New HPV Diagnostic Assay
Presented by
Dr Ivan Brukner
Dr Damian Labuda
Hôpital Sainte-Justine
COMMERCIALIZATION AND TRANSFER
ASSISTANCE PROGRAM
Ministère du Développement Économique,
de l’Innovation et de l’Exportation
Le 26 septembre 2008
Table des matières
CONTENTS OF THE GRANT APPLICATION ........................................................................................ 4
1.
GRANT APPLICATION FORM ................................................................................................ 4
2.
PROJECT SUMMARY MAXIMUM THREE PAGES (in French) ................................................ 4
3.
PRINCIPAL RESEARCHER: PRESENTATION AND CONTACT INFORMATION ......................... 5
4.
DESCRIPTION OF THE TECHNOLOGY ................................................................................... 6
5.
6.
4.1.
Background - HPV as etiological agent of cervical cancer. .......................................... 6
4.2.
Technology developed and scientific basis ................................................................. 7
4.3.
Current development status of the technology – functional prototype ..................... 9
4.4.
Scientific value ........................................................................................................... 11
INTELLECTUAL PROPERTY .................................................................................................. 11
5.1.
Invention disclosure .................................................................................................. 11
5.2.
Previous disclosure and/or anterior art .................................................................... 13
5.3.
Freedom to operate analysis (FTO) ........................................................................... 15
5.4.
Revenue sharing ........................................................................................................ 17
TECHNOLOGY DEVELOPMENT PLAN ................................................................................. 18
6.1.
Identification of current stage of technology development. .................................... 18
6.2.
Remaining Steps and Time to Market ....................................................................... 20
6.3.
Technical and technological challenges to be met and anticipated progress........... 20
6.4.
Objectives sought ...................................................................................................... 25
6.5.
Type of activities to be carried out ............................................................................ 26
6.6.
Work plan and timetable ........................................................................................... 32
6.7.
Deliverables and result indicators ............................................................................. 32
6.8.
Decision-making milestones (go/no go) for measurable and clearly identified results
33
6.9. Proposal for granting the subsidy according to the decision-making milestones
reached .................................................................................................................................. 34
7.
RESEARCH TEAM ............................................................................................................... 34
8.
ESTABLISHMENT’S PROJECT MANAGER (In French) ......................................................... 35
9.
PARTNER ORGANIZATION ................................................................................................. 36
3
9.1. Role, experience and qualifications of the partner organization in conjunction with
the project ............................................................................................................................. 36
9.2.
Description of the partner organization's project selection process ........................ 37
9.3.
Professional who will support the valorization process ............................................ 37
10.
POTENTIAL MARKET ...................................................................................................... 38
10.1.
Target market ........................................................................................................ 38
10.2.
Business opportunity: Cervical cancer prevention program and HPV screening. . 39
10.3.
Technological competition and benchmarking ..................................................... 40
11.
COMMERCIALIZATION STRATEGY ................................................................................. 44
11.1.
Commercialization of the assay in Canada – Warnex partnership........................ 45
11.2.
Commercialization of the assay in Africa – Continental Diagnostic partnership .. 45
11.3.
Other partnership and future development ......................................................... 46
12.
PRO FORMA BUDGET (in French) .................................................................................. 46
12.1.
Coûts du projet de maturation technologique ...................................................... 46
12.2.
Montage financier ................................................................................................. 48
12.3.
Documents démontrant la nature des engagements des partenaires financiers . 48
12.4.
Démonstration que les autres sources de financement possibles ont été prises
en considération .................................................................................................................... 49
13.
OUTSIDE OPINIONS ....................................................................................................... 50
13.1.
Evaluator 1 ................................................................. Error! Bookmark not defined.
13.2.
Evaluator 2 ................................................................. Error! Bookmark not defined.
Letters of support .......................................................................... Error! Bookmark not defined.
Annex 1. Extended patent searches and comments ........................ Error! Bookmark not defined.
Annex 2. Analysis of documents cited in the PCT research report ... Error! Bookmark not defined.
Annex 3. Curriculum vitae – Research team ..................................... Error! Bookmark not defined.
Annex 3.1. Ivan Brukner’s CV .................................................... Error! Bookmark not defined.
Annex 3.2. Damian Labuda’s CV ................................................ Error! Bookmark not defined.
Annex 3.3. Maja Krajinovic’s CV ................................................ Error! Bookmark not defined.
Annex 4. ............................................................................................. Error! Bookmark not defined.
4
CONTENTS OF THE GRANT APPLICATION
1. GRANT APPLICATION FORM
Please see attached form.
2. PROJECT SUMMARY MAXIMUM THREE PAGES (in French)
Le projet proposé par le CHU Sainte-Justine consiste à développer d’un point de vue
commercial leur nouvelle méthode de sélection de sondes spécifiques, nommée
« hybridation itérative ». En particulier, on propose un essai fonctionnel pour le
diagnostic du virus du papillome humain (VPH) pouvant être utilisé au sein d’un
laboratoire de diagnostic clinique au Québec et en Afrique du Sud. Cette approche permet
le typage efficace de génomes montrant de hauts niveaux de similitudes de séquences. À
l’heure actuelle, dans le cas du dépistage et du typage du virus du papillome humain
(VPH), il n’existe aucun système de sondes offrant une solution de diagnostic dont
l’éventail soit complet et satisfaisant. Pour parvenir à améliorer la conception des sondes
d'hybridation, les chercheurs se sont attaqués au problème de leurs spécificités et leur
pouvoir discriminatoire.
Leur méthode peut être étendue pour générer un système diagnostique qui repose sur
l'hybridation d’acides nucléiques de courtes séquences étroitement liées. Au lieu d'ajuster
les conditions d'hybridation à la sonde, un ensemble de sondes est sélectionné de manière
à fonctionner dans les conditions d'hybridation. Cette méthode permettant d'obtenir une
détection spécifique offre une plus grande efficacité et un plus large éventail
d'applications. Un ensemble de 39 sondes spécifiques pour chacun des types de VPH
ciblés a été testé. Les résultats obtenus à partir d’échantillons cliniques positifs pour le
VPH, y compris les six types les plus fréquents (6, 11, 16, 18, 31 et 33), montrent que ces
sondes permettent la discrimination de tous les sous-types. Ainsi, les résultats démontrent
que cette stratégie permet de détecter des différences de 3-7 nucléotides entre les
différents amplicons / types, à température ambiante et dans un tampon PCR adapté.
La preuve de concept préclinique ayant été réussie, la prochaine étape réside dans la
validation à l’aide d’échantillons cliniques. Une fois cette preuve de principe obtenue, la
technologie pourra alors être adaptée dans un outil de détection et de typage plus général.
Le but du projet est donc de développer et de valider la technique de « l’hybridation
itérative » dans le cadre d’un test diagnostic en :
1. Effectuant une validation de l’essai prototype sur des échantillons
cliniques;
2. Élaborant un format de l’essai dont le design facilitera la
commercialisation. L’objectif étant d’en arriver à un essai de typage du
VPH qui soit simple, efficace et peu coûteux;
5
3. S’assurant le respect des exigences réglementaires afin de permettre une
commercialisation rapide de l’essai;
4. Optimisant le format commercial de l’essai pour une plus grande sensibilité
et une plus grande spécificité;
5. Validant la performance de l’essai commercial final à l’aide d’échantillons
cliniques.
Plusieurs raisons justifient que nous entreprenions ce projet, dont les avantages très
concurrentiels de la méthode qui sont:
1er) Capacité d'adaptation et haut pouvoir discriminatoire: Bien que plusieurs trousses de
diagnostic pour la détection et / ou de typage soient actuellement disponibles sur le
marché ou décrits dans la littérature, la grande variété de techniques disponibles pour la
détection et le typage des ~ 40 sous types de virus du papillome humain (VPH) au niveau
des muqueuses démontre le fait qu'aucune autre d’entre elles n’offrent une solution
complète à ce problème.
2e) Faibles coûts et facilité d'emploi: Il existe deux obstacles importants et inter liés
ralentissant le typage universel dans la population en générale, soit la complexité
technique de l'essai et le prix par dosage. Cette technique, lorsqu’intégrée à un outil
diagnostic, ne nécessite ni instruments coûteux, ni personnel hautement qualifié. En
outre, cette méthode offre la possibilité d'unifier les tests de diagnostic pour les différents
agents pathogènes dans les mêmes conditions de réaction qui, à terme, conduira à des
tests de diagnostic qui soient plus simples, rapides et moins coûteux.
3. PRINCIPAL RESEARCHER: PRESENTATION AND CONTACT
INFORMATION
Damian Labuda, Ph.D., D.Sc.
Professor, Pediatrics Department, Montreal University
Sainte-Justine Hospital Research Center, room B-607 b
3175 Cote Sainte-Catherine
Montreal, PQ
Canada H3T 1C5
Telephone: (514) 345-4931 ext.3586 [sec. 3282] fax: (514) 345-4731
damian.labuda@umontreal.ca
Damian Labuda studied biology and biochemistry at Adam Mickiewicz University in
Poznan, Poland, where he also obtained his Ph.D. and D.Sc. He received additional
training in Szeged, Hungary, in Saclay, France, and in Göttingen, Germany (post-doctoral
fellow of Max-Planck Institute). His early works concerned structure-function
relationship in transfer RNA, origin of the genetic code, biochemistry and physicochemistry of nucleic acids. Since 1982, he continued his research on RNA structure and
interactions, in Cedergren’s lab in the Department of Biochemistry, University of
6
Montreal. In 1984 he joined the Research Center of the Sainte-Justine Hospital and the
Department of Pediatrics, University of Montreal, developing DNA based diagnostics
program as well as research in molecular, medical, population and evolutionary genetics.
Presently, he carries studies in human population genetics on the origins and the
evolutionary history of human populations, the founder effects and the genetic history of
French-Canadians. His laboratory is also involved in genetic epidemiology studies
aiming genetic bases of complex diseases, the underlying genetic models, the
identification of cancer susceptibility variants as well as genetic variants influencing the
disease outcome
4. DESCRIPTION OF THE TECHNOLOGY
4.1.
Background - HPV as etiological agent of cervical cancer.
HPVs are genetically diverse; those infecting genital epithelium represent types with low
and high oncogenic potential. Low-risk HPVs, such as types 6, 11, 34, 40 44 and others,
cause benign genital warts, whereas high risk HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 68) lead to cervical cancer. The most frequent 16 and 18 account for about
70% of infections seen in cervical carcinoma (Bosch et al., 2002; Franco et al., 2001);
HPV 16 alone being found in nearly 50% of high grade lesions. There exists a substantial
degree of DNA sequence identity among types as well as between malignant and benign
HPV strains. The difference between particular HPV types in their sequence segments
that are relevant to the HPV molecular testing can be as small as few nucleotides (Villa et
al., 2000; zur Hausen, 2000).
Cervical cancer is presently the second most common cancer affecting women
worldwide, and the most frequent malignancy among women in developing countries
(Ferenczy & Franco, 2002). It was estimated that 493,000 new cases of invasive cervical
cancer were diagnosed worldwide in 2002, representing nearly 10 % of all cancers in
women (Ferenczy & Franco, 2002). Indeed, the HPV-attributable proportion of the
cervical cancer is estimated at more than 95%. The relative risk associating HPV
infection to cervical neoplasia is very high and increases to several hundred in the case of
the persistent infection with types 16 and 18 (Bosch et al., 2002; Clavel et al., 2001;
Franco et al., 2001). Once molecular pathogenesis of cervical cancer was recognized, it
became clear that an accurate type-specific testing of HPV is absolutely required for the
disease prevention and management, as recurrent detection of high-risk HPV types is a
strong predictor of high grade cervical intraepithelial neoplasia (CIN) and invasive cancer
lesions (Brummer et al., 2006; Schlecht et al., 2001; Trottier & Franco, 2005; Aho et al,
2004, Gagnon et al, 2004). Importantly, in the era of HPV vaccination, planning cervical
cancer screening will inevitably have to change, emphasizing need for HPV type-specific
assays. (Franco et al., 2006). Several diagnostic kits are commercially available and
numerous diagnostic systems have been described. However, of significant importance is
the recent study by the World Health Organization (WHO) which carried detection of 24
samples of seven most frequent HPV types, using commercial individual typing kits, such
7
as PGMY line blot (Roche), SPF10-LiPa (Innogenetics), Deg GP5+/6+ reverse line blot
and DNA chip (Biomed Lab Seoul, Korea). The measurements were performed in 29
independent laboratories and 12 different countries. The overall detection rate of HPV16
was 62% and that of HPV18 was 73.9%; approximately, half of the laboratories failed to
identify HPV type 6. In 2008, WHO issued recommendation guidelines for the use of
“reconstructed” clinical samples and therefore make a common cross-reference among
different diagnostics tests and different platforms on the identically “reconstituted”
samples.
