Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 33
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 33
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
VACCINE
Field of the Invention
The present invention relates to human papillomavirus (HPV) vaccines.
Background of the Invention
Papillomaviruses are small DNA tumour viruses, which are highly species
specific. So far, over 100 individual human papillomavirus (HPV) genotypes
have
been described. HPVs are generally specific either for the skin (e.g. HPV-1
and -2) or
mucosal surfaces (e.g. HPV-6 and -11) and usually cause benign tumours (warts)
that
persist for several months or years. Such benign tumours may be distressing
for the
individuals concerned but tend not to be life threatening, with a few
exceptions.
Some HPVs are also associated with cancers, known as oncogenic HPV types.
The strongest positive association between an HPV and human cancer is that
which
exists between HPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is
the
most common malignancy in developing countries, with about 500,000 new cases
occurring in the world each year.
Other HPV types which can cause cancer are types 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 66 and 68 (referred to as "oncogenic-HPV types"). Types 16 and 18
are
those which have the highest association with cervical cancer. Types 31 and 45
are the
types with the next highest association with a cancer risk (Munoz N, Bosch FX,
de
Sanjose S et al. International Agency for Research on Cancer Multicenter
Cervical
Cancer Study Group. NEngl JMed 2003; 348: 518-27.)
HPV virus like particles (VLPs) have been suggested as potential vaccines for
treatment of HPV. Animal studies have shown that VLPs produce no cross
protection
against infection for other HPV types - see, for example Suzich, J. A., et al,
Proc Natl
Acad Sci, 92: 11553-11557, 1995, and Breitburd, Seminars in Cancer Biology,
vo19,
1999, pp 431 - 445.
W02004/056389 discloses that an HPV 16, 18 VLP vaccine can provide cross
protection against infection by HPV types other than 16 and 18. Statistically
significant
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
2
protection was observed against certain groups of HPV types. However, the
level of
cross protection against individual types within groups was not disclosed.
There is still a need for a vaccine that protects against multiple HPV types.
Summary of the invention
The present invention relates to a multivalent HPV vaccine, the vaccine
comprising an L1 protein or immunogenic fragment thereof from at least 3
different
oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the
vaccine
does not comprise an Ll protein or immunogenic fragment thereof from an HPV
type
selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination
thereof.
The present invention further relates to use of a composition comprising an Ll
protein or immunogenic fragment thereof from HPV 16 and HPV 18 in the
manufacture of a medicament for prevention of infection and/or disease by one
or more
of the group consisting of HPV 31, HPV 45 and HPV 52.
The present invention further relates to use of a composition comprising an L1
protein or immunogenic fragment thereof from HPV 16 and HPV 18 in the
manufacture of a medicament for prevention of cytological abnormalities,
and/or
reduction of the frequency of cytological abnormalities, and/or prevention of
CIN
lesions (ASCUS, CIN 1, CIN 2, CIN, cervical cancer) in an individual, the
abnormalities or lesions caused by at least one HPV type other than HPV 16 or
HPV
18, suitably being caused by HPV type 31, or 45, or 52, or a combination
thereof.
The invention further relates to a method of prevention and/or treatment of
HPV
infection and/or disease, the method comprising delivering to an individual in
need
thereof an effective amount of a composition comprising an Ll protein or
immunogenic
fragment thereof from at least 3 different oncogenic HPV types, those types
including
HPV 16 and HPV 18, wherein the vaccine does not comprise an L1 protein or
immunogenic fragment thereof from an HPV type selected from the list
consisting of
HPV 31, HPV 45, HPV 52 or any combination thereof.
The invention further relates to use of a multivalent composition in the
manufacture of a medicament for the prevention and/or treatment of HPV
infection
and/or disease, the multivalent composition comprising an Ll protein or
immunogenic
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
3
fragment thereof from at least 3 different oncogenic HPV types, those types
including
HPV 16 and HPV 18, wherein the vaccine does not comprise an L1 protein or
immunogenic fragment thereof from an HPV type selected from the list
consisting of
HPV 31, HPV 45, HPV 52 or any combination thereof, and wherein the medicament
provides protection against infection and/or disease caused by the omitted HPV
type.
The invention also relates to a method for manufacture of a vaccine, the
method
comprising combining an L1 protein or immunogenic fragment thereof from at
least 3
different oncogenic HPV types, those types including types HPV 16 and HPV 18,
wherein the vaccine does not comprise an Ll protein or immunogenic fragment
thereof
from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52
or any
combination thereof.
Detailed description
The general existence of cross protection afforded by HPV 16 and HPV 18
against both incident and persistent infection, as assessed in relation to
certain groups
of HPV types, has been disclosed in W02004/056389.
We have surprisingly discovered that the cross protection against certain (non
HPV 16, HPV 18) HPV types ( as assessed by the efficacy of an HPV 16 and HPV
18
vaccine against those types), is higher than against certain other (non HPV
16, HPV 18)
HPV types. Cross protection may be considered as the protection afforded by a
vaccine
containing one HPV type against infection (incident or persistent) and/or
disease
caused by a different HPV type. Cross protection may be assessed by
considering the
vaccine efficacy (V.E.), wherein the V.E. is the % improvement in protection
against
infection or disease by the vaccine compared to a placebo group for a given
type.
Infection may be incident or persistent infection. Disease may be abnormal
cytology, ASCUS, CIN1, CIN2, CIN3 or cervical cancer related to HPV infection.
Infection may be assessed by PCR, for example. Disease may be assessed by, for
example, histological examination or analysis of biomarkers such as p16.
Such a finding has potential implications for vaccine design. For example, the
level of cross protection afforded by HPV 16 and HPV 18 L1 containing vaccines
against certain other HPV types, such as HPV 31, HPV 45 and HPV 52, allows L1
components from these HPV types to be omitted from a vaccine comprising HPV 16
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
4
and HPV 18 while still providing a vaccine which provides some protection
against
incident and/or persistent infection and/or disease related to those omitted
types.
After HPV types 16 (found in 53.5% of cervical cancer) and 18 (found in 17.2%
of cervical cancer), types 45 (6.7%) and 31 (2.9%) are the next most
significant in
terms of their frequency in cervical cancers (Munoz et al. supra). HPV 33
(2.6%) is
next, followed by HPV 52 (2.3%). Thus, when designing a multivalent HPV
vaccine
against cervical cancer containing at least HPV 3 types then types 31 and 45
would
generally be included by the skilled person after types 16 and 18 from a
statistical
perspective.
The ability to omit antigens from certain HPV types potentially allows
inclusion
of LI protein from other HPV types, or indeed antigens from other viruses or
pathogens, into a vaccine in a scenario where the total amount of antigen in a
vaccine
may be limited, for example by physical, chemical, regulatory or other
constraints.
In particular other HPV types include oncogenic HPV types such as HPV 31,
33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
The present invention relates to a multivalent HPV vaccine, the vaccine
comprising an L1 protein or immunogenic fragment thereof from at least 3
different
oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the
vaccine
does not comprise an Li protein or immunogenic fragment thereof from an HPV
type
selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination
thereof.
In one aspect of the invention the vaccine does not contain an Ll protein or
immunogenic fragment thereof from HPV 31.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by HPV 31.
In one aspect of the invention the vaccine of the invention does not contain
an
LI protein or immunogenic fragment thereof from HPV 45.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by HPV 45.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
In one aspect of the invention the vaccine does not contain an Ll protein or
immunogenic fragment thereof from HPV 52.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by HPV 52.
