
Example 1 - Epitope-C3d-C4bp fusion protein.
This example illustrates the fusion of an epitope (comprising
amino acids 8-22 of human Cpn10) to human C3d which is itself
fused to the N-terminus of the human C4bp core protein. The
fusion protein was expressed in, and purified from, the
bacterial strain C41(DE3). The protein behaved as an oligomer
on gel filtration.
The methodology illustrated in this example may be extended to
provide a three component product of the invention, for
example by replacing the Cpn10 epitope with other antigen-
encoding DNA in the construct described below. Alternatively,
the recovered protein may be covalently linked to an antigen
provided by other means.
Cloning.
A XbaI-BamHI fragment of 975bp, (encoding the T7 ribosome
binding site, residues 8-22 of human Cpn10 (the epitope) and

residues 995 to 1287 of human C3d) from pAVD 95 (the
expression construct for C3d7(l)in Example 2 below) was
ligated into pAVD 77 (pRSETa-Db-C4bp) previously digested with
XbaI and BamHI. This fused the human Cpn10 and C3d protein
fragments to the C-terminal 57 residues of the alpha chain of
human C4bp. The construction, called pAVD94, was checked by
PCR and double digestion.
The amino acid sequence of the fusion protein of the construct
is as follows:

The residues 2-16 of SEQ ID NO:17 correspond to residues 8-22
of human Cpn10 (the epitope), residues 19-311 of SEQ ID N0:17
to human C3d residues 995 to 1287, and residues 314-370 of SEQ
ID NO:17 to the 57 residues of the human C4bp core protein. A
GS linker sequence, in bold in the sequence above, appears
between the three components.
The protein has an estimated molecular weight of 41,485
Daltons, a theoretical pi of 5.51 and an estimated extinction
coefficient of 45090 M-1cm-1. On this basis, to calculate the
concentration we use: Abs 0,1%(=lg/l)=1.087.
Expression
The plasmid pAVD94 encoding the epitope-C3d-C4bp core protein

was expressed in the E. coli strain C41(DE3). After overnight
growth at 25°C without induction, the protein was well
expressed. After cell lysis in 20 mM Tris-HCl buffer pH8/100
mM NaCl using a French press, almost half of the protein was
found to be in the supernatant.
Purification of C3d-C4bp
The soluble fraction of epitope-C3d-C4bp was purified from 1
litre of culture using three purification steps : an anion
exchange column, a cation exchange column and a gel filtration
column.
Anionic column (Mono Q HR 16/10)
The column was equilibrated in 20 mM Tris-HCl buffer pH 8/100
mM NaCl. The protein was eluted with a gradient of 20 column
volumes from 20 mM Tris-HCl buffer pH 8/100 mM NaCl (Buffer A)
to 20 mM Tris-HCl buffer pH 8/1M NaCl (Buffer B). The protein
eluted at approximately 350 mM NaCl.
The MonoQ fractions containing epitope-C3d-C4bp were dialysed
against 20mM Tris-HCl buffer pH 7/100 mM NaCl before loading
on a cationic column.
Cationic column (Mono S HR 10/10)
The fractions after the column Mono Q containing epitope-C3d-
C4bp were loaded on a cationic column (Mono S HR 10/10)
equilibrated with 20mM Tris-HCl buffer pH 7/100 mM NaCl. The
protein was eluted with a gradient of 20 column volumes from
20 mM Tris-HCl buffer pH 7/100 mM NaCl (Buffer A) to 20 mM
Tris-HCl buffer pH 7/1M NaCl (Buffer B). The protein eluted at
approximately 350 mM NaCl.
The fractions containing the epitope-C3d-C4bp without the
major contaminant (>66 Kda) were pooled, concentrated and

loaded on a gel filtration column.
Gel filtration column (Superdex 200 26/60 prep grade)
The fractions from the Mono S column containing epitope-C3d-
C4bp were loaded on a Gel Filtration column (Superdex 200
26/60 prep grade) equilibrated with 50 mM Na phosphate pH
7.4/150 mM NaCl. The protein eluted with 152.69 ml of buffer
as a nice symmetric peak. This elution volume shows that the
protein is oligomeric. After the column, the protein
concentration was 0.45 mg/ml. The protein was concentrated to
1.5 mg/ml and stored at -70°C with 10% Glycerol. The protein
was at least 90% pure.
Example 2 - Insertion of the human C3d molecule in the mobile
loop of Human Cpn10 (C3d7).
This example describes the purification of the soluble portion
of three similar C3d7 constructs and their expression at 25°C.
C3d7(l)
A 42.85 kDa tri-partite fusion protein, comprising human C3d
replacing the mobile loop of human Cpn10 (truncated at its N-
terminus) and a C-terminal myc tag epitope, with the amino
acid sequence. SEQ ID NO:18, was expressed from the plasmid
pAVD 95 in the E. coli strain C41(DE3) at 25°C.