4.2.
Technology developed and scientific basis
Four years ago we proposed a novel and generally applicable approach consisting of the
development of nucleic acid probes by selection in vitro. This differs from commonly
used approaches based on the rational design of probes. Addressing the common problem
of DNA diagnostics to distinguish between closely related population variants, similar
strains or subtypes, we developed a novel technology that would allow generation of
probes discriminating DNA targets that differ only by few sequence positions. Specific
probes are selected from a random oligonucleotides mixture by a process of iterative
hybridization. Repetitive rounds of forward and subtractive hybridization lead to specific
pools of probes with high discriminatory power from which individual probes can be
cloned.
In the summer 2004, we obtained the funding from CIHR to develop this technology and
show its applicability using a model system of HPV infection. The first task was to
develop proposed technology. For that, we used a specific segment of the viral L1 gene
of six HPV types differing by one to seven sequence positions. Starting from the initial
pool of probes we obtained pooled probes, and subsequently, target-specific cloned
probes. Both pooled and cloned probes were shown to discriminate well between six
HPV types. (Brukner et al, NAR 2007, Brukner et al, Nature Protocols, 2007). Once the
technology was developed, we extended the experiments to 39 HPV types. For the
selection we used the HPV genome region corresponding to the PCR amplified HPV
segment usually used (Jacobs et al., 1997; de Roda et al, 1995; Evans et al, 2005,Schmitt
et al, 2006; van den Brule et al, 2002; Schmitt et all, 2008; Nazarenko et all, 2008) in
the clinical diagnostics (GP5+/6+ region of the viral L1 gene). We have obtained a set of
39 probes specific for each of the targeted HPV type and efficiently discriminating
against the remaining 38 types in a single experiment under the ambient temperature of
hybridization (Brukner et al., J. Clinical Virology, 2007).
In summer 2007 we obtained proof of principle (POP) grant from CIHR to simplify our
assay and render it generally applicable in the research and diagnostic setting. We tested
the probes in the reverse format (as opposed to previously used direct format) in which
the array of the HPV specific probes is immobilized on solid support. This format is more
suitable for diagnostic use and allows simultaneous detection of distinct HPV variants in
the clinical sample. Miniaturization of the assay was achieved by single compartment
8
hybridization (as opposed to previously used 96-well format). For this, the attachment of
probes was performed via streptavidin-biotin connection using SAM membranes
(Promega, WI). For all testing we used full-length GP5+/6+ target oligonucleotides (and
double strand amplicons derived from these oligonucleotides, mimicking thus PCR from
clinical samples) labeled with [32P] for initial optimization (Fig 1).
1
1
HPV 6
HPV 16
HPV 11
HPV 18
4
5
11
HPV 31
HPV39
8
12
HPV 33
HPV 51
9
18
HPV 35
HPV 68
Figure 1. Example of HPV typing probes in reverse format. Hybridization signal between 39 selected HPV
type-specific probes (each 100 pmols), y spotted on SAM membrane (7 cm x 3 cm) and particular intended
targets (HPV 6, HPV 11, HPV 16 and HPV 18 on upper panel and HPV 31, HPV 33, HPV 35, HPV 39
HPV 51 and HPV 68, on lower panel) each presented in the concentration of 3 pmols per 5 mL of
hybridization solution. (P32 autoradiography). Hybridizaton is performed at ambient temperatue (26+/4oC) using buffers compatable with colorimetric detection. No false positives (cross-hybriization) with
remaining 38 HPV types were seen.
As a result of these successful developments, we subsequently focused our assay on four
most relevant HPV types (6, 11, 16 and 18). These types are the most relevant from the
point of view of recently introduced national vaccination programs (duration and liability
of vaccination strategy can be monitored). At the same time, we can guarantee that
probes that we obtained during selection will not produce significant cross-talk with other
relevant HPV types as shown in Fig. 2.
9
25
20
15
6
10
11
16
18
5
6
6
11
13
16
18
26
30
31
33
34
35
39
40
42
43
44
45
51
52
53
54
55
56
58
59
61
62
64
66
67
68
69
70
72
73
76
MM4
MM7
MM8
0
Figure 2 – Hybridization signal (y-axis) between selected 6-FAM labeled type-specific probes (HPV 6, 11, 16
and 18, using 20 pmol of each) and 39 HPV GP56 targets, where HPV types-specific numbering nomenclature is
ranked in the growing order on x-axis. Each target (20 pmol) is bound to one well (using 96-well steptavidincoated Pierce plate) and hybridization is performed in 100L volume (see Brukner et al, 2007, J. Clinical
Virology, for more details).
4.3.
Current development status of the technology – functional prototype
Processes/algorithm for the generation of spectrum of specific nucleic acid (NA) probes
able to discriminate against plurality of similar targets is not yet known. When sequences
that are to be distinguished are similar, the difference in their binding energy is small,
restricting the window of adjustable experimental conditions, which would allow
discrimination between all potentially reacting species. Finding such conditions is usually
problematic in multiplex applications, when many probes and/or many targets are
considered simultaneously. Moreover, if probes with such a requirement have to perform
under robust conditions, the design process is especially prone to failure. Here the term
“specificity” is used as an ability to discriminate NA target in the context of similar NA
targets (differentiating up to 87% sequence identity), while assay is considered “robust”
if stability of the assay performance within a wide range of performing conditions is
preserved.
Following urgent need for developing more robust and more accurate HPV typing assay,
we applied this approach to obtain new generation of probes. These probes are selected to
discriminate among 39 clinically relevant HPV types, based upon the previously
characterized GP5+/6+ L1 segment of the HPV. In a series of hybridization steps, starting
from a mixture of random oligonucleotides, we iteratively enriched mixture in
10
oligonucleotides that selectively recognize each specific HPV type out of the 39 HPV
targets. (Brukner et al, (2007a), Brukner et al, (2007b). A detailed analysis of data
showed clearly that, given the number of variables, the rational design of probes would
not be as efficient and straightforward as selection performed in vitro. Analysis of
sequences of obtained probes showed that specificity of binding between probes and
targets is achieved through a fine balance between non consecutive stretches of basepairs, segments of mismatches and often accommodation of secondary structures. These
combinations allow maximizing the difference in binding energy between probe-specific
target and probe-unspecific target complexes.
In the next phase, we optimized a reverse format of probes (Fig 3). The performance of
the assay was examined using clinical samples containing HPV 16 and HPV 6.
Figure 3. HPV typing of pre-characterized clinical samples containing HPV6 and HPV16 to the array of 39
immobilized type specific clonded probes (CP). (A) the arrangements of CP probes; (B) hybridization with
HPV6; (C) hybridization with HPV16. Arrows indicate the orientation of the probes array. (see Brukner et
al, J Clinical Virol. 2007, for more details)
We confirmed specificity of our probes and stability of our novel assay at the wide range
of temperatures, starting from 20°C to 28°C and in the different spectrum of nondenaturing buffers (classical hybridization buffers, as well as simple, PCR-like and
colorimetric-compatible buffers).
11
Such assay performance is prerequisite for the future point-of-care medical device,
contrary to the present genotyping assays, whose setting performance is not only
challenging for similar NA targets, but also based on sophisticated technological
platforms.
4.4.
Scientific value
The main advantage of our method resides in its enhanced power of identification and
discrimination between multiple short nucleic acid sequences that differ by a few
mutations, as it is the case with different HPV types, in a multiplex hybridization assay.
In other words, instead of adjusting hybridization conditions to the whole set of probe:
target pairs that we want to include in the diagnostic device, by using iterative
hybridization we adjust the probes to the conditions we have chosen.
Example of direct expected outcome using our HPV typing assay in clinical follow-up is
to estimate HPV vaccine performance and its protective time in industrial countries
where HPV vaccine program is available. It will also provide a cost-effective but accurate
and easy to use diagnostic tool, an essential requirement for deployment of HPV
screening program in developing countries.
Finally, the methodology of probe selection is applicable to many other medical
conditions where investigation or diagnosis or detection of resistant strains is based upon
a differentiation of highly similar nucleic acid sequences (HIV, hepatitis virus,
tuberulosis.).
5. INTELLECTUAL PROPERTY
5.1.
Invention disclosure
In January 2006, Dr. Brukner, Dr. Labuda and Dr. Krajinovic filed an invention
disclosure at the research administration of Hôpital Sainte-Justine. The inventorship’s
contribution is presented in Table 1.
Inventors
Îvan Brukner
Damian Labuda
Maja Krajinovic
Status
Research associate
Professor
Associate professor
Institution
%
inventorship
CHU Ste-Justine
45 %
CHU Ste-Justine
35 %
CHU Ste-Justine
20 %
Table 1. List of the inventors and their contribution to the invention
12
The invention describes a new method for generating sequences of hybridization probes,
suitable for multiplex target detection, even if the plurality of targets are very similar at
the sequence level. The method is based on the hybridization of nucleic acids. A
prototype for genotyping HPV virus was built and performance of 6 selected probes was
tested and compared with complementary probes. A set of 39 oligonucleotide probes for
HPV typing was generated and more recently a prototype assay was optimised for 4
probes (see section 6.1.). A provisional patent application entitled “NUCLEIC ACID
PROBES, METHODS FOR THEIR PREPARATION AND USES THEREOF” was filed
in the US in August 2006. We filed a complete PCT application in August 2007 and we
expect to enter the National Phase in different markets in February 2009 (see section
5.3.1.). M. Serge Shahinian from the firm Goudreau Gage Dubuc in Montreal is the
patent agent handling the file.
The data base “Derwent World Patents Index”, where specialist editors provide a
comprehensive summary of the patent contents with its advantages gives the following
description of the invention:
NOVELTY - Identifying an oligonucleotide for discriminating a first nucleic acid from a second
nucleic acid comprises: (a) hybridizing the first nucleic acid with oligonucleotides comprising a
random nucleotide sequence flanked by primer recognition sequences; (b) amplifying the bound
oligonucleotides to obtain amplified oligonucleotide duplexes; and (c) repeating hybridization in the
presence of a second nucleic acid, where an oligonucleotide comprising the random nucleotide
sequence can be used for discriminating the first nucleic acid from the second nucleic acid.
DESCRIPTION - Identifying an oligonucleotide for discriminating a first nucleic acid from a second
nucleic acid comprises:
1.hybridizing the first nucleic acid with a pool of oligonucleotides in a hybridization mixture, the
oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences;
2.removing oligonucleotides which are not bound to the first nucleic acid from the hybridization
mixture; 3.dissociating bound oligonucleotides from the first nucleic acid; 4.amplifying the bound
oligonucleotides using primers capable of binding to the primer recognition sequences to obtain
amplified oligonucleotide duplexes comprising a first strand corresponding to the bound oligonuc
leotides and a second strand corresponding to the complement of the bou nd oligonucleotides;
5.treating the duplexes to remove or degrade the second strand to obtain single-stranded amplified
oligonucleotides; 6.repeating (a) to (e), where the pool of oligonucleotides of (a) is the amplified
oligonucleotides obtained in (e) thus to obtain further ampli fied oligonucleotides; and 7.repeating (a)
to (e), where the hybridization in (a) is performed in th e further presence of the second nucleic acid;
where an oligonucleotide comprising the random nucleotide sequence of the further amplified oli
gonucleotides can be used for discriminating the first nucleic acid fro m the second nucleic acid.
13
INDEPENDENT CLAIMS are:
1.an oligonucleotide (a) identified by the method above, (b) capable of discriminating a first nucleic
acid from a second nucleic acid, where the oligonucleotide is not exactly complementary to the first
nucleic acid, and (c) comprising a nucleotide sequence selected from SEQ ID NO. 1- 43, 100-104, and
116; 2.a method for detecting the presence or absence of a first nucleic acid in a sample; 3.a kit for
detecting the presence of a first nucleic acid in a sample, the kit comprising the oligonucleotide; 4.a
collection of two or more oligonucleotides, where the oligonucleotides comprise a nucleotide
sequence selected from SEQ ID NO. 1-43, 100-104 , and 116; 5.an array comprising the
oligonucleotide or the collection of two or more oligonucleotides; and 6.a kit for identifying an
oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, the kit comprising
the pool of oligonucleotides.
