Field of the Invention
This invention relates to a method of inducing an immune
response in an individual and to novel compositions for performing the method of
the invention and methods of making said compositions. Particularly, but not exclusively,
the present invention relates to plant viral coat proteins as adjuvant sequences
for inducing an immune response.
Background of the Invention
The idiotypic determinants expressed on the cell surface
immunoglobulin (Ig) of B-cell lymphomas can act as tumour-associated antigens (for
review see George and Stevenson, 1989). As such they present an attractive target
for therapy, notably for the administration of passive anti-idiotypic antibody to
patients (Miller et al., 1982 New Engl. J. Med. 306, 517-522). Murine monoclonal
antibodies (MAbs) raised against idiotypic determinants on B cell non-Hodgkin's
Lymphomas (NHLs) have given limited benefit in human therapeutic trials. Partial
and complete responses have been observed, but the murine MAbs tend to recruit human
effector functions inefficiently and are themselves the target of a human anti-mouse
antibody response. Also, outgrowth of surface Ig negative lymphoma cells has been
observed following therapy (Levy et al., 1987 J. Immunol. Rev. 96, 43-; Bahler
and Levy, 1992 PNAS 89, 6770-6774), although the complete loss of immunoglobulin
expression is rare (Meeker, et al., 1985 New Engl. J. Med. 312, 1658-1665;
Zelentz et al., 1990 Ann. Oncol..2, 115-122). In light of these limitations.
coupled with the cost and inconvenience of generating MAbs for individual patients,
the approach has not been widely adopted. However, it is clear that anti-idiotypic
antibodies do have therapeutic potential in lymphomas.
One alternative to passive anti-idiotypic serotherapy is
active immunisation which aims to break tolerance and induce a strong anti-idiotypic
antibody response in the patient. Since the response will be polyclonal, it is more
difficult for the target B-cell to escape selection, and furthermore, the response
will be present on a continuing basis, and so might be able to control residual
disease. An additional advantage of this approach is that it also has the potential
to stimulate T cell mediated immune responses against the lymphoma. Efforts to stimulate
tumour immunity using modified tumour cell vaccines have met with limited success,
but active immunisation with idiotypic Ig prior to tumour challenge has proved effective
in suppressing model B-cell tumours (Stevenson and Gordon, 1983 J. Immunol.
130, 970-973; George et al., 1987 J. Immunol. 138, 628-634; Campbell
et al., 1987 J. Immunol. 139, 2825-2833) in animals and to treat animals
bearing incipient tumour (George et al., 1988 J. Immunol. 141, 2168-2174).
Furthermore idiotypic immunisation with human Ig isolated from patients with lymphoma
has been associated with sustained tumour regression (Kwak et al., 1992 New Engl.
J. Med. 327, 1209-1215).
The problem is how best to present the antigen (the idiotypic
antibody) to break tolerance and stimulate an effective anti-lymphoma immune response,
and this remains a challenge. In addition, for lymphomas, which secrete little immunoglobulin,
making the idiotype is a major problem. To make sufficient idiotypic antibody for
immunisation heterohybridomas must be prepared by fusion with mouse cell lines and
the antibody then purified (Carroll et al., 1985 J. Immunol. Methods 89,
61-67). The yield is frequently low and it must be subsequently confirmed that the
fusion derives from the human B-cell tumour.
This latter problem has now been overcome: the use of recombinant
DNA technology allows the VH and VL genes encoding the idiotypic
determinant to be readily identified in patient biopsy material by PCR and sequencing
(Hawkins et al, 1994 Blood 83:3279). These genes can be assembled as scFv for use
as a DNA vaccine. This approach is based on the data coming from a range of infectious
diseases where it is clear that DNA encoding sequences from pathogens can transfect
cells directly and induce protective immune responses (Ulmer et al, 1993259:1745;
Davis & Whalen 1995, In Molecular and Cell Biology of Human Gene Therapeutics.
Ed. George Dickson. Chapman & Hall, p368).
In previous work, in a mouse model for lymphoma, the V-genes
of the tumour idiotope were cloned and expressed as light and heavy chain fusion
proteins in bacteria. The separate chains were then used as immunogens. However,
the separate chains were denatured, and in any case were not co-expressed to provide
the paratope of the antibody. Indeed the authors suggested that "future work on
peptides with fixed configurations similar to epitopes present in the native protein
may prove useful, as may the co-expression of both VH and VL
genes in bacteria to produce a recombinant Fv protein" (Campbell et al., 1987, cited
above). However, the authors did not teach how to isolate the V-genes of the idiotope.
Nor did the authors teach how to combine the recombinant Fv fragments into a vaccine.
The present inventors have previously shown that nucleic
acid constructs can be prepared which can be delivered into living cells in vivo
and which can then induce an immune response to an idiotypic determinant present
on a malignant B-cell. The construct encodes a fusion protein comprising the idiotypic
determinant and tetanus toxoid fragment C (FrC). Indeed, the inventors have already
specifically shown that genetic fusion of a lymphoma or a myeloma-associated antigen
to the adjuvant sequence FrC of tetanus toxin induces a protective immunity in mice
against challenge with lymphoma or myeloma when used as a DNA vaccine.
However, the human population is immune to FrC due to vaccination
with Tetanus Toxoid. Therefore, when FrC-based vaccine is used in patients it will
work in a setting of pre-existing immunity to FrC. So far the inventors have found
that pre-existing antibody does not reduce the response to the DNA vaccine. However,
it is possible that different immune pathways are activated in this situation.
Summary of the Invention
Given the above, the present inventors have appreciated
that there is a need to provide novel adjuvant sequences which overcome potential
problems related to pre-existing immunity to C fragment of Tetanus Toxoid.
Plant viral coat proteins are highly immunogeneic molecules
when injected into mammals. They have been used as protein vaccines when assembled
into viral particles to present epitopes from infectious agents (Brennan, F. R.
et al. Vaccine 17, 1846-1857 (1999). Such vaccines can induce high levels of neutralising
antibodies and in some cases protection against challenge with the pathogen (Haynes,
J.R. et al Bio/Technology 4, 637-641 (1986); Turpen, T.H. et al Bio/Technology 13,
53-57 (1995)). Potato virus X (PVX) was used to deliver systemically a functional
single chain antibody fragment as a fusion to PVX coat protein into plants (Smolenska,
L. et al FEBS letters 441, 379-382 (1998). However, PVX coat protein-based
fusions were never delivered as DNA into mammalian cells. For DNA delivery, tobacco
mosaic virus coat protein has been used to raise antibody against itself (Hinrichs,
J. et al Journal of Virological Methods 66, 195-202 (1997)) but it has not be used
as part of a fusion so as to induce an immune response against the other member(s)
of the fusion construct.
The inventors have found that the potato virus X coat protein
(PVXCP) works surprisingly well as an adjuvant in the construction of a DNA vaccine
to induce protective immunity. Specifically, the inventors have genetically fused
PVXCP to a lymphoma-associated antigen (scFv-A31) and used the construct for DNA
vaccination of mice. This led to elevated antibody responses and protection against
challenge with A31 lymphoma (see example 4, Fig. 14 A). The inventors have further
shown that this protection was mediated both by antibody to scFv-A31 and CD4 positive
cells. This is in contrast with DNA vaccination using fusion of FrC to ScFv-A31,
where such protection is mediated only by antibody to scFv. This finding could be
significant when considering the disease to be treated, e.g. when the effectiveness
of the treatment depends on a cellular response as opposed to an antibody response.
The inventors have also fused a myeloma-specific antigen
scFv-5T33 to PVXCP. This construct was then used to vaccinate mice. This vaccination
resulted in induction of antibody responses against scFv-5T33 and protection against
challenge with 5T33 myeloma (see example 4, Fig. 14 B). The inventors also determined
that in this example the protection against myeloma is mediated by CD4 positive
Thus, the inventors have surprisingly shown that the plant
viral coat protein PVXCP can act as an adjuvant for induction of protective immunity
against tumour cells, for example, lymphoma and myeloma.
In a first aspect, the present invention provides a nucleic
acid construct for delivery into living cells in vivo for inducing an immune
response in a patient to an antigen; the construct directing the expression of a
fusion protein, said fusion protein comprising said antigen and an adjuvant wherein
said adjuvant is PVXCP.
In one embodiment, the antigen of the invention is derived
from a pathogen, such as a virus (e.g. herpes simplex virus, human immunodeficiency
virus etc) or a bacterium (e.g.staphylococcus, salmonella etc). However, the inventors
have found that the present invention is particularly applicable where the antigen
is a self or altered self polypeptide or is derived from a self or altered self
polypeptide. The self or altered self polypeptide may be associated with an autoimmune
disease or a cancer type. With regard to cancer, the self or altered self polypeptide
is a tumour associated antigen such as an idiotypic determinant of a lymphocyte
Thus, in preferred embodiment, the invention provides a
nucleic acid construct for delivery into living cells in vivo for inducing an immune
response in a patient to a self or altered self polypeptide; the construct directing
the expression of a fusion protein, said fusion protein comprising the self or altered
self polypeptide and an adjuvant, wherein said adjuvant is PVXCP.
Preferably, the adjuvant promotes a helper T cell response
when administered to a patient. More preferably, the adjuvant comprises at least
one T helper cell epitope.
Preferably the fusion protein will comprise a plurality
of PVXCP epitopes, both B and T cell. Typically the PVXCP will be placed on the
carboxy side of the antigen.
It will be apparent to those skilled in the art that the
composition, nucleic acid (DNA) vaccines and methods of the present invention can
be used to either enhance or suppress the immune response to a particular antigen.
Where the antigen is a tumour associated antigen then it will clearly be desirable
to enhance the immune response to the antigen so as to halt or reduce the growth
of the tumour. Enhancement of the immune response is a preferred aspect of the present
invention and preferably the enhancement is to tumour associated antigens in general,
but particularly, idiotypic determinants in the immunoglobulin expressed on the
surface of B cell malignancies, idiotypic determinants of T cell receptors (TCRs)
expressed on the surface of T cell malignancies, mutated oncogenes or other self
polypeptides expressed on the surface of tumours, and oncofoetal antigens.
For convenience, the following description will describe
the invention in relation to idiotypic determinants. However, it will be apparent
to the skilled person that the invention can be applied with ease to any antigen,
e.g. antigen derived from pathogens such as viruses or bacteria, self or altered
self polypeptides and, in particular, tumour associated antigen.