5 In one aspect of the invention the vaccine of the invention does not contain
an
Ll protein or immunogenic fragment thereof from HPV 31 and 45.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by both HPV 31 and 45.
In one aspect of the invention the vaccine does not contain an Ll protein or
immunogenic fragment thereof from HPV 31 and 52.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by both HPV 31 and 52.
In one aspect of the invention the vaccine of the invention does not contain
an
L1 protein or immunogenic fragment thereof from HPV 45 and 52.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by both HPV 52 and 45.
In one aspect of the invention the vaccine is capable of providing protection
against incident and /or persistent HPV infection by HPV 31 and HPV 45 and
HPV52.
Suitably the vaccine is capable of protection against persistent infection.
Suitably the vaccine is capable of protection against incident infection.
Incident and persistent cervical infection are defined in Example 1.
We have also determined that a vaccine comprising HPV 16 Ll and HPV 18 L1
proteins (for example, as described in example 1) provides protection against
cytological abnormalities caused by certain other oncogenic HPV types such as
HPV
52, and is significantly protective with respect to such abnormalities caused
by a group
of HPV high risk types (defined as 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66,
and 68).
Cytological abnormalities are suitably detected by the well known Pap smear
technique.
Thus the invention further relates to use of a combination of an Ll protein or
immunogenic fragment thereof from HPV 16 and HPV 18 in the preparation of a
composition for the prevention of cytological abnormalities or reduction of
the
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
6
frequency of cytological abnormalities in an individual caused by other (non
HPV 16,
HPV 18) HPV types, suitably oncogenic HPV types, and in the prevention of
histologically-confirmed CIN lesions (CIN 1, CIN 2, CIN 3) and cervical cancer
associated with infection by HPV types which are not HPV 16 or 18. Said use is
in
addition to the prevention or reduction of such events caused by the HPV types
in the
vaccine, HPV 16 and 18.
Suitably the prevention of cytological abnormalities, reduction of the
frequency
of cytological abnormalities or prevention of histological-confirmed CIN
lesions is
prevention against those abnormalities or lesions caused by types not included
in the
combination, suitably selected from the list of HPV 31, HPV 45 and HPV 52, or
is
prevention against those abnormalities or lesions caused by the group of 31,
33, 35, 39,
45, 51, 52, 56, 58, 59, 66, and 68. Said use is in addition to the prevention
or reduction
of such events caused by the HPV types in the vaccine, HPV 16 and 18.
Suitably the composition comprising HPV 16 and HPV 18 for use as above is
the multivalent HPV vaccine of the invention, the vaccine comprising an L1
protein or
immunogenic fragment thereof from at least 3 different oncogenic HPV types,
those
types including HPV 16 and HPV 18, wherein the vaccine does not comprise an L1
protein or immunogenic fragment thereof from an HPV type selected from the
list
consisting of HPV 31, HPV 45, HPV 52 or any combination thereof.
The vaccine of the invention comprises Ll or immunogenic fragment from
HPV 16, HPV 18 and at least one other oncogenic HPV type. The oncogenic HPV
types are those types associated with a risk of cervical cancer and those
oncogenic
types that might be included in the vaccine of the invention in addition to
HPV 16 and
HPV 18 include, but are not limited to, HPV 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 66
and 68, with the proviso that the vaccine does not comprise all of HPV 31, 45
and 52.
The vaccine of the invention suitably comprises an HPV 33 L1 protein or
immunogenic fragment thereof.
The vaccine of the invention suitably comprises an HPV 58 Ll protein or
immunogenic fragment thereof.
The vaccine of the invention suitably comprises an HPV 59 L1 protein or
immunogenic fragment thereof.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
7
The vaccine of the invention suitably comprises an HPV 16 LI protein or
immunogenic fragment thereof, HPV 18 L1 protein or inununogenic fragment
thereof,
HPV 33 Li protein or immunogenic fragment thereof and HPV 58 L1 protein or
immunogenic fragment thereof.
L1 proteins or protein fragments from additional HPV types can be included in
the vaccine of the invention, such as skin types (in particular HPV 5 and 8)
and types
associated with genital warts, such as HPV 6 and 11. Types 6 and 11 are not
considered
oncogenic types herein.
In one aspect of the invention the vaccine may include an HPV early antigen,
for example an antigen selected from the list consisting of HPV El, E2, E3,
E4, E5, E6,
E7, or E8. In an alternative aspect the vaccine may lack an HPV early antigen,
for
example an antigen selected from the list consisting of HPV El, E2, E3, E4,
E5, E6,
E7, or E8.
In one aspect the vaccine of the invention is trivalent (contains an HPV L1 or
fragment thereof from 3 different oncogenic HPV types). In a further aspect
the vaccine
is tetravalent. In a further aspect the vaccine is pentavalent. In a further
aspect the
vaccine is heptavalent. In a further aspect the vaccine is septavalent. In a
further
aspect the vaccine is octavalent. Higher order valancies are also contemplated
herein.
In further aspects the vaccine is at least tetravalent, pentavalent,
heptavalent,
septavalent or octavalent with respect to oncogenic HPV types.
Preferably the combination of HPV components within the vaccine does not
significantly impact the immunogenicity of any one HPV component. In
particular it is
preferred that there is no biologically relevant interference between HPV
antigens in
the combination of the invention, such that the combined vaccine of the
invention is
able to offer effective protection against infection or disease caused by each
HPV
genotype represented in the vaccine. Suitably the immune response against a
given
HPV type in the combination is at least 50 % of the immune response of that
same
HPV type when measured individually, preferably 100% or substantially 100%.
For
responses to the HPV 16 and HPV 18, the combined vaccine of the invention
preferably stimulates an immune response which is at least 50% of that
provided by a
combined HPV 16 / HPV 18 vaccine. Suitably the immune response generated by
the
vaccine of the invention is at a level in which the protective effect of each
HPV type is
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
8
still seen. The immune response may suitably be measured, for example, by
antibody
responses, in either preclinical or human experiments. Measurement of antibody
responses is well known in the art, and disclosed in (for example)
W003/077942.
We have determined that a vaccine comprising HPV 16 Ll and HPV 18 Ll proteins
(e.g. see example 1) provides cross protection against infection or disease
caused by
certain HPV types. As well as providing novel compositions, this information
allows
new uses to be developed.
In particular, the invention relates to use of a composition comprising HPV 16
and HPV 18 Ll protein, or immunogenic fragment thereof, in the manufacture of
a
medicament for prevention of infection by HPV 31.
The invention further relates to use of a composition comprising HPV 16 and
HPV 18 L1 protein, or immunogenic fragment thereof, in the manufacture of a
medicament for prevention of infection by HPV 45.
The invention further relates to use of a composition comprising HPV 16 and
HPV 18 L1 protein, or immunogenic fragment thereof in the manufacture of a
medicament for prevention of infection by HPV 52.
In one aspect the invention relates to use of a vaccine comprising HPV 16 Ll
proteins, or immunogenic fragment thereof, in the preparation of a medicament
for the
prevention of infection and/or disease caused by HPV 31, or HPV 52, or any
combination thereof.
In one aspect the invention relates to use of a vaccine comprising HPV 18 L1
proteins, or immunogenic fragment thereof, in the preparation of a medicament
for the
prevention of infection and/or disease caused by HPV 45.