USE - The methods are useful for identifying an oligonucleotide for discriminating a first nucleic acid
from a second nucleic acid, and for detecting the presence or absence of a first nucleic acid in a
sample. The kit is for detecting the presence of the pathogen in the sample, for detection of the
pathogen in the subject, and for diagnosing a disease or condition associated with the pathogen in the
subject (all claimed). The methods can be used for discriminating between closely related or similar
nucleic acids, and for identifying or preparing an oligonucleotide for discriminating a desired or
intended target nucleic acid from other undesired or non-intended non-target nucleic ac ids. The
oligonucleotides, methods, and kits may be used in analytical, diagnostic (e.g. infection of an animal,
plant, or organism by a pathogen), detection, manufacturing/quality control, research, environmental
monitoring (e.g. pollution/contamination of air/water/reagents) intended for use in biological systems
(e.g. culture or animal systems/other materials), microbiology (detection studies of organisms difficult
to c ultivate), and forensic applications.
ADVANTAGE - The method has the capacity of identifying or preparing an oligonucleotide for
discriminating nucleic acids, which share sequence similarities, e.g. similar nucleic acid sequences
from different organisms (e.g. orthologous genes), variants (e.g. polymorphisms, different alleles) of a
given nucleic acid sequence, nucleic acid sequences derived from genes belonging to the same family
or nucleic acids derived from subtypes of a given organism (e. g. virus, bacteria, parasites).
5.2.
Previous disclosure and/or anterior art
In conjunction with Univalor, the organization that provides commercialization services
to the research centre of Hôpital Sainte-Justine (see section 9), we performed a review of
the prior art. The search was carried out using Delphion patent databases, and literature
(PubMed) together with a web-based search.
5.2.1. Method of selection probes
The concept of selecting nucleic acid sequences that specifically bind particular targets
has been developed using an approach called SELEX (systematic evolution of ligands by
14
exponential amplification. However that patented method (see Gold, Table 2a) and other
related methods described in the literature (c.f. Kyung 2003) do not teach the use of
iterative selections for generation of nucleic acid ligands against nucleic acid targets for
the purpose of genotyping or identifying/detecting nucleic acids. Therefore, our iterative
hybridization method to select probes that can discriminate between closely related or
similar nucleic acids is entirely novel.
Patent
(filing date)
US5475096
June 10,
1991
376 family
members
Title
Nucleic
acid
ligands
Inventor
(Assignee)
Gold, et al.
(University
Research
Corporation)
Abstract
A new class of nucleic acid compounds, referred to as
nucleic acid ligands, have been shown to exist that have a
specific binding affinity for three dimensional molecular
targets. In a preferred embodiment the nucleic acid ligands
are identified by the method of the invention referred to as
the Systematic Evolution of Ligands by EXponential
enrichment (SELEX), wherein a candidate mixture of
nucleic acids are iteratively enriched in high affinity nucleic
acids and amplified for further partitioning.
Table 2a. Patent related to the method
5.2.2. HPV probes
Our search revealed, that prior art taught the use of nucleic acid probes to detect HPV
types. Several patents (see examples in table 2b and in Annex 1) and articles (Hwang et
al., 2003; Klaassen et al., 2004; Kleter et al., 1999; Schmitt et al., 2006; van den Brule et
al., 2002) describe typing systems based on PCR/classical hybridization but in all cases
they rely on complementary sequences to discover HPV types (hybridization rules based
on Watson-Crick base-pairing). Our probes do not rely on complementary sequences and
also none of our probes sequences are disclosed or claimed in these documents.
Patent
(filing date)
US6583278
November
14, 1996
11 family
members
US6265154
October 25,
Title
Nucleic acid probes
complementary to human
papillomavirus nucleic
acid and related methods
and kits
Nucleic acid primers and
probes for detecting
Inventor
(Assignee)
Gordon, et al.
(Gen-Probe
Corporation)
Kroeger, et al.
(Abbott
Abstract
The present invention describes oligonucleotides targeted to HPV
Type 16 and/or Type 18 nucleic acid sequences which are
particularly useful in aiding the detection of HPV Type 16 and or
18 by, for instance, acting as hybridization assay probes, helper
probes, and/or amplification primers.
Probe sequences that are useful for detecting oncogenic HPV
types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 are
15
1996
8 family
members
oncogenic human
papillomaviruses
Laboratories)
US5364758
July 16,
1992
20 family
members
Primers and process for
detecting human
papillomavirus genotypes
by PCR
Meijer, et al.
(Stichting
researchfonds
pathologie)
US5712092
July 7, 1994
39 family
members
Papillomavirus probe and
process for in vitro
diagnosis of
papillomavirus infections
Orth, et al.
(Institut Pasteur)
herein provided. These sequences can be used in hybridization
assays or amplification based assays designed to detect the
presence of these oncogenic HPV types in a test sample.
Additionally, the sequences can be provided as part of a kit.
The invention relates to primers and a method of detecting
human papilloma virus (HPV) genotypes by means of the
Polymerase Chain Reaction (PCR). The invention provides such
primers and such PCR conditions that in principle any genital
HPV genotype is detected. The invention enables a sensitive and
reliable preselection of samples to be examined, such as cervical
smears.
The invention relates to human papillomaviruses HPV,
particularly to HPV-DNAs isolated from papillomaviruses HPV-2d,
HPV-10b, HPV-14a, HPV-14b, HPV-15, HPV-17a, HPV-17b,
HPV-19, HPV-20, HPV-21, HPV-22, HPV-23, HPV-24, HPV-28,
HPV-29, HPV-31, HPV-32, HPV-IP2 and HPV-IP4. The invention
also relates to DNA capable of hybridizing with the HPV-DNAs or
fragments thereof, to kits containing distinct groups of probes
containing one or more of these HPV-DNAs or fragments thereof,
and to procedures for detecting and identifying HPV in tissue.
Table 2b. Patent related to our HPV assay
5.2.3. Research report on the PCT patent application
In November 2007, we received the international research report and the written opinion
of the international searching authority.
Seven documents, including four documents published by our inventors were found to
oppose our invention in terms of both novelty and inventive step (nothing was opposed to
the industrial applicability of the invention). We carefully analyzed these documents and
our conclusion, which was validated by our patent agent, is that none of these documents
teach, disclose or can be considered as anterior art of what we claim in our invention (see
Annex 2 for a summary of our analysis).
In conclusion, based on our analysis of the prior art, we do not anticipate any problems
for the patentability of our invention and we are confident that we would be in an
excellent position to argue potential objections from patent Examiners at the regional and
national phases of protection, should there be any. This confidence was validated by the
patent agent handling the file. It might be important to mention that Univalor’s team has
experience in patent prosecution and with interpretation of PCT written search and
opinion reports.
5.3.
Freedom to operate analysis (FTO)
We do not see any FTO problems related to the commercial use of our new method for
generating sequences of hybridization probes concerning the SELEX patent (see section
5.2.1) The SELEX process is described in two issued US Patents, No. 5,270,163 (filed
Aug 1992), which claims the SELEX method itself, and No. 5,475,096 (see table 2a)
which claims the use of the method to identify nucleic acid ligand for target molecules
16
“other than a polynucleotide that binds to said nucleic acid ligand,” and “wherein said
nucleic acid ligand is not a nucleic acid having the known physiological function of being
bound by the target molecule.”
Our preliminary FTO search and analysis gives us no indication that the commercial
production, marketing and use of our new assay for HPV detection would infringe the
intellectual property rights of other patents. Our analysis included the US patents listed in
table 2c and in Annex 1. It appears that these inventions relate mainly to the HPV DNA
isolated from the appropriate strain, the probes containing these DNA sequences, and the
kits containing these probes. Because the intrinsic nature of our probes is not fully
complementary to any of the HPV DNA, our probes are not covered by the claims of
these patents. Our position was validated by our patent agent. Note that most of the HPV
types were covered by patents that are now expired or will expire in the near future.
Consequently, we do not anticipate a need to license any patent on either SELEX or any
specific sequence of HPV variants in order to commercialize our technology.
Patent number (priority date)
No. 6,391,539 (November 1984), No. 5,958,674
(November 1984), No. 5,876,723 (March 1986), No.
5,824,466 (May 1988), No. 5,656,423 (December 1990)
and No. 5,981,173 (February 1996)
No. 4,849,334 (June 1987), No. 4,849,332 (May 1987), No.
4,849,331 (June 1987) and No. 4,908,306 (June 1987)
No. 5,057,411 (April 1985) and No. 5,643,715 (October
1988)
Comments
Patents that are part of the broad HPV
patent portfolio developed at the Institut
Pasteur that were licensed to Roche in June
2002;
Patents were assigned to Life Technologies
(now Invitrogen)
Patents were assigned to Georgetown
University.
Table 2c US patents related to HPV types
5.3.1. Acquired or planned protection
A provisional patent application entitled “NUCLEIC ACID PROBES, METHODS FOR
THEIR PREPARATION AND USES THEREOF” was filed in the US in August 2006.
That patent application claims a method for identifying and preparing probes for selective
detection of nucleic acids. This method is particularly useful in discriminating between
closely related nucleic acid sequences. Such a method may be used in a variety of
analytical and diagnostic research and related applications. Probes selected for 39
different HPV types are covered in the patent application. A complete PCT application
was filed in August 2007.





Priority Application Number (Number Kind Date): US 2006822153 P 20060811
PCT Application Number WO 2008017162
Patent Assignee: SAINTE-JUSTINE UHC
Inventors: BRUKNER I; KRAJINOVIC M; LABUDA D
PCT filing date: 20080214
17
We plan on entering the Regional and National phases in February or March 2009 as
described in the following table (Table 3).
1)
2)
3)
4)
Canada (February 2009)
USA (February 2009)
South Africa (February 2009)
ARIPO (March 2009). ARIPO is considered a regional phase covering the
following countries: Botswana, Gambia, Ghana, Kenya, Lesotho, Malawi,
Mozambique, Namibia, Sierra Leone, Sudan, Swaziland, Tanzania, Uganda,
Zambia, and Zimbabwe.
We will budget another patent filing in a territory that will be chosen between the
following: Europe, Angola (a relevant territory for our financial partner in this
application) and Brazil (the translation of the application into Portuguese for the Angola
application can be subsequently used for the Brazil application and will reduce the cost of
filing in this country by 50%).
The IP protection in Canada, South Africa and ARIPO is justified by the partnership with
the company Continental Diagnostic, based in South Africa (see section 14), and the
interest of Warnex, based in Laval, Québec to provide HPV diagnostic services through
Canada. It is also strategically important to get protection in US which is the largest
market for HPV testing presently. Europe and Brazil are also two relevant territories for
IP protection since vaccines clinical trials were done in these territories. However the
final choice between Europe, Brazil and/or Angola will be decided in January 2009.
5.4.Revenue sharing
The documents attesting to the assignments of titles, rights and revenue sharing between
the researchers, the CHU Sainte-Justine Research Centre (“CHU Ste-Justine”), its
commercialisation entity (i.e. Valorisation-HSJ, limited partnership) and/or the
Université de Montréal (“UdeM”), shall be concluded prior to the start of the project, the
whole according to the provisions of the intellectual property policies and related
agreements in force between these institutions.
As a precision, CHU Ste-Justine is an affiliated institution of UdeM and as such, CHU
Ste-Justine and UdeM jointly own, in equal parts (50%/50%), the undivided ownership
rights in any invention originally disclosed at CHU Ste-Justine by a researcher who holds
an academic qualification or a faculty title of UdeM. CHU Ste-Justine and UdeM also
jointly own, in equal shares (50%/50%), the institutional share of the benefits or revenues
which will be allotted to CHU Ste-Justine, as mutually agreed between these institutions.
The share of the proceeds of commercialization of the invention (the “Proceeds”) shall
therefore be divided and paid as follows:
18
(A) fifty percent (50%) of the Proceeds shall be allotted to the researchers, which
portion shall be divided between each of Îvan Brukner (22,5%), Damian Labuda
(17,5%) and Maja Krajinovic (10%); and
(B) fifty percent (50%) of the Proceeds shall be allotted to Valorisation-HSJ, limited
partnership (the valorisation entity of CHU Ste-Justine), which portion shall be
divided in equal shares (50%/50%) between Valorisation-HSJ, limited
partnership and UdeM.