The idiotypic determinant is preferably present in the
fusion protein in essentially the same conformation as that which it adopts on the
surface of the patient's malignant B cells, thereby optimising the efficiency of
the anti-idiotypic immune response induced by the construct. Conveniently this is
achieved by expression of the idiotypic determinant within the context of a portion
of an immunoglobulin (Ig) molecule or immunoglobulin-like molecule, such as a single
chain Fv (scFv) fragment. The scFv fragment is particularly convenient, providing
the necessary structural features of the idiotypic determinant with few extraneous
amino acid residues. However, if desired additional amino acid residues could be
included in the fusion protein, such as one or more constant domains (e.g. Syrengelas
et al, 1996 Nature Medicine 2, 1038). Thus, for example, one could express the idiotypic
determinant in the context of an entire immunoglobulin molecule.
In a preferred embodiment, the fusion protein is expressed
with a leader sequence (recognised in human cells) which directs the fusion protein
to the endoplasmic reticulum, where the leader sequence is cleaved from the fusion
protein. A large number of suitable leader sequences are known including, for example,
the leader sequences (such as that for VH1 described below) found at
the 5' end of human V genes. Such leader sequences have been found by the present
inventors to increase the immunogenicity of the fusion protein. In principle, any
other leader sequence is likely to exert an equivalent advantageous effect, but
it is probable that those most similar to the natural immunoglobulin-type leader
sequence will be optimal.
For the sake of convenience, the nucleic acid construct
will preferably comprise a number of restriction endonuclease recognition sites.
In particular, one or more such recognition sites may be located 5' of the sequence
encoding the idiotypic determinant (conveniently between the optional leader sequence
and the sequence encoding the idiotypic determinant), and one or more sites may
be located 3' of the sequence encoding the idiotypic determinant (conveniently between
the sequence encoding the idiotypic determinant and the epitope(s) from the PVXCP).
In this way, the same basic construct can readily be adapted to express different
fusion proteins in which the idiotypic determinant may be altered. Thus sequences
encoding idiotypic determinants from different patients can easily be introduced
into the construct.
In a particular embodiment, this invention provides a vaccine
nucleic acid which can be used to elicit an immune response against transformed
human lymphocytes displaying an idiotypic marker, the nucleic acid encoding proteins
comprising the heavy and light chain variable regions of an anti-idiotypic antibody
displayed on surface of a malignant human B-cell and PVXCP.
In a second aspect, the invention provides a method of
making a nucleic acid construct for raising an immune response against an antigen,
the method comprising:
- (a) identifying a nucleic acid sequence encoding the antigen;
- (b) cloning the nucleic acid sequence; and
- (c) introducing the cloned nucleic acid into a vector, said vector allowing
the protein to be expressed as a fusion with an adjuvant, wherein said adjuvant
The antigen may be derived from a pathogen such as a virus
or a bacterium. However, in a preferred embodiment of the invention, the antigen
is a self or altered self polypeptide, e.g. a tumour associated antigen.
In a further aspect, the invention provides a method of
making a nucleic acid construct for treating a patient suffering from a B cell malignancy,
the method comprising:
- (a) identifying a nucleic acid sequence encoding an idiotypic determinant present
on the malignant B cells of the patient by analysis of a sample of cells from the
- (b) cloning the nucleic acid sequence encoding the idiotypic determinant; and
- (c) introducing the cloned nucleic acid into a vector, which vector allowing
the idiotypic determinant to be expressed as a fusion with an adjuvant, wherein
said adjuvant is PVXCP.
Preferably, the adjuvant is able to promote helper T cell
responses in vivo. More preferably, the adjuvant comprises at least one T
helper cell epitope.
Conveniently the nucleic acid encoding the idiotypic determinant
is cloned from a sample of the patient's cells by PCR. A large family of suitable
generic PCR primers, capable of recovering nucleic acid sequences encoding essentially
any B cell idiotypic determinant, is now available (Hawkins & Winter, 1992 Eur.
J. Immunol. 22, 876). Typically the B cell malignancy is a lymphoma or a myeloma.
Generally, the nucleic acid construct made by the method defined above will be in
accordance with the first aspect of the invention.
A method of inducing an immune response in a patient may
comprise the step of administering to said patient a nucleic acid construct in accordance
with the first aspect of the invention defined above. Preferably, the nucleic acid
construct forms part of a nucleic acid expression vector such that the adjuvant
and the antigen can be expressed in the patient. The nucleic acid sequence may be
expressed in the patient without entering the patient's genome.
Preferably, the nucleic acid construct of the invention
forms a naked DNA vaccine. Such a DNA vaccine is used to induce an immune response
against the protein product encoded by the DNA.
DNA vaccination has been used successfully in the past
as a way of inducing an immune response, see for example King et al., Nature Medicine
4: 1381, 1998.
A method of vaccinating a patient against an infectious
disease may comprise administering to the patient a nucleic acid construct in accordance
with the first aspect of the invention, in a physiologically acceptable medium,
wherein the antigen is derived from a pathogen associated with said infectious disease.
The method may be carried out on a patient with said infectious disease or on a
subject at risk of contracting the infectious disease.
A method of treating a patient having or at risk of a cancer
may comprise the step of administering to said patient a nucleic acid construct
in accordance with the first aspect of the invention, in a physiologically acceptable
medium, wherein the antigen is a tumour associated antigen associated with said
cancer. The tumour associated antigen may well have been derived from the patients
own cancer cells.
A method of treating a patient suffering from a B cell
malignancy and/or a method of inducing an immune response in a patient against a
tumour associated antigen may comprise administering to the patient a nucleic acid
construct in accordance with the first aspect of the invention defined above, so
as to induce an immune response to the idiotypic determinant present on the surface
of the patient's malignant B cells. B cell lymphomas or myelomas are the conditions
preferably treated by the method of the invention.
Preferably the nucleic acid sequence encoding the idiotypic
determinant is cloned from samples obtained from the individual to whom it is delivered.
Conveniently the nucleic acid sequence is delivered in unencapsidated form (i.e.
not enclosed within a viral particle or other package). The nucleic acid may, however,
be associated with the external surface of a package or particle (e.g. a liposome
or a viral particle), which allows for the possibility of receptor-mediated delivery
of the nucleic acid.
The fusion protein may direct the expression of the idiotypic
determinant and the PVXCP alone. Alternatively the fusion protein may additionally
comprise further immunomodulatory polypeptide sequences, such as other foreign immunogenic
proteins, or cytokines. Indeed it may be valuable to use several antigenic fusion
partners to help prevent the theoretical problem that the immune response to the
highly immunogenic moiety of the fusion protein could ultimately overwhelm any response
to the relatively weakly immunogenic idiotypic determinant. Other coat proteins
of enveloped viruses and immunogenic cell surface or secreted proteins derived from
any pathogenic organism or non-human species may be suitable for inclusion in the
An alternative modification is to design the nucleic acid
construct so as to allow for the co-expression of the further immunomodulatory polypeptides
as separate entities rather than as fusions with the idiotypic determinant/plant
viral coat protein epitope. Less preferably, the method of the invention could employ
the use of a separate nucleic acid construct to express the further immunomodulatory
A number of cytokines are known to improve aspects of antigen
presentation and the direct delivery of expression vectors containing cytokine genes
could enhance vaccine efficacy. Interferon gamma is one example that could be useful
due to the property of up-regulating MHC expression (Gaczynska et al. 1993 Nature
365, 2, 264-267). Another polypeptide that could be expressed by the vaccine
nucleic acid is granulocyte/macrophage-colony stimulating factor (GM-CSF). The relevant
gene could be encoded on the same, or on a separate, vector and the amount of polypeptide
expressed varied independently.
One advantage of the genetic approach to vaccination is
that it potentially allows efficient use of the natural method of presenting antigen,
which should therefore engage a wide range of effector systems. Moreover, manipulation
and improvement of the response obtained should be relatively easy; for example,
it may be possible to improve the efficiency of presentation of antigen to T cells
by expressing molecules with co-stimulatory activity together with the immunogen.
One important molecule involved in co-stimulation is B7, which interacts with CD28
expressed by T cells thereby providing accessory signals for T-cell activation (Galvin
et al., 1992 J. Immunol. 12, 3802-3808). Vectors could be constructed which
express both B7 and the idiotypic Ig. Sequences of both mouse and human 87 are published
(Freeman et al., 1989 J. Immunol. 8, 2714-2722) and the genes may readily be cloned
The therapeutic methods described may further comprise
the delivery of a second nucleic acid sequence to the individual, the second nucleic
acid sequence directing the expression of a further immunomodulatory polypeptide
for the purpose of further modulating the immune response to the idiotypic determinant.
This second nucleic acid sequence may be comprised on the same nucleic acid molecule
as the first nucleic acid sequence, or may be present on a second nucleic acid molecule.
Methods of introducing the nucleic acid construct into
living cells in vivo are now well known to those skilled in the art. Conveniently
the nucleic acid is simply injected as naked DNA into the patient (typically intramuscularly)
as a mixture with a physiologically acceptable diluent, such as saline solution.
Details of some suitable methods and preferred embodiments of the administration
of the nucleic acid construct into a patient are described in US patent No.s 5,580,859
and 5,589,466. More involved methods of gene transfer include the use of viral vectors,
encapsulating the DNA into liposomes, coupling of DNA to cationic liposomes or to
the outside of viruses (for review see Miller, 1992 Nature 357, 45-46). These
have the advantage of increased efficiency of transfer but, by comparison with direct
injection of purified plasmid DNA, these alternative approaches are somewhat involved
and raise more safety issues.
In a further aspect the invention therefore provides a
composition for use in the method of treating a patient suffering from a B cell
malignancy or a method of inducing an immune response against a tumour associated
antigen in a patient, the composition comprising a nucleic acid construct directing
the expression of a fusion protein, said fusion protein comprising an idiotypic
determinant or a tumour associated antigen which is present on the malignant B cells
of the patient and PVXCP, together with a physiologically acceptable diluent or
In a particular embodiment, the present invention entails
the isolation of the V-genes from the tumour B-cells to express the idiotope (and
paratope) of the tumour antibody. Thus, starting with a sample of B-cells from the
patient, the rearranged V-genes of both heavy and light chains are amplified using
the polymerase chain reaction and generic "universal" primers (Orlandi et al., 1989
PNAS 86, 3833-3837; Marks et al., 1991 J. Mol. BioI. 222, 581-597;
see also Table 1, Seq. ID Nos. 1-48). The amplified V-genes are cloned and then
sequenced (Sanger et al., 1977 PNAS 74., 5463-5467). Those from the malignant
B-cells are identified as predominant repeated VH and VL gene sequences. In several
patients, and for both heavy and light chains, it was possible to identify a common
repeated sequence. The combination of the heavy and the light chains identifies
the idiotope of the tumour (Example 1). In principle, it would also be possible
to amplify and link the rearranged V-genes within the same cell (Embleton et al.,
1992 Nucl. Acids Res. 20, 3831-3837) to identify a major combination of linked
heavy and light chain sequences that identify the idiotope but here the VH and VL
are separately identified and then linked by PCR assembly. The VH and VL genes are
cloned into vectors for the expression of both V-domains as a functional antibody
fragment, for example as a linked single chain Fv fragment (see below).