The composition for said use may lack an antigenic component from the HPV
type for which cross protection is provided. Alternatively the composition for
said use
may comprise such an antigenic component, e.g. the Ll protein or fragment
thereof
from said cross protected type. In the latter case the use of the composition
comprising
HPV 16 and HPV 18 Ll protein, or immunogenic fragment thereof, provides both
cross
protection (e.g. against HPV 31, 45 and 52) and homologous protection (e.g.
against
HPV 16 and HPV 18).
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
9
The composition in one aspect is a multivalent composition comprising
L1 proteins or immunogenic fragments thereof from HPV 16, HPV 18 and at least
one
other oncogenic HPV type, wherein an Ll protein or immunogenic fragment
thereof
from one or more HPV types selected from the group consisting of HPV 31, HPV
45,
and HPV 52 is omitted from the vaccine and wherein the vaccine provides
protection
against infection caused by the omitted HPV type.
Where the vaccine or composition of the invention comprises an immunogenic
fragment of L1, then suitable immunogenic fragments of HPV Ll include
truncations,
deletions, substitution, or insertion mutants of Ll. Such immunogenic
fragments are
suitably capable of raising an immune response (if necessary, when
adjuvanted), said
immune response being capable of recognising an Ll protein such as a virus
like
particle, from the HPV type from which the L1 protein was derived.
A suitable immunogenic fragment of HPV 16 is capable of cross protection
against at least one of HPV 31 and HPV 52, and in an aspect of the invention,
capable
of cross protection against both.
A suitable immunogenic fragment of HPV 18 is capable of cross protection
against HPV 45.
Cross protection obtainable by immunogenic fragments of HPV 16 and/or HPV
18 can be assessed by trials in humans, for example as outlined in Example 1.
Similarly, different vaccines according to the present invention can be tested
using standard techniques, for example as in Example 1, or in standard
preclinical
models, to confirm that the vaccine is immunogenic.
Suitable immunogenic L1 fragments include truncated L1 proteins. In one
aspect the truncation removes a nuclear localisation signal. In another aspect
the
truncation is a C terminal truncation. In a further aspect the C terminal
truncation
removes fewer than 50 amino acids, such as fewer than 40 amino acids. Where
the L1
is froin HPV 16 then in another aspect the C terminal truncation removes 34
amino
acids from HPV 16 L1. Where the Ll is from HPV 18 then in a further aspect the
C
terminal truncation removes 35 amino acids from HPV 18 L1. Truncated Ll
Proteins
are described in US 6,060,324, US 6,361,778, and US 6,599,508 incorporated
herein by
reference.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
In one aspect the HPV 16 amino acid sequence is the following sequence: (SEQ
ID
NO: 1)
MSLWLPSEATVYLPPVPVSKV V STDEYVARTNIYYHAGTSRLLA VGHPYFPIKKPNNNKI 60
LVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGH 120
5 PLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAV 180
NPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSE 240
PYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLASSNYFPTPSGSMV 300
TSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNF 360
KEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRF 420
10 VTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQ 471
In another aspect the invention relates to virus like particles consisting
only of HPV 16
L1 having the amino sequence above, and to compositions containing such VLPs.
The HPV 16 sequence may also be that disclosed in WO9405792 or
US6649167, for example, suitably truncated. Suitable truncates are truncated
at a
position equivalent to that shown above, as assessed by sequence comparison.
In one aspect the HPV 18 amino acid sequence is the following sequence: (SEQ
ID
NO: 2)
MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ 60
DIPKVSAYQYRVFRVQLPDPNKFGLPDNSIYNPETQRLVWACVGVEIGRGQPLGVGLSGH 120
PFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPL 180
SQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSAD 240
PYGDSMFFCLRREQLFARHFWNRAGTMGDTVPPSLYIKGTGMRASPGSCVYSPSPSGSIV 300
TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQSPVPGQYDATK 360
FKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYR 420
FVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQ 472
In another aspect the invention relates to virus like particles consisting
only of HPV 18
L1 having the amino sequence above, and to compositions containing such VLPs.
An alternative HPV 18 sequence is disclosed in WO9629413, which may be
suitably truncated. Suitable truncates are truncated at a position equivalent
to that
shown above, as assessed by sequence comparison.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
11
Other HPV 16 and HPV 18 sequences are well known in the art and may be
suitable for use in the present invention.
Suitable truncations of HPV 31, HPV 45 and HPV 52 may also be made,
suitably removing equivalent C terminal portions of the Ll protein to those
described
above as assessed by sequence alignment.
Truncated Ll proteins are disclosed in, for example, WO9611272 and
US6066324, herein incorporated by reference.
The Ll protein or fragment of the invention may optionally be in the fonn of a
fusion protein, such as the fusion of the L1 protein with L2 or an early
protein.
The HPV L1 protein is suitably in the form of a capsomer or virus like
particle
(VLP). In one aspect HPV VLPs may be used in the present invention. HPV VLPs
and methods for the production of VLPs are well known in the art. VLPs
typically are
constructed from the L1 and optionally L2 structural proteins of the virus,
see for
example W09420137, US5985610, W09611272, US6599508B1, US6361778B1, EP
595935. Any suitable HPV VLP may be used in the present invention which
provides
cross protection, such as an LI or Ll + L2 VLP.
Suitably the VLP is an L1-only VLP.
In one aspect of the invention the vaccine comprises HPV 16 and HPV 18 Ll
only VLPs, suitably in combination with an L1 VLP selected from HPV 31, 33,
35, 39,
45, 51, 52, 56, 58, 59, 66 and 68, with the proviso that the vaccine does not
comprise
VLPs from all of HPV 31, 45 and 52.
VLP formation can be assessed by standard techniques such as, for example,
electron microscopy and dynamic laser light scattering.
The VLP may comprise full length Ll protein. In one aspect the LI protein
used to form the VLP is a truncated LI protein, as described above.
VLPs may be made in any suitable cell substrate such as yeast cells or insect
cells e.g. baculovirus cells, and techniques for preparation of VLPs are well
known in
the art, such as W09913056, US 6416945B1 , US 6261765B1 and US6245568, and
references therein, the entire contents of which are hereby incorporated by
reference.
VLPS are suitably made by disassembly and reassembly techniques, which can
provide for more stable and/or homogeneous papillomavirus VLPs. For example,
McCarthy et al, 1998 "Quantitative Disassembly and Reassembly of Human
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
12
Papillomavirus Type 11 Virus like Particles in Vitro" J. Virology 72(1):33-41,
describes the disassembly and reassembly of recombinant Ll HPV 11 VLPs
purified
from insect cells in order to obtain a homogeneous preparation of VLPs.
W49913056
and US6245568 also describe disassembly/reassembly processes for making HPV
VLPs.
In one aspect HPV VLPS are made as described W09913056 or US6245568.
The HPV Ll the invention may be combined with an adjuvant or
imunostimulant such as, but not limited to, detoxified lipid A from any source
and non-
toxic derivatives of lipid A, saponins and other reagents capable of
stimulating a TH1
type response.
It has long been known that enterobacterial lipopolysaccharide (LPS) is a
potent
stimulator of the immune system, although its use in adjuvants has been
curtailed by its
toxic effects. A non-toxic derivative of LPS, monophosphoryl lipid A (MPL),
produced
by removal of the core carbohydrate group and the phosphate from the reducing-
end
glucosamine, has been described by Ribi et al (1986, Immunology and
Immunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407-419)
and
has the following structure:
~6.