Party
Share of Proceeds
Îvan Brukner
22,5%
Damian Labuda
17,5%
Maja Krajinovic
10%
Valorisation-HSJ, limited partnership (CHU Ste-Justine)
25%
UdeM
25%
TOTAL:
100%
Table 3. Final sharing of the Proceeds between the parties
6. TECHNOLOGY DEVELOPMENT PLAN
The proposed development plan will allow optimizing and validating the efficacy of a
new HPV assay and will provide us with convincing arguments that it can fulfill all
principal requirements of clinical and market needs: multiplex detection, detection of
different HPV types in the same patient (super infection), low production cost and ease of
use.
6.1.
Identification of current stage of technology development.
The actual prototype kit is composed of 4 probes designed to detect 4 HPV vaccinerelevant types (6, 11, 16 and 18), including two positive controls: one reflecting presence
of any HPV in the sample and the other controlling for sample DNA integrity (see Figure
4). HPV targets are 150 nucleotides long GP5+6+ DNA segments, derived through
amplification or chemical synthesis.
19
6
11
100
100
16
18
100
100
UP
CD
0.5
10
Figure 4: Schematic presentation of 4 cm x 2cm Steptavidin-coated Membrane (SAM, Promega) with
biotinilated oligonucletide probes Optimal number of picomoles (right panel) spotted on the membrane
(left panel) for each HPV probe and controls (UP, universal HPV probe, CD, control DNA designed for the
control of the quality of DNA extraction
HPV 16 and HPV 18 are the most frequent virus types found in cervical cancer. In fact,
HPV types 16 and 18 account for 70% of HPV infections. HPV 6 and 11 are found
frequently in genital and upper respiratory tract condylomas. Accurate and affordable
HPV genotyping for these strains will also be in high demand for the next few decades
due to HPV vaccination studies. Recently, two experimental vaccines to prevent infection
with HPV 6, 11, 16 and 18 became available. HPV screening is needed here: i) to identify
individuals that are eligible for vaccination and ii) to monitor the efficiency of vaccines.
The Advisory Committee of Immunization Practices (ACIP, US) recommended the
continuation of HPV screening protocols until (i) other type-dependent vaccines are
developed and (ii) the protective time period of the vaccine is fully characterized. The
advantage of our assay is in the selection of probes in the context of 39 viruses.
Therefore, the probes are highly specific for these types and do not cross-hybridize with
other HPV amplicons (see Figure 2).
We also developed an HPV universal probe, which is designed to detect any HPV
infection (Fig. 5, probe UP). This “promiscuous probe” allows for the follow-up of HPV
positive cases (but HPV 16 and 18 negative ones), which is of particular interest to
researchers and to public health. Despite typing only 4 viruses, all cases of HPV-positive
cases could be clinically registered. Further type-specific analysis can be then performed
by complementary methods.
For estimation of DNA quality and quantity of a clinical sample, we also
introduced a new probe hybridizing with monomer repeat of 29 adenines (A29), present
in a single copy locus of human genome of well-described BAT26 amplicon (Fig. 5
probe CD).
The performance of each type-specific probe and its corresponding
intended targets is presented in Figure 6A.
20
6.2.
Remaining Steps and Time to Market
Class II, III and IV medical devices need to have Canadian licenses in order to be sold in
Canada. After speaking with Sarah Chandler, Acting Head of the Regulatory and
Scientific Section at the Device Licensing Services Division of the Medical Devices
Bureau at Health Canada, we believe our HPV test is most likely a Class III product. We
plan to validate the classification status of our device before the end of 2008(please
introduce a new date 2009). This is the first step toward getting a medical device license.
The regulatory process in South Africa starts with a letter to the authority mentioning the
intention to file clinical information for registration of a new test. The authority can call
for local clinical information and trials if deemed necessary. When the information has
been submitted, questions could follow on the proof of concept and clinical validation
methodology results. The whole process takes up to 3 years right now, but when the new
authority steps in it might be shortened to 18 months and if fast tracked, 9 months.
Our strategy is to partner with a company already established in the molecular diagnostic
market who will be responsible for the final stages of our product development, including
all aspects of clinical validation and regulatory affairs. We will have the opportunity to
work with the Quebec company Warnex (see letter of support) to define the regulatory
path for our HPV assay.
6.3.
Technical and technological challenges to be met and anticipated progress
21
1)
Preservation of assay specificity and sensitivity under different assay
conditions
Our results indicate that we will be able to develop a new format of HPV typing kit that
will preserve clinical sensitivity (genome equivalent, GE, 1000 or more), but will have
unique specificity features preserved in a wider-than-usual range of assay conditions.
What remains to prove is that our type-specific probes will produce better assay stability
than any other probes on the market. It is well known from literature data that small
variations in HPV genotyping assay conditions can lead to the wrong interpretation of
hybridization intensity patterns when using known commercial kits. The most critical
issues are in the domain of specificity of hybridization among multiplicity of similar
targets, where even a 1oC deviation from the intended temperature, or small variations in
buffer conditions can be detrimental. In fact, recent data (Steinau et al., 2008) showed
that even intra-laboratory repetitions with the same clinical samples (but different
DNAextractions) did not produce the same results using Roche Linear Array assay (83%
concordance). We believe that our HPV typing probes have inherent (sequencedependent) features which preserve stability under a wider range of assay conditions.
Considering that this issue presents a major barrier to the current accuracy of
hybridization-based assays, overcoming it by confirming our data in a clinical setting
would be important accomplishment. As an example of anticipated progress, we recently
perform hybridizations between HPV 6, 11, 16 and 18 single stranded targets (GP5+
strand) at 4 different temperatures (20, 25, 30 and 35oC) and recorded hybridization
pattern, as presented on Figure 6B. While, as expected the efficiency of the assay (as
measured by signal intensity) is affected by the temperature it preserves its specificity in
a wide range of temperature conditions.
22
2)
Optimization of a prototype assay format and developing it into its final
product
We already have a functional prototype assay format which will be challenged with
reconstructed samples having 1,000 and 10,000 genome equivalents (GE) of different
types of HPV in the background of 1,000 GE of human DNA (see Figure 7).
23
We do not foresee obstacles in detecting 1,000 genome equivalents of HPV in the
hybridization assay. In fact, our present developments indicate that a hybridization
assay is not an obstacle per se, since 1,000 GE of HPV 6, 11, 16 and 18 are
amplifiable by PCR (using oligonucleotide targets as substrates) in sufficient quantity
to guarantee detection of the hybridization signal. We will have to do HPV GP56
amplification in the context of human DNA, where we will have to satisfy “normal”
PCR yield requirements (1-10pmols) and at the same time be able to perform CD
PCR (i.e. produce amplicons which reflect DNA quality of sample).
24
3)
PCR amplification from clinical samples
Protocols designed to collect, store and purify DNA from cervical swabs are welldescribed. In addition, the expertise of Dr Gòrska-Flipot Izabella from the Hotel-Dieu
diagnostics laboratory, guarantees that this challenge will be addressed in a professional,
effective fashion given the long clinical experience and expertise she has in the domain of
molecular diagnostics.
4)
Technology transfer with the group from South Africa
The present assay format is functional at Saint-Justine Hospital and we have to be sure
that all parameters of protocol are well defined and universal, so the assay can be
reproduced, without technical help from the person who developed the protocol.
Therefore, we will establish a technological transfer procedure and a corresponding
manual, which will guarantee that the assay can be independently repeated with the same
quality of assay performance as originally described. Here is the summary of current
25
optimal conditions for each procedural step: (the details of protocol 4a to 4d might as
well go to Appendix if you think it is a good idea) May be the whole description can be
moved to Appendix (DL)
4a) PCR The 50 l volume PCR was performed as originally suggested (van den
Brule, et al., 2002) with minor modifications including shortening elongation and
denaturation time to 20 seconds. Yield of PCR was monitoring by loading 10l reaction
mix over agarose gel and EtBr staining.
4b) Conversion of PCR product to single stranded (ss) DNA and labeling The
40L were digested for 15 minutes at 37C, with EXO1 (NEB), following enzyme
inactivation (20min, 85C), and lambda exonuclease (15 min, 37C, following 20 min,
85C). Sample was passed over S25 column for buffer exchange reaction compatibility
with downstream T4 Polynucleotide Kinase labeling step. The conversion of double
stranded to single stranded form of PCR product was monitored by disappearance of EtBr
stained bend before and after digestion.
4c) Membranes Streptavidine-coated Promega membranes (SAM, Biotin Capture
Membrane, Medison, WI) were used in the following manner. The 1 l of 100pmols/l
of type specific (CP) 5’ biotinilated oligonculeotide probes were manually sported
(HPV6, HPV11, HPV 16 and HPV 18) on the surface of 3cm x 2cm membrane (see
Figure 1 for spotting schematic). The HPV universal probes (UP) were spotted using 1 l
of 0.3 pmols/l of oligonucleotide. Spotted drops were dried at ambient temperature for
5 minutes, membrane was washed in ddH2O for 1-2 minutes and pre-hybridized in 2 mL
hybridization buffer SSPE (150 mM NaCl, 10 mM NaH2PO, 1.1 mMEDTA, pH 7.4),
0.75MNaCl, 70mMTris–HCl, pH 7.4) containing 1% SDS and 200 mg/ml heparin
(hybridization oven, Model 400, Robbins Scientific ) for 1-12h at 55oC. These
membranes were either stored at room temperature for couple of days, or immediately
used for hybridization assay.
4d) Hybridization Labeling of 2-20 pmols of PCR and/or oligonucleotide was
perfomed using 5 l of [32P] ATP (6000 Ci/ mmol) and 1 L of T4 Polynucleotide
Kinase to a specific activity of 105 to 106 cpm/pmol, following produce manual
recommendation (Invitorogen). Hybridization was carried out for 1-12 hours. The
membranes were then washed with 1x SSPE containing 0.1% SDS for 10 min at room
temperature and either exposed overnight at -80°C with intensifying screens, or exposed
in Cyclone Storage Phosphore Screen (Perkin Elmer) for 10-30 minutes and read by
Cyclones software (OptiQuant, version 4.00).
6.4.
Objectives sought
The overall goal of this project is to develop a functional HPV assay that will be
implemented in a clinical diagnostic laboratory in Quebec and in South Africa.
The specific objectives required to reach this goal are
1. Clinical validation of the prototype assay
26
2. Design of the commercial format of the assay (simple and cost-effective HPV
typing assay)
3. Establish the regulatory pathway for the commercial use of the assay
4. Optimization of the commercial format of the assay for high sensitivity and
specificity
5. Validation of the performance of the commercial assay with clinical samples.
6.5.
Type of activities to be carried out
The point by point summary given below refers to major issues needed for assay
optimization as well as validation of sensitivity and specificity of assay with standardized
and clinical samples.
1. Preparation of the test: Spotting of 4 type-specific DNA probes (6, 11, 16, 18) on
SAM membranes (Promega); Inclusion of the positive control that is universal
control positive for any HPV type; Introduction of the control of DNA extraction
probe (DNA integrity) for clinical sample collection. To this end, independent PCR
will be subsequently performed from reconstructed and clinical samples (see
below). This PCR is amplifying single copy human genome segment known as
BAT26 locus. Preliminary data demonstrating the performance of recently designed
universal HPV probe are shown in the figures 3 to 8. The preliminary data using
oligonucleotide mimicking BAT26 PCR product (without primers) are given in
Figures 5.
2. Verification of the amplification sensitivity of standardized (in vitro reconstructed)
samples. Range of genome equivalents (GE) of HPV (1000-10 000) will be tested.
Multiplex PCR that is simultaneously amplifying any HPV genome and BAT26
locus from human DNA will be developed.
3. Evaluate the performance of different variant of GP56 primers. This would be done
to see performance of different variant of GP56 primers in the context of different
number of HPV genome equivalents and particularly in the context of different
combinations of mixed infections (GE 1000, 10 000 and 100 000), spiked with
human DNA (1000 GE)) and co-amplification of other HPVs except those here
tested. Amplification products should enter into the yield range of 1-10pmols for all
39 types, where HPV16 amplicon (known to perform well) will be used as a
reference point (producing 100% “standard” yield).