Ideally a vaccine should be capable of stimulating antigen-specific
B cells, cytotoxic T lymphocytes (CTLs) and helper T cells. B cell stimulation requires
that the target antigen should bind with sufficiently high affinity to specific
antigen receptors (surface Ig) on the B-cell surface. Certain multivalent antigens
can stimulate B cell proliferation directly but more often, and to provide an effective
anamnestic response, there is a requirement for additional signals provided by helper
T cells (see below). In the present invention, T cell help is recruited by expressing
the idiotypic determinant as a fusion protein with PVXCP.
The T cell receptors (TCRs) of CTLs recognise specific
MHC class I-associated peptides displayed at the target cell surface. Such peptides
are generally derived by processing of larger polypeptides or proteins manufactured
within the target cell. Thus, for efficient CTL stimulation the target antigen should
be synthesised intracellularly in MHC class I-expressing cells. The level of expression
should be high enough to generate sufficient peptide to displace those self peptides
which are normally bound (possibly with higher affinity) in the MHC peptide-binding
groove (Ohno, 1992 PNAS 89, 4643-4647). Similar to antigen-specific B cells,
for proliferation and increased cytotoxic capability, CTLs require additional signals
(in the form of cytokines) following antigen recognition and these are provided
by helper T cells.
CD4-positive helper T cells interact (via their unique
TCRs) with specific cell surface MHC class II-associated peptides and such peptides
are generally derived by proteolytic cleavage of protein antigens internalised by
specialised antigen presenting cells (APCs). Macrophages, dendritic cells and B
lymphocytes are amongst the cells which can present antigen in this way. Thus, B
lymphocytes internalise and process antigen bound to their surface Ig and subsequently
present MHC class II-associated derivative peptides. CD4-positive T helper cells
recognising the surface peptide can then release various immunostimulatory cytokines
and stimulate further B cell activation, proliferation and antibody production.
Similarly, macrophages present at the site of a local inflammatory response can
process phagocytosed antigen and stimulate cytokine release by T helper cells, leading
to enhanced activation, proliferation and cytotoxicity of locally resident CTLs.
The vaccine antigen should therefore ideally be (1) synthesised
intracellularly by MHC class I-positive host cells, (2) give rise to peptides which,
when displayed by host cell class I MHC can stimulate a subset of host CTLs via
their TCRs, (3) give rise to peptides which, when displayed by host cell class II
MHC can stimulate a subset of host helper T cells via their TCRs, (4) be internalised
and processed by host APCs including both macrophages and antigen-specific B cells,
(5) be available in its native form for interaction with host B lymphocytes.
In a further specific embodiment, the invention provides
for the expression of the rearranged VH and VL genes of the idiotope of the tumour
antibody within mammalian cells, allowing the production of peptides for display
on the cell surface in combination with host MHC, and for display (or secretion)
of the paratope (as a folded antibody fragment), to trigger the production of anti-idiotypic
antibodies. The antibody fragments could be introduced into mammalian cells by infection
with a recombinant virus encoding the antibody fragments.
In principle, the antibody fragments could be provided
with a signal sequence for their secretion or display on the surface of the infected
(transfected) cell. Alternatively the fragments could be linked to another protein
that is displayed on the surface of the cell, for example a viral coat protein,
as described in Example 2. The antibody fragments (as single chain Fv fragments)
are displayed in a functional form attached to the coat protein of a virus, indicating
that they are also folded and in a native form on the surface of an infected cell
(Russell et al., 1993 Nucl. Acids Res. 21, 1081-1085).
The antibody fragments could also be introduced into mammalian
cells using nucleic acid encoding the antibody fragments. For example, a gene encoding
a fusion protein between PVXCP and the antibody fragments may be used to immunise
mice by direct injection (e.g. subcutaneous or intramuscularly).
Aspects and embodiments of the present invention will now
be illustrated, by way of example, with reference to the accompanying figures. Further
aspects and embodiments will be apparent to those skilled in the art. All documents
mentioned in this text are incorporated herein by reference.
Brief Description of the Drawings
Detailed Description of the Invention
Example 1 - Indentification of V-genes from biopsies of B-cell lymphoma.
Preparation of biopsy material.
Figure 1 is a schematic representation of the method of PCR assembly of DNA
Figure 2 shows the sequence of vectors used to express and purify scFv idiotypic
Figure 3 is a schematic representation of the method used to produce plasmid
Figure 4 is a schematic representation of the plasmid constructs pNipenv,
pSV2 Nipenv, pSV2 Nip stop env, and pSV2 BCL env;
Figure 5 shows the sequence of a HindIII - Xbal fragment of the vector
Figure 6 is a graph showing the results of an immunisation experiment described
in example 3;
Figure 7 shows the entire sequence of the vector pVAC1;
Figure 8 shows a pVAC1 restriction map;
Figure 9 shows a schematic representation of the main features of pVAC1;
Figure 10 is a graph showing the results of an immunisation experiment described
in example 3;
Figures 11a and 11b show the results of FACS analysis described in
Figure 12 illustrates the assembly of scFv-PVXCP fusions.
Figures 13 A-D show graphs illustrating the levels of antibody produced in
an experiment described in example 4.
Figures 14 A and B show graphs illustrating the protection against
challenge with the A31 lymphoma or the 5T33 myleoma following DNA vaccination.
Figure 15 shows a graph illustrating protection against challenge with 5T33
myeloma following vaccination with fused and separate DNA sequences.
Figures 16 A and B show graphs illustrating protection against challenge
with A31 lymphoma in mice treated or untreated with anti-CD4+ monoclonal antibodies
Figure 17 show a graph illustrating protection against challenge with 5T33
myeloma in mice treated or untreated with anti-CD4+ monoclonal antibodies after
Figures 18 A and B show the results of analysis of scFv5T33-PVXCP and PVXCP
proteins in experiments described in example 4. (In B, bar = 200 nM.)
Figure 19 shows the results of an experiment described in example 5. The
graph shows protection after vaccination with p.scFvBCL1-PVXCP (3x vaccinated then
challenge with 5 x 104 BCL1 cells).
Biopsy specimens were obtained from five patients with
pathologically confirmed Follicular Lymphoma. They were obtained during routine
diagnostic procedures. The light chains were identified as kappa or lambda by immunohistochemistry.
As non-malignant controls, a small bowel lymph node from a patient with Crohn's
disease and a sample of spleen from a patient undergoing splenectomy were obtained.
Biopsy material was prepared as a single cell suspension and the cells subsequently
frozen and stored at -70°C.
Preparation of DNA for PCR.
For PCR the DNA was prepared using a simple proteinase
K/Tween 20 lysis method (Innis et al., 1990 PCR Protocols: A Guide to Methods and
Applications; Academic Press Inc., pI47). Briefly the cells were pelleted by centrifugation
for 20 seconds at 13,000 rpm in a microcentrifuge. The cells were then washed twice
with 1 ml PBS before resuspending at approximately 106/ml in K-buffer
(10mM Tris.Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCI2, 0.5% Tween 20, 100mg/ml
proteinase K) and incubated at 56°C for 60 minutes to lyse the cells and release
DNA. The proteinase K was then inactivated by incubation for 30 minutes at 95°C.
DNA thus released was used directly in the PCR reactions or stored for subsequent
use at -20°C.
PCR primers were designed to amplify re-arranged heavy
chain kappa and lambda light chain genes. The 5' primers are based on framework
1 of the V-genes. The VH and Vk primers are similar to those described by Marks
et al. (1991). However, for amplification from genomic DNA (as opposed to cDNA)
the product was found to be cleaner if primers shortened by one base at the 3' end
were used (data not shown). In addition, the number of primers used was reduced
by combining similar primers as one consensus primer. With the exception of one
change in the JH primers, to introduce a common BstEII site, changes were not made
to introduce restriction sites.
Limited DNA sequence information was available on which
to base the V&lgr; primers but primers were made to V&lgr;1, V&lgr;2, V&lgr;3
and V&lgr;4 families from the available sequence data (Songsivilai, et al. 1990
Eur. J. Immunol. 20, 2661-2666; Alexandre et al., 1989 Nucl. Acids Res.
17, 3975; Bernard et al. 1990 Nucl. Acids Res. 18, 7139; Chuchana
et al., 1990 Eur J. Immunol. 20, 1317-1325). Other families are known to
exist (Chuchana et al., 1990) but there were no nucleotide sequence data available
and so primers were not made. J-region primers were made to be complementary to
the genomic sequence of the germline J-regions for heavy chain (Ravetch et al.,
1981 Cell 27, 583-591), kappa chain (Hieter et al., 1982 I. BioI. Chem.
257, 1516-1522) and lambda chain (Udey and Blomberg 1987 Immunogenetics
25., 63- 70; Dariavach 1987 PNAS 84, 9074-9078; Bauer and Blomberg
1991 J. Immunol. 146, 2813-2820; Combriato and Klobeck, 1991 Eur. J. Immunol.
21, 1513-1522; Frippiat, 1990 Nucl. Acids Res. 18, 7134). The J&lgr;
genes combine with their respective C&lgr; genes and thus since C&lgr;4, C&lgr;5
(Dariavach, 1987) and probably C&lgr;6 (Bauer and Blomberg, 1991; Combriato and
Klobeck, 1991) are pseudogenes they should not appear as expressed protein. As a
result primers to these J&lgr; genes were not made. By combining two J region
primers, in all three Jl primers were made J&lgr;, J&lgr;2/3, J&lgr;7. Table
1 gives a full list of the primary PCR primers used in example 1.
