U.S. patent application number 13/017885 was filed with the patent office on 2013-08-22 for peptide ii.
The applicant listed for this patent is Miles Carroll, Richard Harrop, Susan Kingsman. Invention is credited to Miles Carroll, Richard Harrop, Susan Kingsman.
Application Number | 20130217116 13/017885 |
Document ID | / |
Family ID | 9931005 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130217116 |
Kind Code |
A1 |
Carroll; Miles ; et
al. |
August 22, 2013 |
Peptide II
Abstract
There is provided an MHC class II peptide epitope from 5T4
antigen. In particular, there is provided a peptide epitope of 5T4
which comprises one of the following: (i) the minimal epitope from
the amino acid sequence shown as SEQ ID No. 2; (ii) the minimal
epitope from the amino acid sequence shown as SEQ ID No. 3; (iii) a
minimal epitope from the region 281-420 of 5T4. There is also
provided a vaccine comprising such a peptide (or precursor thereof)
and its use to treat and/or prevent a disease, in particular a
cancerous disease.
Inventors: |
Carroll; Miles; (Oxon,
GB) ; Harrop; Richard; (Oxford, GB) ;
Kingsman; Susan; (Oxon, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Carroll; Miles
Harrop; Richard
Kingsman; Susan |
Oxon
Oxford
Oxon |
|
GB
GB
GB |
|
|
Family ID: |
9931005 |
Appl. No.: |
13/017885 |
Filed: |
January 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10504603 |
Aug 12, 2004 |
7910109 |
|
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PCT/GB03/00618 |
Feb 13, 2003 |
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13017885 |
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Current U.S.
Class: |
435/325 ;
530/324; 530/326; 530/350; 536/23.4; 536/23.5 |
Current CPC
Class: |
A61K 2039/5158 20130101;
C07K 14/4748 20130101; C12N 2799/021 20130101; A61K 39/00 20130101;
A61K 38/00 20130101; C07K 7/08 20130101 |
Class at
Publication: |
435/325 ;
530/326; 530/324; 530/350; 536/23.5; 536/23.4 |
International
Class: |
C07K 7/08 20060101
C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 13, 2002 |
GB |
0203420.5 |
Claims
1. An isolated peptide consisting of the amino acid sequence of SEQ
ID NO: 3.
2. (canceled)
3. A polyepitope string comprising two or more peptides, wherein
each of the peptides consists of the amino acid sequence of SEQ ID
NO: 3.
4. A composition comprising the isolated peptide according to claim
1 in association with a cell penetrator.
5. A composition comprising the polyepitope string according to
claim 3 in association with a cell penetrator.
6. An isolated nucleic acid sequence encoding a peptide, wherein
said peptide has an amino acid sequence consisting of the amino
acid sequence of SEQ ID NO: 3.
7. An isolated nucleic acid sequence encoding a polyepitope string
comprising two or more peptides, wherein each of the consists of
the amino acid sequence of SEQ ID NO: 3.
8. A cell pulsed with the isolated peptide according to claim
1.
9. An antigen presenting cell according to claim 8.
10-21. (canceled)
22. A composition comprising an isolated peptide consisting of the
amino acid sequence of SEQ ID NO: 3 that is bound to an adjuvant.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/504,603 filed Aug. 12, 2004, which is the
U.S. National Phase under 35 U.S.C. .sctn.371 of PCT/GB03/00618,
filed Feb. 13, 2003, which claims the benefit of priority to
British Patent Application No. 0203420.5, filed on Feb. 13, 2002.
The disclosures of all priority applications are incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to peptide epitopes of 5T4
antigen, and their use in immunotherapy.
BACKGROUND TO THE INVENTION
[0003] Prior to the identification of specific human tumour
antigens, many clinical trials were performed attempting to
immunise cancer patients against either whole cancer cells or
subcellular fractions form cancer cells. The identification of
genes encoding tumour antigens, however, has made it possible to
develop specific immunotherapies based on attacking tumour cells
bearing the identified antigens. A variety of clinical approaches
utilising these genes or gene products are possible as summarised
in the following table.
TABLE-US-00001 Active immunotherapy ("Cancer vaccines") 1.
Immunisation with: i) purified antigen ii) immunodominant peptide
(native or modified) iii) "naked" DNA encoding the antigen iv)
recombinant viruses encoding the antigen v) antigen presenting
cells pulsed with protein or peptide (or transfected with genes
encoding the antigen) 2. Use of cytokine adjuvants such as IL-2 and
IL-12 administered systemically or encoded by the immunising vector
Passive immunotherapy ("Adoptive immunotherapy") 1. Transfer of
cells sensitized in vitro to the specific antigen (bulk or cloned
populations) 2. Transduction of effector cells (or stem cells) with
genes encoding T cell receptors that recognise sepcific
antigens.
[0004] Immunisation with intact protein has the potential advantage
of simultaneously immunising against both class I and class II
epitopes but requires extensive and time-consuming efforts to
purify large amounts of tumour antigen. The identification of class
I and class II peptides within a tumour antigen makes it possible
to immunise with high levels of pure synthetic peptide. The peptide
approach also has the advantage that one can choose between a class
I and a class II type response (or mixture) by choosing which
epitopes to use. Immunisation with peptide also means that
subdominant and/or cryptic epitopes can be chosen (as the need for
antigen processing may be bypassed or reduced to a "trimming" role)
in order to stimulate a different subset of T cells. Also the
peptide may be modified (for example at their HLA class I or II
anchor sites) to increase their immunogenicity.
[0005] In the past few years, much attention has been given to the
role of CD8+ T cells in tumour immunity. Tumour-specific CD8+ CTLs
have been shown to be capable of lysing tumour cells directly and
eradicating tumour masses in vivo in animal models. However, CD4+ T
cells are also thought to play a critical role (Wang and Rosenberg
(1999) Immunological Reviews 170:85-100) and it may be that optimal
cancer vaccines require the participation of both CD4+ and CD8+ T
cells.
[0006] A number of oncofoetal or tumour-associated antigens (TAAs)
have been identified and characterised in human and animal tumours.
In general, TAAs are antigens expressed during foetal development
which are downregulated in adult cells, and are thus normally
absent or present only at very low levels in adults. Tumour cells
have been observed to resume expression of TAAs, and the
application of TAAs for tumour diagnosis, targeting and
immunotherapy has therefore been suggested.
[0007] The TAA 5T4 (see WO 89/07947) has been previously
characterised. It is a 72 kDa glycoprotein expressed widely in
carcinomas, but having a highly restricted expression pattern in
normal adult tissues (see Table 1). It appears to be strongly
correlated to metastasis in colorectal and gastric cancer. The full
nucleic acid sequence of human 5T4 is known (Myers et al., 1994 J
Biol Chem 169: 9319-24).
TABLE-US-00002 TABLE 1 Distribution of Human 5T4 Tumour Type 5T4
Frequency (%) Breast 84 Ovarian 71 Gastric 74 Colorectal 85
[0008] (Starzynska et al., Eur J Gastroenterol Hepatol 1998 June;
10(6):479-84; Starzynska et al., Br J Cancer 1994 May;
69(5):899-902; Starzynska et al., Br J Cancer 1992 November;
66(5):867-9)
[0009] 5T4 has been proposed as a marker, with possible mechanistic
involvement, for tumour progression and metastasis potential
(Carsberg et al., (1996) Int J Cancer 1996 Sep. 27; 68(1):84-92).
5T4 has also been proposed for use as an immunotherapeutic agent
(see WO 00/29428).
SUMMARY OF THE INVENTION
[0010] The present inventors have identified a number of MHC class
I and II restricted epitopes of 5T4. The identification of
particular antigenic peptides provides new opportunities for the
development of diagnostic and therapeutic strategies against
cancer.
[0011] Thus the first aspect of the present invention relates to
MHC class II epitopes of 5T4 antigen. The invention provides
peptide epitopes of 5T4 antigen which are capable of being
presented in conjunction with an MHC class II molecule such that
they are specifically recognised by a T cell.
[0012] In particular, the present invention provides a peptide
epitope of 5T4 which comprises one of the following: [0013] (i) the
amino acid sequence shown as SEQ ID No. 2; [0014] (ii) the minimal
epitope from the amino acid sequence shown as SEQ ID No. 3; [0015]
(iii) a minimal epitope from the region 281-420 of 5T4.
[0016] Further aspects of the invention relate to: [0017] a
polyepitope string comprising such a peptide. [0018] such a peptide
epitope, or such a polyepitope string in association with a cell
penetrator. A such a peptide epitope, or such a polyepitope string
associated with a tetramer. [0019] a nucleic acid sequence capable
of encoding such a peptide epitope or polyepitope string (and
optionally an associated cell penetrator). [0020] a vector system
capable of delivering such a nucleic acid sequence to a cell.
[0021] a cell pulsed with such a peptide epitope (or a precursor
thereof). [0022] a vaccine comprising such a peptide epitope, a
polyepitope string, nucleic acid sequence, vector system and/or
cell. [0023] the use of such a vaccine in the manufacture of a
medicament for use in the prevention and/or treatment of a disease.
[0024] a method for treating and/or preventing a disease in a
subject in need of same which comprises the step of administering
an effective amount of such a vaccine to the subject. [0025] an
agent capable of binding specifically to such a peptide and/or
nucleic acid sequence. [0026] a method which comprises the step of
detecting the presence of such a peptide, nucleic acid or agent in
a subject. [0027] a T cell line or clone capable of specifically
recognising such a peptide epitope in conjunction with an MHC class
II molecule.
[0028] Other aspects of the present invention are presented in the
accompanying claims and in the following description and
discussion. These aspects are presented under separate section
headings. However, it is to be understood that the teachings under
each section heading are not necessarily limited to that particular
section heading.
DETAILED DESCRIPTION OF THE INVENTION
Epitopes
[0029] The present invention relates to peptide epitopes.
[0030] The term "peptide" is used in the normal sense to mean a
series of residues, typically L-amino acids, connected one to the
other typically by peptide bonds between the .alpha.-amino and
carboxyl groups of adjacent amino acids. The term includes modified
peptides and synthetic peptide analogues.
[0031] A T cell epitope is a short peptide derivable from a protein
antigen. Antigen presenting cells can internalise antigen and
process it into short fragments which are capable of binding MHC
molecules. The specificity of peptide binding to the MHC depends on
specific interactions between the peptide and the peptide-binding
groove of the particular MHC molecule.
[0032] Peptides which bind to MHC class I molecules (and are
recognised by CD8+ T cells) are usually between 6 and 12, more
usually between 8 and 10 amino acids in length. The amino-terminal
amine group of the peptide makes contact with an invariant site at
one end of the peptide groove, and the carboxylate group at the
carboxy terminus binds to an invariant site at the other end of the
groove. The peptide lies in an extended confirmation along the
groove with further contacts between main-chain atoms and conserved
amino acid side chains that line the groove. Variations in peptide
length are accommodated by a kinking in the peptide backbone, often
at proline or glycine residues.
[0033] Peptides which bind to MHC class II molecules are usually at
least 10 amino acids, for example about 13-18 amino acids in
length, and can be much longer. These peptides lie in an extended
confirmation along the MHC II peptide-binding groove which is open
at both ends. The peptide is held in place mainly by main-chain
atom contacts with conserved residues that line the peptide-binding
groove.
[0034] The peptide of the present invention may be made using
chemical methods (Peptide Chemistry, A practical Textbook. Mikos
Bodansky, Springer-Verlag, Berlin). For example, peptides can be
synthesized by solid phase techniques (Roberge J Y et al (1995)
Science 269: 202-204), cleaved from the resin, and purified by
preparative high performance liquid chromatography (e.g., Creighton
(1983) Proteins Structures And Molecular Principles, WH Freeman and
Co, New York N.Y.). Automated synthesis may be achieved, for
example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in
accordance with the instructions provided by the manufacturer.
[0035] The peptide may alternatively be made by recombinant means,
or by cleavage from a longer polypeptide. For example, the peptide
may be obtained by cleavage from full-length 5T4.