4. Amplification of DNA from 40 clinical samples for which custom HPV typing is already
available. Samples from Department of Pathology of Centre Hospitalier de l’Université de
Montréal will be used. Cervical biopsies were evaluated by service pathologists as cervical
intraepithelial neoplasia of a different grade. Custom HPV detection was performed by
PCR amplification with PGMY09/11 primers designed to amplify a product from the L1
open reading frame of a wide spectrum of HPV types. The HPV types were assigned by
27
restriction fragment length polymorphism based on comparison with known HPV
sequences. Two restriction enzymes, RsaI and Dde I, were used which gave characteristic
restriction patterns for most HPV types (Fig .9) (Gorska-Flipot et al. Ann Biochem Clin,
1996, 35, 66-70). To avoid confusion, we will call this assay the CHUM HPV assay. We
are aware of the fact that each primer set used in amplification has its own bias toward
particular type of viruses (see also Sabol et al., 2008). Therefore, we will try to minimize
sequence-dependent differential effect of primers on production (Schmitt et al, 2008) and
interpretation of results.
5. Analysis of the same reconstructed and clinical samples with commercial Roche
HPV typing kit.
6. Comparative analysis with custom and commercial HPV typing. This analysis will
be done in collaboration with Dr Eduarod Franco (Professor of Epidemiology and
Oncology Director, Division of Cancer Epidemiology, McGill University), which
for many years leads the study in HPV epdemilology (see letter).
Cloning and sequencing of HPV PCR products will be performed in the case of
discordant results.
Activities which are currently performed to reach each objective are described in the
following sections.
6.5.1. Clinical validation of the prototype assay
As presented in section 6.1, the actual prototype kit is composed of 4 probes designed to
detect 4 HPV vaccine- relevant types (6, 11, 16 and 18) including two positive controls,
one reflecting presence of any HPV in the sample and second, controlling for sample
DNA integrity. HPV targets are 150 nucleotides long GP5+6+ DNA segments, derived
through amplification or chemically synthesized. We use of in vitro “reconstructed
clinical samples” of HPV 6, 11, 16 and 18 following suggestion of Word Health
organization for optimizing HPV tests. The variable input of GP5+/6+ HPV
oligonucleotides or HPV plasmids is spiked prior to PCR with a constant amount of
human DNA, originating from HPV negative cervical samples, to mimic molecular
complexity of biological samples. In particular, range of genome equivalents (GE) of
HPV (1000-10000) is added to human DNA. The concentration of human genomic DNA
in reconstructed samples is comparable to the amount of DNA that is generally found in
cervical scrape specimens (~106 human genomes/ml), (Quint et al., 2006). These
“sample-reconstruction” experiments are allowing simulation of single and double HPV
infection and controllable measure of analytical performance of our type-specific probes.
In the next step, clinical samples (n=40) with known HPV status, as confirmed by the
CHUM HPV assay (Fig. 9), will be used for estimating the performance of HPV assay.
The results of HPV typing obtained with our probes and custom approach will be
compared with the results of Roche LA HPV Genotyping Test.
28
HPV - PCR-RLFP
16
31
6
11
?
Rsa
16
31
6
11
Figure 9. Schematic presentation of the CHUM HPV assay (a custom PCR-RFLP used for HPV typing)
6.5.2.
Design of the commercial format of the assay
There are multiple formats of multiplex hybridization kits on the market and custom
assays in research or clinical laboratories. Several options for commercial format exist for
our technology including solid support for DNA probe attachement and for hybridization
signal detection. As previously stated, our prototype assay cannot reach the market in its
current format, with its large SAM membrane, radioactive detection (P32), and high cost
of production.
As presented in Table 4, our rough estimation of the cost of production per kit is $26
which is highly expensive for such a kit. It should be noted that our kits have actually
been manufactured in the researcher’s laboratory and there is no economy of scale. The
membrane cost is clearly the main issue if we want to substantially reduce production
cost.
Materials
Oligo probe 6*
Cost/ kit (CDN$) comments
0.04
umol scale needs 100pmol per spot
29
Oligo probe 11*
Oligo probe 16*
Oligo probe 18*
0.04
0.04
0.04
umol scale needs 100pmol per spot
umol scale needs 100pmol per spot
umol scale needs 100pmol per spot
Two DNA control oligos to be
spotted
0.08
umol scale needs 100pmol per spot
Membrane SAM-Promega
25.00
$200 per membrane/8 HPV kits of
2cmX3cm
0.08
reduce background signal; increase
ratio signal/background
Blocker (needed to block
membrane)
Technician time to prepare
membrane ($50/hr) - cut
membrane, spot DNA, etc)
Package (plastic )
total=
* 67 mer single strands with biotin
0.50
0.50
26.32
100 membranes prepared per hour
plastic 15mL Falcon tubes
Table 4. Cost breakdown for the current production of our HPV prototype assay
Our data obtained so far shows that other membrane-based hybridization (Immunodyne
ABC membrane, Pall) could replace SAM membranes (Promega). Preliminary data
showing performance using modified streptavidin coated Immunodyne ABC membranes
are given in Fig. 10. This might be an excellent alternative for reducing the cost of the
final assay given the 100 fold lower price of Immunodyne ABC versus SAM membranes.
Immunodyne ABC membrane (Pall)
Example of HPV 18
6
11
16
18
UP
CD
Figure 10. Example of possible reduction of final product prize (100x) using Immunodyne ABC-Pall
instead SAM-Promega, Immunodyne membrane can replace SAM (Promega) membranes used in the
experiments shown in previous slides reducing thus the most significant material cost by 100 times (20$ to
0.2$)
30
Development of an alternative system of visualization of probe–target hybridization is
also essential for the simplicity and commercialization of the assay. One of the
possibilities is a colorimetric assay with the use of 6-FAM labeled primers to produce
PCR and a corresponding anti-6FAM-antibody with downstream alkaline phosphatecoupled colorimetric detection.
Another important aspect will be to reduce the size of the assay. A 10X reduction of
hybridization volume and corresponding solid support surface (beads, or array) will
reduce PCR yield requirement to 50-100ng, typical for PCR volumes of 10uL. Detection
of hybridization will be adequately adjusted. For example, the Luminex platform would
require Cy3 fluorophore, while a slide micro-array format would allow use of DIG
(digoxigenin-modified nucleic acids), or 6-FAM and the corresponding horseradish
peroxidase-conjugated anti-fluorescein, or anti-DIG antibodies (Roche, Invitrogen and
number of other suppliers). It is worthwhile mentioning that gold-nanoparticles are the
simplest alternative for colorimetric detection of DNA (post-PCR) with robust chemistry
and existing expertise in Canada (Yingfu Li, Department of Chemistry and Department
of Biochemistry and Biomedical Sciences, School of Biomedical Engineering, McMaster
University).
In order to define the best format for the assay for commercialization, a deep analysis of
the available options will be done. We plan to outsource this activity to a consultant with
strong expertise in molecular diagnostics. This consultant has yet to be identified, but one
possible candidate is Dr Yvan Côté, VP at Warnex, who has not only industrial
experience but also who has expressed his interest in performing this analysis as a private
consultant (independently from Warnex). It is worthwhile to mention that Yvan Coté has
suggested that Warnex provides support to help in the design of the final format of the
assay (see letter of support).
Briefly, the analysis will have to take into account the scientific aspects (specificity &
sensitivity), the commercial aspects (cost, license from third parties, others), as well as
the regulatory requirement for the commercialization of the assay in Canada and South
Africa.
At least three meetings will be held with a consultant over the 8 week mandate.
Participating in the meeting will be the inventors, Anne-Marie Larose from Univalor, and
one representative from our potential commercial partners (Continental Diagnostic and
Warnex). Meetings will include:
1) An initial meet and greet of the inventors, Anne-Marie Larose from Univalor, one
representative from each of our potential commercial partners (Continental
Diagnostic and Warnex).
2) A follow-up meeting midway through the mandate.
3) An oral presentation of the final report and recommendations (a written report
will also be produced).
31
Experimental testing of the new format design will be performed before the beginning of
the second phase of the project.
6.5.3.
Establish the regulatory pathway for the commercial use of the assay
The objective to get clinical validation of our HPV assay, in a commercial format, is to
conclude licensing agreement with commercial partners. It is clearly not the scope of this
project to get regulatory approvals in Canada or South Africa. However, we consider it is
important to establish what the regulatory requirements to reach the market are. These
aspects have to be taken in account for the final design of the assay and in the licensing
negotiation (will help both parties to better define license milestones). This task will be
driven by Anne-Marie Larose from Univalor.
6.5.4. Optimization of the commercial format of the assay for high sensitivity and
specificity
The outcome of phase 1 (first 20 weeks, i.e. five months) will define the choice of
technological platform. Sensitivity and specificity of the assay will be re-assessed on the
new assay format.
This optimization step will required the following:
1) Evaluation of the right amount of DNA probes to be spotted on the new support
2) Optimization of the hybridization conditions to reduce background
3) Optimization of the signal detection
4) Development of a new reproductive procedures to perform the analysis, from the
PCR product to the detection
5) Optimization of the control probes, including the HPV control probe
Since we anticipate a 10X reduction of hybridization volume and corresponding solid
support surface (beads or array) this will allow reduction of total PCR yield (50-100ng),
typical to the volume of 10uL. At this phase of the project, technique for visualization of
the hybridization signal will be adjusted to best serve the chosen platform. Comparison
with results obtained with the prototype assay will help the development and optimization
of the new format of the assay.
Optimization will be performed with the in vitro reconstructed sample, before testing
clinical samples (see section 6.5.5).
Some testing will also be conducted in at least one external laboratory, ideally by one of
our commercial partners, in order to validate the reproducibility of new protocols. These
experiments are not covered by this proposal and will be performed at the company’s
expenses.
32
6.5.5. Validation of the performance of the commercial assay with clinical samples.
The performance (sensitivity and specificity) of the probes using the new platform will be
compared with the typing results obtained in the first phase of the project. More
precisely, the commercial format of the test will be used for HPV typing of the same 40
samples tested with our prototype assay, as described in section 6.5.1. Additional 100
clinical samples that are already charactrized by commercial tests like Roche or Hybrid
Capture will be tested in this phase. The samples will be obtained through collaboration
with Dr Francis Coutlée, Molecular Virology Laboraty at the Research Center of CHUM
(see letter) who were running and partipating in variety of projects addressing biology,
epidemilogy and diagnostics of HPV. The concordance analysis will follow.
6.6.
Work plan and timetable
The maturation project will require 12 months for its completion. As illustrated in table 5
the activities to meet the first objectives will start immediately while the activities for the
second and third objectives will be performed concomitantly at the end of phase 1, most
likely from week 10 to week 32. The activities for the last two objectives will be done
successively.
Dr Damian Labuda, Ms Sylvie Cossette and Dr Anne-Marie Larose, respectively the
principal investigator with the inventors, the project manager and the representative of
the partner organization, will meet on a regular basis, every 3 months to evaluate the
progress of the project and the achievement of the milestones. The same committee will
meet if necessary to evaluate any project of disclosure generated within the framework of
this project in order not to reveal information that could preclude the filling of patent
application.
Phase 1 - 32
weeks
Objectives/Activities
1 Clinical validation of the prototype assay
2 Design of the commercial format of the assay
Establish the regulatory pathway for the commercial use of the
3 assay
Go/No go decision
4 Optimization of the commercial format of the assay
5 Validation of the performance of the commercial assay
Table 5: Gantt Chart for work plan
6.7.
Deliverables and result indicators
Phase 2 - 20
weeks
33
At the end of phase 1 we should have reach the first 3 objectives:
1. Clinical validation of the prototype assay
2. Design of the commercial format of the assay (simple and cost-effective HPV
typing assay)
3. Establish the regulatory pathway for the commercial use of the assay
More specifically this means that we will get specificity and sensitivity data with clinical
samples with the prototype assay and obtain the following results:



Ability to detect HPV 6, 11, 16 and 18 alone (1-10 pmols) or in a combination, where
individual types are presented in final PCR product in the low picomolar ranges.
No cross-hybridization with non-specific probes, under 10 pmols of one HPV types
(from reconstructed samples) which represent a scenario where PCR yield is very
abundant (10 pmols in total is around 1000 ng of GP56 PCR).
Universal HPV probe will detect any HPV type present in PCR, while control DNA
(CD) probe would be indicator of clinical sample DNA quality.