PCR amplification of rearranged immunoglobulin variable regions
The V-gene family and J-region primers were used as equimolar
mixes of the individual primers shown in Table1. VHBACK and JHFOR mixes were used
for the heavy chain PCR reaction. Similar mixes were used for kappa or lambda chain
PCR amplification was performed in 50ml volume using Hybaid
Thermal Reactor (Hybaid). Reaction mixtures containing 20pmol of each primer mix,
250mM dNTPs (Pharmacia, Uppsala, Sweden) in 1 x PCR buffer (Promega, 10mM Tris.Cl
[pH8.8], 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100). To minimise any
risk of contamination extensive precautions were taken. The mixes were set up in
a laminar flow hood in a room designated specifically for setting up PCR reactions.
The samples were then UV treated for 5 minutes in a UV oven (Amplirad, Genetic Research,
Dunmow, UK). The template (5 ml) was then added, the reaction mix was then overlaid
with mineral oil (Sigma) and the sample heated to 94°C for 5 minutes. At this
stage Taq DNA polymerase (Promega), 2.5 units, was added. Amplification was performed
using 35 cycles, 94°C 1 min, 65°C, 1 min annealing; 72°C, 1 min elongation.
Amplified variable regions were analysed on a 1.5% LMP
agarose/TAE gel and visualised with ethidium bromide. The band of size 320/350 base
pairs was excised and purified using a GENECLEAN II kit (Bio101) according to the
manufacturers instructions. At least two independent PCR amplifications of V regions
were performed from the sample of every patient and the PCR of lymph node DNA was
performed before the corresponding PCR from the heterohybridomas (which were also
Cloning and sequencing of PCR products
The T-vector cloning system described by Marchuk (Marchuk
et al, 1991 Nucl. Acids Res. 19, 1154) was used. In brief, the vector was
prepared from pBluescript II KS+ (Stratagene) by digestion with EcoRV (from NBL)
to produce blunt ends and then treatment with Taq DNA polymerase (Promega) in PCR
buffer (Promega) containing 2mM dTTP at 70°C for 2 hours. The purified V-gene
PCR product was ligated into the T vector and transformed into competent
E. coli - strain TG1 (Gibson, 1984 Ph.D. thesis, University of Cambridge,
United Kingdom). Recombinant clones were identified by blue/white selection using
isopropyl-&bgr;-thiogalactosidepyranoside (IPTG, Sigma). Random recombinant clones
were picked and ssDNA prepared after superinfection with helper phage (MI3KO7, Stratagene)
(Vieira and Messing, 1987 Methods Enzymol. 153, 3-11). The clones were sequenced
by the dideoxy method (Sanger et al., 1977) using T7 DNA polymerase (Sequenase,
USB, Cleveland, USA). A number of clones from each patient were sequenced and the
Assembly of Tumour V-genes as scFv
The assembly method, illustrated in figure 1, is based
on that described by Davis et al., (1991 Bio/Technology 9, 165-169). The
assembly process uses a second set of primers. The VHSfiBAK primers encode
SfiI cloning site and also hybridise to the original set of VHBAK primers.
The scJHFOR and scVk/V&lgr; BAK primers hybridise to their respective initial
primers but also encode the sc linker to allow production of a single chain Fv (scFv)
(Huston et al., 1988 PNAS 85, 5879-5883).
The NotJk/IFOR primers hybridise to their respective initial
primers but also include the NotI restriction site. These primers are also
summarised in Table 1. The assembly is carried out in two stages. First the V-genes
(heavy and light chains) were amplified from the sequencing template using the new
set of oligonucleotides. The PCR mixture was made up as above but the primers used
were only the relevant V-gene family and J-region primers identified by previous
sequencing. Template was 100ng of ssDNA sequencing template. The PCR conditions
were 94°C for 1 minute, 50°C for 1 minute, 74°C for 1 minute with
10 cycles of amplification. At the completion of the PCR the further dNTPs were
added (5 ml of 2.5 mM stock solution) with Klenow polymerase (Boehringer, 2.5 units)
and then incubated at 20°C for 15 minutes to produce blunt ends.
Following this step the product was gel purified as above
and resuspended in 25ml water. Then 5ml from the heavy and 5ml from the light chain
product were used in the assembly. For this process the PCR reaction was carried
out in two steps. Initially no primers were added and the following cycles were
used: 94°C for 1 minute, 50°C for 1 minute, 74°C for 1 minute for
7 cycles to join the heavy and light chains. During the secondary PCR described
above the heavy chain and light chain are tagged with primers encoding the single
chain linker. This tag contains 15 nucleotides on each of the heavy and light chains
complementary to each other and thus allows them to anneal to each other. During
the extension reaction full length joined scFv molecules are formed. At the end
of these 7 cycles the temperature is held at 94°C for 3 minutes and the relevant
outer primers (SfiVHBACK/NotJFOR) added for the "pull-through" amplification. This
amplification consists of 10 cycles: 94°C for 1 minute, 74°C for 2 minutes
an serves to amplify the small amount of linked product formed.
Cloning for Expression, and Expression and Purification of scFv
After assembly the scFv was digested with SfiI/NotI
as described (Marks et. al., 1992) and cloned into a scFv expression vector (Hawkins
et al., 1992 I. Mol. BioI. 226, 889-896) based on pUCl19 (Vieira and Messing,
1987). A new expression vector, pRH2, which has the Myc Tag replaced by a hexahistidine
tag was made to allow purification using metal affInity chromatography. This was
made by inverted PCR site directed mutagenesis (Hemsley et al., 1989 Nucl. Acids
Res. 17, 6545-6551). The vectors are shown in Figure 2 (Seq. ID Nos. 59-62).
To check for expression of full length scFv individual
colonies were picked and grown for four hours with constant shaking in 1 ml 2xTY/0.1%
Glucose/100mg/ml Ampicillin at 30°C. At that stage IPTG was added to a final
concentration of 1 mM and shaking continued for 18 hours. Supernatant was harvested
by centrifugation at 13,000 rpm in a microcentrifuge for 5 minutes. The bacterial
pellet was frozen at -20°C for preparation of plasmid DNA and the supernatant
was analysed by Western blotting using the 9E10 anti-Myc antibody (Ward et al.,
1989 Nature.341, 544-546). Plasmid DNA was then prepared from the bacterial
pellet of colonies thus shown to express full length scFv. From this plasmid preparation
the scFv was subcloned as an SfiI/NotI fragment into pRH2. One litre
cultures of bacteria were grown with constant shaking in 2 litre flasks containing
2xTY/0.1% Glucose/100mg/ml Ampicillin at 30°C to an A600nm of 0.9. At this
stage IPTG was added to a final concentration of 1mM and the incubation continued
for a further 4 hours. The bacteria were then pelleted by centrifugation and the
periplasmic fraction was prepared as described by Skerra et al., (1991 Bio/Technology
The scFv antibody fragment was purified from the periplasmic
fraction utilising the hexahistidine tag. The method is based on that described
by Skerra et al., (Skerra et al., cited above) but it was found that the use of
six histidines and nickel rather than five histidines and zinc was preferable (data
not shown). The periplasmic preparation from a 1 litre culture was loaded onto a
1ml column of Chelating Sepharose Fast Flow (Pharmacia) previously coupled with
nickel ions according to the manufacturers instructions. The column was then washed
with 10 ml of PBS/1M NaCl (pH 7.2) followed by 5ml PBS/1M NaCl/75 mM Imidazole (pH7.2).
The retained scFv was then eluted with 5ml PBS/1M NaCl/300 mM Imidazole (pH 7.2)
and collected as 1ml fractions. The peak protein fractions were identified by determining
the A280nm and these were then dialysed against PBS before analysis by SDS-PAGE.
PCR, cloning and sequencing of V-genes from Follicular Lymphoma and normal Lymph
The PCR amplification from the DNA of biopsy specimens
was successful in all cases apart from the lambda light chain from patient number
5. A number of clones from each patient were sequenced. Analysis of the sequences
derived from the reactive lymph node and from the normal spleen revealed that there
were no repeated sequences. From each of the tumour bearing lymph nodes there were
single repeated sequences. A summary of the sequencing results is shown in Table
2. Amongst the repeated sequences there were up to two base changes which were presumed
to result from PCR errors. Nevertheless a consensus sequence was readily apparent
and in each case there were clones with this consensus sequence. To confirm the
sequence a second independent amplification was performed and further V -genes sequenced.
The same consensus sequence was identified. The repeated V-gene sequences suggest
clonal expansion and thus identifies the tumour V -gene. For three of the five tumour
biopsies analysed here a heterohybridoma was available. PCR amplification, cloning
and sequencing confirmed the sequence identified direct from the lymph node.
The absolute percentage of the tumour derived V-gene varied
and there are several reasons for this. First, the biopsies vary in the degree of
tumour infiltration (although in all cases examined here malignant B-cells comprise
> 50% of the total cells present). Second, the primers will vary in the efficiency
with which they amplify any particular gene - in extreme cases as with the lambda
light chain in patient 4 a chain may not amplify at all. Third, some pseudogenes
can be amplified by these primers and this may reduce the overall percentage of
tumour derived V -genes.
Assembly, expression and purification
The use of PCR assembly avoids the use of multiple restriction
enzymes which may cut V-genes at internal sites. This process used here appears
efficient and does not require the separate preparation of a linker fragment (Clackson
et al., 1991 Nature 352, 624-628). To check the assembly process the linked
product was cloned into an expression vector which included the Myc Tag (Figure
2). Randomly picked clones were grown up and induced as previously described (Hawkins
and Winter, 1992)..Western Blotting using a monoclonal antibody, 9E10, against the
Myc Tag (Ward et al., 1989) demonstrated that 80% of the clones correctly expressed.
For ease of purification the scFv fragment was subcloned into the expression vector
pRH2 containing a hexahistidine tag. A clone from patient 5 was grown up in a 1L
volume the scFV fragment purified from the periplasm. The yield was estimated as
0.5 mg/L/OD600 based on an A280nm of 1.4 for a 1mg/ml solution.