[0036] The composition of a peptide may be confirmed by amino acid
analysis or sequencing (e.g., the Edman degradation procedure).
[0037] The term "peptide epitope" encompasses modified peptides.
For example 5T4 peptides may be mutated, by amino acid insertion,
deletion or substitution, so long as the MHC binding-specificity of
the wild-type 5T4 peptide is retained. In a preferred embodiment
the modified epitope has greater affinity for the peptide binding
groove. Preferably the peptide contains 5 or fewer mutations from
the wild-type sequence, more preferably 3 or fewer, most preferably
1 or 0 mutations.
[0038] Alternatively (or in addition) modifications may be made
without changing the amino acid sequence of the peptide. For
example, D-amino acids or other unnatural amino acids can be
included, the normal amide bond can be replaced by ester or alkyl
backbone bonds, N- or C-alkyl substituents, side chain
modifications, and constraints such as disulphide bridges and side
chain amide or ester linkages can be included. Such changes may
result in greater in vivo stability of the peptide, and a longer
biological lifetime.
[0039] Modification of epitopes may be performed based on
predictions for more efficient T-cell induction derived using the
program "Peptide Binding Predictions" devised by K. Parker (NIH)
which may be found at
http://www-bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform
(see also Parker, K. C et al. 1994. J. Immunol. 152:163).
[0040] A "modified" 5T4 peptide epitope includes peptides which
have been bound or otherwise associated to transporter peptides or
adjuvants, in order to increase their ability to elicit an immune
response. For example, peptides may be fused to TAP independent
transporter peptides for efficient transport to HLA and interaction
with HLA molecules to enhance CTL epitopes (for review see Yewdell
et al., 1998 J Immunother 21:127-31; Fu et al., (1998) J Virol
72:1469-81).
[0041] In a further embodiment, 5T4 or 5T4 peptides may be fused to
hepatitis B core antigen to enhance T helper and antibody responses
(Schodel et al., 1996 Intervirology 39:104-10).
[0042] To be an epitope, the peptide should be capable of binding
to the peptide-binding groove of a MHC class I or II molecule and
be recognised by a T cell.
[0043] Cell surface presentation of peptides derived from a given
antigen is not random and tends to be dominated by a small number
of frequently occurring epitopes. The dominance of a particular
peptide will depend on many factors, such as relative affinity for
binding the MHC molecule, spatio-temporal point of generation
within the APC and resistance to degradation. The epitope hierarchy
for an antigen is thought to change with progression of an immune
response. After a primary immune response to the immunodominant
peptides, epitope "spreading" may occur to sub-dominant
determinants (Lehmann et al (1992) Nature 358:155-157).
[0044] For any given antigen, cryptic epitopes may also exist.
Cryptic epitopes are those which can stimulate a T cell response
when administered as a peptide but which fail to produce such a
response when administered as a whole antigen. It may be that
during processing of the antigen into peptides in the APC the
cryptic epitope is destroyed.
[0045] The peptide of the invention may be an immunodominant
epitope, a sub-dominant epitope or a cryptic epitope of 5T4.
[0046] Epitopes for an antigen may be identified by measuring the T
cell response to overlapping peptides spanning a portion of the
antigen (see below) when presented by APC. Such studies usually
result in "nested sets" of peptides, and the minimal epitope for a
particular T cell line/clone can be assessed by measuring the
response to truncated peptides.
[0047] The minimal epitope for an antigen may not be the best
epitope for practical purposes. It may well be that amino acids
flanking the minimal epitope will be required for optimal binding
to the MHC.
Identification of Epitopes
[0048] There are a number of methods known in the art to identify
the T cell epitopes within a given antigen.
[0049] Naturally processed epitopes may be identified by mass
spectrophotometric analysis of peptides eluted from antigen-loaded
APC. These are APC that have either been encouraged to take up
antigen, or have been forced to produce the protein intracellularly
by transformation with the appropriate gene. Typically APC are
incubated with protein either in solution or suitably targeted to
the APC cell surface. After incubation at 37.degree. C. the cells
are lysed in detergent and the class II protein purified by, for
example, affinity chromatography. Treatment of the purified MHC
with a suitable chemical medium (for example, acid conditions)
results in the elution of peptides from the MHC. This pool of
peptides is separated and the profile compared with peptide from
control APC treated in the same way. The peaks unique to the
protein expressing/fed cells are analysed (for example by mass
spectrometry) and the peptide fragments identified. This procedure
usually generates information about the range of peptides (usually
found in "nested sets") generated from a particular antigen by
antigen processing.
[0050] Another method for identifying epitopes is to screen a
synthetic library of peptides which overlap and span the length of
the antigen in an in vitro assay. For example, peptides which are
15 amino acids in length and which overlap by 5 or 10 amino acids
may be used. The peptides are tested in an antigen presentation
system which comprises antigen presenting cells and T cells. For
example, the antigen presentation system may be a murine splenocyte
preparation, a preparation of human cells from tonsil or PBMC.
Alternatively, the antigen presentation system may comprise a
particular T cell line/clone and/or a particular antigen presenting
cell type.
[0051] T cell activation may be measured via T cell proliferation
(for example using .sup.3H-thymidine incorporation) or cytokine
production. Activation of TH1-type CD4+ T cells can, for example be
detected via IFN.gamma. production which may be detected by
standard techniques, such as an ELISPOT assay.
[0052] Overlapping peptide studies usually indicate the area of the
antigen in which an epitope is located. The minimal epitope for a
particular T cell can then be assessed by measuring the response to
truncated peptides. For example if a response is obtained to the
peptide comprising residues 1-15 in the overlapping library, sets
which are truncated at both ends (i.e. 1-14, 1-13, 1-12 etc. and
2-15, 3-15, 4-15 etc.) can be used to identify the minimal
epitope.
Polyepitope String
[0053] It has been found that it is particularly effective way to
induce an immune response to an antigen is by the use of a
polyepitope string, which contains a plurality of antigenic
epitopes from one or more antigens linked together. For example,
for malaria, a polyepitope string of mainly malaria (P. falciparum)
CD8 T cell peptide epitopes has been described which also expresses
CD4 T cell epitopes from tetanus toxoid and from the 38 Kd
mycobacterial antigen of various strains of M. tuberculosis and M.
bovis.
[0054] The present invention also provides a polyepitope string
comprising at least one peptide according to the present invention.
The string may also comprise another epitope derivable from the 5T4
antigen or an epitope from another antigen--such as another TAA--or
combinations thereof.
[0055] TAAs have been characterised either as membrane proteins or
altered carbohydrate molecules of glycoproteins and glycolipids,
however their functions remain largely unknown. One TAA family, the
transmembrane 4 superfamily (TM4SF), usually has four
well-conserved membrane-spanning regions, certain cysteine residues
and short sequence motifs. There is evidence that TM4SF antigens
exist in close association with other important membrane receptors
including CD4 and CD8 of T cells (Imai & Yoshie (1993) J.
Immunol. 151, 6470-6481). It has also been suggested that TM4SF
antigens may play a role in signal transduction which in turn,
affects cell development, activation and motility. Examples of
TM4SF antigens include human melanoma-associated antigen ME491,
human and mouse leukocyte surface antigen CD37, and human
lymphoblastic leukemia-associated TALLA-1 (Hotta, H. et al. (1988)
Cancer Res. 48, 2955-2962; Classon, B. J. et al. (1989) J. Exp.
Med. 169: 1497-1502; Tomlinson, M. G. et al. (1996) Mol. Immun. 33:
867-872; Takagi, S. et al. (1995) Int. J. Cancer 61: 706-715).
[0056] Further examples of TAAs also include, but are not limited
to, TAAs in the following classes: cancer testis antigens
(HOM-MEL-40), differentiation antigens (HOM-MEL-55), overexpressed
gene products (HOM-MD-21), mutated gene products (NY-COL-2), splice
variants (HOM-MD-397), gene amplification products (HOM-NSCLC-11)
and cancer related auto-antigens (HOM-MEL-2.4) as reviewed in
Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverley and
Carroll, Cambridge University Press, Cambridge. Further examples
include, MART-1 (Melanoma Antigen Recognised by T cells-1) MAGE-A
(MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A10,
MAGE-A12), MAGE B (MAGE-B1-MAGE-B24), MAGE-C (MAGE-C1/CT7, CT10),
GAGE (GAGE-1, GAGE-8, PAGE-1, PAGE-4, XAGE-1, XAGE-3), LAGE
(LAGE-1a(1S), -1b(1L), NY-ESO-1), SSX (SSX1-SSX-5), BAGE, SCP-1,
PRAME (MAPE), SART-1, SART-3, CTp11, TSP50, CT9/BRDT, gp100,
MART-1, TRP-1, TRP-2, MELAN-A/MART-1, Carcinoembryonic antigen
(CEA), prostate-specific antigen (PSA), MUCIN (MUC-1) and
Tyrosinase. TAAs are reviewed in Cancer Immunology (2001) Kluwer
Academic Publishers, The Netherlands.
Cell Penetrators
[0057] The present invention also provides a peptide epitope, or a
polyepitope string in association with a cell penetrator.
[0058] Antigen presenting cells (such as dendritic cells) pulsed
with peptides have proven effective in enhancing antitumour
immunity (Celluzzi et al (1996) J. Exp. Med. 183 283-287; Young et
al (1996) J. Exp. Med. 183 7-11). It has been shown that it is
possible to prolong the presentation of a peptide by dendritic
cells (and thus enhance antitumour immunity) by linking it to a
cell penetrating peptide (CPP) (Wang and Wang (2002) Nature
Biotechnology 20 149-154).
[0059] A cell penetrator may be any entity which enhances the
intracellular delivery of the peptide/polyepitope string to the
antigen presenting cell. For example, the cell penetrator may be a
lipid which, when associated with the peptide, enhances its
capacity to cross the plasma membrane. Alternatively, the cell
penetrator may be a peptide. Several cell penetrating peptide
(CPPs) have been identified from proteins, including the Tat
protein of HIV (Frankel and Pabo (1988) Cell 55 1189-1193), the
VP22 protein of HSV (Elliott and O'Hare (1997) Cell 88 223-233) and
fibroblast growth factor (Lin et al (1995) J. Biol. Chem. 270
14255-14258).
[0060] The term "associated with" is intended to include direct
linkage, for example by a covalent bond. Examples of covalent bonds
for linking amino acids include disulphide bridges and peptide
bonds. In a preferred embodiment, the peptide/polyepitope string
and a CPP are linked by a peptide bond to create a fusion
protein.
[0061] The term also includes non-covalent linkage, such as
association by electrostatic bonding, hydrogen bonding and van der
Waals forces. The cell penetrator and peptide/polyepitope string
may be associated without covalent or non-covalent bonding. For
example the cell penetrator may be a lipid which encapsulates the
peptide/polyepitope string (e.g. a. liposome). 5T4
[0062] 5T4 has been previously characterised, for example, in
WO89/07947. The sequence of human 5T4 which appears in GenBank at
accession no. Z29083. The peptide may also be derived from a 5T4
antigen from a different species, such as murine 5T4 (WO00/29428),
canine 5T4 (WO01/36486) or feline 5T4 (SEQ ID No 1 presented
herein). The peptide may also be derived from a naturally occurring
variant of 5T4 found with a particular species, preferably a
mammal. Such a variant may be encoded by a related gene of the same
gene family, by an allelic variant of a particular gene, or
represent an alternative splicing variant of the 5T4 gene.
[0063] A peptide derived from 5T4 from a different species or a
splice variant may have a different amino acid sequence from the
analogous human wild-type 5T4 peptide. However, as long as the
peptide retains the same qualitative binding specificity as the
human peptide (i.e. it binds in the peptide binding groove of an
MHC molecule of the same haplotype) then it is still an epitope in
accordance with the present invention.
Nucleic Acid
[0064] The present invention also relates to a nucleic acid
sequence capable of encoding a peptide epitope or polyepitope
string according to the first aspect of the invention.
[0065] A "nucleic acid", as referred to herein, may be DNA or RNA,
naturally-occurring or synthetic, or any combination thereof.