The design of the final format of the commercial product will need to be defined as well
as the main regulatory requirement for the commercialization of the HPV kit. We will
also need to get recommendations for the final design of the project. At the end of the
second and last phases of the project we should have reach the 4th and 5th objectives:
4. Optimization of the commercial format of the assay for high sensitivity and
specificity
5. Validation of the performance of the commercial assay with clinical samples:
Anticipated results in terms of specificity and sensitivity should be at least as
good as what we get at the end of phase I with the prototype assay: specific
detection of the 4 types of HPV, no cross-hybridization and positive results with
the Universal HPV probes and de CD probe. Importantly, our comparative study
should demonstrate that the performance of our assay is as good or superior to the
Roche assay. The stability of both assays (reproducibility) using repeated typing
of critical samples with PCR yield approaching 2 extreme situations (very low
and very high yield) will be performed. We expect that this comparative test
produce more stabile typing results with our assay format, then with Roche assay.
Implementation of the new assay format in at least one clinical laboratory in Quebec, or
South Africa will be performed.
6.8.
Decision-making milestones (go/no go) for measurable and clearly
identified results
At the end of phase 1, the project will be pursued only if we get positive results on
clinical samples with the current prototype (SAM membrane and P32). Herein, clinical
34
sample will have to give yield of GP56 PCR product in the minimal range of 100-1000ng
(150nt long), which is in fact 1-10 pmols in total, the quantity typically detected in our
present developmental experiments. This is, according to our observation and literature
data, an average PCR yield of 50uL reaction volume of “classical” GP56 PCR after 40-45
cycles. We know that present solution for GP56 primers perform well for majority of
HPV types, using 39 artificial targets. If our hybridization assay does not work in this
range of concentrations we will consider that there is a significant failure in the process
which will justify first NO GO.
6.9.
Proposal for granting the subsidy according to the decision-making
milestones reached
Total disbursement
Valorization-HSJ
MDEIE
Phase 1 32 weeks
Phase 2 20 weeks
End of phase 2
114 000 $
70 000 $
16 000 $
25 000 $
15 000 $
89 000 $
55 000 $
16 000 $
Table 6. Proposed distribution of granting
7. RESEARCH TEAM
In 2004, Ivan Brukner (see CV in Annex 3) joined our team to develop the iterative
technology in its practical application focusing on the small sequence segments. His
knowledge in nucleic acid chemistry matched the expertise of other members of the team
in the physical chemistry of nucleic acids, in diagnostic application, in the development
of genotyping tools, in the genetic epidemiology and pharmacogenetics, and importantly
in the HPV DNA testing and in the epidemiology of HPV infections.
Damian Labuda (see CV in Annex 3) has considerable experience in physical chemistry
of nucleic acids, in genetic diagnostics, genetic epidemiology and in vitro selection. Ivan
Brukner is the principal inventor of the proposed technology; he will be responsible for
the technology implementation and daily follow-up of the experiments. Maja Krajinovic
(see CV in Annex 3) has experience with the HPV DNA analysis, as well as in genetic
epidemiology and pharmacogenetics; she will be involved in design of reconstructed
sample experiments, selection of clinical samples (with Izabella Gorska-Flipot) and
between tests concordance analysis. Izabella Gorska-Flipot (see CV in Annex 3), at the
Hospital Hotel-Dieu, has a longstanding experience in the molecular diagnostics and in
the HPV cervical infections in particular; she will be responsible for the analysis of
clinical samples using custom approach and commercial kit.
Our achievements are summarized in 4 published manuscripts Nucleic Acids Research,
2007; Journal of Clinical Virology, 2007; Nature Protocols, 2007 and Int. J Cancer.,
35
2008. In addition, intellectual property related to our technology is protected. The present
application is to finalize the prototype of the HPV diagnostic device and to validate its
use in the clinical setting.
8. ESTABLISHMENT’S PROJECT MANAGER (In French)
Gestionnaire de projet au Centre hospitalier universitaire Sainte-Justine
(CHU Sainte-Justine)
Le CHU Sainte-Justine est un des plus grands centres hospitaliers universitaires pédiatriques du
Canada. Il a pour mission d’améliorer la santé des enfants, des adolescents et des mères du
Québec. Afin d’arriver à assumer pleinement sa mission et surtout son rôle en tant que centre
universitaire d’enseignement et de recherche, le CHU Sainte-Justine compte sur l’excellence de la
recherche de ses chercheurs regroupés dans son Centre de recherche.
Au cours des dix dernières années, le Centre de recherche du CHU Sainte-Justine a connu une
croissance sans égal parmi les dix-neuf centres et instituts de recherche subventionnés par le
FRSQ. Depuis sa fondation en 1973, le Centre de recherche s'est transformé en un réseau d'axes
de recherche pluridisciplinaire allant de la recherche biomédicale à la recherche clinique en
passant par la recherche sur les soins et le système de santé, la santé des populations et
l'évaluation des technologies. Le leadership du CHU Sainte-Justine en recherche est aussi fondé
sur un partenariat actif et engagé dans des réseaux de recherche. Depuis 2003, 30 nouveaux
chercheurs ont été recrutés portant le nombre de chercheurs à temps complet à 90. La progression
du nombre d'étudiants qui est passé de 276 à 402 au cours des cinq dernières années témoignent
du pouvoir attractif du Centre, de la qualité de ses chercheurs et de sa mission de former du
personnel hautement qualifié au niveau académique de même que pour l'industrie biomédicale et
pharmaceutique. Les publications témoignent de notre productivité scientifique comme centre de
recherche. De 2004-2005 à 2007-2008, le nombre d'articles avec comité de pairs a augmenté
annuellement de façon constante passant de 364 à 385.
Parallèlement à cette productivité scientifique du Centre de recherche, le CHU Sainte-Justine aura
été le foyer d'une importante activité en valorisation de la recherche et des connaissances dans le
domaine de la santé. Au cours des cinq dernières années, plus d'une trentaine de chercheurs ont
soumis une soixantaine de déclarations d'inventions menant à trente-deux demandes de brevets,
aboutissant à une dizaine d'accords commerciaux, dont neuf licences et la création d'une
entreprise dérivée. Également, le Centre de recherche a assisté à une croissance continue des
contrats de recherche en partenariat avec l'industrie et autres centres d’enseignement, totalisant
plus de 100 nouveaux contrats en 2006 dont la valeur atteint plus de 22 millions de dollars,
témoignage tangible de la valorisation du savoir-faire et connaissance de la communauté des
chercheurs du CHU Sainte-Justine.
Le Centre de recherche, sous la direction du Dr Guy A. Rouleau depuis près de trois ans s’est
doté d’une structure administrative qui facilite le support à la recherche et la valorisation des
résultats. Sous la direction de Madame Sylvie Cossette CA, adjointe au directeur et comptant plus
de 15 ans d’expérience comme gestionnaire au Centre de recherche, deux professionnels
spécialisées en ententes de recherche et en valorisation sont à la disposition des chercheurs. De
plus, un chercheur clinicien est délégué par le Centre de recherche pour siéger sur le comité
d’évaluation des technologies de sa société de valorisation Univalor.
Madame Cossette sera la gestionnaire de projet de maturation technologique du CHU SainteJustine et sera responsable de s’assurer de son bon déroulement. Elle devra notamment :
36

S’assurer que l’équipe de chercheurs et le partenaire financier respectent leurs
engagements;

S’assurer que les objectifs poursuivis dans le projet soient en harmonie avec la stratégie
de commercialisation;

S’assurer avec les chercheurs que toute nouvelle propriété intellectuelle développée dans
le cadre du projet soit protégée adéquatement et avec diligence;

S’assurer que les ententes à intervenir entre les partenaires soient conformes aux
politiques institutionnelles.
9. PARTNER ORGANIZATION
9.1. Role, experience and qualifications of the partner organization in conjunction
with the project
Gestion Univalor, Limited partnership ("Univalor") is a limited partnership whose
mission is to commercialize discoveries made by researchers at the Université de
Montréal, École Polytechnique de Montréal, CHU Sainte-Justine-Mother and Child
University Hospital Center, HEC Montréal, Hôpital Maisonneuve-Rosemont, Hôpital du
Sacré-Coeur de Montréal, Institut de recherches cliniques de Montréal and the Institut
universitaire de gériatrie de Montréal. Univalor also provides commercialization services
to the limited partnerships of the Montréal Heart Institute.
Univalor strives to develop profitable and long term business relationships with
companies wishing to maintain or improve their competitive position in their industry by
having access to cutting edge technologies developed by internationally acclaimed
researchers.
Univalor's multidisciplinary team is made up of 12 professionals in business development
and commercialization with expertise in life sciences, engineering, intellectual property,
legal affairs, and finance.
Achievements
Between 2001 and 2008, about 520 discoveries from Université de Montréal and its
affiliated institutions have been evaluated by Univalor and more than 900 patents
applications have been filed. Univalor currently has approximately 265 patents and
patents pending in its portfolio, has signed over 33 license agreements and has been
involved in the creation of 23 spin-off companies over the years.
Since 2001, funding provided for commercial maturation from Valorisation-Recherche
Québec and the Ministère du Développement Économique, de l’Innovation et de
l’Exportation (“MDEIE”) have allowed many technologies to reach maturity and
demonstrate operational capabilities. More than 8.2 million dollars have been invested in
37
our most commercially promising technologies and 130 million dollars into our spin-off
companies.
9.2.
Description of the partner organization's project selection process
Over time, Univalor has developed its own methods and tools for evaluating technologies
emerging from research laboratories and for helping researchers who wish to
commercialize their inventions. Internal evaluation is conducted by the business
development manager and the project leader using 5-point criteria: scientific and
technological quality, intellectual property, market size, business opportunity, and
relevancy for Univalor. Different databases such as Dialog Pro Competitive Intelligence,
Medtrack, and Delphion are used to accomplish this evaluation. Meetings with the
inventor(s) and a literature review are also very important steps in order to thoroughly
evaluate the technology on its scientific basis.
Results of the internal evaluations are discussed weekly during our evaluation committee,
which is comprised of all of Univalor’s professionals. This allows us to take full
advantage of our multidisciplinary team. Relevant questions regarding intellectual
property, technical issues, time-to-market, etc. often arise. The manager of business
development may either pursue the evaluation after this meeting or retain the services of
one or more experts. These experts, skilled in the specific technology, are asked to give
their opinion about the technology on both a scientific and commercial basis under a
confidential agreement. If the technology is recommended for commercialization, a
technology transfer strategy is then drawn up by the evaluation committee and rapidly
implemented.
When a technology shows a real commercial potential, Univalor’s business development
manager, along with the help of the researcher and the research administrator at the
institution, looks for financial resources to perform proof of concept or validation when
the development status of the technology is not mature enough to start licensing
discussions with a company. Once a grant is procured and the maturation project is
started, the manager of business development carefully follows the progression of the
project. Results are communicated to potential partners if discussions have already been
initiated and interest in the technology has been shown. Business development activities
start (if they have not already started) once the maturation project begins and intensifies
once the proof of principal is accomplished.
9.3.Professional who will support the valorization process
Anne-Marie Larose, Ph.D, MBA, Business Development Manager, is currently
responsible of this technology at Univalor. Ms. Larose has more than ten years of
experience in the life sciences industry. After having obtained her doctoral degree in
cellular and molecular biology, she joined a young biotech company at the pre-startup
38
stage. During the five years that followed, Ms. Larose was directly involved in the
different aspects of the company's corporate and technological development, notably in
the elaboration of business and commercial strategies, the protection of intellectual
property, the planning and management of R&D projects, and the management of the
quality control department. Afterwards, as commercial attaché for the British consulate in
Montréal, Ms. Larose was responsible for commercial exchanges with the Canadian
biopharmaceutical sector. More recently, Ms. Larose has contributed to the fulfillment of
various industrial and institutional development mandates in the Life Sciences sector as
main advisor in an advisory office. As of March 2004, Ms. Larose has been developing
business relations with corporations in the Life Sciences field who wish to commercialize
technologies with the greatest growth potential.
10. POTENTIAL MARKET
Why typing HPV in Canada now?
There are few distinct, but inter-related reasons why having “home”, provincial, or
national DNA HPV typing assay, presents advantageous situation over not having it.
Commercial impact is for example one of the resons. In Canada, Roche is the only assay
approved by Health Canada with prize of 80-100$CA per screening (material cost), while
FDA approved HC2 assay is reaching value of 42$ per single patient (QIAGEN Product
Guide, 2009, listing prices, for high volume request QIAGEN offers around 50% price
off). Both assays are developed in US. In Europe, the situation is more complex and
diversified with more small companies trying to get part of the market. On the contrary,
to our knowledge, Canada does not have any force to address this challenge.