Example 2 - Construction of a Fusion Protein
The BamHI/ClaI env fragment (nt 6537-7674,
nt numbering from Shinnick et al, 1981 Nature 293, 543-548) from pCRIP (gift
from O. Danos, Danos & Mulligan 1988 PNAS 85, 6460-6464) was cloned into
the BamHI/Clal backbone fragment of pZipNeoSV (X) (gift from R. Mulligan,
Cepko et al., 1984 Cell 37, 1053-1062) to generate an intermediate plasmid
A SfiI/NotI cloning site was introduced beyond
the leader peptide sequence between codons corresponding to the 6th and 7th amino
acids (from the N-terminus) in the mature MoMLV env polypeptide. The oligonucleotide
pair envNotrev (5'-CTG CAG GAG CTC GAG ATC AAA CGG GCG GCC GCA CCT CAT CAA GTC TAT
AAT ATC-3', Seq ID No. 49, complementary to MoMLV env nts 5894-5914 with a 33nt
5' overhang encoding a Not1 site and 21nt complementary to the 5' tail of
envSfifor) and envseq 7 (5'-GCC AGA ACG GGG TTT GGC C-3', Seq ID No.50, reverse
complement of MoMLV env nts 6581-6600) was used to prime amplification (from plasmid
pCRIP) of a 739bp fragment downstream ofenv codon 6. A second oligonucleotide pair,
envSfifor (5'-TTT GAT CTC GAG CTC CTG CAG GGC CGG CTG GGC CGC ACT GGA GCC GGG CGA
AGC AGT-3', Seq ID No.51, reverse complement of MoMLV env nts 5873-5893 with a 36nt
5' overhang encoding a SfiI site and 21nt complementary to the 5' tail of envNotrev)
and revMLVpol (5'-AAT TAC ATT GTG CAT ACA GAC CC-3', Seq ID No.52, complementary
to MoMLV pol nts 5277-5249) was used to prime amplification (from pCRIP) of a 702bp
fragment upstream of env codon 7. Amplifications were carried out using Vent polymerase
and reactions were subjected to 15 PCR cycles at 94°C for 1 min, 60°C
for 1 min and 72°C for 1 min. The complementary 21nt tails of the 702 and 739bp
gel-purified PCR products allowed PCR linkage to generate an env gene fragment incorporating
a SfiI/NotI cloning site at the desired position. The two fragments
were mixed and subjected to three PCR cycles (94°C, 40°C, 72°C for
1, 1, and 2 minutes respectively) followed by 17 further cycles of amplification
(94°, 60°, 72° for 1, 1 and 2 minutes respectively) after addition
of oligonucleotides envseq7 and Bglenvrev (5'- TAA TCA CTA CAG ATC TAG ACT GAC ATG
GCG CGT-3', Seq ID No.53, complementary to MoMLV pol nucleotides 5766 to 5785 with
the 5' tail incorporating a BglII restriction site).
The product, a 905bp fragment, was digested with
BglII and BamHI and cloned in forward orientation into the
BamHI site of penvBam/Cla (see above) giving the plasmid pSfi/Notenv. Correct
assembly of this plasmid was confirmed by restriction analysis and dideoxy sequencing
(Sanger et al., 1977 PNAS 74., 5463-5467). A functional B1.8 scFv antibody
was then subcloned from prokaryotic expression vector (Hawkins et al., 1992 J. Mol,
Biol. 226, 889-896) as an Sfi/lNotI fragment into the SfiI/NotI
cloning site of pSfi/Not.Env to generate the plasmid pNIP.env (Fig. 3). The sequence
across the junctions of pNIPenv is shown in Figure 4 (Seq. ID No.63 to 66, including
translation of nucleotide sequence).
Finally, the modified retroviral envelope expression cassette
was subcloned as a HindIII/EcoRI fragment into a modified pSV2Neo plasmid (a gift
from Ashok Venkitaraman, MRC Centre, Cambridge) to generate the plasmid pSVNIPenv
NIH3T3 fibroblasts and the ecotropic retroviral packaging
cell line psi2 (Mann et al., 1983 Cell 33, 153-159) were maintained in DMEM/10%FBS
supplemented with 60µg/ml benzylpenicillin and 100µg/ml streptomycin at
37°C in atmosphere of 5%CO2. The cells were replated twice weekly
using EDT A without trypsin to disrupt the monolayer.
Plasmid pNIPenv was transfected (with pDCneo, a plasmid
containing a neomycin resistance marker) into psi2 cells by calcium phosphate precipitation.
Briefly, 2x105 cells were plated in 90mm tissue culture plates (Nunc),
cultured overnight, washed and fed with 10mls new medium. 10µl plasmid DNA
and 50µl 2M CaCl2 (0.2µm-filtered) were diluted in sterile
water to a volume of 400µl. The CaCl2/DNA mix was added drop wise
to an equal volume of 0.2µm-filtered 2xHEPES-buffered saline (280mM NaCl, 10mM
KCl, 1.5mM Na2HPO4.2H2O, 12mM dextrose, 50mM HEPES,
pH adjusted to 7.05 with 0.5N NaOH) and left to stand for 20 minutes at RT. The
transfection solution (800ml) was added to the cells which were cultured for 16hrs,
washed and refed. G418 selection (lmg/ml) was commenced 24 hrs later and continued
for approximately 2 weeks.
Transfected colonies expressing surface B1.8 single chain
antibody were identified by panning with NIP.BSA-coated beads. Briefly, Tosyl activated
paramagnetic beads (Dynal, Oslo, Norway, Prod. no. 14004) were coated with NIP10.BSA
(approximately 10 NIP-caproate-O-succinimide molecules coupled to each bovine serum
albumin molecule, Hawkins et al., 1992), washed extensively in PBS and blocked with
DMEM/10%FBS. 90mm tissue culture plates containing up to 50 G418-resistant psi2
colonies were rocked gently for 1 hr at 40°C followed by 1 hr at room temperature
with 2x107 (50µl) beads in 5mls DMEM/10%FBS. After 5 washes in PBS,
positive colonies (heavily coated with paramagnetic beads) were easily identified
and were transferred individually for further expansion, cryopreservation and harvest
of cell supernatants.
Therefore it was shown that the specificity of the antibody
is displayed on the surface of the cells, and therefore that the antibody is folded.
Construction of a soluble protein expression vector
To create a soluble expression vector a stop codon and
frame shift mutation were inserted between the antibody gene and the 3' portion
of the MoMLV Envelope gene. The Bl.8 scFv fragment was PCR-amplified (10 cycles,
94°C 1 min, 55°C 1 min, 74°C 1 min) using SfiVHBAK (5'-TAC TCG CGG
CCC AAC CGG CCA TGG CCC AGG TSM ARC TGC AGS AGT C-3', Seq ID No.54) and a forward
primer Not.STOP (5'- AAC AGT TTC TGC GGC CGC CTC CTC AGA GGA C-3', Seq ID No.55)
encoding the nucleotide insertion to create a stop codon and frameshift 5' of the
NotI site. The fragment was then digested with SfiI/NotI and
cloned into pSfi/Not.Env to create the plasmid pNIPstop (Fig 4). Plasmid pSVBCLenv
(Fig 4) was derived from pSVNIPenv by replacing the B1.8 scFv with a control scFv
gene which was PCR-cloned from the BCL1 mouse lymphoma. VH and V&lgr; genes were
PCR cloned and assembled from BCL-derived DNA using standard protocols.
Preparation of plasmid DNA
Plasmids were amplified in E. coli strain TG1 (Gibson,
1984), extracted by alkaline lysis and column purified using the Promega Magic Maxipreps™
DNA purification system (Promega, Madison, WI, USA). The DNA was eluted in water.
The purity of the plasmid prep was confirmed by agarose gel electrophoresis and
by measuring the A260nm/A280nm ratio (in all cases the ratio was >1.7). The purified
plasmid was stored at -20°C. Prior to use the plasmid was adjusted to 160mg/ml
in 200mM NaCl.
Preparation of B1.8 scFv protein
For bacterial expression, the B1.8 scFv gene was cloned
as a PstI/NotI fragment into the vector pRH2, which links a tail of
six histidines to the C-terminus of the scFv and was derived by inverse PCR mutagenesis.
This plasmid was transformed into E. coli, strain TG1 and the scFv protein
expressed and purified on an NP-sepharose column as previously described (Hawkins
and Winter, 1992 Eur. J. Immunol. 22, 867-870). The purified protein was shown to
bind strongly to NIP-BSA using a previously described BLISA (Hawkins and Winter,
1992). As a negative control scFvD1.3 (Hawkins et al., 1992) was cloned into the
expression vector pRH2 and expressed and then purified on a lysozyme column as described
by Hawkins et al., 1992, (cited previously).
Vaccination protocol Male BALB/c mice 10 weeks of age were
used for immunisation. Pre-immunisation blood samples were obtained by tail bleeds.
The blood was centrifuged at 13,000 rpm for 2 minutes in a microcentrifuge to separate
the serum. The serum then stored at -20°C for subsequent assay. Two groups
of mice were immunised some with DNA and some with protein. The two groups were
immunised as described below.
Analysis of immune response
- a) Protein vaccine: Bl.8 scFv protein was adjusted to a concentration of 250
mg/ml in PBS and mixed with an equal volume of CFA. Mice were challenged subcutaneously
with 100ml of this vaccine (12.5mg scFv) at two separate sites. Identical boosts
were administered two and four weeks later. 200ml tail bleeds were obtained 10 days
after the final boost. Blood samples were processed as above.
- b) DNA vaccine: Two groups of three mice were challenged with 50ml (8mg) DNA,
either subcutaneously (sc) in both flanks, or by the intramuscular (im) route (right
and left quadriceps, total DNA for each mouse 16mg). Two identical booster inoculations
were given at one week intervals. 200ml tail bleeds were obtained immediately prior
to the first, second and third challenge and one week after the final boost. Serum
was separated and stored as above.
Individual flat-bottomed wells in flexible 96 well assay
plates (Falcon 3912 MicroTest III) were coated with Bl.8.His or control (Dl.3.His
anti-lysozyme) scFv protein at 25mg/ml overnight in PBS at room temperature. The
use of the histidine tagged scFv to coat the plates has been found to result in
more of the protein retaining its antigen binding capacity. Plates were washed x
3 with PBS, blocked for 2 hrs at 37°C with 3% BSA in PBS and washed x 3 in
PBS. Test serum was added (diluted 1: 100 or 1: 1000 in PBS/3 % B SA) and incubated
for one hour at room temperature. Plates were washed x 3 in PBS and incubated for
one hour at room temperature with a second layer HRP-conjugated polyclonal goat
anti-mouse Fc antibody at 1: 1000 dilution (Sigma, cat. no. AO 168). Plates were
washed x 4 in PBS, developed with ABTS and the A405nm measured after 30 minutes
using a Thermomax™ microplate reader (Molecular devices, Menlo
Immune response to protein vaccine
It was first sought to establish whether mice could mount
an effective anti-idiotypic humoral immune response when challenged with a scFv
murine antibody in CFA. Six mice were challenged subcutaneously with 25mg of the
B1.8 anti-NIP scFv in CFA, with booster doses two and four weeks later. Ten days
after the final challenge, serum from these animals contained insufficient anti-
B 1.8 antibody to give a positive BLISA signal at 1: 100 dilution of the serum.