Nucleic acids according to the invention are limited only in that
they serve the function of encoding a 5T4 peptide in such a way
that it may be translated by the machinery of the cells of a host
organism. Thus, natural nucleic acids may be modified, for example
to increase the stability thereof. DNA and/or RNA, but especially
RNA, may be modified in order to improve nuclease resistance of the
members. For example, known modifications for ribonucleotides
include 2'-O-methyl, 2'-fluoro, 2'--NH.sub.2, and 2'-O-allyl. The
modified nucleic acids according to the invention may comprise
chemical modifications which have been made in order to increase
the in vivo stability of the nucleic acid, enhance or mediate the
delivery thereof, or reduce the clearance rate from the body.
Examples of such modifications include chemical substitutions at
the ribose and/or phosphate and/or base positions of a given RNA
sequence. See, for example, WO 92/03568; U.S. Pat. No. 5,118,672;
Hobbs et al., (1973) Biochemistry 12:5138; Guschlbauer et al.,
(1977) Nucleic Acids Res. 4:1933; Schibaharu et al., (1987) Nucleic
Acids Res. 15:4403; Pieken et al., (1991) Science 253:314, each of
which is specifically incorporated herein by reference.
[0066] The present invention also encompasses nucleic acids which
will hybridise to a nucleic acid sequence capable of encoding a
peptide epitope or polyepitope string according to the first aspect
of the invention.
[0067] Stringency of hybridisation refers to conditions under which
polynucleic acid hybrids are stable. Such conditions are evident to
those of ordinary skill in the field. As known to those of skill in
the art, the stability of hybrids is reflected in the melting
temperature (Tm) of the hybrid which decreases approximately 1 to
1.5.degree. C. with every 1% decrease in sequence homology. In
general, the stability of a hybrid is a function of sodium ion
concentration and temperature. Typically, the hybridisation
reaction is performed under conditions of higher stringency,
followed by washes of varying stringency.
[0068] As used herein, high stringency refers to conditions that
permit hybridisation of only those nucleic acid sequences that form
stable hybrids in 1 M Na+ at 65-68.degree. C. High stringency
conditions can be provided, for example, by hybridisation in an
aqueous solution containing 6.times.SSC, 5.times.Denhardt's, 1% SDS
(sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml
denatured salmon sperm DNA as non specific competitor. Following
hybridisation, high stringency washing may be done in several
steps, with a final wash (about 30 min) at the hybridisation
temperature in 0.2-0.1.times.SSC, 0.1% SDS.
[0069] Moderate stringency refers to conditions equivalent to
hybridisation in the above described solution but at about
60-62.degree. C. In that case the final wash is performed at the
hybridisation temperature in 1.times.SSC, 0.1% SDS.
[0070] Low stringency refers to conditions equivalent to
hybridisation in the above described solution at about
50-52.degree. C. In that case, the final wash is performed at the
hybridisation temperature in 2.times.SSC, 0.1% SDS.
[0071] It is understood that these conditions may be adapted and
duplicated using a variety of buffers, e.g. formamide-based
buffers, and temperatures. Denhardt's solution and SSC are well
known to those of skill in the art as are other suitable
hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York or Ausubel, et al., eds. (1990) Current
Protocols in Molecular Biology, John Wiley & Sons, Inc.).
Optimal hybridisation conditions have to be determined empirically,
as the length and the GC content of the probe also play a role.
[0072] Given the guidance provided herein, the nucleic acids of the
invention are obtainable according to methods well known in the
art. For example, a DNA of the invention is obtainable by chemical
synthesis, using polymerase chain reaction (PCR) or direct cleavage
from a longer polynucleotide, such as the entire 5T4 coding
sequence or a fragment thereof.
[0073] Chemical methods for synthesis of a nucleic acid of interest
are known in the art and include triester, phosphite,
phosphoramidite and H-phosphonate methods, PCR and other autoprimer
methods as well as oligonucleotide synthesis on solid supports.
These methods may be used if the entire nucleic acid sequence of
the nucleic acid is known, or the sequence of the nucleic acid
complementary to the coding strand is available. Alternatively, if
the target amino acid sequence is known, one may infer potential
nucleic acid sequences using known and preferred coding residues
for each amino acid residue.
[0074] It is envisaged that the nucleic acid of the invention can
be modified by nucleotide substitution, nucleotide deletion,
nucleotide insertion or inversion of a nucleotide stretch, and any
combination thereof. Such mutants can be used e.g. to produce a 5T4
peptide that has an amino acid sequence differing from the
wild-type 5T4 epitope. Such a peptide is still a peptide in
accordance with the present invention if it retains the capacity to
act as a T cell epitope. Mutagenesis may be predetermined
(site-specific) or random. A mutation which is not a silent
mutation should not place sequences out of reading frames and
preferably will not create complementary regions that could
hybridise to produce secondary mRNA structure such as loops or
hairpins.
Variants/Fragments/Homologues/Derivatives
[0075] The present invention encompasses the use of nucleotide and
amino acid sequences and variants, homologues, derivatives and
fragments thereof.
[0076] The term "variant" is used to mean a naturally occurring
polypeptide or nucleotide sequence which differs from a wild-type
sequence.
[0077] The term "fragment" indicates that a polypeptide or
nucleotide sequence comprises a fraction of a subject sequence.
Preferably the sequence comprises at least 50%, more preferably at
least 65%, more preferably at least 80%, more preferably at least
90%, most preferably at least 90% of the subject sequence. If the
fragment is a fragment of an amino acid then preferably the
fragments are 6-12 amino acids in length.
[0078] The term "homologue" means an entity having a certain
homology with the subject amino acid sequences and the subject
nucleotide sequences. Here, the term "homology" can be equated with
"identity".
[0079] In the present context, a homologous sequence is taken to
include an amino acid sequence, which may be at least 75, 85 or 90%
identical, preferably at least 95 or 98% identical to the subject
sequence. Typically, the homologues will comprise the same activity
as the subject amino acid sequence. Although homology can also be
considered in terms of similarity (i.e. amino acid residues having
similar chemical properties/functions), in the context of the
present invention it is preferred to express homology in terms of
sequence identity.
[0080] In the present context, a homologous sequence is taken to
include a nucleotide sequence, which may be at least 75, 85 or 90%
identical, preferably at least 95 or 98% identical to the subject
sequence. Typically, the homologues will comprise the same activity
as the subject sequence. Although homology can also be considered
in terms of similarity (i.e. amino acid residues having similar
chemical properties/functions), in the context of the present
invention it is preferred to express homology in terms of sequence
identity.
[0081] Homology comparisons may be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0082] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0083] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0084] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example, when using the GCG Wisconsin
Bestfit package the default gap penalty for amino acid sequences is
-12 for a gap and -4 for each extension.
[0085] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (University of
Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research
12:387). Examples of other software than can perform sequence
comparisons include, but are not limited to, the BLAST package (see
Ausubel et al., 1999 ibid--Chapter 18), FASTA (Atschul et al.,
1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison
tools. Both BLAST and FASTA are available for offline and online
searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).
However, for some applications, it is preferred to use the GCG
Bestfit program. A new tool, called BLAST 2 Sequences is also
available for comparing protein and nucleotide sequence (see FEMS
Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999
177(1): 187-8).
[0086] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual
for further details). For some applications, it is preferred to use
the public default values for the GCG package, or in the case of
other software, the default matrix--such as BLOSUM62.
[0087] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0088] The sequences may also have deletions, insertions or
substitutions of amino acid residues, which produce a silent change
and result in a functionally equivalent substance. Deliberate amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
secondary binding activity of the substance is retained. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine, valine,
glycine, alanine, asparagine, glutamine, serine, threonine,
phenylalanine, and tyrosine.
[0089] Conservative substitutions may be made, for example,
according to the Table below. Amino acids in the same block in the
second column and preferably in the same line in the third column
may be substituted for each other:
TABLE-US-00003 ALIPHATIC Non-polar G A P I L V Polar - uncharged C
S T M N Q Polar - charged D E K R AROMATIC H F W Y
[0090] The present invention also encompasses homologous
substitution (substitution and replacement are both used herein to
mean the interchange of an existing amino acid residue, with an
alternative residue) may occur i.e. like-for-like
substitution--such as basic for basic, acidic for acidic, polar for
polar etc. Non-homologous substitution may also occur i.e. from one
class of residue to another or alternatively involving the
inclusion of unnatural amino acids--such as ornithine (hereinafter
referred to as Z), diaminobutyric acid ornithine (hereinafter
referred to as B), norleucine ornithine (hereinafter referred to as
O), pyriylalanine, thienylalanine, naphthylalanine and
phenylglycine.
[0091] Replacements may also be made by unnatural amino acids
include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino
acids*, lactic acid*, halide derivatives of natural amino
acids--such as trifluorotyrosine*, p-Cl-phenylalanine*,
p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*,
.beta.-alanine*, L-.alpha.-amino butyric acid*, L-.gamma.-amino
butyric acid*, L-.alpha.-amino isobutyric acid*, L-.epsilon.-amino
caproic acid.sup.#, 7-amino heptanoic acid*, L-methionine
sulfone.sup.#*, L-norleucine*, L-norvaline*,
p-nitro-L-phenylalanine*, L-hydroxyproline.sup.#, L-thioproline*,
methyl derivatives of phenylalanine (Phe)--such as 4-methyl-Phe*,
pentamethyl-Phe*, L-Phe (4-amino).sup.#, L-Tyr (methyl)*, L-Phe
(4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl
acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*. The
notation * has been utilised for the purpose of the discussion
above (relating to homologous or non-homologous substitution), to
indicate the hydrophobic nature of the derivative whereas # has
been utilised to indicate the hydrophilic nature of the derivative,
#* indicates amphipathic characteristics.
[0092] Variant amino acid sequences may include suitable spacer
groups that may be inserted between any two amino acid residues of
the sequence including alkyl groups--such as methyl, ethyl or
propyl groups--in addition to amino acid spacers--such as glycine
or .beta.-alanine residues. A further form of variation involves
the presence of one or more amino acid residues in peptoid form
will be well understood by those skilled in the art. For the
avoidance of doubt, "the peptoid form" is used to refer to variant
amino acid residues wherein the .alpha.-carbon substituent group is
on the residue's nitrogen atom rather than the .alpha.-carbon.
Processes for preparing peptides in the peptoid form are known in
the art, for example, Simon R J et al., PNAS (1992) 89(20),
9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4),
132-134.
[0093] The nucleotide sequences for use in the present invention
may include within them synthetic or modified nucleotides. A number
of different types of modification to oligonucleotides are known in
the art. These include methylphosphonate and phosphorothioate
backbones and/or the addition of acridine or polylysine chains at
the 3' and/or 5' ends of the molecule. For the purposes of the
present invention, it is to be understood that the nucleotide
sequences may be modified by any method available in the art. Such
modifications may be carried out to enhance the in vivo activity or
life span of nucleotide sequences useful in the present
invention.
Vector System
[0094] The nucleic acid sequence of the present invention may be
delivered to a cell by way of a vector system.
[0095] As used herein, a "vector" may be any agent capable of
delivering or maintaining nucleic acid in a host cell, and includes
viral vectors, plasmids, naked nucleic acids, nucleic acids
complexed with polypeptide or other molecules and nucleic acids
immobilised onto solid phase particles. Such vectors are described
in detail below. It will be understood that the present invention,
in its broadest form, is not limited to any specific vector for
delivery of the 5T4 peptide-encoding nucleic acid.
[0096] The vector may be a prokaryotic or eukaryotic vector.
[0097] Nucleic acids encoding 5T4 epitopes and polyepitope strings
in accordance with the present invention can be delivered by viral
or non-viral techniques.
[0098] Non-viral delivery systems include but are not limited to
DNA transfection methods. Here, transfection includes a process
using a non-viral vector to deliver a 5T4 gene to a target
mammalian cell.