H--0 H ''CH2
F~0 0H Q
H-~-0
''O r NH H
OiliC~ H< rHO-?,- H O H
cmO
CH2 " t x
C)E12
MH
CH 3
O(CHf?to ~mo H C,~o H
O=~ ~s o (H2)t0 ~3
{CHiltt O= 1 cH3 CH-OH N2
~' (C'H2)to tCHi)to 1
CH3 ~3 ( I Hto O
CHs C=O
(C.H23ts
CI'13
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
13
A further detoxified version of MPL results from the removal of the acyl chain
from the 3-position of the disaccharide backbone, and is called 3-0-Deacylated
monophosphoryl lipid A(3D-I\.PL). It can be purified and prepared by the
methods
taught in GB 2122204B, which reference also discloses the preparation of
diphosphoryl
lipid A, and 3-0-deacylated variants thereof.
A suitable form of 3D-MPL is in the form of an emulsion having a small
particle size less than 0.2 m in diameter, and its method of manufacture is
disclosed in
WO 94/21292. Aqueous formulations comprising monophosphoryl lipid A and a
surfactant have been described in W09843670A2.
The bacterial lipopolysaccharide derived adjuvants to be formulated in the
compositions of the present invention may be purified and processed from
bacterial
sources, or alternatively they may be synthetic. For example, purified
monophosphoryl
lipid A is described in Ribi et al 1986 (supra), and 3-0-Deacylated
monophosphoryl or
diphosphoryl lipid A derived from Salmonella sp. is described in GB 2220211
and US
4912094. Other purified and synthetic lipopolysaccharides have been described
(Hilgers et al., 1986, Int.Arch.Allergy.Immunol., 79(4):392-6; Hilgers et al.,
1987,
Immunology, 60(1):141-6; and EP 0 549 074 B1). In one aspect the bacterial
lipopolysaccharide adjuvant is 3D-MPL.
Accordingly, the LPS derivatives that may be used in the present invention are
those immunostimulants that are similar in structure to that of LPS or MPL or
3D-
MPL. In another aspect of the present invention the LPS derivatives may be an
acylated monosaccharide, which is a sub-portion to the above structure of MPL.
Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review of
the biological and pharmacological activities of saponins. Phytomedicine vol 2
pp 363-
386). Saponins are steroid or triterpene glycosides widely distributed in the
plant and
marine animal kingdoms. Saponins are noted for forming colloidal solutions in
water
which foam on shaking, and for precipitating cholesterol. When saponins are
near cell
membranes they create pore-like structures in the membrane which cause the
membrane to burst. Haemolysis of erythrocytes is an example of this
phenomenon,
which is a property of certain, but not all, saponins.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
14
Saponins are known as adjuvants in vaccines for systemic administration. The
adjuvant
and haemolytic activity of individual saponins has been extensively studied in
the art
(Lacaille-Dubois and Wagner, supra). For example, Quil A (derived from the
bark of
the South American tree Quillaja Saponaria Molina), and fractions thereof, are
described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R.,
Crit Rev
Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279 B 1. Particulate
structures, termed Immune Stimulating Complexes (ISCOMS), comprising fractions
of
Quil A are haemolytic and have been used in the manufacture of vaccines
(Morein, B.,
EP 0 109 942 Bl; WO 96/11711; WO 96/33739). The haemolytic saponins QS21 and
QS 17 (HPLC purified fractions of Quil A) have been described as potent
systemic
adjuvants, and the method of their production is disclosed in US Patent
No.5,057,540
and EP 0 362 279 B I. Other saponins which have been used in systemic
vaccination
studies include those derived from other plant species such as Gypsophila and
Saponaria (Bomford et al., Vaccine, 10(9):572-577, 1992).
An enhanced system involves the combination of a non-toxic lipid A derivative
and a saponin derivative particularly the combination of QS21 and 3D-MPL as
disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is
quenched with cholesterol as disclosed in WO 96/33739.
A particularly potent adjuvant formulation involving QS21 and 3D-MPL in an
oil in water emulsion is described in WO 95/17210 and use of this adjuvant
forms an
aspect of the invention.
Accordingly in one embodiment of the present invention there is provided a
vaccine adjuvanted with detoxified lipid A or a non-toxic derivative of lipid
A, more
suitably adjuvanted with a monophosphoryl lipid A or derivative thereof.
In one aspect the vaccine additionally comprises a saponin, for example QS21.
In one aspect the vaccine formulation comprises an oil in water emulsion. The
present invention also provides a method for producing a vaccine formulation
comprising mixing an Ll peptide of the present invention together with a
pharmaceutically acceptable excipient, such as 3D-MPL.
Additional components that may be included present in an vaccine formulation
according to the invention include non-ionic detergents such as the octoxynols
and
polyoxyethylene esters as described herein, particularly t-octylphenoxy
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
polyethoxyethanol (Triton X-100) and polyoxyethylene sorbitan monooleate
(Tween
80); and bile salts or cholic acid derivatives as described herein, in
particular sodium
deoxycholate or taurodeoxycholate. Thus, in one aspect of the invention a
formulation
comprises 3D-MPL, Triton X-100, Tween 80 and sodium deoxycholate, which may be
5 combined with an L2 antigen preparation to provide a suitable vaccine.
In one embodiment of the present invention, the vaccine comprises a vesicular
adjuvant formulation comprising cholesterol, a saponin and an LPS derivative.
In this
regard the adjuvant formulation suitably comprises a unilamellar vesicle
comprising
cholesterol, having a lipid bilayer suitably comprising dioleoyl phosphatidyl
choline,
10 wherein the saponin and the LPS derivative are associated with, or embedded
within,
the lipid bilayer. In one aspect these adjuvant formulations comprise QS21 as
the
saponin, and 3D-MPL as the LPS derivative, wherein the ratio of
QS21:cholesterol is
from 1:1 to 1:100 weight/weight, and in one aspect, a ratio of 1:5
weight/weight. Such
adjuvant formulations are described in EP 0 822 831 B, the disclosure of which
is
15 incorporated herein by reference.
Suitably the vaccines of the invention are used in combination with aluminium,
and are suitably adsorbed or partially adsorbed onto aluminium adjuvants.
Suitably the
adjuvant is an aluminium salt, which may be in combination with 3D MPL, such
as
aluminium phosphate and 3D MPL. Aluminium hydroxide, optionally in combination
with 3D MPL is also suitable.
In another aspect of the present invention the vaccine comprises the
combination of HPV VLPs with an aluminium salt or with an aluminium salt + 3D
MPL. Aluminium hydroxide is suitable as the aluminium salt.
The vaccine may also comprise aluminium or an aluminium compound as a
stabiliser.
In another aspect the adjuvant may be a combination of an oil-in-water
emulsion adjuvant and 3D MPL. In one aspect the oil-in-water emulsion
comprises a
metabolisable oil, a sterol and an emulsifying agent.
