NZ626124B2 - Vaccines against hpv - Google Patents
Vaccines against hpv Download PDFInfo
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- NZ626124B2 NZ626124B2 NZ626124A NZ62612412A NZ626124B2 NZ 626124 B2 NZ626124 B2 NZ 626124B2 NZ 626124 A NZ626124 A NZ 626124A NZ 62612412 A NZ62612412 A NZ 62612412A NZ 626124 B2 NZ626124 B2 NZ 626124B2
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- 239000011778 trisodium citrate Substances 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
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Classifications
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Abstract
Disclosed is a nucleic acid molecule encoding an amino acid chain comprising (1) a signal peptide, (2) a targeting unit, (3) a dimerization motif, and (4) an antigenic unit, said targeting unit comprising an amino acid sequence having at least 80 % sequence identity to the amino acid sequence 24-93 of SEQ ID NO:1, and an antigenic unit comprising an amino acid sequence of human papillomavirus (HPV) HPV16 and/or HPV18, preferably an antigenic unit derived from early proteins E6 and/or E7 of HPV16 and/or HPV18, which amino acid chain is able to form a homodimeric protein. of SEQ ID NO:1, and an antigenic unit comprising an amino acid sequence of human papillomavirus (HPV) HPV16 and/or HPV18, preferably an antigenic unit derived from early proteins E6 and/or E7 of HPV16 and/or HPV18, which amino acid chain is able to form a homodimeric protein.
Description
VACCIN ES AGAINST H PV
FIELD OF THE INVENTION
The present invention relates to eutic compounds, such as vaccines against human
papi||omavirus (HPV) and in particular to DNA vaccines against HPV16 and/or HPV18. The
ion further relates to protein uct encoding homodimeric peptides, which peptides
may be released from a DNA vaccine or used separately. Further bed are
pharmaceutical formulations, host cells and methods for ing the vaccines, as well as
methods for the treatment of various HPV induced diseases, such as cancers and infectious
diseases by ation.
BACKGROUND OF THE INVENTION
It is now well established that human papi||omavirus (HPV) is the cause of cervical cancer
and other HPV—associated malignancies such as anogenital (anus, vulvar, vaginal and penile)
cancers and a subset of head and neck cancers. In particular, HPV16 and HPV 18 are
sible for about 70% of all cervical cancers ide.
To date, two prophylactic HPV es are on the market (Gardasil and Cervarix). The aim of
the prophylactic vaccines is to induce humoral immune responses by stimulating the
production of neutralizing antibodies specific for the HPV viral capsid proteins, L1 and L2.
Although the preventive vaccines are an important milestone for the control of HPV induced
cervical cancer and possibly other HPV—associated malignancies, the effect of these es
will not be significantly observed for 20—40 years (Ma B et al., Current Cancer Therapy
Reviews, 2010). er, since the coverage of mass vaccination for the prophylactic
vaccines are to date limited in addition to a substantial population worldwide that already are
HPV infected, HPV—associated malignancies will continue to progress. Thus, it will be
important to develop ecific therapeutic vaccines in order to reduce the mortality and
morbidity of HPV—associated malignancies and its precursor lesions (Ma B et al., Current
Cancer Therapy Reviews, 2010).
The development of various cancer vaccines and cancer immunotherapy strategies has
throughout the last two decades expanded. Still, only one therapeutic cancer vaccine, called
Provenge (Dendreon INC) has so far been approved to be applied as standard therapy for
prostate cancer. y, due to ethical reasons the majority of therapeutic cancer vaccines
are tested on a patient group bearing a late stage tumor. This patient group is substantially
immunosuppressed meaning that the tumor cells have for long escaped the immune system
and contributed to induce immunological tolerance to the tumor along carcinogenesis. In
addition, the choice of antigens (tumor—specific vs. tumor—associated) applied as vaccines are
critical in order to induce tumor—specific immune responses and avoid killing of healthy cells
in the patients which may lead to serious adverse events. Thus, the major nges in
cancer immunotherapy are to break the immunological tolerance and activate tumor—specific
effector functions to recognize and kill tumor cells. gh some case s show clinical
response to therapeutic cancer vaccines in late stage tumor patients, the most common
primary endpoint is to observe the impact on overall survival compared to conventional
therapy (surgery, chemo and radiation therapy). However, most s are either not
conclusive or that they completely fail to show this. One reason for the negative results lies in
the patient group carrying age tumors that are challenging to treat in the first place. A
possible gy could be to include patients with early—stage tumors in therapeutic vaccine
trials.