[0099] Typical transfection methods include electroporation,
nucleic acid biolistics, lipid-mediated transfection, compacted
nucleic acid-mediated transfection, liposomes, immunoliposomes,
lipofectin, cationic agent-mediated, cationic facial amphiphiles
(CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations
such as spermine, cationic lipids or polylysine,
1,2,-bis(oleoyloxy)-3-(trimethylammonio) propane
(DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature
Biotechnology 16: 421) and combinations thereof.
[0100] Non-viral delivery systems may also include, but are not
limited to, bacterial delivery systems. The use of bacteria as
anticancer agents and as delivery agents for anticancer drugs has
been reviewed in Expert Opin Biol Ther 2001 March;
1(2):291-300.
[0101] Suitable bacteria include, but are not limited to, bacterial
pathogens and non-pathogenic commensal bacteria. By way of example,
suitable genera may be selected from Salmonella, Mycobacterium,
Yersinia, Shigella, Listeria and Brucella. Recent advances in the
pathogenesis and molecular biology of these bacteria have allowed
the rational development of new and improved bacterial carriers and
more effective gene expression systems. These advances have
improved the performance and versatility of these delivery
systems.
[0102] The bacteria may be invasive intracellular bacteria that are
able to transfer eukaryotic expression plasmids into mammalian host
cells in vitro and in vivo. Plasmid transfer may take place when
the recombinant bacterium dies within the host cell, either due to
metabolic attenuation or induction of autolysis. Alternatively,
antibiotics may be used and spontaneous transfer has also been
observed, indicating that this phenomenon might also occur under
physiological conditions. Plasmid transfer has been reported for
Shigella flexneri, Salmonella typhimurium, S. typhi, Listeria
monocytogenes and recombinant Escherichia coli, but other invasive
bacteria may also be used.
[0103] Bacteria may be used for DNA vaccine delivery. Such bacteria
may enter the host cell cytosol after phagocytosis, for example,
Shigella and Listeria, or they remain in the phagosomal
compartment--such as Salmonella. Both intracellular localisations
may be suitable for successful delivery of DNA vaccine vectors.
[0104] The bacterial delivery systems may utilise Mycobacterium in
the form of non pathogenic Mycobacterium strains, genetic transfer
systems in the form of cloning and expression vectors, and related
technologies to provide products containing, for example, non toxic
immuno-regulating Mycobacterium adjuvants, non toxic
immuno-stimulating exogenous antigens specific for a variety of
diseases, and non toxic amounts of cytokines that boost the TH-1
pathway (Tunis Med 2001 February; 79(2):65-81).
[0105] Salmonella strains--such as attenuated strains--which
comprise defined gene deletions, may be used as suitable delivery
systems--such as the delivery of antigens. A number of strategies
for delivery by these strains have been attempted, ranging from
plasmid-based to chromosomal integration systems. By way of
example, Rosenkranz et al. Vaccine 2003, 21(7-8), 798-801 describe
eukaryotic expression plasmids encoding cytokines, and assessed
their capacity to modulate immune responses in different
experimental models. Plasmids encoding mouse IL-4 and IL-18 under
cytomegalovirus promoter were constructed and transformed into live
attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA,
and Salmonella enterica serovar Typhimurium strain SL3261.
[0106] The use of attenuated Salmonella typhimurium as a potential
gene delivery vector has been reviewed in Anticancer Res 2002,
22(6A):3261-6.
[0107] Brucella abortus may also be used as a suitable delivery
system as described by Vemulapalli et al. Infect Immun (2000)
68(6):3290-6. Brucella abortus strain RB51 is a stable, rough,
attenuated mutant widely used as a live vaccine for bovine
brucellosis. This strain may be used as a delivery vector, for
example, in the delivery of protective antigens of other
intracellular pathogens to which the induction of a strong Th1 type
of immune response is needed for effective protection.
[0108] Boyd et al. Eur J Cell Biol (2000) 79 (10) 659-71 describe
the use of Yersinia enterocolitica for the delivery of proteins
into a wide range of cell types. Y. enterocolitica translocates
virulence proteins, called Yop effectors, into the cytosol of
eukaryotic cells. No limit to the range of eukaryotic cells into
which Y. enterocolitica can translocate Yops was reported. The Yop
effectors YopE, YopH and YopT were each cytotoxic for the adherent
cell types tested, showing that not only is Y. enterocolitica not
selective in its translocation of particular Yop effectors into
each cell type, but also that the action of these Yop effectors is
not cell type specific. To use the Yersinia translocation system
for broad applications, a Y. enterocolitica translocation strain
and vector for the delivery of heterologous proteins into
eukaryotic cells was constructed. This strain and vector
combination lacks the translocated Yop effectors and allows
delivery into eukaryotic cells of heterologous proteins fused to
the minimal N-terminal secretion/translocation signal of YopE.
[0109] U.S. Pat. No. 5,965,381 describes a recombinant Yersinia for
the delivery of proteins into eukaryotic cells. Such Yersinia are
deficient in the production of functional effector proteins, but
are endowed with a functional secretion and translocation
system.
[0110] Cell adhesion molecules are a large group of molecules
involved in a variety of cell-to-cell and cell-to-extra-cellular
matrix (ECM) interactions and are exploited by a number of
pathogenic micro-organisms as receptors for cell entry. These
molecules may be used for the targeting and uptake of both gene and
drug delivery systems. Cell adhesion molecules and their use in
gene transfer has been reviewed in Adv Drug Deliv Rev 2000 Nov. 15;
44(2-3):135-52.
[0111] The gene gun delivery system may also be used for the
delivery of DNA, which is a highly reliable method compared to
intramuscular inoculation (Jpn J Pharmacol 2000 July;
83(3):167-74).
[0112] Viral delivery systems include but are not limited to
adenovirus vectors, adeno-associated viral (AAV) vectors, herpes
viral vectors, retroviral vectors, lentiviral vectors or
baculoviral vectors, venezuelan equine encephalitis virus (VEE),
poxviruses such as: canarypox virus (Taylor et al 1995 Vaccine
13:539-549), entomopox virus (Li Y et al 1998 XII.sup.th
International Poxvirus Symposium p 144. Abstract), penguine pox
(Standard et al. J Gen Virol. 1998 79:1637-46) alphavirus, and
alphavirus based DNA vectors.
[0113] Examples of retroviruses include but are not limited to:
murine leukaemia virus (MLV), human immunodeficiency virus (HIV),
equine infectious anaemia virus (EIAV), mouse mammary tumour virus
(MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV),
Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma
virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson
murine leukaemia virus (A-MLV), Avian myelocytomatosis virus-29
(MC29), and Avian erythroblastosis virus (AEV).
[0114] A detailed list of retroviruses may be found in Coffin et al
("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: J M
Coffin, S M Hughes, H E Varmus pp 758-763).
[0115] Lentiviruses can be divided into primate and non-primate
groups. Examples of primate lentiviruses include but are not
limited to: the human immunodeficiency virus (HIV), the causative
agent of human auto-immunodeficiency syndrome (AIDS), and the
simian immunodeficiency virus (SIV). The non-primate lentiviral
group includes the prototype "slow virus" visna/maedi virus (VMV),
as well as the related caprine arthritis-encephalitis virus (CAEV),
equine infectious anaemia virus (EIAV) and the more recently
described feline immunodeficiency virus (FIV) and bovine
immunodeficiency virus (BIV).
[0116] A distinction between the lentivirus family and other types
of retroviruses is that lentiviruses have the capability to infect
both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11:
3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516). In
contrast, other retroviruses--such as MLV--are unable to infect
non-dividing cells such as those that make up, for example, muscle,
brain, lung and liver tissue.
[0117] The vector of the present invention may be configured as a
split-intron vector. A split intron vector is described in PCT
patent applications WO 99/15683 and WO 99/15684.
[0118] If the features of adenoviruses are combined with the
genetic stability of retroviruses/lentiviruses then essentially the
adenovirus can be used to transduce target cells to become
transient retroviral producer cells that could stably infect
neighbouring cells. Such retroviral producer cells engineered to
express 5T4 antigen can be implanted in organisms such as animals
or humans for use in the treatment of angiogenesis and/or
cancer.
[0119] The vector of the present invention may be configured as a
psuedotyped vector.
[0120] In the design of retroviral vectors it may be desirable to
engineer particles with different target cell specificities to the
native virus, to enable the delivery of genetic material to an
expanded or altered range of cell types. One manner in which to
achieve this is by engineering the virus envelope protein to alter
its specificity. Another approach is to introduce a heterologous
envelope protein into the vector particle to replace or add to the
native envelope protein of the virus.
[0121] The term pseudotyping means incorporating in at least a part
of, or substituting a part of, or replacing all of, an env gene of
a viral genome with a heterologous env gene, for example an env
gene from another virus. Pseudotyping is not a new phenomenon and
examples may be found in WO 99/61639, WO-A-98/05759, WO-A-98/05754,
WO-A-97/17457, WO-A-96/09400, WO-A-91/00047 and Mebatsion et al
1997 Cell 90, 841-847.
[0122] Pseudotyping can improve retroviral vector stability and
transduction efficiency. A pseudotype of murine leukemia virus
packaged with lymphocytic choriomeningitis virus (LCMV) has been
described (Miletic et al (1999) J. Virol. 73:6114-6116) and shown
to be stable during ultracentrifugation and capable of infecting
several cell lines from different species.
Poxvirus Vectors
[0123] TAAs are weakly immunogenic, being recognised as "self" by
the immune system and thus tolerated to a large extent. The use of
poxvirus vectors is sometimes able to cause the antigens to be
presented such that this tolerance may be overcome at least in
part, (especially if immune evasion genes are deleted--see below)
thus enabling a host to raise an immune response.
[0124] Poxvirus vectors are preferred for use in the present
invention. Pox viruses are engineered for recombinant gene
expression and for the use as recombinant live vaccines. This
entails the use of recombinant techniques to introduce nucleic
acids encoding foreign antigens into the genome of the pox virus.
If the nucleic acid is integrated at a site in the viral DNA which
is non-essential for the life cycle of the virus, it is possible
for the newly produced recombinant pox virus to be infectious, that
is to say to infect foreign cells and thus to express the
integrated DNA sequence. The recombinant pox virus prepared in this
way can be used as live vaccines for the prophylaxis and/or
treatment of pathologic and infectious disease.
[0125] Expression of 5T4 peptide(s) in recombinant pox viruses,
such as vaccinia viruses, requires the ligation of vaccinia
promoters to the nucleic acid encoding the 5T4 peptide(s). Plasmid
vectors (also called insertion vectors), have been constructed to
insert nucleic acids into vaccinia virus through homologous
recombination between the viral sequences flanking the nucleic acid
in a donor plasmid and homologous sequence present in the parental
virus (Mackett et al 1982 PNAS 79: 7415-7419). One type of
insertion vector is composed of: (a) a vaccinia virus promoter
including the transcriptional initiation site; (b) several unique
restriction endonuclease cloning sites located downstream from the
transcriptional start site for insertion of nucleic acid; (c)
nonessential vaccinia virus sequences (such as the Thymidine Kinase
(TK) gene) flanking the promoter and cloning sites which direct
insertion of the nucleic acid into the homologous nonessential
region of the virus genome; and (d) a bacterial origin of
replication and antibiotic resistance marker for replication and
selection in E. Coli. Examples of such vectors are described by
Mackett (Mackett et al 1984, J. Virol. 49: 857-864).
[0126] The isolated plasmid containing the nucleic acid to be
inserted is transfected into a cell culture, e.g., chick embryo
fibroblasts, along with the parental virus, e.g., poxvirus.
Recombination between homologous pox DNA in the plasmid and the
viral genome respectively results in a recombinant poxvirus
modified by the presence of the promoter-gene construct in its
genome, at a site which does not affect virus viability.
[0127] As noted above, the nucleic acid is inserted into a region
(insertion region) in the virus which does not affect virus
viability of the resultant recombinant virus. Such regions can be
readily identified in a virus by, for example, randomly testing
segments of virus DNA for regions that allow recombinant formation
without seriously affecting virus viability of the recombinant.