Human Cpn10 amino acid sequence of SEQ ID NO: 18 are residues
1-16 and 311-377. The human C3d amino acid sequence is from
17-310 of SEQ ID NO: 18, and the myc-tag epitope amino acid
sequence from 378-387.
The DNA sequence (an NdeI-HindIII restriction fragment)
encoding this fusion protein was cloned between the NdeI-
HindIII sites of a pRSET derived plasmid, placing the coding
sequence under the control of the T7 promoter.
C3d7(2)
A second fusion protein, differing only in the positioning of
human C3d insertion in place of the mobile loop of human Cpn10
was similarly constructed. This has the sequence of SEQ ID
NO:19:

Amino acid residues 1-20 and 315-378 are derived from human
Cpn10, flanking the human C3d amino acid sequence. The myc-
tag epitope amino acid sequence is from 379-388.
C3d7 (3)
Likewise, a third fusion protein called C3d7(3) with the


The human Cpn10 amino acid sequence is 1-18 and 313-373
flanking the human C3d amino acid sequence, and the myc-tag
epitope amino acid sequence is 374-383.
Expression of C3d7(l).
To have this protein in a soluble form, we expressed pAVD95 in
the E. coll strain C41(DE3) at 25°C. After an overnight
induction at 25°C with 0.5 mM IPTG the protein was expressed
and almost half of the protein was found to be in the
supernatant.
Purification
The soluble fraction of C3d7(l) was purified using two
purification steps, namely an anionic column, followed by a
gel filtration column.
Anionic column (Mono Q HR 16/10)
The column was equilibrated in 20 mM Tris pH 8. The protein
was eluted with a gradient of 20 column volumes from 20 mM
Tris pH 8 to 20 mM Tris pH 8, 1M NaCl. The protein eluted with
approximately 350 mM NaCl in one fraction (E3) of 5 ml.

Gel filtration column (Superdex 200 26/60 prep grade)
The fraction E3 of the column Mono Q was loaded on a gel
Filtration column (Superdex 200 26/60 prep grade) equilibrated
with 50 mM Na phosphate pH 7.4, 150 mM NaCl. The protein was
eluted with 150 ml of buffer. The elution volume of ovalbumin
(MW=43 Kd) on the same column was 167 ml. This indicates that
the protein is oligomeric.
Circular Dichroism
Analysis of the protein by Far UV Circular Dichroism indicated
the presence of secondary structure. The deconvolution of the
spectrum gave a percentage of alpha-helix around 49 %. This
percentage is in agreement with the percentage determined by
modelling (48% of alpha-helix) . This -is a good indication
that the protein is correctly folded.
The protein was concentrated to 1.2 mg/ml in 50mM sodium
phosphate, pH7.4, 150 mM NaCl.
Example 3 - CR2 binding activity of C3d7(l) and epitope-C3d-
C4bp.
ELISA Assay Method
The epiotope-C3d-C4bp molecule prepared as in Example 1 and
the C3d7(l) prepared as in Example 2 were assayed over a
concentration range from 500nM - O.OlnM and compared against
human C3d (Calbiochem) and a linear trimer of human C3d,
called C3d3 or APT2029, constructed and prepared as described
in WO99/35260. The results are shown in Figures 2 and 4.
Briefly, the assay method was as follows:
A IgG constant region-CD21 fusion protein, was expressed and
purified in tissue culture cells and the purified protein was

used to coat the wells of an ELISA plate. The various C3d
molecules were added, in a range of concentrations, to these
wells and incubated. After incubation, the wells were
extensively washed, before adding a biotinylated anti-C3d
monoclonal antibody. After incubation and washing, a
horseradish peroxidase(HRP)-labelled anti-biotin antibody was
added. Following a further incubation and washing step, a
substrate for HRP was added and the generation of a coloured
product from the substrate by the HRP was measured at an
absorbance of 450 nanometres. The assay is illustrated as a
cartoon in Figure 3.
Clearly the epitope-C3d-C4bp molecule binds to the C3d
receptor CD21 much better than the monomeric C3d does and
better than even the linear trimer C3d3 at several
concentrations, as seen in Figure 2.
The assay was repeated three times with C3d7(l) and the
gradients of each response averaged and compared. Figure 4
shows the results of one of these assays.
In comparing the results between C3d7(l) and the Calbiochem
C3d (which is in monomeric form), the Abs 450 of the C3d7(l)
increases at a lower concentration than the monomeric C3d.
This indicates that the C3d is in a multimeric form.
Example 4 - CR2 binding activity of C3d7(l), (2) & (3).
The second binding experiment of example 3 was repeated as
described in Example 3 with C3d7(l), (2) and (3), all of which
were prepared as described above in Example 2.
The binding of these three proteins in an ELISA assay is shown