The vaccines of the invention may be provided by any of a variety of routes
such as oral delivery (e.g. see W09961052 A2), topical, subcutaneous, mucosal
(typically intravaginal), intraveneous, intramuscular, intranasal, sublingual,
intradermal
and via suppository.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
16
Optionally the vaccine may also be formulated or co-administered with other
HPV antigens or non-HPV antigens. Suitably these non-HPV antigens can provide
protection against other diseases, such as sexually transmitted diseases such
as herpes
simplex virus, EBV, chlamydia and HIV. We particularly prefer that the vaccine
comprises gD or a truncate thereof from HSV. In this way the vaccine provides
protection against both HPV and HSV.
The dosage of the vaccine components will vary with the condition, sex, age
and weight of the individual, the administration route and HPV of the vaccine.
The
quantity may also be varied with the number of VLP types. Suitably the
delivery is of
an amount of vaccine suitable to generate an immunologically protective
response.
Suitably each vaccine dose comprises 1-100 g of each VLP, in one aspect 5-80
g, in
another aspect 5- 30 g each VLP, in a further aspect 5-20 g of each VLP, in
a yet
further aspect 5 g, 6 g, 10 g, 15 g or 20 g.
In one aspect the vaccine can comprise HPV L1 protein components, preferably
as virus like particles, in different amounts. In one aspect, HPV 16 and HPV
18 VLPs
may be provided at a higher dose than other oncogenic types, such as HPV 33 or
58. In
one aspect HPV 16 and HPV 18 Ll only VLPs are provided at 20 g per dose for
human use. Other HPV VLPs may be used at a lower dose, such as 15 or 10 g per
dose for human use.
For all vaccines of the invention, in one aspect the vaccine is used for the
vaccination of adolescent girls aged 10-15, such as 10-13 years. However,
older girls
above 15 years old and adult women may also be vaccinated. The vaccine may
also be
administered to women following an abnormal pap smear or after surgery
following
removal of a lesion caused by HPV, or who are seronegative and DNA negative
for
HPV cancer types.
The vaccine of the invention may be used in men.
In one aspect the vaccine is delivered in a 2 or 3 dose regime, for example in
a
0, 1 month regime or 0,1 and 6 month regime respectively. Suitably the
vaccination
regime incorporates a booster injection after 5 to 10 years, such as 10 years.
In one aspect the vaccine is a liquid vaccine formulation, although the
vaccine
may be lyophilised and reconstituted prior to administration.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
17
The teaching of all references in the present application, including patent
applications and granted patents, are herein fully incorporated by reference.
The vaccines of the invention comprise certain HPV components as laid out
above. In a further aspect of the invention the vaccine consists essentially
of, or
consists of, said components.
The term 'vaccine', as used in the present invention, refers to a composition
that
comprises an immunogenic component capable of provoking an immune response in
an
individual, such as a human, optionally when suitably formulated or adjuvant.
A
vaccine suitably elicits a protective immune response against incident
infection, or
persistent infection, or cytological abnormality such as ASCUS, CINI, CIN2 ,
CIN3, or
cervical cancer caused by one or more HPV types.
The present invention is now described with respect to the following examples
which serve to illustrate the invention.
Example 1
Precise details of the experiment carried out are provided in Harper et al,
the
Lancet. 2004 Nov 13;364(9447):1757-65, incorporated herein by reference.
In summary, healthy women between the ages of 15 and 25 years were
immunised with a mixture of HPV 16 and HPV 18 L1 VLPs. The women at enrolment
were: 1) seronegative for HPV-16 and HPV-18; 2) negative for high risk HPV
infection
of the cervix (detected by HPV PCR); 3) had 6 or fewer lifetime sexual
partners and 4)
had normal PAP smears.
The mixture comprised, per 0.5 ml dose, 20 g of HPV-16 L1 VLP, 20 g of
HPV-18 L1 VLP and was adjuvanted with 500 g of aluminum hydroxide and 50 g
of
3D MPL. The placebo group was injected with 500 g of aluminum hydroxide
alone.
The vaccine efficacy (V.E.) against certain cancer HPV types was assessed,
wherein the V.E. is the % improvement in protection against infection or
disease by the
vaccine compared to a placebo group.
Cross protection was assessed by detecting the presence of nucleic acid
specific
for various oncogenic types in the vaccinees and control group. Detection was
carried
out using techniques as described in W003014402, and references therein,
particularly
for non-specific amplification of HPV DNA and subsequent detection of DNA
types
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
18
using a LiPA system as described in WO 99/14377, and in Kleter et al, [Journal
of
Clinical Microbiology (1999), 37 (8): 2508-2517], the whole contents of which
are
herein specifically incorporated by reference.
Any suitable method can, however, be used for the detection of HPV DNA in a
sample, such as type specific PCR using primers specific for each HPV type of
interest.
Suitable primers are known to the skilled person, or can be easily constructed
given that
the sequences of the oncogenic HPV types are known.
In detail, the methods section of the Lancet paper is reproduced here below,
for
completeness (continues until section entitled "Initial analysis and results")
The primary objective of this study was to assess vaccine efficacy in the
prevention of
infection with HPV-16, HPV-18, or both (HPV-16/18), between months 6 and 18 in
participants who were initially shown to be seronegative for HPV- 16/18 by
ELISA and
negative for HPV-16/18 DNA by PCR. Secondary objectives included: evaluation
of
vaccine efficacy in the prevention of persistent infection with HPV-16/18, and
the
evaluation of vaccine efficacy in the prevention of cytologically confirmed
low-grade
squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial
lesions
(HSIL), and histologically confirmed LSIL (CIN 1), HSIL (CIN 2 or 3) squamous
cell
cancer, or adenocarcinoma associated with HPV-16/18 infection between months 6
and
18, and months 6 and 27. The prevention of atypical squamous cells of
undetermined
significance (ASCUS) cytology associated with HPV-16/18 infection was added
post-
hoc to the outcome analyses.
We also did an exploratory analysis of the histopathological endpoints CIN 1
and 2 associated with HPV-16/18 DNA detected by PCR in lesional tissue. Other
objectives included the assessment of vaccine immunogenicity, safety, and
tolerability.
Investigators in North America (Canada and the USA) and Brazil recruited women
for
this efficacy study through advertisements or previous participation in an HPV
cross-
sectional epidemiology study that took place between July and December, 2000.
For each of the 32 study sites, an institutional review board approved the
protocol,
consent forms, and amendments. Women signed separate written consents for
study
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
19
participation and colposcopy. For those under 18 years, parental consent and
assent
from the participant were obligatory.
There were two study phases: an initial phase for vaccination and follow-up
that
concluded at month 18; and a blinded follow-up extension phase that concluded
at
month 27.
Women eligible for the initial phase (months 0-18) included healthy women
aged 15-25 years, who had had no more than six sexual partners, no history of
an
abnormal Pap test or ablative or excisional treatment of the cervix, and no
ongoing
treatment for external condylomata; and who were cytologically negative,
seronegative
for HPV- 16 and HPV- 18 antibodies by ELISA, and HPV-DNA-negative by PCR for
14
high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and
68) no more
than 90 days before study entry.
Women who completed the initial phase of the study earliest, and who did not
have ablative or excisional therapy of the cervix, or hysterectomy after
enrolment, were
eligible to participate in the extension phase of the study (months 18-27).