[0128] One region that can readily be used and is present in many
viruses is the thymidine kinase (TK) gene. For example, the TK gene
has been found in all pox virus genomes examined [leporipoxvirus:
Upton, et al J. Virology 60:920 (1986) (shope fibroma virus);
capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989) (Kenya
sheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983)
(vaccinia); Esposito, et al Virology 135:561 (1984) (monkeypox and
variola virus); Hruby, et al PNAS, 80:3411 (1983) (vaccinia);
Kilpatrick, et al Virology 143:399 (1985) (Yaba monkey tumour
virus); avipoxvirus: Binns, et al J. Gen. Virol 69:1275 (1988)
(fowlpox); Boyle, et al Virology 156:355 (1987) (fowlpox);
Schnitzlein, et al J. Virological Method, 20:341 (1988) (fowlpox,
quailpox); entomopox (Lytvyn, et al J. Gen. Virol 73:3235-3240
(1992)].
[0129] In vaccinia, in addition to the TK region, other insertion
regions include, for example, HindIII M.
[0130] In fowlpox, in addition to the TK region, other insertion
regions include, for example, BamHI J [Jenkins, et al AIDS Research
and Human Retroviruses 7:991-998 (1991)] the EcoRI-HindIII
fragment, BamHI fragment, EcoRV-HindIII fragment, BamHI fragment
and the HindIII fragment set forth in EPO Application No. 0 308 220
A1. [Calvert, et al J. of Virol 67:3069-3076 (1993); Taylor, et al
Vaccine 6:497-503 (1988); Spehner, et al (1990) and Boursnell, et
al J. of Gen. Virol 71:621-628 (1990)].
[0131] In swinepox preferred insertion sites include the thymidine
kinase gene region.
[0132] A promoter can readily be selected depending on the host and
the target cell type. For example in poxviruses, pox viral
promoters should be used, such as the vaccinia 7.5K, or 40K or
fowlpox C1. Artificial constructs containing appropriate pox
sequences can also be used. Enhancer elements can also be used in
combination to increase the level of expression. Furthermore, the
use of inducible promoters, which are also well known in the art,
are preferred in some embodiments.
[0133] Foreign gene expression can be detected by enzymatic or
immunological assays (for example, immuno-precipitation,
radioimmunoassay, or immunoblotting). Naturally occurring membrane
glycoproteins produced from recombinant vaccinia infected cells are
glycosylated and may be transported to the cell surface. High
expressing levels can be obtained by using strong promoters.
[0134] Other requirements for viral vectors for use in vaccines
include good immunogenicity and safety. MVA is a
replication-impaired vaccinia strain with a good safety record. In
most cell types and normal human tissue, MVA does not replicate.
Replication of MVA is observed in a few transformed cell types such
as BHK21 cells. Carroll et al (1997) have shown that the
recombinant MVA is equally as good as conventional recombinant
vaccinia vectors at generating a protective CD8+ T cell response
and is an efficacious alternative to the more commonly used
replication competent vaccinia virus. The vaccinia virus strains
derived from MVA, or independently developed strains having the
features of MVA which make MVA particularly suitable for use in a
vaccine, are also suitable for use in the present invention.
[0135] Preferably, the vector is a vaccinia virus vector such as
MVA or NYVAC. Most preferred is the vaccinia strain modified virus
ankara (MVA) or a strain derived therefrom. Alternatives to
vaccinia vectors include avipox vectors such as fowlpox or
canarypox known as ALVAC and strains derived therefrom which can
infect and express recombinant proteins in human cells but are
unable to replicate.
[0136] In one aspect of the present invention at least one immune
evasion gene is deleted from the poxvirus vector.
[0137] Viruses, especially large viruses such a poxviruses which
have an extensive coding capacity and can thus encode a variety of
genes, have developed a number of techniques for evading the immune
system of their hosts. For example, they are able to evade
non-specific defences such as complement, interferons and the
inflammatory response, as well as to interfere with or block the
function of cytokines. A number of these immune evasion
polypeptides have been deleted from MVA, with the exception of the
interferon resistance protein in the left terminal region.
[0138] Poxviruses in general, being large DNA viruses which
establish acute, rather than latent, infections. They encode so
many antigenic proteins that antigenic variation is difficult, thus
relying on active immune evasion to protect themselves from the
mammalian immune system. They possess a number of genes which
encode polypeptides which are responsible for interfering with a
number of aspects of the immune system: they disrupt interferon
action, interfere with complement, cytokine activity, inflammatory
responses and CTL recognition (for a review, Smith et al., (1997)
Immunol Rev 159:137-154). Removal of these proteins is beneficial
in promoting the ability of weak immunogens encoded on a poxvirus
vector to elicit an immune response in a subject.
[0139] An immune evasion gene or polypeptide is a gene, or its
product, which assists the virus in evading the mammalian immune
system. Preferably, the gene or gene product interferes with the
working of the immune system, at least one level. This may be
achieved in a number of ways, such as by interfering in signalling
pathways by providing competitors for signalling molecules, by
providing soluble cytokine receptor mimics and the like.
[0140] Immune evasion genes include, but are not limited to, the
following:
[0141] Interferon Evasion Genes.
[0142] Vaccinia possesses at least three genes which interfere with
IFN action. The E3L gene expresses a 25 Kd polypeptide which
competes with P1 protein kinase for binding to dsRNA, an event
which leads to activation of P1, phosphorylation of eIF2.alpha. and
resultant failure of translation initiation complex assembly. This
pathway is ordinarily responsive to IFN activation, but is impeded
by E3L expression thus allowing translation initiation to proceed
unimpeded.
[0143] The K3L gene expresses a 10.5 Kd polypeptide which also
interferes with P1 activity, since it is effectively an eIF2 mimic
and acts as a competitor for P1 protein kinase. Its mode of action
is thus similar to E3L.
[0144] The A18R gene is predicted to encode a helicase, which
appears to interfere with the 2',5'-oligoadenylate pathway, which
is in turn IFN responsive. 2',5'-A activates RNAse L, which acts to
prevent viral translation. Expression of A18R appears to reduce
2',5'-A levels in infected cells.
[0145] Complement.
[0146] The product of the B5R gene of vaccinia is known to be
highly related to factor H, a regulator of the alternative
complement pathway. This pathway may be activated by antigen alone,
unlike the classical pathway. The B5R gene product thus may
interfere with the alternative complement pathway.
[0147] The C21L gene is in turn related to C4b-binding protein in
humans, and interacts with cells bearing C4b on the surface to
prevent binding to the CR1 complement receptor.
[0148] Soluble Cytokine Receptors.
[0149] The product of the vaccinia WR B15R gene (B16R in Copenhagen
strain vaccinia) is related to IL1-R.
[0150] The WR gene ORF SalF19R, A53R in Copenhagen strain vaccinia,
encodes a TNF receptor. However, in wild-type virus both of these
genes are believed to be inactive due to fragmentation of the
ORFs.
[0151] The B8R gene is believed to encode a soluble IFN-.gamma.
receptor, providing the virus with yet another IFN evasion
mechanism.
[0152] Inflammation.
[0153] A number of genes are believed to be involved in the
prevention of inflammatory responses to viral infection. These
include A44L, K2L, B13R and B22R.
[0154] In one aspect of the present invention, the majority of the
immune evasion genes are deleted from the recombinant poxvirus
vector. Preferably, all the immune evasion genes are deleted. Thus,
in one aspect of the present invention, the recombinant poxvirus
vector is a recombinant MVA vector in which the K3L interferon
resistance protein gene has been disrupted or deleted.
[0155] Preferred are poxviruses which are non-hazardous to the
intended subject. Thus, for example, for use in humans, poxviruses
which are either host-range restricted, such as avipox viruses, or
otherwise attenuated, such as attenuated strains of vaccinia
(including NYVAC and MVA) are preferred. Most preferred are
attenuated vaccinia virus strains, although non-vaccinia strains
are usefully employed in subjects with pre-existing smallpox
immunity.
[0156] A construct which contains at least one nucleic acid which
codes for 5T4 epitope(s) flanked by MVA DNA sequences adjacent to a
naturally occurring deletion, e.g. deletion II, within the MVA
genome, is introduced into cells infected with MVA, to allow
homologous recombination.
[0157] Once the construct has been introduced into the eukaryotic
cell and the 5T4 epitope DNA has recombined with the viral DNA, the
desired recombinant vaccinia virus, can be isolated, preferably
with the aid of a marker (Nakano et al Proc. Natl. Acad. Sci. USA
79, 1593-1596 [1982], Franke et al Mol. Cell. Biol. 1918-1924
[1985], Chakrabarti et al Mol. Cell. Biol. 3403-3409 [1985], Fathi
et al Virology 97-105 [1986]).
[0158] The construct to be inserted can be linear or circular. A
circular DNA is preferred, especially a plasmid. The construct
contains sequences flanking the left and the right side of a
naturally occurring deletion, e.g. deletion II, within the MVA
genome (Altenburger, W., Suter, C. P. and Altenburger J. (1989)
Arch. Virol. 105, 15-27). The foreign DNA sequence is inserted
between the sequences flanking the naturally occurring
deletion.
[0159] For the expression of at least one nucleic acid, it is
necessary for regulatory sequences, which are required for the
transcription of the nucleic acid to be present upstream of the
nucleic acid. Such regulatory sequences are known to those skilled
in the art, and includes for example those of the vaccinia 11 kDa
gene as are described in EP-A-198,328, and those of the 7.5 kDa
gene (EP-A-110,385).
[0160] The construct can be introduced into the MVA infected cells
by transfection, for example by means of calcium phosphate
precipitation (Graham et al Virol. 52, 456-467 [1973; Wigler et al
Cell 777-785 [1979] by means of electroporation (Neumann et al EMBO
J. 1, 841-845 [1982]), by microinjection (Graessmann et al Meth.
Enzymology 101, 482-492 (1983)), by means of liposomes (Straubinger
et al Methods in Enzymology 101, 512-527 (1983)), by means of
spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA 77, 2163-2167
(1980)) or by other methods known to those skilled in the art.
Transfection by means of liposomes is preferred.
[0161] The recombinant priming and boosting vectors of the present
invention can have a tropism for a specific cell type in the
mammal. By way of example, the recombinant vectors of the present
invention can be engineered to infect professional APCs such as
dendritic cells and macrophages. Dendritic cells are known to be
orchestrators of a successful immune response especially that of a
cell mediated response. It has been shown that ex vivo treatment of
dendritic cells with antigen or viral vectors containing such a
target antigen, will induce efficacious immune responses when
infused into syngeneic animals or humans (see Nestle F O, et al.
Vaccination of melanoma patients with peptide- or tumor
lysate-pulsed dendritic cells, Nat Med. 1998 March; 4(3):328-32 and
Kim C J, et al. Dendritic cells infected with poxviruses encoding
MART-1/Melan A sensitize T lymphocytes in vitro. J Immunother. 1997
July; 20(4):276-86. The recombinant vectors can also infect tumour
cells. Alternatively, the recombinant vectors are able to infect
any cell in the mammal.
[0162] Other examples of vectors include ex vivo delivery systems,
which include but are not limited to DNA transfection methods such
as electroporation, DNA biolistics, lipid-mediated transfection and
compacted DNA-mediated transfection.
[0163] The vector may be a plasmid DNA vector. As used herein,
"plasmid" refers to discrete elements that are used to introduce
heterologous DNA into cells for either expression or replication
thereof. Selection and use of such vehicles are well within the
skill of the artisan.
Pulsed Cells
[0164] The present invention also provides cells pulsed with
peptides of the first aspect of the invention.
[0165] Preferably the cells to be pulsed are capable of expressing
MHC class I or class II.
[0166] MHC class I molecules can be expressed on nearly all cell
types, but expression of MHC class II molecules is limited to
so-called "professional" antigen presenting cells (APCs); B cells,
dendritic cells and macrophages. However, expression of MHC class
II can be induced on other cell types by treating with
IFN.gamma..
[0167] Expression of MHC class I or MHC class II molecules can also
be achieved by genetic engineering (i.e. provision of a gene
encoding the relevant MHC molecule to the cell to be pulsed). This
approach has the advantage that an appropriate MHC haplotype(s) can
be chosen which bind specifically to the peptide(s).