One strategy is to target pre—cancerous lesions. The nges for this strategy are mainly
the lack of le biomarkers that are specifically expressed by precancerous lesions for
many tissues and poor medical screening (either non—existing or that the existing method
suffers from lack of sensitivity). Exceptionally, this is not the case for HPV—induced
malignancies. For instance, the majority of western countries have good screening programs
for cervical sia and cervical cancer by performing the papanicolaou test (Pap smear
test). If there are unclear or abnormal results from Pap smear test, colposcopy will be
performed (National Cervical Cancer Coalition). HPV—testing may also be recommended for
some patients to detect the presence of “high—risk” HPV—type in the precancerous lesion.
Thus, HPV represents a potential biomarker for HPV—associated precancerous s, in
particular cervical pithelial dysplasia (CIN).
DNA vaccines have shown increasing promise for the treatment of human diseases, in
particular cancer. DNA vaccines induce strong antigen—specific immune responses and can be
repeatedly administered to maintain the —specific immune responses. Such vaccines are
considered to be safe and simple and cheap to produce on a large scale compared to other
cancer therapeutic formats. Numerous immunotherapeutic interventions fail to induce
immunological memory. Exceptionally, DNA vaccination ensures sustained release of the
e product in vivo which enhances antigen—specific logical . Direct
delivery of antigens to professional antigen—presenting cells (APCs) stimulates both CD4+
and CD8+ T cell immune responses in vivo. Such strong cellular immune responses have
been demonstrated to specifically recognize and kill n—positive malignant cells
efficiently both in vitro and in vivo.
There is still a need in the art for improved es for inducing strong and specific immune
responses against HPV responsible for both ious es and cancers.
OBJECT OF THE ION
It is an object of embodiments of the invention to provide specific and highly effective
therapeutic compounds, such as DNA vaccines against diseases and conditions caused by
HPV.
SUMMARY OF THE INVENTION
It has been found by the present inventors that by combining the antigens of the early gene
products E6 and E7 from HPV, such as from HPV16 and/or HPV18 with the targeting module
of hMIP-lCl, therapeutic es are provided, wherein the strong immunogenic epitopes of
HPV gene products are presented with high efficiency to APCs to induce a specific and strong
immune response. The products according to the present invention is primarily envisioned as
therapeutic nucleic acid es, such as DNA vaccines, wherein a nucleic acid uct
encoding the vaccibody construct is used as the eutic compound leading to in vivo
production of the protein product within the person receiving the vaccine. However, as an
alternative the protein product itself may be formulated and used directly in the vaccine.
Accordingly, in a first aspect the present invention relates to a meric protein of two
identical amino acid chains, each amino acid chain comprising (1) a signal peptide, (2) a
targeting unit, (3) a dimerization motif, and (4) an antigenic unit, said targeting unit
comprising an amino acid sequence having at least 80 % sequence identity to the amino acid
sequence 24—93 of SEQ ID NO:1, and an antigenic unit comprising an amino acid sequence of
human papillomavirus (HPV), such as an antigenic unit comprising an amino acid sequence of
HPV16 and/or HPV18, such as an nic unit derived from early proteins E6 and/or E7 of
HPV16 and/or HPV18.