Procedures
Each dose of the bivalent HPV-16/18 virus-like particle vaccine
(G1axoSmithKline Biologicals, Rixensart, Belgium) contained 20 Pg of HPV-16 Ll
virus-like particle and 20 Pg of HPV-18 LI virus-like particle. Each type of
virus-like
particle was produced on Spodopterafrugiperda Sf-9 and Trichoplusia ni Hi-5
cell
substrate with ASO4 adjuvant containing 500 Pg aluminum hydroxide and 50 Fig 3-
deacylated monophosphoryl lipid A (MPL, Corixa, Montana, USA) provided in a
monodose vial. The placebo contained 500 Pg of aluminum hydroxide per dose,
and
was identical in appearance to the HPV- 16/18 vaccine. Every study participant
received
a 0=5 mL dose of vaccine or placebo at 0 months, 1 month, and 6 months.
Health-care providers obtained cervical specimens with a cervical brush and
spatula
(washed in PreservCyt, Cytyc Corporation, Boxborough, MA, USA) for cytology
and
HPV DNA testing at screening and months 6, 12, and 18. At months 0 and 6, and
subsequently every 3 months, women self-obtained cervicovaginal samples with
two
sequential swabs (placed in PreservCyt) for HPV DNA testing.[ DM Harper, WW
Noll,
DR Belloni and BF. Cole, Randomized clinical trial of PCR-determined human
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
papillomavirus detection methods: self-sampling versus clinician-directed-
biologic
concordance and women's preferences. Am J Obstet Gyneco1186 (2002), pp. 365-
373]
A central laboratory (Quest Diagnostics, Teterboro, NJ, USA) reported cytology
results
(ThinPrep, Cytyc Corporation) by use of the 1991 Bethesda classification
system.
5 Protocol guidelines recommended colposcopy after two reports of ASCUS, or
one
report of atypical glandular cells of undetermined significance, LSIL or HSIL,
squamous cell carcinoma, adenocarcinoma in situ, or adenocarcinoma. These
guidelines also recommended biopsy for any suspected lesions.
The central histology laboratory made an initial diagnosis from the formalin-
fixed
10 tissue specimens for clinical management. A panel of three pathologists
made a
subsequent consensus diagnosis for HPV- 16 and HPV- 18 associated lesions with
the
CIN system. This consensus diagnosis also included review of the sections
taken at the
time of microdissection for PCR detection of lesional HPV DNA.
HPV DNA isolated from the cytology specimen (MagNaPure Total Nucleic Acid
15 system, Roche Diagnostics, Almere, Netherlands) and from the cervical
biopsy
specimen (proteinase K extraction) was amplified from an aliquot of purified
total
DNA with the SPF10 broad-spectrum primers that amplify a 65 bp region of the
L1
gene.[ B Kleter, LJ van Doom, J ter Schegget et a1., Novel short-fragment PCR
assay
for highly sensitive broad-spectrum detection of anogenital human
papillomaviruses.
20 Am JPatho1153 (1998), pp. 1731-1739: LJ van Doom, W Quint, B Kleter et al.,
Genotyping of human papillomavirus in liquid cytology cervical specimens by
the
PGMY line blot assay and the SPF(10) line probe assay. J Clin Microbiol 40
(2002),
pp. 979-983 and WG Quint, G Scholte, LJ van Doom, B Kleter, PH Smits and J.
Lindeman, Comparative analysis of human papillomavirus infections in cervical
scrapes and biopsy specimens by general SPF(10) PCR and HPV genotyping.
JPathol
194 (2001), pp. 51-58] The amplification products were detected by a DNA
enzyme
immunoassay. A line probe assay (LiPA Kit HPV INNO LiPA HPV genotyping assay,
SPF-10 system version 1, Innogenetics, Gent, Belgium, manufactured by Labo Bio-
medical Products, Rijswijk, Netherlands) detected 25 HPV genotypes (6, 11, 16,
18, 31,
33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, and
74). [B Kleter,
LJ van Doom, L Schrauwen et al., Development and clinical evaluation of a
highly
sensitive PCR-reverse hybridization line probe assay for detection and
identification of
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
21
anogenital human papillomavirus. JClin Microbiol 37 (1999), pp. 2508-2517] Any
specimen that was positive by DNA enzyme immunoassay was tested by type-
specific
HPV-16 and HPV-18 PCR. HPV-16 type-specific PCR primers amplified a 92 bp
segment of the E6/E7 gene and HPV-18 type-specific PCR primers amplified a 126
bp
segment of the Ll gene. [MF Baay, WG Quint, J Koudstaal et al., Comprehensive
study of several general and type-specific primer pairs for detection of human
papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin
Microbiol 34 (1996), pp. 745-747]
We defined incident cervical infection with HPV-16/18 as at least one positive
PCR
result for HPV-16 or HPV-18 during the trial, and persistent infection with
HPV-16/18
as at least two positive HPV-DNA PCR assays for the same viral genotype
separated by
at least 6 months.[ H Richardson, G Kelsall, P Tellier et al., The natural
history of type-
specific human papillomavirus infections in female university students. Cancer
Epidemiol Biomarkers Prev 12 (2003), pp. 485-490 and AB Moscicki, JH
Ellenberg, S
Farhat and J. Xu, Persistence of human papillomavirus infection in HIV-
infected and -
uninfected adolescent girls: risk factors and differences, by phylogenetic
type. Jlnfect
Dis 190 (2004), pp. 37-45] HPV-DNA test results were concealed from
investigators
during the study and cytological and histological diagnoses were only revealed
for
clinical management purposes. Analyses included HPV-16/18 DNA results for
cervical
specimens and combined cervical and self-obtained cervicovaginal specimens.
We collected serum from study participants at months 0, 1, 6, 7, 12, and 18
for
assessment of immunogenicity. Serological testing for antibodies to HPV-16 and
HPV-
18 virus-like particles was by ELISA. Recombinant HPV- 16 or HPV- 18 virus-
like
particles were used as coating antigens for antibody detection (see
webappendix
http://image.thelancet.com/extras/04art10103webappendix.pdf). Seropositivity
was
defined as a titre greater than or equal to the assay cut-off titre
established at 8 ELISA
units/mL for HPV-16 and 7 ELISA units/mL for HPV-18. Typical natural titres
were
determined by use of blood samples obtained from women in the preceding
epidemiology study who were found to be seropositive for HPV-16 or HPV-18 by
ELISA.
Women recorded symptoms experienced during the first 7 days after
vaccination on diary cards with a three-grade scale of symptom intensity.
Additionally,
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
22
they reported to study personnel by interview all adverse events within the
first 30 days
after vaccination. Information on serious adverse events and pregnancies was
collected
throughout the study.
Statistical methods
Assuming a 6% cumulative incidence rate of both HPV- 16 and HPV- 18 type
infections over 12 months, we estimated that 500 women per treatment group
would
provide 80% power to assess a lower limit of the 95% CI of the vaccine
efficacy above
zero. We assumed an 80% retention rate over 18 months. Interim analyses for
efficacy,
safety, and immunogenicity were done for future study planning purposes only;
the
O'Brien and Fleming method was used to adjust the cevalue for the final
analysis after
interim analyses occurred (overall c-r.=0=05; two-sided test).[ PC O'Brien and
TR.
Fleming, A multiple testing procedure for clinical trials. Biometrics 35
(1979), pp. 549-
556]
Stratified, block randomisation according to validated algorithms was
centralised with
an internet randomisation system. Stratification was according to age (15-17,
18-21,
and 22-25 years) and region (North America and Brazil). Each vaccine dose was
attributed a randomly chosen number based on specific participant information
entered
into the computerised randomisation system by study personnel. Treatment
allocation
remains concealed from investigators and the women participating in a long-
term
follow-up study.