[0168] Preferably the cell to be pulsed is an antigen presenting
cell, i.e. a cell which, in a normal immune response, is capable of
processing an antigen and presenting it at the cell surface in
conjunction with an MHC molecule. Antigen presenting cells include
B cells, macrophages and dendritic cells. In an especially
preferred embodiment, the cell is a dendritic cell.
[0169] Preferably the cell is capable of expressing an MHC molecule
which binds a peptide according to the first aspect of the
invention in its peptide binding groove. For example, the cell may
express one of the following HLA restriction elements: B8, Cw7 or
A2 (for MHC class I).
[0170] Peptide pulsing protocols are known in the art (see for
example Redchenko and Rickinson (1999) J. Virol. 334-342; Nestle et
al (1998) Nat. Med. 4 328-332; Tjandrawan et al (1998) J.
Immunotherapy 21 149-157). For example, in a standard protocol for
loading dendritic cells with peptides, cells are incubated with
peptide at 50 .mu.g/ml with 3 .mu.g/ml .beta.-2 microglobulin for
two hours in serum free medium. The unbound peptide is then washed
off.
[0171] The pulsed cell of the present invention may be used as a
vaccine, for example to stimulate a prophylactic or therapeutic
anti-5T4 immune response.
[0172] The present invention therefore also provides a method for
treating and/or preventing a disease which comprises the step of
administering a peptide-pulsed cell to a subject in need of
same.
Vaccine/Pharmaceutical Composition
[0173] The present invention also provides a vaccine/pharmaceutical
composition comprising a peptide epitope, a polyepitope string, a
nucleic acid sequence, a vector system and/or a cell according to
previous aspects of the invention.
[0174] The vaccine/pharmaceutical composition may be for
prophylactic or therapeutic use.
[0175] The vaccine may by prepared as an injectable, either as
liquid solution or suspension; solid form suitable for solution in,
or suspension in, liquid prior to injection may also be prepared.
The preparation may also be emulsified, or the protein encapsulated
in liposomes. The active immunogenic ingredients are often mixed
with excipients which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, or the like
and combinations thereof.
[0176] In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine. Examples of adjuvants which may be
effective include but are not limited to: aluminium hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred
to as MTP-PE), and RIBI, which contains three components extracted
from bacteria, monophosphoryl lipid A, trehalose dimycolate and
cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80
emulsion.
[0177] Further examples of adjuvants and other agents include
aluminium hydroxide, aluminium phosphate, aluminium potassium
sulphate (alum), beryllium sulphate, silica, kaolin, carbon,
water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide,
bacterial endotoxin, lipid X, Corynebacterium parvum
(Propionobacterium acnes), Bordetella pertussis,
polyribonucleotides, sodium alginate, lanolin, lysolecithin,
vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked
copolymers or other synthetic adjuvants. Such adjuvants are
available commercially from various sources, for example, Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,
Detroit, Mich.).
[0178] Typically, adjuvants such as AMPHIGEN.TM. (oil-in-water),
ALHYDROGEL.TM. (aluminium hydroxide), or a mixture of AMPHIGEN.TM.
and ALHYDROGEL.TM. adjuvants are used. Only aluminium hydroxide is
approved for human use.
[0179] The proportion of immunogen and adjuvant can be varied over
a broad range so long as both are present in effective amounts. For
example, aluminium hydroxide can be present in an amount of about
0.5% of the vaccine mixture (Al.sub.2O.sub.3 basis). Conveniently,
the vaccines are formulated to contain a final concentration of
immunogen in the range of from 0.2 to 200 .mu.g/ml, preferably 5 to
50 .mu.g/ml, most preferably 15 .mu.g/ml.
[0180] After formulation, the vaccine may be incorporated into a
sterile container which is then sealed and stored at a low
temperature, for example 4.degree. C., or it may be freeze-dried.
Lyophilisation permits long-term storage in a stabilised form.
[0181] The vaccine may be administered in a convenient manner such
as by the oral, intravenous (where water soluble), intramuscular,
subcutaneous, intranasal, intradermal or suppository routes or
implanting (e.g. using slow release molecules).
[0182] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1% to 2%. Oral formulations include such normally
employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10% to 95% of
active ingredient, preferably 25% to 70%. Where the vaccine
composition is lyophilised, the lyophilised material may be
reconstituted prior to administration, e.g. as a suspension.
Reconstitution is preferably effected in buffer.
[0183] Capsules, tablets and pills for oral administration to a
patient may be provided with an enteric coating comprising, for
example, EUDRAGIT.TM. coating "S", EUDRAGIT.TM. coating "L",
cellulose acetate, cellulose acetate phthalate or
hydroxypropylmethyl cellulose.
[0184] 5T4 peptides may be formulated into the vaccine as neutral
or salt forms. Pharmaceutically acceptable salts include the acid
addition salts (formed with free amino groups of the peptide) and
which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids such as
acetic, oxalic, tartaric and maleic. Salts formed with the free
carboxyl groups may also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine and procaine.
Heterologous Vaccination Regimes
[0185] Regimes for administration of vaccines/pharmaceutic
compositions according to the present invention may be determined
by conventional efficacy testing. Especially preferred, however,
are regimes which include successive priming and boosting steps. It
is observed that such regimes achieve superior breaking of immune
tolerance and induction of T cell responses (see Schneider et al.,
1998 Nat Med 4:397-402).
[0186] Prime-boost regimes may be homologous (where the same
composition is administered in subsequent doses) or heterologous
(where the primimg and boosting compositions are different). For
example, the priming composition may be a non-viral vector (such as
a plasmid) encoding a 5T4 antigen and the boosting composition may
be a viral vector (such as a poxvirus vector) encoding a 5T4
antigen, wherein either or both of said "5T4 antigens" is an
epitope or polyepitope string of the present invention.
Diagnostic Methods
[0187] The present invention also provides an agent capable of
binding specifically to a peptide according to the present
invention and/or a nucleic acid sequence which encodes such a
peptide.
[0188] An agent is considered to "bind specifically" to a
peptide/nucleic acid sequence of the present invention if there is
a greater than 10 fold difference, and preferably a 25, 50 or 100
fold difference between the binding of the agent to a
peptide/nucleic acid sequence of the present invention and another
peptide/nucleic acid sequence.
[0189] The agent may be any compound capable of binding
specifically to a peptide and/or a nucleic acid sequence. The term
"compound" refers to a chemical compound (naturally occurring or
synthesised), such as a biological macromolecule (e.g., nucleic
acid, protein, non-peptide, or organic molecule), or an extract
made from biological materials such as bacteria, plants, fungi, or
animal (particularly mammalian) cells or tissues, or even an
inorganic element or molecule.
[0190] Preferably the agent is identifiable by screening a library
of candidate compounds. Libraries of compounds may be screened in
multi-well plates (e.g., 96-well plates), with a different test
compound in each well. In particular, the library of candidate
compounds may be a combinatorial libraries. A variety of
combinatorial libraries of random-sequence oligonucleotides,
polypeptides, or synthetic oligomers have been proposed and number
of small-molecule libraries have also been developed. Combinatorial
libraries of oligomers may be formed by a variety of solution-phase
or solid-phase methods in which mixtures of different subunits are
added stepwise to growing oligomers or parent compound, until a
desired oligomer size is reached (typically hexapeptide or
heptapeptide). A library of increasing complexity can be formed in
this manner, for example, by pooling multiple choices of reagents
with each additional subunit step. Alternatively, the library may
be formed by solid-phase synthetic methods in which beads
containing different-sequence oligomers that form the library are
alternately mixed and separated, with one of a selected number of
subunits being added to each group of separated beads at each step.
Libraries, including combinatorial libraries are commercially
available from pharmaceutical companies and speciality library
suppliers.
[0191] Where the agent recognises a nucleic acid according to the
present invention, the agent may comprise an antisense
sequence.
[0192] Where the agent recognises a peptide according to the
present invention, the agent may comprise an MHC molecule or part
thereof which comprises the peptide binding groove. Alternatively
the agent may comprise an anti-peptide antibody.
[0193] As used herein, "antibody" includes a whole immunoglobulin
molecule or a part thereof or a bioisostere or a mimetic thereof or
a derivative thereof or a combination thereof. Examples of a part
thereof include: Fab, F(ab)'.sub.2, and Fv. Examples of a
bioisostere include single chain Fv (ScFv) fragments, chimeric
antibodies, bifunctional antibodies.
[0194] The term "mimetic" relates to any chemical which may be a
peptide, polypeptide, antibody or other organic chemical which has
the same binding specificity as the antibody.
[0195] The term "derivative" as used herein in relation to
antibodies includes chemical modification of an antibody.
Illustrative of such modifications would be replacement of hydrogen
by an alkyl, acyl, or amino group.
[0196] A whole immunoglobulin molecule is divided into two regions:
binding (Fab) domains that interact with the antigen and effector
(Fc) domains that signal the initiation of processes such as
phagocytosis. Each antibody molecule consists of two classes of
polypeptide chains, light (L) chains and heavy (H) chains. A single
antibody has two identical copies of the L chain and two of the H
chain. The N-terminal domain from each chain forms the variable
regions, which constitute the antigen-binding sites. The C-terminal
domain is called the constant region. The variable domains of the H
(V.sub.H) and L (V.sub.L) chains constitute an Fv unit and can
interact closely to form a single chain Fv (ScFv) unit. In most H
chains, a hinge region is found. This hinge region is flexible and
allows the Fab binding regions to move freely relative to the rest
of the molecule. The hinge region is also the place on the molecule
most susceptible to the action of protease which can split the
antibody into the antigen binding site (Fab) and the effector (Fc)
region.
[0197] The domain structure of the antibody molecule is favourable
to protein engineering, facilitating the exchange between molecules
of functional domains carrying antigen-binding activities (Fabs and
Fvs) or effector functions (Fc). The structure of the antibody also
makes it easy to produce antibodies with an antigen recognition
capacity joined to molecules such as toxins, lymphocytes or growth
factors.
[0198] Chimeric antibody technology involves the transplantation of
whole mouse antibody variable domains onto human antibody constant
domains. Chimeric antibodies are less immunogenic than mouse
antibodies but they retain their antibody specificity and show
reduced HAMA responses.
[0199] In chimeric antibodies, the variable region remains
completely murine. However, the structure of the antibody makes it
possible to produce variable regions of comparable specificity
which are predominantly human in origin. The antigen-combining site
of an antibody is formed from the six complementarity-determining
regions (CDRs) of the variable portion of the heavy and light
chains. Each antibody domain consists of seven antiparallel
.beta.-sheets forming a .beta.-barrel with loops connecting the
.beta.-strands. Among the loops are the CDR regions. It is feasible
to more the CDRs and their associated specificity from one
scaffolding .beta.-barrel to another. This is called CDR-grafting.
CDR-grafted antibodies appear in early clinical studies not to be
as strongly immunogenic as either mouse or chimaeric antibodies.
Moreover, mutations may be made outside the CDR in order to
increase the binding activity thereof, as in so-called humanised
antibodies.
[0200] Fab, Fv, and single chain Fv (ScFv) fragments with VH and VL
joined by a polypeptide linker exhibit specificities and affinities
for antigen similar to the original monoclonal antibodies. The ScFv
fusion proteins can be produced with a non-antibody molecule
attached to either the amino or carboxy terminus. In these
molecules, the Fv can be used for specific targeting of the
attached molecule to a cell expressing the appropriate antigen.
Bifunctional antibodies can also be created by engineering two
different binding specificities into a single antibody chain.
Bifunctional Fab, Fv and ScFv antibodies may comprise engineered
domains such as CDR grafted or humanised domains.
[0201] Procedures for identifying, characterising, cloning and
engineering polyclonal and monoclonal antibodies and their
derivatives are well established, for example using hybridomas
derived from mice or transgenic mice, phage-display libraries or
scFv libraries. Genes encoding immunoglobulins or
immunoglobulin-like molecules can be expressed in a variety of
heterologous expression systems. Large glycosylated proteins
including immunoglobulins are efficiently secreted and assembled
from eukaryotic cells, particularly mammalian cells. Small,
non-glycosylated fragments such as Fab, Fv, or scFv fragments can
be produced in functional form in mammalian cells or bacterial
cells.