as Figure 5. The data show that C3d7(l), C3d7(2) and C3d7(3)
all conclusively bind to CR2. The gradient of the linear
portion of the binding curve gives an indication of
multimerisation of the proteins as shown by the 3.4 fold
increase in gradient between the monomeric C3d (supplied by
Calbiochem) and the linear trimer of C3d3, called APT2029. The
gradient for the linear portion of the three C3d7 constructs
suggests that they are all multimerised.
Example 5 - Analysis by immunofluorescent flow cytometry.
The CR2 binding activity of the C3d7 constructs on the
immortalised human CD21+ Raji lymphoblastoid cell line and
human CD20+/CD4- / CD8- peripheral blood lymphocytes was
tested. Flow cytometry analysis was carried out using a
Becton Dickenson FACSCalibur; 10,000 events were acquired.
Immortalised Raji (B cells) and Jurkat (T cells) cells were
washed in PBS and incubated with optimised dilutions of FITC
conjugated, anti-human, CD3 (pan-T cell marker), CD20 (pan-B
cell marker) and CD21 (CR2 marker) (DAKO) monoclonal
antibodies (Mabs). This verified that Raji cells are CD21+,
CD20+ whereas Jurkat cells are CD21dim, CD3+.
Binding of C3d7(1-3) is detected on Raji (CD21+/CD20+) cells,
but not Jurkat (CD3+/CD21dim) cells.
Using a single staining immunofluorescence assay format,
washed Raji and Jurkat cells (lxl06/ml) were incubated with
100nM (final dilution) of C3d7(l), C3d7(2), C3d7(3), human
monomeric C3d (Calbiochem) and human linear trimer C3d3
(APT2029) for 30 minutes at room temperature, washed in ice-
cold PBS and then incubated with an optimised dilution of Cy3
(a pink fluorophore) conjugated anti-human C3d monoclonal

antibody (Mab) for 30 minutes at 4°C in the dark, washed again
and resuspended in 0.5ml ice-cold PBS. Figure 6 shows the
results of this analysis.
C3d7(l) and C3d7(2) conclusively bind to CD21+ cells and not to
CD3+/CD21dim cells. The increase in signal intensity gives an
indication of multimerisation shown by the 7 and 9 fold signal
intensity increase between C3d (Calbiochem) , C3d7(l) and
C3d7(2) respectively compared with the 6.4 fold increase with
C3d3 (APT2029) .
Binding of C3d7(1) on the surface of CD20+/CD4-/CD8- human
peripheral blood lymphocytes (PBLs)
This experiment was conducted using a double staining
immunofluorescence assay format. Human PBLs were isolated from
blood by density gradient centrifugation through Ficoll.
Contaminating erythrocytes were removed by lysis. Washed PBLs
at lxl06/ml were incubated with 200nM (final dilution) C3d7(l)
or human linear trimer C3d3 (APT2029) for 30 minutes at room
temperature, washed in ice-cold PBS and then incubated with
optimised dilutions of Cy3-anti-human C3d Mab and FITC-anti
CD4 (Th cell marker), anti CD8 (CTL marker) anti-CD20 (B cell
marker) Mabs (DAKO) for 30 minutes at 4°C in the dark, washed
and resuspended in 0.3ml ice-cold PBS prior to flow cytometry
analysis; 5,000 events were acquired.
Analysis of the data indicated that C3d7(l) conclusively binds
to the PBL B (CD20+) cell population (presumed to be CD21+), in
a similar manner to that seen with the linear trimer human
C3d3 called APT2029.

WE CLAIM:
1. A product comprising:
a first component which is a scaffold;
a second component which is an adjuvant; and
a third component which is an antigen,
wherein the adjuvant is a ligand for CD21.
2. A product as claimed in claim 1, wherein the
adjuvant is different from the scaffold.
3. A product as claimed in claim 1 or claim 2, wherein
the ligand for CD21 is C3d.
4. A product as claimed in claim 1 or 2 wherein the
third component is a polypeptide antigen.
5. A product as claimed in claim 1 or 2 wherein the
third component is a non-polypeptide antigen.

6. A product as claimed in any one of claims 1 to 3
wherein the scaffold and antigen are the same.
7. A product as claimed in claim 5 wherein the scaffold
and antigen are a viral coat protein.
8. A product as claimed in claim 6 wherein the viral
coat protein is Hepatitis B surface antigen.
9. A product as claimed in claims 1 to 3, wherein the
scaffold is C4bp core protein.
10. A pharmaceutical composition comprising the product
of any one of claims 1 to 9 together with a
pharmaceutically acceptable carrier or diluent.
11. A product as claimed in any one claims 1 to 10 in
the preparation of a medicament for inducing an
immune response to an antigen.