Another important factor is timing, as: 1) HPV vaccination era requires more rigorous
screening activity with new and specific HPV typing tests, as suggested by Canadian
and international authorities (see Rogosa et al, (2008). The urgent need for new HPV
screening guidelines and new biomarkers is also well documented (Kiviat et al., 2008).
The current and future necessity and role of HPV screening in the era of HPV vaccination
is also well described by Myers et al., (2008). All these arguments are pointing out that
HPV screening has to be continued in the new form - if not inforsed by government,
because of the following reasons:
1) Vaccine is not clinically tested on already infected women,
2) The mother-child frequency of transition is not known,
3) The vaccine protective time is not yet defined and
4) 30% of cervical cancer will not be protected by the vaccine.
Target market
Sugestion:I would make few comments about local Montreal-Quebec-Canada market,
including private laboratories, clinics, hospitals and general public awareness for
diagnostics of STD.
39
The US molecular diagnostics field is the most dynamic segment of the in vitro
diagnostic (IVD) market, with an expected growth rate of approximately 17% through
2012. Infectious disease testing accounts for the bulk of this market, and HPV testing is
one of the fastest growing. As the second most prevalent molecular test, HPV testing has
been estimated in 2006 to have reached approximately 55 million tests that year alone,
representing an IVD market size of $500 million (Frost & Sullivan, 2006). Recently, the
company Third Wave Technologies has provided projections which suggest that the
global market for HPV testing could reach $250 million in 2008 with a market
penetration of only 28% but a growth rate in excess of 25%. This includes more than 10
million HPV tests being performed in the United States each year. Finally, the HPV
testing market in the EU is just emerging, as studies are underway to evaluate the use of
HPV tests as a primary screen for cervical cancer in women, replacing Pap testing.
10.1.
Business opportunity: Cervical cancer prevention program and HPV
screening.
Cervical cancer is the second largest cause of cancer deaths in women worldwide. The
estimated prevalence of HPV infection in women is 10.5%. Persistent infection with HPV
is a trigger for cervical cancer, and preventing this infection can avert this deadly disease.
The tests used in routine screening and clinical prevention of cervical cancer are so far
based mostly on a cytological examination known as the Papanicolaou (Pap) smear. In
2005, more than 60 million Pap smears were performed in the US, and it is estimated that
such screening programs and interventions have reduced the incidence of cervical cancer
by ~80% in the United States, but at a cost of more than $6 billion US a year (Wu et al.,
2006). Although the application of the conventional Pap test led to a dramatic reduction
in the incidence rate of cervical carcinoma in the last fifty years, this test has significant
limitations, including a sensitivity of only 50% to 60% in a routine screening setting
(Fahey et al., 1995; Nanda et al., 2000). Because of this, an improved cytological test has
been developed called the thin-layer liquid based cytology, or ThinPrep Pap test. This
new test is far from infallible, failing to detect from 15% to 35% of cervical
intraepithelial neoplasia (CIN) or cancer (Kulasingam et al., 2002). Due to the marginal
clinical benefits of liquid based cytology (ThinPrep Pap) and the major cost increase with
the adoption of this technology compared to standard Pap smear testing, the Joint
Technology Assessment Unit of MUHC and CHUM recommends that both institutions
NOT switch to the systematic use of liquid-based cytology at this time.
http://www.mcgill.ca/files/tau/Liquid_Based_Cytology_2008.pdf
Recently published results of ASCUS/LSIL Triage Study for Cervical Cancer (ALTS)
underline the importance of HPV DNA testing to improve specificity and reduce costs
(ALTS, 2000; Sherman et al., 2001; Solomon et al., 2002a; Solomon et al., 2001;
Solomon et al., 2002b; and http://www.cancer.gov/prevention/alts/results.html).
Likewise, accurate and affordable HPV genotyping will be in high demand for the next
few decades for ongoing HPV vaccination studies. The US Food and Drug
Administration (FDA) approved the first preventive HPV vaccine on June 8, 2006 for the
immunization of women between 9–26 years of age (Merck's quadrivalent vaccine
40
Gardasil, targeting HPV 6, 11, 16 and 18 ). A second vaccine, Cervarix from
GlaxoSmithKline (targeting HPV 16 and 18), is already available in Australia and in
Europe. So far, these vaccines have shown to offer 100% protection against persistent
homologous HPV infections (Stanley et al, 2006). In the current experimental phase of
vaccine trials, HPV screenings are needed to: i) identify HPV status of individuals before
vaccination and ii) monitor the efficacy of vaccines. Furthermore, the Advisory
Committee of Immunization Practices (ACIP) recommends continuation of HPV
screening protocols until other type-dependent vaccines are developed and until the
protective time period of the vaccines has been fully characterized. In spite of the above
recommendations and the introduction of a number of commercial HPV genotyping kits
(see table 1), a DNA test of satisfactory specificity and sensitivity to detect HPV types is
still unavailable.
10.2.
Technological competition and benchmarking
Available HPV DNA tests, sensitivity and specificity.
Key industry participants in the molecular infectious disease diagnostics market in North
America are Roche, Gen-Probe, QIAGEN (QIAGEN-Digene), bioMerieux, and Becton
Dickinson. QIAGEN-Digene is the sole provider of molecular HPV tests in the US. The
company offers two HPV tests which distinguish two groups of HPV subtypes (benign
and malignant) and offers no subtypes differentiation. The Roche HPV genotyping test
(LA) and the new Genomica CLART® HPV2 test, are the only commercially available
test kits for the detection of 35 or more variants of HPV. The Roche test was expected to
hit the US market in 2008, but is still unavailable to date (Frost & Sullivan, 2006).
A recent WHO international collaborative study (Quint et al., 2006) addressed the
question of standardization of HPV molecular typing and detection. The methodology
used in this study was performed by twenty-four participating laboratories included
QIAGEN-Digene Hybrid Capture II, and by a variety of PCR/hybridization typing
systems. The results demonstrate that the sensitivity of detection and the specificity of
typing varied considerably among participating laboratories working with the eight most
frequent high-risk types (16, 18, 31, 33, 35, 45, 52 and 58) and one low risk HPV 6. The
sensitivity and specificity of the detection of HPV 16 and HPV 18 was only 62% and
74% respectively. For the other seven types, the adequate assessment varied from 95%
(HPV 33 and 45) to 43% (HPV 31). Some false positive results were also reported.
Clearly, an alternative approach to HPV typing is needed. Existing HPV DNA analyses
rely either on hybridization techniques using type-specific HPV DNA probes, or are
based on direct identification of HPV DNA PCR-products (see table 7). Several
hybridization based tests and their prototypes have been developed that either require the
viral DNA to be pre-amplified by PCR (Innogenetics, Roche Diagnostics, Genomica,
Greiner Bio One) or do not require it (QIAGEN-Digene and Ventana). The main
41
difficulty of hybridization approaches that use “classical probes” in these tests, is
obtaining specific results when multiple similar targets have to be assayed simultaneously
(note that here we use the term “classical” to distinguish from probes obtained by our
method of iterative hybridization). Probes that fully match their targets (Roche,
Genomica, Greiner Bio One) compromise accurate detection of multiple targets because
they cross-hybridize ((Sandri et al, 2006) and our unpublished observations). Another
group of hybridization-based tests that does not require prior amplification of HPV DNA
include QIAGEN-Digene’s Hybrid Capture 2 (HC2) assay and Ventana’s Inform HPV.
QIAGEN-Digene’s FDA-approved assay is widely used in clinical studies due to its
relative simplicity and sensitivity. However, several recent reports pointed out two main
disadvantages with HC2, questioning its use as a screening tool. First, it lacks individual
HPV type identification. Second, there is a significant degree of cross-reactivity between
the oncogenic and non-oncogenic types that leads to false-positive results 10% to 20% of
the time (Cox et al., 2000; Gravitt et al., 1998; Poljak et al., 2002; Schneede et al., 2001;
Terry et al., 2001; Yamazaki et al., 2001). In brief, the Ventana Inform HPV in situ
hybridization assay seems to be more specific and sensitive than HC2, but its efficacy in
predicting cervical lesions (positive predictive value) is no more than 48% (Qureshi et
al., 2003). Several companies offer PCR-based kits (see table 3) like GenoID which
became a small participant in the European molecular diagnostics market (Frost &
Sullivan, 2006).
It should be noted that another option exists for the sequencing of HPV PCR products.
This is performed by Visible Genetics (Toronto) (Mahony et al., 2003) and is also an inhouse test at the Hôpital Hôtel-Dieu de Montréal and at the Hôpital Sainte-Justine de
Montréal. Although these sequencing tests are highly specific, they require trained
personnel, expensive technology, and most significantly, are rendered useless when
multiple variants of the virus are present in the same patient. Numerous other typing
systems have been described (Hwang et al., 2003; Klaassen et al., 2004; Kleter et al.,
1999; Schmitt et al., 2006; van den Brule et al., 2002, Schmitt et al., 2006) and they
await a detailed clinical evaluation.
Competitive advantages
The main advantage of our method resides in its enhanced power of identification and
discrimination of multiple short nucleic acid sequences that differ by a few mutations, as
with the different HPV types in a multiplex hybridization assay. Because we select the
most specific probes in predefined hybridization conditions we considerably reduce
cross-hybridization problems inherent to the conventional hybridization approach used by
Roche and Genomica (such as requiring a 52oC (+/-2) special hybridization buffer). Other
important benefits are described below:

Market need: The American Cancer Society (ACS) and the American Society of
Colposcopy and Cervical Pathology (ASCCP) have issued new guidelines which
incorporate DNA testing for high-risk HPV types, along with Pap tests, as a
42
primary screen for women aged 30 and over. The capacity of our probes to detect
and also to identify HPV types is clearly an asset if we consider that prevention
by vaccine will address only specific HPV types.

Multiplex detection: Finding universal experimental conditions for hybridization
becomes problematic in multiplex applications where many DNA targets are
considered simultaneously. Our method overcomes this problem. Instead of
adjusting the hybridization conditions to the probes, a set of probes is selected in
such a way as to function under the intended hybridization conditions.

Detection of different types in the same patient: According to GeneticLab, the
frequency of HPV patient superinfection is 28%, where 71% are infected with 2
subtypes, 21% with 3 subtypes, and 8% with 4 subtypes. Kits that can not
discriminate HPV types are unable to differentiate new and persistent infections
and have poor diagnostic potential in regard to superinfection.

Low production cost and ease of use: A kit based on our technology would
require neither expensive instrumentation nor skilled personnel to use. Production
of our diagnostic is not expected to be expensive, which would allow the licensor
to get a high profit margin. This is crucial for commercial success in the
diagnostic field.
Even though QIAGEN-Digene presently dominates the HPV DNA diagnostic market and
there will be strong competition between new kits—such as the ones from Roche and
Genomica—we strongly believe there is an opportunity for these innovative kits to enter
this large, underpenetrated, growing market. By fulfilling all the main requirements of the
clinical and market needs, our superior kit could quickly become the test of choice for
HPV detection.
Company
Test Name
Technology
Total number
Detection of
Comments
Market
43
of subtypes
detected
QIAGEN
(Digene)
Hybrid
Capture 2
RNA-DNA
hybridization
2
multiple
subtypes in
same patient
No
Ventana
Medical
Systems
Ventana
Inform
PCR+ In situ
hybridization
2
No
Genomica
CLART® HPV low density
2
microarray
35
Yes
Cross-reactivity of probes
US, Canada,
Europe,
Japan, Asia,
Australia
Europe
Greiner Bio
One
PapilloCheck® DNA-Chip
(microarray)
18 high-risk
and 6 low-risk
types of HPV
Yes
Cross-reactivity of probes
Europe
GenoID
Reveal HPV
19
not in one
assay
Semi-quantitative analysis; Very complex Europe
and requires RealTime PCR
Roche
Amplicor
PCR+
Molecular
beacons
PCR +
hybridization
13 high-risk
types
No
No individual identification of HPV type;
Detects only high risk cases.