[0202] The agent may recognise the peptide/nucleic acid of the
present invention alone, or in conjunction with another compound.
For example, the agent may be capable or binding specifically to
the peptide when presented by an MHC molecule. In this case, the
agent of the present invention may comprise a T cell receptor
molecule or part thereof.
[0203] The T cell receptor may be associated with another molecule
such as CD4 (for MHC class II epitopes) or CD8 (for MHC class I
epitopes). Alternatively, or in addition, the receptor may be
associated with CD3.
[0204] If the agent occurs naturally in the human body, then
preferably the agent of the present invention is in a substantially
isolated form.
[0205] The present invention also provides a method which comprises
the step of detecting the presence of a peptide, nucleic acid or
agent of the present invention in a subject.
[0206] In a preferred embodiment, the method is used to detect the
presence of T cells capable of specifically recognising a peptide
epitope according to the present invention in conjunction with an
MHC molecule.
[0207] The diagnostic method may, for example, be for diagnosing or
monitoring the progression of a disease or for monitoring the
progression of an immune response in a subject.
[0208] As mentioned above, as an immune response progresses, the
dominance of particular epitopes may change, and sub-dominant
epitopes can predominate. Thus by detecting the presence of a
particular epitope, or a TCR/T cell capable of recognising such an
epitope, information can be gained about the progression of the
immune response.
[0209] The method may be carried out in vivo, or more preferably on
an ex vivo sample.
[0210] Thus the present invention also provides a diagnostic method
which comprises the following steps: [0211] (i) isolating a sample
from a subject; [0212] (ii) detecting in the sample ex vivo the
presence of T cells capable of specifically recognising a peptide
epitope according to the present invention in conjunction with an
MHC molecule.
[0213] In a preferred embodiment, the method is for diagnosing or
monitoring the progression of a cancerous disease.
[0214] The nature of the method will depend on whether a peptide,
nucleic acid or agent of the present invention is being detected
(and if it is an agent, on the nature of that agent).
[0215] In order to detect a peptide of the present invention, an
agent of the present invention (such as an antibody or an MHC
molecule) may be used. Methods of screening with antibodies (such
as ELISAs, immunoblotting, western blotting, competitive assays,
two site capture assays) are well known in the art.
[0216] In order to detect peptides or specific T cells, an antigen
presentation assay may be used. When a T cell successfully
recognises an MHC:peptide complex, it is stimulated. This
stimulation can be monitored by proliferation of the T cells (for
example by incorporation of .sup.3H) and/or by production of
cytokines by the T cells (for example by an ELISPOT assay). Thus it
is possible to detect the presence of a specific peptide by using
appropriate APCs and T cells lines, and to detect the presence of a
specific T cell by using appropriate APCs and peptide/antigen.
[0217] The presence of a particular cell surface molecule (such as
a TCR or MHC molecule) can also be investigated using fluorescence
activated cell scanning (FACS).
[0218] Where the method is to detect the presence of a nucleic
acid, numerous methods are known in the art such as PCR, southern
blotting (for DNA) and northern blotting (for RNA).
T Cells
[0219] The present invention also relates to a T cell, such as a T
cell clone, or line, which is capable of specifically recognising a
peptide epitope according to the present invention in conjunction
with an MHC molecule. Several methods for generating T cell lines
and clones are known in the art. One method for generating T cell
lines is as follows:
[0220] Mice are primed with antigen (usually subcutaneously in the
rear footpad), and the draining lymph nodes (in this case the
popliteal and inguinal) are removed 1 week later and set up in
co-culture with the antigen and with syngeneic feeder cells i.e.
cells from mice of the same inbred line (e.g. normal thymocytes or
splenocytes). After 4 days the lymphoblasts are isolated and
induced to proliferate with IL-2. When the population of cells has
expanded sufficiently, they are checked for antigen and MHC
specificity in a lymphocyte transformation test, and are maintained
by alternate cycles of culture on antigen-treated feeder cells and
culture in IL-2-containing medium.
[0221] The definitive T-cell lineage marker is the T-cell receptor
(TCR). There are presently two defined types of TCR, both of which
are heterodimers of two disulphide-linked polypeptides. One type
consist of .alpha. and .beta. chains, the other type consists of
.gamma. and .delta. chains. Approximately 90-95% of blood T cells
express .alpha./.beta. TCR, the other 5-10% expressing
.gamma./.delta. TCR.
[0222] T cells can be divided into two distinct populations: a
subset which carries the CD4 marker and mainly "helps" or "induces"
immune responses (T.sub.H) and a subset which carries the CD8
marker and is predominantly cytotoxic (T.sub.C). CD4+ T cells
recognise peptides in association with MHC class II molecules,
whereas CD8+ T cells recognise peptides in association with Class I
molecules, so the presence of CD4 or CD8 restricts the types of
cell with which the T cell can interact.
[0223] The CD4 set has been functionally sub-divided into two
further subsets: [0224] (i) T cells that positively influence the
response of T cells and B cells (the helper T cell function) are
CD29+. Practically all the cells in this population also express a
low molecular weight isoform of the CD45 leucocyte common antigen,
designated CD45RO. [0225] (ii) Cells that induce the
supressor/cytotoxic functions of CD8+ cells (the suppressor/inducer
function) express a different form of the CD45 molecule,
CD45RA.
[0226] Functional diversity has also been demonstrated by
functional analysis of T.sub.H clones for cytokine secretion
patterns. The T.sub.H1 subset of CD4+ T cells secrete IL-2 and
IFN-.gamma., whereas the T.sub.H2 subset produces IL-4, IL-5, IL-6
and IL-10. T.sub.H1 cells mediate several functions associated with
cytotoxicity and local inflammatory reactions. Consequently these
cells are important for combatting intracellular pathogens,
including viruses, bacteria and parasites. T.sub.H2 cells are more
effective at stimulating B cells to proliferate and produce
antibodies, and therefore in normal immune responses function to
protect against free-living organisms.
[0227] Expression of all of the markers described above can readily
be detected using specific antibodies, so the type of T cell can be
selected/determined using FACS. Expression of particular cytokines
can also be detected by methods known in the art, such as ELISPOT
assay.
Prophylactic/Therapeutic Methods
[0228] The present invention also provides the use of a vaccine
according to the present invention in the manufacture of a
medicament for use in the prevention and/or treatment of a
disease.
[0229] There is also provided a method for treating and/or
preventing a disease in a subject which comprises the step of
administering an effective amount of a vaccine according to the
present invention.
[0230] Administration of the vaccine may elicit an immune response
in the subject. In a preferred embodiment, administration of the
vaccine breaks immune tolerance to 5T4 in the subject.
[0231] Where the peptide is a class I epitope, the immune response
elicited may involve the activation of 5T4 specific cytotoxic
T-lymphocytes. Where the peptide is a class II epitope, the immune
response elicited may involve the activation of T.sub.H1 and/or
T.sub.H2 cells.
[0232] Advantageously, the response is an anti-tumour
immunotherapeutic response which is effective to inhibit, arrest or
reverse the development of a tumour in a subject.
Combination Therapies
[0233] The invention further relates to the use of 5T4 targeting
molecules, such as anti-5T4 antibodies, for example anti-5T4 scFvs.
These antibodies may be used to (i) to target natural or exogenous
5T4 in situ and/or (ii) deliver immune enhancer molecules, such as
B7.1, to natural or exogenous 5T4 in situ (Carroll et al. (1998) J
Natl Cancer Inst 90(24):1881-7). This potentiates the
immunogenicity of 5T4 in the subject.
[0234] The present invention thus also relates to the sequential
use of a vaccine according to the present invention and anti-5T4
antibodies, for example anti-5T4 scFvs. The anti-5T4 scFvs
antibodies may be administered as naked DNA encoding the antibodies
(for example, in a plasmid comprising the encoding DNA together
with a short promoter region to control its production), in an
expression vector (which may be viral or non-viral) comprising the
encoding sequence or in a protein form. Thus, the invention
provides a vector encoding a 5T4 peptide antigen and an agent
capable of binding 5T4 which is optionally fused with an
immunostimulatory molecule, for separate, such as sequential use,
in the treatment of tumours.
[0235] In a further embodiment, the invention encompasses a
combination therapy including enzyme/prodrug therapy and
immunotherapy with 5T4. For example, the enzyme/prodrug therapy may
comprise intratumoural or systemic delivery of P450, delivered
optionally using an retroviral or lentiviral vector, and
cyclophosphamide (CPA) followed by systemic immunotherapeutic
induction with 5T4.
[0236] Thus, the invention further relates to a vector encoding 5T4
peptide antigen and a prodrug/enzyme combination, for separate,
simultaneous separate or combined use in the treatment of
tumours.
Diseases
[0237] 5T4 is a tumour associated antigen. Presence of 5T4 on
cancer cells is associated with metastasis and has been shown to be
an independent indicator of prognosis in a number of different
cancers.
[0238] In a preferred embodiment, the disease (which is
preventable/treatable using a vaccine according to the present
invention) is a cancer. In particular the disease may be a
carcinoma of, for example, the breast, lung, stomach, pancreas,
endometrium, cervix, colorectal, renal or prostate.
[0239] WO89/07947 describes an immunohistochemical screen of
neoplastic tissues using an anti-5T4 monoclonal antibody (see
Tables II and VI). Preferably, the disease is a cancer which can be
shown to be 5T4 positive by diagnostic testing (such as with an
anti-5T4 antibody), for example: an invasive carcinoma of the
Ampulla of Vater, breast, colon, endometrium, pancreas, or stomach;
a squamous carcinoma of the bladder, cervix, lung or oesophagus; a
tubulovillous adenoma of the colon; a malignant mixed Mullerian
tumour of the endometirem; a clear cell carcinoma of the kidney; a
lung cancer (large cell undifferentiated, giant cell carcinoma,
broncho-alveolar carcinoma, metastatic leiomyosarcoma); an ovarian
cancer (a Brenner tumour, cystadenocarcinoma, solid teratoma); a
cancer of the testis (seminoma, mature cystic teratoma); a soft
tissue fibrosarcoma; a teratoma (anaplastic germ cell tumours); or
a trophoblast cancer (choriocarcimoma (e.g. in uterus, lung or
brain), tumour of placental site, hydatidiform mole).
Tetramers
[0240] The present invention also provides 5T4 peptide epitope
associated with (eg. folded with) tetramers and uses thereof.
[0241] Tetramers are fluorescent reagents that allow for the direct
visualisation of antigen-specific T-cells (Altman et al. (1996)
Science 271, 94-96). They consist of individual peptides epitopes
refolded with HLA class I protein and bind to T cells that are
specific for that particular epitope. They allow for the direct
quantification of antigen specific lymphocytes and have been
applied widely in human and murine immunology.
[0242] The tetramers may be prepared using the methods described by
Altman et al. (1996) Science 271, 94-96. Briefly, tetramers may be
prepared by adding biotinylated protein to streptavidin PE at a
ratio of 4:1. Tetramer bound cells may be selected using magnetic
activated cell sorting (MACS). MACS has been described in Radbruch
et al. (1994) Methods in Cell Biology 42, 387-403.
[0243] Advantageously, the use of tetramers allows for the tracking
of a 5T4-specific immune response before, during and after
vaccination; to purify autologous CD4+ T cells from individual
patients and expand/manipulate them ex vivo for possible
re-infusion; as a diagnostic indicator, for example, in subjects
prone to colorectal and other 5T4-positive cancers. Accordingly,
the present invention also relates to the use of a 5T4 peptide
epitope tetramer for monitoring a 5T4-specific immune response
before, during or after vaccination. The present invention further
relates to the use of a 5T4 peptide epitope tetramer for the
purification of autologous CD4+ T cells from individual patients.
The present invention still further relates to the use of a 5T4
peptide epitope tetramer as a diagnostic indicator in subjects
prone to 5T4-positive cancers--such as colorectal cancers.