The intention-to-treat and according-to-protocol cohorts are shown in the
figure,
in which the reasons for exclusion from analyses are listed in rank order;
women who
met more than one exclusion criterion were only counted once according to the
highest
ranking criterion. We refer to the sets of participants entered in the
intention-to-treat
and according-to-protocol analyses as cohorts, although the infon nation used
to restrict
subject inclusion in the according-to-protocol was only known after follow-up.
We did both according-to-protocol and intention-to-treat analyses for
efficacy.
Calculation of vaccine efficacy in the according-to-protocol 18-month analysis
was
based on the proportion of participants with HPV-16/18 infection in the
vaccinated
versus placebo groups. Vaccine efficacy was defined as 1 minus the ratio
between these
two proportions; 95% CIs measured the precision of the efficacy estimates. p
values
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
23
were calculated with the two-sided Fisher's exact test. Corresponding rates
were
expressed as the numbers of cases with the outcome divided by the numbers of
participants at risk. The according-to-protocol 18-month cohort included
enrolled
women who received three scheduled doses of vaccine and complied with the
protocol
as described in the figure.
Calculation of vaccine efficacy in the intention-to-treat and according-to-
protocol 27-month analyses was based on the Cox proportional hazard model
using the
time-to-occurrence of cases with HPV-16/18 infection in the vaccinated versus
placebo
groups. This allowed controlling for the accrued person-time data in each
group.
Vaccine efficacy was calculated using 1 minus the hazard ratio and p values
calculated
using the log rank test. Corresponding rates were expressed as the number of
cases
divided by the total person-time. All enrolled women who received at least one
dose of
vaccine or placebo, were negative for high-risk HPV-DNA at month 0, and had
any
data available for outcome measurement were included in the intention-to-treat
cohort.
The according-to-protocol 27-month cohort included outcome results from the
according-to-protocol 18-month cohort and results that occurred during the
extension
phase (from 18 months to 27 months).
Calculation of p values for the safety analysis was performed using Fisher's
exact test comparisons. The cohort for safety analysis included all enrolled
women who
received at least one dose of vaccine or placebo and complied with specified,
minimal
protocol requirements (see protocol below:)
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
24
4939 assessed for eligibility
t 113 randomised
560 randomized to vaccine 553 randomized to placebo
F 560 included in ITT cohort 553 included in ITT cohort
1 1
540 included in ATP cohort (saftey analysis) 541 included in ATP cohort
(saftey analysis)
20 excluded 12 excluded
concomitant of placebo dose as 11 concomitant vaccine adminstration
replacement for lost/damaged vial I randomisation code broken at site
I randomisation code broken at site
366 included in ATP cohort (vaccine efficacy 355 included in ATP cohort
(vaccine efficacy
analysis) for months 6-18, 27 analysis) for months 6-18, 27
Primary analysis incident HPV-16/18 infections Primary analysis incident HPV-
16/18 infections
174 excluded from month 6-18 analysis 186 excluded from month 6-18 analysis
2 eligibility criteria not met 6 eligibility criteria not met
79 initially seropositive for HPV- 16/18 positive 73 initially seropositive
for HPV- 16/18 positive
for high-risk HPV DNA; or abnormal cytology for high-risk HPV DNA; or abnormal
cytology
0 medication administration violating protocol 1 medication administration
violating protocol
41 non-compliance with vaccine schedule administration of blood product
9 missing HPV DNA results or serology results 45 non-compliance with vaccine
schedule
at screening 12 missing HPV DNA results or serology results
7 had positive HPV-16/18 DNA results at 6 at screening
months 18 had positive HPV-16/18 DNA results at 6
36 dropped out before month 18 months
36 dropped out before month 18
E 316 completed month 21 visit 291 completed month 21 visit
209 completed month 24 visit 188 completed month 24 visit
81 completed month 27 visit 59 completed month 27 visit
384 included in ATP cohort for months 6-18 344 included in ATP cohort for
months 6-18
Secondary analysis immunogenisity Secondary analysis imrnunogenisity
156 excluded 197 excluded
2 eligibility criteria not met 6 eligibility criteria not met
23 initially seropositive or unknown antibody 20 initially seropositive or
unknown antibody
status status
0 medication administration violating protocol I medication administration
violating protocol
40 had positive HPV-16/18 DNA results during of blood product
the study period 85 had positive HPV-16/18 DNA results during
52 non-compliance with vaccine schedule the study period
35 non-compliance with blood sampling 51 non-compliance with vaccine schedule
schedule 29 non-compliance with blood sampling
4 serological data missing schedule
5 serological data missing
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
Immunogenicity was assessed in a subset of the according-to-protocol safety
cohort,
which included women with serology results at months 0, 7, and 18, who
received all
5 three doses of study vaccine or placebo according to schedule, complied with
the blood
sampling schedule, and did not become positive for HPV-16/18-DNA during the
trial.
Seropositivity rates between the vaccine and placebo groups were compared with
Fisher's exact test (p<0=001 judged significant). Geometric mean titres were
compared
with ANOVA and Kruskal-Wallis test.
10 Block randomisation and statistical analyses were done with SAS version 8.2
(SAS
Institute, Cary, North Carolina).
Initial analysis and results
Results of the initial analysis on cross protection are presented in patent
15 application W02004/056389, the whole contents of which herein incorporated
by
reference.
An initial analysis was carried out on an "ITT" (Intention To Treat cohort,
representing all individuals who received at least one dose of vaccine). This
data is
shown in Table A.
20 The results presented in Tables B and C- relate to the "ATP" (According To
Protocol) group for those patients who complied with all the criteria of the
trial. Table
B is a midpoint analysis with data taken from all patients at the timepoint at
which at
least 50% of the cohort were 18 months after their first vaccination. Table C
gives the
final results, all data being from subjects at 18 months post first
vaccination (month 0).
25 In the ATP group all patients received 3 doses of vaccine at 0, 1 and 6
months and were
seronegative at 6 months.
As demonstrated by the data presented in table A, immunization with a mixture
of HPV 16 and HPV 18 VLPs provided apparent cross-protection against other HPV
types. At this point the sample sizes are too small to provide for a rigorous
statistical
analysis, however the data demonstrate a positive trend and suggest that
immunization
with HPV 16 and HPV 18 VLPs will be efficacious against infection with other
HPV
types.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
26
This was confirmed as the study progressed.
Table B demonstrates that HPV 16 and HPV 18 provide statistically significant
cross protection against the group of high risk cancer types
31,33,35,39,45,51,52,56,58,59, 66 and 68.
Table C demonstrates that, except for the HPV- 18 related types (which show a
very strong trend), there is statistically significant cross-protection
against the groups
of: HPV 31, 35, 58; HPV 31, 33, 35, 52, 58; and the 12 high risk (non HPV-
16/18)
types evaluated.
Further analysis was carried out on the specific cross protection against
specific
types.
Vaccine efficacy was assessed against infections and diseases related to the
12
high risk cancer types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, HPV-
16
phylogenetic-related types (the groups of; 31, 35, and 58; 31, 33, 35, 52 and
58) and
HPV-18 phylogenetic related types (45 and 59).