Immune response to DNA vaccine
Plasmid pNIPenv (Fig 3, see materials and methods for details
of construction) encodes a chimeric fusion protein consisting of the ecotropic MoMLV
envelope polypeptide Pr80env with a scFv anti-NIP antibody fragment (Kd 4x10-8M)
inserted 6 amino acids from the N-terminus. The 33 amino acid MoMLV env leader sequence
is retained, without disruption of the leader cleavage site. As well as the 6 N-terminal
amino-acids from the MoMLV envelope protein the scFv also has a further 6 amino-acids
derived from the pelB leader remaining at the N-terminus. Expression is driven from
promoter/enhancer sequences in the 5' MoMLV long terminal repeat (LTR). Polyadenylation
signal sequences are provided by the 3' MoMLV LTR. When transfected into mouse fibroblasts
(described above), it was found that pNIPenv gave stable cell-surface expression
of functional Bl.8 scFv in fusion with the MoMLVenv protein.
Mice were primed via the subcutaneous (three mice) or intramuscular
(three mice) route with 16mg of pSVNIPenv in 200 mM NaCl, with booster doses one
and two weeks later. Control mice were vaccinated pSVBCLenv. Pre-vaccination, pre-booster
and one week post-vaccination serum samples were tested by ELISA for a humoral response
to B1.8scFv. Prior to the second booster, anti-B1.8 scFv antibodies were detected
at 1:100 dilution of the serum in three of the six pSVNIPenv-vaccinated mice, two
inoculated im and one sc. One week after the second booster, all six mice had easily
detectable anti-B1.8 scFv antibodies which did not cross-react with the D1.3 scFv.
Sera from control pBCLenv-vaccinated mice remained negative in the anti-B1.8 ELISA.
Anamnestic response to protein vaccine after DNA vaccine
After 8 weeks, anti-Bl.8 antibody titres had fallen in
the pSVNIPenv immunised mice. At this point the three mice, originally inoculated
intramuscularly with pNIPenv, were challenged intravenously with 20mg purified Bl.8
scFv protein in PBS. Five days later, serum from these mice contained a greatly
increased titre of anti-Bl.8 antibodies - the average rise was 12 fold and all had
antibodies clearly detectable at 1:1000 dilution.
Boosting with soluble scFv expression vector (pNIPstop)
To test whether boosting with a soluble protein expression
vector would also boost the antibody titre, mice were inoculated with pNIPstop.
Ten weeks after the primary immunisation, the three mice immunised subcutaneously
with pNIPenv were inoculated with 8 mg sc and 8 mg im in 200mM NaCl. Five days after
boosting, test bleeds were obtained and assayed for antibody activity. The serum
titre increased an average of 10 fold and again all were now positive at 1:1000
Comparison of soluble pNIPstop and pNIPenv in generating primary immune response
To demonstrate the importance of the fusion protein to
enhance the immune response the inventors carried out a control experiment to compare
the efficacy of two vectors at stimulating a primary immune response. Two groups
each consisting of two BALB/c mice were used as before. Serum was obtained by tail
bleed as before and then the mice were inoculated weekly x 3 with the appropriate
plasmid. Twenty-eight days after the start of immunisation serum was again obtained
following tail bleeds and assayed for anti-Bl.8 activity. 2/2 from the group inoculated
with the pNIPenv plasmid were positive at 1:100 dilution and 2/2 in the pNIPstop
group were positive. Clearly the env tag is not necessary to stimulate an immune
Confirmation that immune response recognises the native antigen.
A group of five mice immunised four times with DNA (pSV2-BCL1)
encoding the unfused BCL1 scFv fragment also generated humoral responses to the
idiotype, as detected by binding to the BCL1 IgM fragment in an ELISA (not shown).
Moreover, as a clear demonstration of their ability to recognise native antigen
in the form required for therapy, these anti-idiotypic antisera were shown by FACS
analysis to bind to lymphoma cells bearing surface BCL1 Ig. BCL1 cells were pre-incubated
with serum at a 1:20 dilution before staining with FITC conjugated anti-mouse IgG
(Sigma) and followed by FACS analysis. Indeed the immune response was comparable
to that for the BCL1 IgM antibody in CFA (Fig. 11). This was in contrast to antisera
derived from mice immunised with pSV2-B1.8 which bound only weakly to the BCL1 lymphoma
Example 3- Construction of a Vector suitable for use in Human Recipients
The initial vectors used in example 2 above were based
on Moloney Murine Leukaemia virus vectors and contained large stretches of unmodified
viral sequences (Russell et al., 1993 Nucl. Acids Res. 21, 1081-1085). These
vectors were shown to be effective in raising anti-idiotype responses and gave no
untoward effects in the mice inoculated. Although there is no evidence of danger
to man from such vectors it was decided to modify the vectors to avoid any potential
risks. There was some concern about two features of the original vectors: the retroviral
envelope gene (as it could theoretically be recombined into another retrovirus thus
changing its tropism) and the packaging signal (which might allow packaging of the
injected DNA into existing human retroviruses). Whilst changing the vector it was
decided to incorporate changes which improve the vectors for use in man: - the promoter
used to drive expression of the idiotypic scFv was changed to the Rous Sarcoma Virus
(RSV) promoter as that has been shown to give expression when directly injected
into non-human primate muscle (Jiao et al., 1992 Hum. Gene Ther. 3, 21-33).
The present inventors also used a vector which contains a bacterial single strand
origin of replication to allow the production of ssDNA which will facilitate sequencing
the scFv portion (which is specific for that individual patient) of the vector before
injecting into the patient. The vector used is based on a commercially available
vector pRc/RSV (British Biotechnology /Invitrogen).
To convert this vector backbone into a vector suitable
for genetic immunisation it was desirable to introduce leader sequences, termination
signals and to allow for the production of fusion proteins. Fusion proteins do not
appear to be necessary for the production of anti-idiotype responses but one way
of enhancing the immune response might be to attach suitable proteins - perhaps
foreign proteins or perhaps cytokines (Tao & Levy, 1993 Nature. 362, 755-758).
As fusion proteins were not necessary in animal models the initial human trial will
use only a short peptide tag but this is one area for potential future modifications
to the protocol.
The vector pSfi/Not.Tag1 was modified to replace the pelB
leader with the human immunoglobulin VH1 leader sequence which permits the encoding
of an SfiI cloning site without modification of the amino-acid sequence. This was
introduced with oligonucleotides using the HindIII/PstI cloning sites and confirmed
This was then cloned as an EcoRI/Blunt -
HindIII fragment into the NotI/Blunt - HindIII cut vector pRc/RSV
to give the sequence (Seq ID No.56) between the HindIII/Xbal sites
as shown in Figure 5. The scFv for an individual patient can be inserted at the
sites shown by the symbol ^.
The vector was then tested in two ways:
- (i) The scFv B1.8 was cloned into the vector and then the resultant construct
transfected into two cell lines - NSO (a myeloma cell line) and NIH 3T3 (a fibroblast
cell line). Utilising the neomycin resistance gene in pRc/RSV stable transfectants
were isolated and the supernatant assayed for scFv B1.8 antigen binding activity.
In both cases the antibody fragment was expressed and bound to the hapten NIP -
the antigen recognised by the monoclonal antibody B1.8. Clones were isolated from
the NSO transfected cells and shown to produce 1-3 mg/L of functional scFv in spent
- (ii) The plasmid was used in a genetic immunisation experiment and compared
to pSV2-B1.8. They gave comparable results and appear to be superior to a further
vector which encodes the Fd bacteriophage gene 8 protein between the NotI
and XbaI site (Figure 6). The likely explanation is that in transfection
experiments the level of expression was 10-100 fold lower for the scFv B1.8-Gene
8 fusion. Other investigators have found a strong correlation of level of expression
and immune response when using genetic immunisation to raise immune responses to
viral proteins (G. Rhodes, personal communication).
Figure 6 (Immunisation of mice with vectors utilising the
RSV promoter) shows the results of idiotypic immunisation against the scFv B1.8.
The response for individual mice as determined by ELISA at 1:100 serum dilution
are shown. The mice were immunised intra muscularly at weeks 0, 1, 2. Note one mouse
with the stop vector (pSV2 - B1.8) had a poor response and the response of mice
immunised with the gene 8 fusion vector were poor (VIII 1 and VIII 2) whereas both
those with the peptide tag vector gave good responses (Tag1 and Tag2).
The sequence (Seq ID No.58) of the final vector pVAC1 is
given in Figure 7 together with a map of the unique restriction sites (Figure 8).
The sequence in lower case letters in Figure 7 corresponds to the sequence shown
in Figure 5 through to the two stop codons, The vector pVAC1 is available from the
present inventors (at the Cambridge Centre for Protein Engineering, Cambridge, United
Figure 9 shows a diagrammatic representation of the vector
pVAC1 indicating important restriction sites and important genes.
Figure 10 is a graph of O.D. (405nm) against time for male
and female mice immunised (by direct injection of DNA) with the pVAC vector expressing
B1.8scFv (pVAC1.Bl-8). The graph shows that, for individual mice, there was a clear
increase in titre following immunisation.
Although the neomycin resistance gene is useful for in
vitro testing it is unnecessary for human immunisation. The SV40 promoter used to
drive the neomycin resistance gene is also associated with the same risks as other
strong promoters. The plasmid to be used for human trials thus has the neomycin
gene deleted by an SfiI/Bst BI digest and then blunt ligation. This
prevents any such risk.
Discussion of Examples 1- 3
The present inventors have demonstrated that a plasmid
vaccine encoding a single chain murine antibody/retroviral envelope fusion protein
induces a strong humoral immune response to the antibody moiety in BALB/c mice,
whereas vaccination with the purified scFv protein mixed with Complete Freunds Adjuvant
gives no detectable response. Induction of B-cell memory appears to occur as boosting
with either soluble protein or a soluble scFv expression vector was effective at
producing a rapid rise in antibody titre.
The humoral anti-B1.8 response to the plasmid vaccine pNIPenv
was clearly superior to that raised against purified B1.8 scFv protein mixed with
Complete Freunds Adjuvant. The approach disclosed herein has several advantages.
Following gene transfer, there is likely to be a continuous supply of the target
antigen, diminishing over a period of days or weeks, whereas injected protein may
have a very short half-life. This prolonged exposure to newly synthesised antigen
may be important for an optimal immune response and this argument has been used
to explain the superiority of live compared to killed viral vaccines.