In a second aspect the present invention relates to an amino acid chain comprising (1) a
signal peptide, (2) a targeting unit, (3) a dimerization motif, and (4) an antigenic unit, said
targeting unit comprising an amino acid sequence having at least 80 % ce identity to
the amino acid sequence 24—93 of SEQ ID NO:1, and an antigenic unit comprising an amino
acid sequence of human papillomavirus (HPV), such as an nic unit comprising an amino
acid sequence of HPV16 and/or HPV18, such as an antigenic unit derived from early proteins
E6 and/or E7 of HPV16 and/or HPV18, which amino acid chain is able to form a homodimeric
n according to the invention.
2012/076404
In a third aspect the present invention relates to a nucleic acid molecule, such as a DNA,
encoding an amino acid chain comprising (1) a signal peptide, (2) a targeting unit, (3) a
dimerization motif, and (4) an antigenic unit, said targeting unit comprising an amino acid
sequence having at least 80 % sequence identity to the amino acid sequence 24—93 of SEQ
ID NO:1, and an antigenic unit comprising an amino acid sequence of human papillomavirus
(HPV), such as an antigenic unit sing an amino acid sequence of HPV16 and/or HPV18,
such as an antigenic unit derived from early proteins E6 and/or E7 of HPV16 and/or HPV18,
which amino acid chain is able to form a homodimeric protein according to the invention.
In a further aspect the present invention relates to a homodimeric protein according to the
invention, or an amino acid chain according to the invention, or the nucleic acid molecule
according to the invention for use as a ment.
In a further aspect the present invention relates to a pharmaceutical composition comprising
a homodimeric n according to the invention, or an amino acid chain according to the
invention, or the nucleic acid molecule ing to the ion.
In a further aspect the present invention relates to a host cell comprising the nucleic acid
molecule according to the invention.
In a further aspect the t invention s to a method for preparing a meric
protein according to the invention, or an amino acid chain of the invention, the method
comprising a) transfecting the c acid molecule according to the invention into a cell
population; b) culturing the cell population; c) collecting and purifying the homodimeric
protein, or amino acid chain expressed from the cell population.
In a further aspect the present invention s to a method for preparing a vaccine, such as
a DNA vaccine, comprising an immunologically effective amount of a nucleic acid molecule
according to the invention, the method comprising a) preparing a nucleic acid molecule
according to the ion; b) dissolving the nucleic acid molecule obtained under step a) in a
pharmaceutically acceptable carrier, diluent, or buffer.
In a further aspect the present invention relates to a e against HPV comprising an
immunologically effective amount of a homodimeric protein according to the invention, or an
amino acid chain according to the invention, or nucleic acid molecule, such as a DNA,
according to the invention, wherein said vaccine is able to trigger both a T—cell— and B—cell
immune response.
In a further aspect the present invention relates to a method of treating or preventing a HPV
induced disease or condition, such as a cancer or an infectious e caused by HPV in a
patient, the method comprising stering to the patient in need thereof, a homodimeric
n according to the invention, or an amino acid chain according to the invention, or the
nucleic acid molecule, such as a DNA, according to the invention.
LEGENDS TO THE FIGURE
Figure 1: The overall structure of vaccibody vaccines with E7/E6 fusion antigen. Shown are
both DNA and protein formats. The vaccibody consist of three functional modules; the
chemokine human MIP-lCl (LD7SB) in the targeting module, hinge and CH3 sequences from
human IgG3 in the zation module and full—length E7 and/or E6 fusion in the vaccine
module.
Figure 2: The ted mode of action for a Vaccibody DNA vaccine against HPV —induced
malignancies. Naked DNA plasmid encoding vaccibody is injected intradermal followed by
electroporation. The plasmid is taken up by local cells and vaccibody proteins are produced
and secreted. The actic targeting s attract CCR1 and CCR5 expressing antigen
presenting cells (APC) and ensure binding and uptake into dendritic cells (DC). The DC will
present antigenic peptides to CD4+ and CD8+ T cells and the CD8+ T cells will kill HPV
infected and transformed cells in the cervix.