Roche (LA)
Linear Array
PCR +
hybridization
37
Yes
BioMerieux
Protect HPVProofer assay
(based on
NucliSens
Easy Q
platform of
bioMerieux)
Detection of
oncogenic
HPV gene
expression
5 high risk
types
n/d
Involves amplification of a portion of the Europe
L1 gene by PCR, coding for the major
capsid protein, and a subsequent
hybridization of the amplification
products with the HPV type-specific
probes; Unable to distinguish hr-HPV 52
from other high-risk genotypes (33, 35,
and 58) presenting 2.2% of all cervical
cancers; Less sensitive if a sample has a
single infection with some specific HPV
genotypes that are poorly amplified by
PGMY (HPV 33 and 52); Hybridization
probe for HPV 51 not sensitive enough;
Requires very controlled temperature of
hybridization 52+/-2°C; Presently the
most accurate typing kit on the market.
Aims at the early detection of cervical
Europe
carcinogenesis. Technology licensed
from the company NorChip in January
2007.
The HC2 test uses specific antibodies
and chemiluminescent signal
amplification to measure the presence of
RNA (DNA hybrids formed between a
specific RNA probe and the viral DNA).
Cross-reactivity of probes; No individual
identification of HPV type; Detects only
high risk HPV
Cytogenetic HPV testing; Allows choice
of sample types; No individual
identification of HPV type; Poor accuracy
US, Canada,
Brazil, Europe,
Asia, Australia
Europe
44
Innogenetics
Inno-LiPA
HPV
Genotyping
CE
PCR +
hybridization
QIAGEN
(through
acquisition of
Shenzhen
P.G. Biotech
Co.)
Third Wave
Technologies
HPV detection PCR -based
kit
25
Yes, but have
problems with
sensitivity, due
to the bias
induced by
'universal'
primers
only 4 most
n/d
common types
(6, 11, 16 and
18)
No
Involves amplification of a portion of the
L1 gene by PCR, coding for the major
capsid protein, and a subsequent
hybridization of the amplification
products with the HPV type-specific
probes; Problems detecting multiple
infections reported, related to typespecific sensitivity of amplification; Full
set of HPV types not included; Crosshybridization reported; Requires
controlled temperature of hybridization.
n/d
HPV
n/d
screening test
(14 high-risk
types of HPV)
Third Wave HPV
n/d
Technologies genotyping
test (HPV—16
and 18)
14
2
Yes
n/d
GeneticLab
PapiPlexTM
Multiplex PCR
16
Yes
SensiGen
AttoSense ™
HPV Test
MassArray
assay (mass
spectrometry
coupled with
competitive
PCR)
15 high risk
types
n/d
Multiplex detection; Complex handling
and assay performance, (requires gel
manipulation); Only 16 types; not clear
how non-including types will perform
Ultrasensitive detection kit (1 to 3 copies
of HPV DNA in blood or tissue sample).
The test is still in development. Licensed
from University of Michigan in February
2007
Europe
China
n/d
FDA
application for
approval
expected in
2008-2009
Japan
Not on the
market
Table 7. HPV molecular diagnostic kits
11.
COMMERCIALIZATION STRATEGY
Our short-term goal is to grant licenses of the HPV test to companies or clinical
laboratories. We are in a position to conclude many license agreements within specific
fields of use and/or specific territories, since the inventors have developed both, a method
for generating specific probes for multiplex diagnostic assay and a specific application of
this method which is the HPV assay (both protected by the current patent application).
So far we have got serious interest from Warnex, a company based in Québec and
Continental Diagnostic, a company based in South Africa. They both would like to get
exclusive rights for the HPV assay, in their respective territories. We have also got
interest from Dr Gorska at CHUM to implement the HPV assay in their clinical
laboratory. It is clear that the clinical validation of the commercial assay will be decisive
to consolidate the interest of these groups and to conclude licensing agreement.
45
11.1.
Commercialization of the assay in Canada – Warnex partnership
Warnex is a life sciences company devoted to protecting public health by providing
laboratory services to the pharmaceutical and healthcare sectors. With its three divisions
(analytical, bioanalytical and medical laboratory), Warnex provides, to clients in the
pharmaceutical and biotechnology industries, a variety of quality control services as well
as method development and validation. They also conduct bioavailability and
bioequivalence studies for clinical trials, and perform contract R&D. Finally, Warnex
provides specialized genetic and biochemical testing for the healthcare industry. This
division also has extensive expertise in genetic testing for human identification,
molecular diagnostics, and pharmacogenetics.
They focus on the development of innovative assays as well as the refinement of existing
diagnostic tests to produce assays with greater clinical value and relevance for reliable
and cost-effective patient assessment and management. Warnex Medical Laboratories is
an important source of specialized testing for hospitals, private laboratories, and medical
specialists. As a development-driven medical laboratory, they work extensively in
collaboration with medical discovery companies to adapt their research into viable
clinical tools for improved diagnosis and monitoring of disease states.
Warnex is clearly a relevant partner to bring the HPV assay on the Canadian market.
Also, as mentioned in their letter of support, Warnex has accepted to provide in kind
contribution for the design of the final format of the assay. Their expertise will also be
highly valuable for the regulatory aspect of assay development.
11.2.
Commercialization of the assay in Africa – Continental Diagnostic
partnership
Continental Diagnostics is a Sales and Marketing outfit that would be taking the HPV to
the market once the clinical validity of the HPV assay will be established. Their business
plan is to boost their current income with the HPV test, which represents a large market
in South Africa: cervical cancer accounts for about 25 percent of cancer deaths among
black women, there is no formal PAP smear program and women seeking PAP smears in
the public health sector are only eligible for one every 10 years after the age of 30. Also
the new vaccines are out of reach for most of these women and 15 out of every 100 000
die of Cervical Cancer every year.
There are four partners in Continental Diagnostics as presented in Table 8. The company
outsources research (validation studies and experience programs) to scientists at the
University of Natal who work under Professor Indres Moodley. Prof. Moodley has
satellite sites in most African countries through his work with the SA government
Department of Science and Technology, where he can conduct validity assays for the
HPV test.
46
The company’s customers include pharmacies, private doctors (including specialists and
physicians), government and private hospitals, private and government laboratories. Also,
the company has a distinct advantage over their competitors in this area as they are a
100% black-owned company and therefore qualify for 60% in the Black Economic
Empowerment (BEE) point system that the government uses to award tenders to those
who are wholly or partly black-owned. They can also infer points to groups who source
their products from us and therefore have a good standing with private companies
(laboratories and hospitals) that source from us.
Co-funding for the MDEIE project will come from the Industrial Development
Corporation in SA, whose brief from the government is to partner African entrepreneurs
who create sustainable businesses and jobs in South Africa.
Continental Diagnostics
Members
Masikana Millan
Mdleleni.PhD,MBA,LLB
Mbulelo Godffery Tabata.
Med.Tech
Samuel Ndaxola
Nkalashe.Med.Tech,MAP
Indres Moodley.PhD
Expertise
Biochemistry Research and
Development
Responsibility
Business Evaluation and Legal
Counsel
Sales Management
Sales Director
New Business Development and
Marketing
Product Research
Product Management
Pharmaceutical Research
Table 8. Continental Diagnostics
11.3.
Other partnership and future development
Business development activities carried by Anne-Marie Larose at Univalor already bring
business relations with two relevant potential industrial partners, as described in the
previous section. Ms Larose will continue seeking for companies that would be interested
to commercialize the HPV kit in their own territory and/or to develop a new diagnostic
kit using this innovative method of generating highly specific probes for multiplex assay.
Worthwhile to mention, the proposed project focuses on the clinical validation of an HPV
assay for the most frequent HPV types. This will be the first generation of our HPV
assay. If we get success in developing a specific and sensitive assay and conclude
agreement with at least one company for the commercialization of the assay, we will
certainly pursue the development of a second generation of the HPV assay, to diagnose
all types of HPV, since we already have probes for 39 types.
12.
PRO FORMA BUDGET (in French)
12.1.
Coûts du projet de maturation technologique
47
Le budget global requis pour accomplir le projet de recherche et les activités de
protection de la propriété intellectuelle est de 200 000$. Les tableaux suivants présentent
les budgets requis.
R&D (salaries and material ) and indirect
expenses
Institution
research associate
HSJ
research assistant
CHUM
laboratory expenses
HSJ
laboratory expenses
CHUM
External
(Outsource)
Phase 1
Phase 2
Total
32 weeks
20 weeks
52
semaines
45 000 $
30 000 $
75 000 $
4 500 $
8 000 $
12 500 $
16 000 $
21 000 $
37 000 $
6 000 $
6 500 $
12 500 $
15 000 $
0$
15 000 $
Sous-total (RDI)
86 500 $
65 500 $
152 000 $
Indirect expenses (on the 30% from external
contribution)
7 500 $
4 500 $
12 000 $
Total
94 000 $
70 000 $
164 000 $
consultant for product design
Table 9 - Estimé budgétaire pour la réalisation du projet de R-D - 12 mois
La réalisation du projet nécessitera le travail de 2 personnes sous la supervision du
Dr Damian Labuda du CHU Ste-Justine et la collaboration du Dr Isabelle Gorska du
CHUM (voir section 7). Il est à noter que ces salaires incluent les bénéfices marginaux.
Territory
Phase 1
Phase 2
32
semaines
20
semaines
Total
End of the
project
52
semaines
Canada
3 000 $
3 000 $
US
6 500 $
6 500 $
South Africa
5 500 $
5 500 $
ARIPO
6 000 $
6 000 $
15 000 $
15 000 $
(16 000 $)
(16 000 $)
Other territory (Europe, Brazil and/or
Angola)
Contribution of Univalor for patent cost
Reimbursement of Univalor for patent cost
Total
20 000 $
0$
16 000 $
16 000 $
16 000 $
36 000 $
Table 10 - Estimé budgétaire pour la protection de la propriété intellectuelle
48
Un budget de 36 000$ est requis pour poursuivre le maintient de la propriété intellectuelle
dans les différents territoires. Pour ne pas réduire le budget de recherche dans la
première phase du projet de recherche, Univalor va supporter des dépenses de brevet de
16 000$, qui lui seront toutefois rembourser à la fin du projet lorsque nous recevrons la
dernière contribution du MDEIE dans le cas ou le projet franchise la phase 2 du projet.
12.2.
Montage financier
Le montant total requis dans ce projet de maturation technologique est de 200 000 $. Une
partie de cette somme, c'est-à-dire 164 000$ servira à accorder un contrat de recherche au
Dr Damian Labuda du CHU Ste-Justine. Un sous-contrat pour effectuer des travaux dans
le laboratoire du Dr Gorska au CHUM est prévu. Le restant du budget servira à défrayer
les frais du consultant ainsi que les coûts de protection de la propriété intellectuelle.
Source de financement
Valorisation-HSJ, société en commandite
PMT du MDEIE
Total
Montant ($)
40 000
160 000
200 000
Table 11 - Montage financier pour financer le projet de maturation technologique
12.3.
Documents démontrant la nature des engagements des partenaires
financiers
La contribution externe au financement du MDEIE, pour un montant de quarante mille
dollars (40 000 $), proviendra de Valorisation-HSJ, société en commandite. Cette
contribution est conditionnelle aux éléments suivants :
1) l’approbation du financement du projet de recherche annexé à la présente
lettre par le MDEIE;
2) la présentation du projet à la satisfaction des membres du Conseil
d’administration d’Univalor inc.;
3) la signature d’une option pour négocier des droits exclusifs d’exploitation de
la technologie utilisée pour la réalisation du Projet avec notre partenaire
Continental Diagnostics.
4) la réception par Valorisation-HSJ de la contribution financière de quarante
mille dollars (40 000 $) de notre partenaire Continental Diagnostics;
La contribution de Valorisation-HSJ sera décaissée en versements, selon l’avancement du
projet, au prorata des ratios de financement requis par le MDEIE et conformément à une
entente à intervenir entre le CHU Ste-Justine et Gestion Univalor, sec, au moment de
49
l’acceptation du Projet par le MDEIE. La propriété intellectuelle développée dans le
cadre du Projet sera propriété du CHU Ste-Justine, le tout tel que décrit dans les
politiques de propriété intellectuelle de l’établissement. Elle sera ensuite cédée à
Valorisation-HSJ.
Une copie de la lettre signée par Valorisation HSJ, commandité du CHU Ste-Justine,
société en commandite est présenté à l’Annexe 4.
12.4.
Démonstration que les autres sources de financement possibles ont été
prises en considération
Le projet soumis dans cette demande ne fait l’objet d’aucune autre source de financement
ni d’aucune autre demande de financement. Le financement du projet par le programme
de maturation technologique est essentiel pour la réalisation du projet de recherche.
50
13.
OUTSIDE OPINIONS
51
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