Antigen-specific T helper cells can amplify both humoral
and cellular immune responses by direct cell interaction and by providing appropriate
stimulatory cytokines. Several mechanisms can be envisaged whereby the plasmid vaccine
may recruit helper T cells more efficiently.
Regardless of the mechanisms involved, the vaccination
strategy employed in this study gave a strong humoral immune response to a weakly
immunogenic single chain antibody fragment and was superior to vaccination with
purified protein plus adjuvant. The scFv gene used in this study could be replaced
with a variety of genes or gene fragments encoding other weakly immunogenic idiotypic
Episomal vectors such as those based on EBV or papova virus
may have advantages over current vectors. These should allow high copy number episomal
replication and may be more effective. New vectors utilising the pVAC1
HindIII/Xba1 insert have been constructed with the expression plasmid
pCEP4 (Invitrogen) which contains the EBV origin of replication and BBNA. These
have given enhanced levels of expression and greater stability of expression in
cell culture experiments using the human osteosarcoma line 791T and may be more
efficient in vivo although they also raise new safety issues for human use. More
efficient methods of transfection such as liposome mediated and receptor mediated
delivery may also improve the efficiency of the process.
Example 4 - CD4+ T cell mediated immunity against B cell malignancies
ScFvPVXCP construct assembly and characterisation
Figure 12 illustrates the assembly of scFv-PVXCP fusions.
The scFv from A31 lymphoma and 5T33 myeloma as well as A31FrC were assembled and
cloned into pcDNA3 previously (King C.A. et al, Nature Medicine,
4 1281-1286 1998). ScFvs were fused in frame to the fourth codon of PVXCP
(isolate CP4) via four amino-acid linker the same as in scFv fused to fragment C
(King et al, 1998). PVXCP cDNA was kindly provided by Dr. K.Kanyuka (Long Ashton,
LACR, Somerset, and U.K.) A31PVXCP and 5T33PVXCP fusion genes were made by two-step
PCT as previously described (Spellerberg, 1997) using HF polymerase Kit (Clontech,
Basingstoke, U.K.). To assemble A31PVX gene primers were as following: for the first
step to amplify A31 gene 5' taatacgactcactatagggagac 3' (referred to as T7), was
forward and 5' ggctggaggtccgggtccacgtttgatctccacctt 3' was reverse and to amplify
PVXCP gene 5' aaacgtggacccggacctccagccaacaccactcaagct 3' forward and 5' accgcggccgctagttatggtgggggtagtgaa
3' reverse. T7 primer was corresponded to sequences derived from pcDNA3 plasmid.
For the second step T7 was used as a forward primer and a reverse primer for PVXCP
gene assembly was used as reverse.
To assemble 5T33PVXCP gene in the first step for the 5T33
gene T7 was also used as a forward primer and 5' ggctggaggtccgggtcctttgatttccagcttggt
3' as reverse; for the PVXCP gene 5' atcaaaggacccggacctccagccaacaccactcaagct 3'
was used as a forward primer, the reverse primer was the same as for A31PVXCP gene
assembly. For PVXCP (further referred to as PVXCP alone) alone gene construct 5'
3' was a forward primer and the reversed primer was as for the fusion constructs.
The forward primer contained an Ig leader sequences, the same as in A31 scFv. Assembled
constructs were cloned into pcDNA3 (invitroGen BV, Leek, Netherlands) plasmid using
HindIII and Notl restriction sites. The expression of the assembled
constructs was checked by TNT (Promega) and by transfection of the plasmids into
Supernatants were assayed for PVXCP by ELISA using a polyclonal
anti-PVX IgG coating antibody and an alkaline phosphatase-conjugated polyclonal
anti-PVX IgG detecting antibody, according to manufacturer's instructions (Bioreba,
Peterborough, U.K.). All PVXCP-containing constructs produced detectable protein
in the supernatants, whereas constructs containing scFv alone were negative. SDS-PAGE
with Western blotting was carried out, and probed with antiserum derived from p.scFv5T33-PVXCP
vaccinated mice in 1/100 dilution with a subsequent incubation with anti-mouse-HRP
conjugate (Binding Site, Birmingham, UK) and developing using ECL plus reagent.
Vaccination and tumor challenge Mice were vaccinated at
6-10 weeks of age with 50 µg of plasmid DNA in saline
in two sites in the quadriceps muscles, on days 0, 21 and 42, unless indicated otherwise.
The A31 lymphoma was passaged as described previously (King, C. A. et al.
Nat. Med. 4, 1281-1286. 1998). The 5T33 myeloma was passaged in C57BL/KaLwRij
mice by intravenous injection. Challenge was on day 63 by intravenous injection
with 104 5T33 cells or with 5x104 A31 cells.
Depletion experiments Mice were vaccinated 3 times with p.scFv5T33-PVXCP
or p.scFvA31-PVXCP as described above. On day 56 mice were bled and the sera were
assayed for the presence of anti-5T33 or anti-A31 antibody. On day - 7, -4, -1 before
challenge and on day +2, +5 mice received intraperitoneal injection of 100 µg
of either anti-CD4 (YTS 191.1.2) or anti-CD8 antibody (YTS126.96.36.199) (Cobbold, S.
P., Jayasuriya, A., Nash, A., Prospero T. D. and Waldmann, H. Therapy with monoclonal
antibodies by elimination of T-cell subsets in vivo. Nature 312 548-551
1984), or normal rat IgG as a control. On day -1 before or day +6 after challenge,
peripheral blood mononuclear cells were collected and double-stained with anti-CD4/anti-CD3
or anti-CD8/anti-CD3 to assess depletion and analysed by fluorescence-activated
cell sorting. It was also checked that none of the depleting antibodies affected
the behaviour of the tumours. Animals were observed for survival.
Antibody responses Anti-idiotypic antibody levels in serum against IgM from
the A31 lymphoma or IgG from the 5T33 myeloma were measured by ELISA as described
(King C.A. et al, Nature Medicine, 4 1281-1286 1998). For anti-PVXCP
antibody, PVXCP isolated from CsCl gradient purified virions (kindly provided by
Dr. S. M. Angel) using the LiCl method of Goodman et al. (Goodman, R. M.,
Horne, R. W. and Hobart, J. M. Reconstitution of potato virus X in vitro. Virology
68 299-308 1975) was coated at 10 µg/ml, using the same ELISA conditions.
Measurement of IgG1 and IgG2a subclasses in mouse serum antibodies was as described
(King C.A. et al, Nature Medicine, 4 1281-1286 1998). To assay sera
from normal human individuals for anti-PVXCP antibody, a similar ELISA was used,
with goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Sigma,
Poole, UK) at 1:2000 dilution for detection.
Electron microscopy Immunosorbent electron microscopy was used with grids
coated with anti-PVX IgG (Bioreba). The grids were then negatively stained with
1% (w/v) uranyl acetate and examined using a JEOL 1200X microscope.
Vaccination with the DNA fusion construct (scFv-PVXCP) promotes anti-Id antibody
production in lymphoma (A31) and myeloma (5T33) models. The constructs containing
scFv from the mouse lymphoma (A31) or myeloma (5T33) were tested in syngeneic mice
either alone (p.scFv) or fused to PVXCP sequence (p.scFv-PVXCP). In each case, scFv
sequence alone failed to induce significant levels of anti-Id able to recognize
whole immunoglobulin (Fig. 13 (A)(B)). However, the scFv-PVXCP fusion genes induced
anti-Id antibody in both models (A)(B). Levels were variable, with only 30-50% of
mice responding in the A31 lymphoma model, and 90-100% in the 5T33 model. These
results were similar in repeated experiments. As expected, the control plasmid containing
PVXCP sequence alone did not induce anti-Id antibody. Constructs containing PVXCP
sequence either alone, or in fusion, induced antibodies against PVXCP (Fig. 13 (C)(D)),
with the p.scFv5T33-PVXCP fusion consistently being most efficient (D), possibly
due to a higher level of expression. For all responses, there was an increase in
antibody following the second injection on day 21, and a further smaller rise following
the third injection on day 42. Analysis of pooled sera for immunoglobulin subclasses
of antibodies against either scFv or PVXCP showed dominance of IgG2a, with only
very low levels of IgG1. In the A31 model, IgG1 could not be detected, and in the
5T33 model, the ratios of IgG1: IgG2a for anti-Id or anti-PVXCP were 0.08:1 and
DNA fusion constructs induce protective immunity against lymphoma and myeloma
Vaccination of syngeneic mice with the p.scFv-PVXCP fusion
constructs induced protective immunity against both lymphoma (A31) and myeloma (5T33)(Fig.
14 A and 16B). In each case, p.scFv alone was ineffective, and the p.PVXCP control
was negative. For the lymphoma, in which anti-Id antibodies were detected in only
~50% of mice, all showed evidence for protection, indicating a requirement for very
little or no antibody. Protection experiments have been repeated three times with
similar results. In the A31 model, some mice developed lymphoma at a late stage
(>90 days), but there was no clear correlation between long-term survival and
antibody levels at challenge. Further investigation is required to assess the mechanism
of escape at this late stage. In both models, injection of separate plasmids encoding
scFv or PVXCP failed to induce protection, demonstrating a requirement for fusion
(data shown for the 5T33 model in Fig.15).
Protection against lymphoma and myeloma involves CD4+ T cells
For the A31 lymphoma, depletion of CD4+ T cells post-vaccination,
by repeated injection of the anti-CD4 monoclonal antibody (mAb), completely abrogated
protection (Fig.16 A). This contrasts with results using a construct containing
scFv sequence fused to a sequence encoding the Fragment C of tetanus toxin (scFv-FrC).
As reported previously (King C.A. et al, Nature Medicine, 4 1281-1286
1998), and confirmed in Fig.16 B, this construct induces protection against the
A31 lymphoma. However, this protection is not abrogated by depletion of CD4+ T cells
(Fig. 16 B), and is therefore probably depends on antibody. It appears that antibody
may be less critical for the protection induced by scFv-PVXCP. In the myeloma model,
protection induced by the scFv-PVXCP construct was again abrogated by depletion
of CD4+ T cells (Fig. 17). This result is consistent with the ineffectiveness of
anti-Id antibody in protection against a surface Ig-ve tumor, and with the involvement
of a cellular mechanism. There was no effect on protection against myeloma when
CD8+ T cells were depleted (Fig. 17). The role of CD8+ T cells in protection against
the A31 lymphoma was more difficult to evaluate since, in contrast to 5T33, depletion
had some effect on the growth of the A31 tumor.