An analysis was carried out on an"ATP" (According To Protocol) group for
those patients who complied with all the criteria of the trial. In the ATP
group all
patients received 3 doses of vaccine at 0, 1 and 6 months and were
seronegative at 6
months.
As demonstrated by the data presented in Table D, immunization with a mixture
of HPV16 and HPV18 VLPs provided statistically significant cross protection
against
incident infection by HPV types 31, 52 and 45 compared to the control.
Statistically significant cross protection against incident infection was also
observed against the group of all HPV 16 related types (HPV-31, 33, 35, 52 and
58)
and the group of all high risk types, excluding 16 and 18 (HPV 31, 33, 35, 39,
45, 51,
52, 56, 58, 59, 66, and 68).
Statistically significant cross protection against persistent infection was
also
observed against types 31 and 52 and was also observed against the group of
all HPV
16 related types (see Table E).
Statistically significant cross protection was observed against cytological
abnormalities associated with HPV 52 and was also observed against cytological
abnormalities associated with the group of all HPV 16 related types (HPV-31,
33, 35,
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
27
52, and 58) and the group of all high risk types, excluding 16 and 18 (31, 33,
35, 39, 45,
51, 52, 56, 58, 59, 66, and 68) (Table F).
Table A
HPV types analysed HPV 31, 35, HPV 31, 33, HPV 45, 59 HPV 31, 33, 35,
58 35, 52, 58 39, 45, 51, 52, 56,
58, 59, 66, 68.
Number of women 5 17 3 27
infected (vaccine group)
% women infected 1.1 3.8 0.7 6.3
(vaccine group)
=A
Number of women 11 24 6 40
infected (placebo group)
% women infected 2.4 5.4 1.3 9.4
(placebo group)
=B
% vaccine efficacy 55.1 30.3 50.6 34.6
1 - (A/B) x 100, adjusted
for relative size of
vaccine and placebo
group
95% confidence limits -29.1 -29.7 -97.7 -6.5
-lower limit
95% confidence limits 84.4 62.6 87.6 59.9
-upper limit
P 0.127 0.252 0.309 0.086
Samples were taken at 9, 12, 15 and 18 months from patients and tested for HPV
infection by the types
specified above.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
28
Table B - vaccine efficacy after three doses in preventing incident
heterologous
infections.
Vaccine efficacy against infection with HPV-16 phylogenetically related types,
HPV-18 phylogenetically related types, HPV-16 and/or HPV-18 phylogenetically
related types and all high-ri sk types exclusive of HPV- 16 and HPV- 18 - ATP
cohort
(month 6-18)
Infection Type Attack rate Vaccine efficacy
Vaccine Placebo
N n AR N n AR % 95% CI p-value
HPV-16 related 433 12 2.8 438 24 5.5 49.4 0.2 74.4 0.060
HPV-16 related* 423 29 6.9 423 46 10.9 37.0 1.6 59.6 0.052
HPV-18 related 442 9 2.0 449 16 3.6 42.9 -27.9 74.5 0.223
HPV-16/18 related 433 21 4.9 438 41 9.4 48.2 13.8 68.9 0.012
HPV-16/18 423 34 8.0 423 56 13.2 39.3 9.0 59.5 0.019
related*
High-risk** 385 53 13.8 386 88 22.8 39.6 17.7 55.7 0.001
N = number of subjects in specific cohort
n = number of subjects with incident HPV infection
AR = Attack rate = n / N
95% CI = 95% confidence interval
lower limit = 1- exp ( log (arv / arp) + 1.96 * sqrt (1/nv - 1/Nv + 1/np -
1/Np))
upper limit = 1- exp ( log (arv / arp) - 1.96 * sqrt (1/nv - 1/Nv + 1/np -
I/Np))
when number of cases in vaccine = 0:
lower limit* = 1- exp ( log (arv* / arp*) + 1.96 * sqrt (1/(nv+0.5) -
1/(Nv+0.5) +
1/(np+0.5) - 1/(Np+0.5)))
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
29
upper limit* = 1- exp ( log (arv* / arp*) - 1.96 * sqrt (1/(nv+0.5) -
1/(Nv+0.5) +
1/(np+0.5) - 1/(Np+0.5)))
with: arv = attack rate in vaccine recipients
arp = attack rate in placebo recipients
nv = number of cases in vaccine recipients
Nv = number of cases and non-cases in vaccine recipients
np = number of cases in placebo recipients
Np = number of cases and non-cases in placebo recipients
HPV-16 related: HPV-16 phylogenetically related types 35, 31, 58 without
considering other HPV
types
HPV-16 related*: HPV-16 phylogenetically related types 35, 31, 58, 33, 52
without considering other
HPV types
HPV-18 related: HPV-18 phylogenetically related types 45, 59 without
considering other HPV types
HPV-16 and/or HPV-18 related: HPV-16 and/or HPV-18 phylogenetically related
types 35, 31, 58,
45, 59 without considering other HPV types
HPV-16 and/or HPV-18 related*: HPV-16 and/or HPV-18 phylogenetically related
types 35, 31, 58,
33, 52, 45, 59 without considering other HPV types
** = High-risk types exclusive of HPV-16 and HPV-18
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
Table C
HPV types analysed HPV 31, HPV 31, 33, HPV 45, 59 HPV 31, 33, 35, 39,
35, 58 35, 52, 58 45, 51, 52, 56, 58, 59,
66, 68.
Total number of number of 412 403 421 368
subjects with information
available per group
Number of women infected 11 28 10 58
(vaccine group)
% women infected (vaccine 2.7 6.9 2.4 15.8
group)
=A
Number of women infected 26 48 15 90
(placebo group).
% women infected (placebo 6.3 12.2 3.6 25.3
group)
=B
% vaccine efficacy 57.9 43.0 33.5 37.7
1 - (A/B) x 100, adjusted for
relative size of vaccine and
placebo group
95% confidence limits 15.9 11.0 -46.3 16.2
-lower limit
95% confidence limits 78.9 63.5 69.8 53.6
-upper limit
P 0.012 0.015 0.319 0.002
Samples were taken at 18 months from patients and tested for HPV infection by
the types specified
above.
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
31
Table D
~~~~~ = .
: - . -. ~0
= ~ ~ .
't ' '1~iL7~t)~ = ~ ~
- = - ' ' = = '
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
32
Table E
i~ c~ in~t ~~~~~~~ ent I ic=~n
i =/1: ' Ic=a ~= YRe~
- . ~~~3=~ ~
' = - ' ~ = =
CA 02606206 2007-10-16
WO 2006/114273 PCT/EP2006/003809
33
Table F
E iCQC~O Q= r l 5 ~/~~~0~! ~~
IQ
= . . .
= 0~ ~ '~~
~0 ~ = - - = = . :.
= - ~ ~ ~~ ~ ..
: - . -. ~
.= ~=- ~- ~=
~ : - . -. ~~~~~ ~ = ~
' = = = '
In tables D, E and F,
N = number of subjects in specific cohort
AR = Attack rate = n (number of subjects with HPV either incident infection,
persistent infection or
cytological abnormality, as appropriate for the table) / N
% Vaccine efficacy is 1-(A/B) x 100, adjusted for relative size of vaccine and
placebo group,
wherein
A = % women in vaccine group with incident infection, persistent infection or
cytological abnormality, as appropriate for the table
B = % women in placebo group with incident infection, persistent infection or
cytological abnormality, as appropriate for the table.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 33
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 33
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