12. A method of making a product comprising:
a first component which is a polypeptide scaffold;
a second component which is a polypeptide which is a
ligand for CD21; and
a third component which is a polypeptide antigen,
the method comprising expressing nucleic acid
encoding the three components in the form of a
fusion protein, and recovering the product.
13. A method of making a product comprising:
a first component which is a polypeptide scaffold;
a second component which is a polypeptide which is
ligand for CD21; and
a third component which is a non-polypeptide
antigen, the method comprising expressing, nucleic
acid encoding the first and second components in the
form of a fusion protein, joining said fusion
protein to the third component, and recovering the
product.

14. The method of claim 12 or 13 wherein the nucleic
acid is expressed in a prokaryotic host cell.
15. A method as claimed in claim 14 wherein the fusion
protein is recovered in multimeric form.
16. A method as claimed in claim 15 wherein the
recombinant protein is present at least a
concentration of at least 2 mg/1 of cell culture.
17. A method as claimed in claim 15 or claim 16 wherein
the host prokaryotic cell is E. coli.
18. An expression vector comprising a nucleic acid
sequence encoding a fusion protein of
a first component which is a polypeptide scaffold;
a second component which is a polypeptide which is a
ligand for CD21; and optionally
a third component which is a polypeptide antigen,
operably linked to a promoter functional in a host
cell.

19. A bacterial host cell transformed with the
expression vector of claim 18.
20. A eukaryotic host cell transformed with the vector
of claim 18.
21. The expression vector of claim 18 in the preparation
of a medicament for inducing an immune response.

The present invention provides a product comprising: a first component which is a scaffold; a second component
which is an adjuvant, preferably a polypeptide which is a ligand for CD21 or a cell surface molecule on B cells or T cells or follicular
dendritic or other antigen presenting cells; and a third component which is an antigen.

Documents:

00232-kolnp-2005-abstract.pdf

00232-kolnp-2005-claims.pdf

00232-kolnp-2005-correspondence.pdf

00232-kolnp-2005-correspondence_1.1.pdf

00232-kolnp-2005-correspondence_1.2.pdf

00232-kolnp-2005-correspondence_1.3.pdf

00232-kolnp-2005-correspondence_1.4.pdf

00232-kolnp-2005-description(complete).pdf

00232-kolnp-2005-drawings.pdf

00232-kolnp-2005-form-1.pdf

00232-kolnp-2005-form-18.pdf

00232-kolnp-2005-form-2.pdf

00232-kolnp-2005-form-26.pdf

00232-kolnp-2005-form-3.pdf

00232-kolnp-2005-form-5.pdf

00232-kolnp-2005-international publication.pdf

00232-kolnp-2005-international search authority report.pdf

00232-kolnp-2005-others document.pdf

00232-kolnp-2005-pct demand form.pdf

00232-kolnp-2005-pct others.pdf

00232-kolnp-2005-pct request.pdf

232-kolnp-2005-granted-abstract.pdf

232-kolnp-2005-granted-claims.pdf

232-kolnp-2005-granted-correspondence.pdf

232-kolnp-2005-granted-description (complete).pdf

232-kolnp-2005-granted-drawings.pdf

232-kolnp-2005-granted-examination report.pdf

232-kolnp-2005-granted-form 1.pdf

232-kolnp-2005-granted-form 18.pdf

232-kolnp-2005-granted-form 2.pdf

232-kolnp-2005-granted-form 26.pdf

232-kolnp-2005-granted-form 3.pdf

232-kolnp-2005-granted-form 5.pdf

232-kolnp-2005-granted-reply to examination report.pdf

232-kolnp-2005-granted-specification.pdf


Patent Number 227314
Indian Patent Application Number 232/KOLNP/2005
PG Journal Number 02/2009
Publication Date 09-Jan-2009
Grant Date 06-Jan-2009
Date of Filing 22-Feb-2005
Name of Patentee AVIDIS SA
Applicant Address BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
Inventors:
# Inventor's Name Inventor's Address
1 ANDREOLETTI, PIERRE AVIDIS SA BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
2 DUMON, LAURENCE AVIDIS SA BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
3 HILL, FERGAL AVIDIS SA BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
4 JULIEN, MICHEL AVIDIS SA BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
5 MARCHAND, JEAN BAPTISTE AVIDIS SA BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
6 RISSE, EMMANUEL AVIDIS SA BIOPOLE CLERMONT-LIMAGNE, F-63360 SAINT BEAUZIRE
PCT International Classification Number A61K 39/385
PCT International Application Number PCT/EP2003/008926
PCT International Filing date 2003-08-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 02292042.5 2002-08-14 EPO