ScFv-PVXCP fusion protein forms self aggregating particles To investigate
the molecular nature of the expressed scFv-PVXCP fusion protein, constructs were
transfected into Cos-1 cells and supernatants collected. The fusion protein from
the 5T33 myeloma was expressed at the highest level (~50ng/ml), and this was therefore
selected for further investigation. Using ELISA to detect PVXCP protein, we found
that centrifugation at 40,000 rpm (135,000 x g) for 2 hours sedimented all detectable
PVXCP reactivity, indicating that the fusion protein was of large molecular size.
A similar result was obtained using plant-derived PVXCP alone. Since the fusion
protein or the free PVXCP could be resolved by SDS-PAGE to monomers of the expected
sizes of 54KD or 24 KD respectively (Fig. 18 A), the expressed proteins must be
undergoing self aggregation, presumably mediated by the PVXCP component. The supernatants
containing expressed scFv-PVXCP or PVXCP proteins were examined by electron microscopy
using immunotrapping with anti-PVX IgG. Aggregates were detectable (Fig. 18 B) with
an appearance was similar to that of PVXCP isolated from virus particles, reflecting
the well-documented tendency of Potexvirus coat proteins to self-aggregrate. Under
physiological conditions, PVXCP forms aggregates comprising two-layer disks with
9 subunits per layer, and stacks thereof (Goodman, R. M., Horne, R. W. and Hobart,
J. M. Reconstitution of potato virus X in vitro. Virology 68 299-308
1975, Erikson, J. W., Bancroft, J. B. and Stillman, M. J. Circular dichroism studies
of papaya mosaic virus coat protein and its polymers. J. Mol. Biol.
147 337-349 1981). It appears that fusion of scFv does not change the shape
or size of the aggregates, and that the scFv fused to PVXCP will present multiple
copies on the PVXCP aggregates. As expected, under the conditions of expression,
in the absence of viral RNA, the PVX CP subunits do not form virus-like helices.
Discussion of Example 4
DNA constructs provide a platform for manipulation of tumor-derived
genes, with the aim of optimising presentation of the encoded antigen to the immune
system (Gurunathan, S., Klinman, D.M. & Seder, R.A. DNA vaccines: immunology, application,
and optimization. Annu. Rev. Immunol. 18, 927-974 2000). For cancer,
DNA vaccines allow rapid testing of the ability of chosen sequences to induce effector
mechanisms able to suppress tumor growth in model systems. For idiotypic antigens
of B-cell tumors, it was already clear that simple constructs containing scFv only
were unable to induce significant anti-Id responses (Stevenson, F.K. et al.
Idiotypic DNA vaccines against B-cell lymphoma. Immunol. Rev. 145,
211-228 1995), but that fusion of the FrC sequence of tetanus toxin led to promotion
of protective immunity against lymphoma and myeloma (King C.A. et al,
Nature Medicine, 4 1281-1286 1998). However, the majority of patients
will have pre-existing immunity against tetanus toxin, and, although this does not
appear to prevent induction of immunity against the scFv-FrC fusion gene (King C.A.
et al, Nature Medicine, 4 1281-1286 1998), it could affect the pathways
induced. The inventors therefore chose to investigate an alternative promotional
gene, derived from a plant viral coat protein (PVXCP). In contrast to FrC, no antibody
against PVXCP was detected in human sera (data not shown). Therefore, there would
be no pre-existing antibody at the priming stage, although such antibody would develop
after vaccination. However, independently of the fact that this is a primary antigen
in humans, the inventors have found that the scFv-PVXCP fusion gene generates a
distinct CD4+ T-cell mediated protective mechanism against B-cell tumours.
The requirement for fusion of the scFv and PVXCP proteins
to promote a specific T-cell response appears to be an example of "linked T-cell
help". This phenomenon, in which an immunologically silent determinant can be rendered
immunogenic if linked to a dominant pathogen-derived antigen has been described
for a mucin (MUC-1) peptide linked to a parasite-derived peptide, delivered within
a whole Ig molecule (Gerloni, M. et al. Functional cooperation between T
helper cell determinants. Proc. Matl. Acad. Sci. USA 97, 13269-13274
2000). Although in that model both epitopes were foreign, and tumor protection was
not assessed, activation of the CD4+ T cells by linkage was clearly demonstrated,
with the mechanism likely to involve up-regulation of the costimulatory ability
of antigen-presenting cells (Gerloni, M. et al. Functional cooperation between
T helper cell determinants. Proc. Natl. Acad. Sci. USA 97, 13269-13274
2000). Here, the inventors have shown that CD4+ T cells against a linked autologous
tumor antigen can also be promoted by this mechanism. The CD4+ T-cell population
appears able to protect against lymphoma, and is clearly critical for protection
against surface Ig-ve myeloma.
One feature of the expressed scFv-PVXCP fusion protein
is that it forms aggregates, including stacked-disk structures (Goodman, R.M., Horne,
R.W. & Hobart, J.M. Reconstitution of potato virus X in vitro. Virology
68, 299-308 1975). Activation of the immune response is known to be influenced
by the molecular form of the antigen, with large aggregates being particularly immunogenic,
presumably because pathogens commonly have this structure (Zinkernagel, R.M. Immunology
and autoimmunity studied with viruses. in The molecular basis of cellular defence
mechanisms 105-129 Wiley, Chichester, 1997). For DNA vaccines, it is not yet
known whether the major route of presentation in vivo involves export from
transfected muscle cells (Ulmer, J.B., Deck, R.R., DeWitt, C.M., Donnely, J.J. &
Liu, M.A. Generation of MHC class I-restricted cytotoxic T lymphocytes by expression
of a viral protein in muscle cells: antigen presentation by non-muscle cells.
Immunology 89, 59-67 1996, Corr, M., von Damm, A., Lee, D.L. & Tighe,
H. In vivo priming by DNA occurs predominantly by antigene transfer. J.
Immunol. 163, 4721-4727 1999) or direct presentation by transfected
APCs (Condon, C., Watkins, S.C., Celluzzi, C.M., Thompson, K. & Falo, L.D., Jr.
DNA-based immunization by in vivo transfection of dendritic cells. Nat. Med.
2, 1122-1128 1996, Casares, S., Inaba, K., Brumeanu, T.D., Steinman, R.M.
& Bona, C.A. Antigen presentation by dendritic cells after immunization with DNA
encoding a major histocompatibily complex class II-restricted viral epitope.
J. Exp. Med. 186, 1481-1486 1997). All the constructs incorporate
a leader sequence, and fusion proteins can be exported from COS-1 cells. However,
fusion of PVXCP gives rise to a different immune outcome as compared to Fragment
C of tetanus toxin. For scFv-PVXCP, the anti-Id antibody response is almost entirely
of the IgG2a subclass, whereas fusion of scFv-FrC induces a high level of IgG1 (King
C.A. et al, Nature Medicine, 4 1281-1286 1998). In the 5T33 model,
PVXCP fusion induced anti-Id with an IgG1:IgG2a ratio of 0.08:1, dramatically contrasting
with the ratio of 28:1 induced by scFv-FrC (King C.A. et al, Nature Medicine,
4 1281-1286 1998). This suggests that the aggregated antigen amplifies the
TH1-dominated response already activated by the cytokine environment
generated by injection of DNA (Roman, M. et al. Immunostimulatory DNA sequences
function as T helper-1-promiting adjuvants. Nat. Med. 3, 849-854 1997).
Induction of an IgG2a-dominant response has also been noted using peptides linked
to chimeric virus particles (McInerney, T.L., Brennan, F.R., Jones, T.D. & Dimmock,
N.J. Analysis of the ability of five adjuvants to enhance immmune responses to a
chimeric plant virus displaying an HIV-1 peptide. Vaccine 17, 1359-1368
The principles of amplification of immune responses against tumor antigens by genetic
linkage may be widely applicable. For DNA vaccines, the inventors have already shown
that promotion of antibody responses against several tumour antigens, including
MUC-1 and carcinoembryonic antigen, can be achieved by fusion with FrC 30. Stevenson,
F.K. et al. Genetic vaccination against defined tumour antigens of B-cell
malignances. Rev. Clin. Exp. Hematol. 9 2-21 1999). It appears that
PVXCP may be superior in amplifying CD4+ T cell responses against linked tumor antigens,
and these are being tested. Promotion of CD8+ T-cell responses may also occur via
linkage, and although the murine scFv sequences contain few or no candidate MHC
Class I-binding peptides (Zhu, D. et al. Immunoglobulin VH gene
sequence analysis of spontaneous murine immunoglobulin-secreting B-cell tumours
with clinical features of human disease. Immunology 93, 162-170 1998),
these can be found in other tumour antigens. It is clear that there are many effector
pathways that could act against cancer cells, and that fusion genes can be selected
to activate those most appropriate for the target antigen.
In summary, the present inventors have genetically fused
PVXCP to a lymphoma-associated antigen (scFv-A31) and used the construct for DNA
vaccination of mice. This led to elevated antibody responses and protection against
challenge with A31 lymphoma. Moreover, the inventors have shown that this protection
was mediated by CD4 positive cells. This is in contrast with DNA vaccination using
fusion of FrC to scFv-A31, where such protection is mediated only by antibody to
scFv. The inventors have also fused a myeloma-specific antigen scFv-5T33 to PVXCP
with subsequent vaccination of mice with the construct. This resulted in induction
of antibody response against scFv-5T33 and protection against challenge with 5T33
myeloma. The inventors have also determined that such protection against myeloma
was mediated by CD4 positive T cells. This depletion was achieved by using anti-CD4+
antibodies to remove cells prior to challenge by the tumour.
The inventors have concluded that plant viral coat proteins,
e.g. PVXCP, can act as an adjuvant sequence for induction of protective immunity
against at least lymphoma and myeloma. They have further noted that there is some
distinction between PVXCP and FrC, both in terms of molecular structure and in terms
of the pathways of immunity induced via DNA vaccination.
Example 5 - Immunogenic properties of PVXCP-fusion vaccine in BCL1 lymphoma model
The inventors evaluated the immunogenic properties of PVXCP-based
DNA vaccines in another, BCL1 lymphoma model, which is similar to the A31 lymphoma
model of example 4 and also displays a tumour-specific immunoglobulin on the cell
scFv derived from BCL1 tumour immunoglobulin was fused
to PVXCP following the procedure described in example 4. Mice were vaccinated with
the resulting p.scFvBCL1-PVXCP vaccine, which generated antibody responses against
BCL1 immunoglobulin and protected the mice upon challenge with BCL1 tumour. The
results are shown in Fig. 19.