[0244] The invention is further described, for the purposes of
illustration only, in the following examples in which reference is
made to the following Figures.
[0245] FIG. 1 is a schematic diagram illustrating the proliferation
assay for measuring cellular responses
[0246] FIG. 2 is a schematic diagram illustrating the ELISPOT assay
for measuring antigen-specific IFN.gamma. production.
[0247] FIG. 3 shows the proliferative responses of breast cancer
patients OBCR24 and OBCR25 following stimulation in vitro with h5T4
peptide pools
[0248] FIG. 4 shows proliferative responses of PBMCs from OBCR25
following stimulation in vitro with h5T4 peptides
[0249] FIG. 5 shows an ELISPOT assay to show IFN-.gamma. secretion
by PBMCs from OBCR24 and OBCR25 following stimulation in vitro with
antigens
[0250] FIG. 6 shows an ELISPOT assay to show IFN-.gamma. secretion
by PBMCs from OBCR25 following stimulation in vitro with single
h5T4 peptides
[0251] FIG. 7 shows proliferative responses of PBMCs from OBCR27
following stimulation in vitro with h5T4 peptide pools.
[0252] FIG. 8 shows an ELISPOT assay to show IFN-.gamma. secretion
by PBMCs from OBCR27 following stimulation in vitro with h5T4
peptide pools.
[0253] FIG. 9 shows proliferative responses of PBMCs from TV1-104
at 2 and 4 weeks post-vaccination following stimulation in vitro
with h5T4 peptides
[0254] FIG. 10 shows an ELISPOT assay to show IFN-.gamma. secretion
by PBMCs from TV1-104 at 2 and 4 weeks post-vaccination following
stimulation in vitro with antigens
EXAMPLES
Example 1
Non-TROVAX.RTM. Patients
Example 1A
Breast Cancer Patients OBCR25
[0255] A h5T4 peptide library was produced consisting of 51 20mer
peptides overlapping by 8 amino acids. Class II peptide pools
containing 5 peptides per pool were incubated with PBMCs from
breast cancer patients OBCR24 and OBCR25 and proliferation was
measures using the assay illustrated in FIG. 1. After 6 days
incubation, cells were pulsed overnight with .sup.3H-thymidine and
incubated overnight at 37.degree. C. It was found that peptides
present in pool #10 elicit a strong proliferative response with
OBCR25 cells (FIG. 3).
[0256] Dissection of the proliferative response seen to peptide
pool 10 revealed that the positive response resides in the 20mer
peptide #51 (FIG. 4).
[0257] To measure antigen-specific IFN.gamma. production, an
ELISPOT assay was conducted following the general scheme
illustrated in FIG. 2. IFN.gamma. ELISPOT of PBMCs from OBCR25
following restimulation in vitro with h5T4 peptides shows that the
positive ELISPOT response observed in pool #10 resides entirely in
peptide #51 (FIG. 5).
[0258] The "pepset" peptide comprise the overlapping peptides
spanning the entire sequence of 5T4. As they are mass produced, the
level of purity is not very high. To rule out the possibility of
contamination, the present inventors repeated the experiment using
peptide 51 resynthesised to high purity (FIG. 6).
[0259] Peptide .about.51 comprises the extreme C-terminal 20 amino
acids of h5T4:
TABLE-US-00004 (SEQ ID. NO. 5)
.sup.401YRYEINADPRLTNLSSNSDV.sup.420-
[0260] This sequence shows no significant homology to any other
protein sequence deposited in accessible databases.
[0261] By using truncated versions of peptide #51, it is possible
to determine the minimal epitope of this peptide.
HLA Typing of OBCR25
[0262] PBMCs from patient OBCR25 were HLA typed, giving the
following results:
TABLE-US-00005 OBCR25: HLA-A *2402/5/9/11/14-15/20/
*2601/11-12/14-15 25-27/30 HLA-B *5001/4 *5101/3/9/11/14/16-18/21
HLA-Cw *0602-3/7 *1502-6/9 HLA-DRB1 *0701 *1301/28/35 HLA-DRB3
*0201-3/5/11/13 HLA-DRB4 *0101-5 HLA-DQB1 *0201-2
*0602-4/7-8/10-11/13-16
Example 1B
Duke's B Colon Cancer Patient OBCR27
[0263] PBMCs from OBCR27 were stimulated in vitro with h5T4 class
II peptide pools and proliferative responses were measured using
the assay outlined in FIG. 1. Strong proliferative responses were
seen to peptide pools 8, 9 and 10 (FIG. 7).
[0264] An IFN.gamma. ELISPOT assay (as outlined in FIG. 2) was also
conducted using class II peptide pools. Positive ELISPOT responses
were seen to peptide pools 8, 9 and 10 (FIG. 8).
[0265] By analysing the individual peptides from each pool, it is
possible to establish which one(s) are responsible for the observed
positive proliferative and ELISPOT responses, and which therefore
are or comprise an h5T4 class II epitope.
Example 2
TROVAX.RTM. Colorectal Patients
[0266] Throughout the phase I/II TROVAX.RTM. clinical trial,
patients were monitored for their ability to mount an immune
response to the h5T4 overlapping peptide library.
[0267] Of 8 people recruited to the trial, 7 have shown a response
to one or more peptides post-boost, but not prior to
vaccination.
[0268] Looking at one patient in particular (TV1-104) proliferation
of PBMCs from this patient to different antigen preparations were
examined at 2 and 4 weeks post-vaccination, using the assay
outlined in FIG. 1. Potent proliferative responses are observed to
peptides present in pool #9 and, in particular its constituent
peptide 45 (FIG. 9). A potent IFN.gamma. ELISPOT response was also
induced by peptide present in pool #9 and in particular its
constituent peptide #45 (FIG. 10) using the assay shown in FIG.
2.
[0269] By using truncated versions of peptide #45, it is possible
to determine the minimal epitope of this peptide.
HLA Typing of TV1-104
[0270] PBMCs from patient TV1-104 were HLA typed, giving the
following results:
TABLE-US-00006 TV1-104: HLA-A *0206-7/10/14-15/17-18/21/33/ *0301-2
39/41 HLA-B *0702/4/15/22-23/25 *4001/22/30/32/34 HLA-Cw
*0702/13/15 *0304-6/8-9/14 HLA-DRB1 *0404/8/19/22-23/25/31-32/36/42
*1301/28/35 HLA-DRB3 *0101/3-6 HLA-DRB4 *0101-5 HLA-DQB1 *0302/7-8
*0602-4/7-8/10-11/ 13-16
[0271] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology or related
fields are intended to be within the scope of the following claims.
Sequence CWU 1
1
511260DNAFelis catus 1atgcctgggg ggtgctcccg gggccccgcc gccggagacg
ggcggctgcg gctggcgcgg 60ctggcgctgg tcctcctggg ctgggtctct tcgtcttctc
tcacttcctc ggcgccctcc 120acctcctcca cgtcgttcct ggcctccgcg
gtgtccgccc agcccccgct gccgggccaa 180tgcccccagc tttgcgagtg
ctccgaggcg gcgcgcactg tcaagtgcgt taaccgcaac 240ctgaccgagg
tgcccgcgga cctgcccccc tacgtgcgca acctcttcct caccggcaat
300cagctggccg tgctccccgc cggcgccttc gcccgccggc cgccgctggc
ggagctggcc 360gcgctcaacc tcagcggcag ccgcctgcag gaggtgcgcg
ccggcgcctt cgagcaactg 420cccagcctgc ggcagctcga cctcagccac
aacccgctgg cccacctcag ccccttcacc 480ttctcgggca gcaacgccag
cttctcggcc cccagccccc tggtggaact gatgctgaac 540cacatcgtgc
cccctgagga ccaccggcac aaccggagct tcgagggtat ggtggcggcg
600tccctacgcg ccggccatgc gcttcgcggg ctccagcgcc tygaactggc
cagcaaccac 660ttcctcttct tgcctcggga cgtactggcc cacctaccgg
gcctcaggca cctggacctg 720cgcaacaact cgctggtgag cctaacttac
gtgtccttcc gcaacctgac acacctacaa 780agcctccacc tggaggacaa
cgccctcaag gtccttcaca acggcaccat ggcggagttg 840cagagcctgc
cccacgtcag ggtcttcctg gacaacaatc cctgggtctg cgactgtcac
900atggtggaca tggtggcctg gctcaaggag acagaggtag tgcagggcaa
agccaggctc 960gcctgtgcat tcccggaaaa aatgaggaat cgggcccttt
tggaactcaa cagctcccac 1020ctggagtgtg accctatcct ccctccatcc
ctgcagactt cttatgtctt tctaggtatt 1080gttttagccc tgataggtgc
cattttctta ctggttttgt acttgaaccg caaggggata 1140aaaaagtgga
tgcataacat cagagatgcc tgcagggatc acatggaagg gtatcactac
1200agatatgaaa tcaacgcgga ccccaggtta acaaacctca gttctaattc
ggatgtctga 1260220PRTHomo sapiens 2Tyr Arg Tyr Glu Ile Asn Ala Asp
Pro Arg Leu Thr Asn Leu Ser Ser 1 5 10 15 Asn Ser Asp Val 20
320PRTHomo sapiens 3Gln Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala
Leu Ile Gly Ala 1 5 10 15 Ile Phe Leu Leu 20 4419PRTFelis catus
4Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu 1
5 10 15 Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser
Ser 20 25 30 Ser Leu Thr Ser Ser Ala Pro Ser Thr Ser Ser Thr Ser
Phe Leu Ala 35 40 45 Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Gly
Gln Cys Pro Gln Leu 50 55 60 Cys Glu Cys Ser Glu Ala Ala Arg Thr
Val Lys Cys Val Asn Arg Asn 65 70 75 80 Leu Thr Glu Val Pro Ala Asp
Leu Pro Pro Tyr Val Arg Asn Leu Phe 85 90 95 Leu Thr Gly Asn Gln
Leu Ala Val Leu Pro Ala Gly Ala Phe Ala Arg 100 105 110 Arg Pro Pro
Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser Arg 115 120 125 Leu
Gln Glu Val Arg Ala Gly Ala Phe Glu Gln Leu Pro Ser Leu Arg 130 135
140 Gln Leu Asp Leu Ser His Asn Pro Leu Ala His Leu Ser Pro Phe Thr
145 150 155 160 Phe Ser Gly Ser Asn Ala Ser Phe Ser Ala Pro Ser Pro
Leu Val Glu 165 170 175 Leu Met Leu Asn His Ile Val Pro Pro Glu Asp
His Arg His Asn Arg 180 185 190 Ser Phe Glu Gly Met Val Ala Ala Ser
Leu Arg Ala Gly His Ala Leu 195 200 205 Arg Gly Leu Gln Arg Leu Glu
Leu Ala Ser Asn His Phe Leu Phe Leu 210 215 220 Pro Arg Asp Val Leu
Ala His Leu Pro Gly Leu Arg His Leu Asp Leu 225 230 235 240 Arg Asn
Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn Leu 245 250 255
Thr His Leu Gln Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val Leu 260
265 270 His Asn Gly Thr Met Ala Glu Leu Gln Ser Leu Pro His Val Arg
Val 275 280 285 Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met
Val Asp Met 290 295 300 Val Ala Trp Leu Lys Glu Thr Glu Val Val Gln
Gly Lys Ala Arg Leu 305 310 315 320 Ala Cys Ala Phe Pro Glu Lys Met
Arg Asn Arg Ala Leu Leu Glu Leu 325 330 335 Asn Ser Ser His Leu Glu
Cys Asp Pro Ile Leu Pro Pro Ser Leu Gln 340 345 350 Thr Ser Tyr Val
Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala Ile 355 360 365 Phe Leu
Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp Met 370 375 380
His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr His Tyr 385
390 395 400 Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser
Ser Asn 405 410 415 Ser Asp Val 520PRTHomo sapiens 5Tyr Arg Tyr Glu
Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser 1 5 10 15 Asn Ser
Asp Val 20
* * * * *
References