Figure 3: T s showing the number of E7 and E6 specific T cell responses as a
function of different amounts of vaccine administered. C57BL/6 mice were injected i.d. with
naked DNA plasmids encoding VBlOO9 and VBlOl6 and their corresponding controls followed
by electroporation (Cellectis, France) on day 0 and day 7. Splenocytes were harvested at day
21 and stimulated with MHC class ricted E7 or E6 peptide for 24h. The number of IFNy
secreting splenocytes was calculated by ELISPOT. (A)E7—specific responses after i.d.
vaccination with 25pg of VBlOO9, control 1 (antigen alone) and pUMVC4a (empty vector).(B)
E7—specific responses after i.d. vaccination with 12.5 and 1.4pg of VBlOl6, l 2 (antigen
alone) and a (empty vector). (C) E6—specific responses after i.d. vaccination with
12.5 and 1.4pg of VBlOl6, control 2 (antigen alone) and pUMVC4a (empty vector).
Figure 4.Therapeutic effect of VBlOl6 shown by measured tumor volume. 6 mice
were injected s.c. with 5x105 TC—1 cells at day 0. At day 3 and day 10, the mice were
injected i.d. with 12.5pg naked DNA plasmids encoding VBlOl6, l 2 or empty vector
followed by electroporation (Cellectis, France). The tumor sizes were measured by caliper two
to three times a week and tumor volume calculated.
Figure 5.Therapeutic effect of VBlOl6 shown by measured tumor volume. C57BL/6 mice
were injected s.c. in the neck area with 5x104 TC—1 cells at day 0. At day 3,7 and day 10, the
mice were ed i.d. with 20pg or 2pg naked DNA plasmids encoding VBlOl6, control 2 or
empty vector followed by electroporation (Cellectis, France). The tumor sizes were measured
by caliper two to three times a week and tumor volume calculated.
Figure 6.Therapeutic effect of VBlOZO and VBlOZl shown by measured tumor volume.
C57BL/6 mice were injected s.c. in the thigh with 5x104 TC—1 cells at day 0. At day 3 and day
, the mice were injected i.d. with 10pg naked DNA plasmids encoding VBlOl6, VBlOZO,
VBlOZl or empty vector followed by electroporation ctis, France). The tumor sizes were
measured by caliper two to three times a week and tumor volume calculated.
DETAILED DISCLOSURE OF THE INVENTION
The ucts and DNA vaccine technology described herein by the inventors of the t
invention (also referred to as body” molecules/vaccines/constructs) represents a novel
vaccine strategy to induce strong and specific immune responses for both infectious diseases
and cancer. The HPV E6/E7, such as HPV16 or HPV18 E6/E7 vaccine described herein may be
stered as a DNA vaccine by intradermal injection, preferably followed by
electroporation. This results in the uptake of the DNA—construct encoding the vaccibody—
HPV16 and/or HPV18 E6/E7 e in cells at the site of injection s) including
dendritic cells (Langerhans cells), leading to in vivo tion of the vaccibody—E6/E7
molecule.
The early gene products E6 and E7 from “high—risk” HPV types such as HPV16 and 18 may be
responsible for transformation of the basal—epithelium cells and induction of precancerous
lesions. Both proteins consist of highly immunogenic epitopes and are shown herein to induce
strong immune responses g to ic eradication of “high—risk” HPV positive tumor
cells both in vitro and in vivo.
The vaccibody molecule described herein is a homodimer consisting of three modules;
targeting , dimerization module and the vaccine module (Figure 1). Genes encoding the
three modules are genetically engineered to be expressed as one gene. When expressed in
vivo, the vaccibody molecule targets antigen presenting cells (APCs) which results in an
enhanced vaccine potency compared to identical, non—targeted antigens. In vivo expression of
the chemokine human macrophage inflammatory protein 1 alpha (hMIP-lCl/ LD788) leads to
attraction of DCs, neutrophils and other immune cells carrying the CCR1 and CCR5 receptors
to the site of expression. Thus, the vaccibody molecule consisting of hMIP-lCl as the targeting
WO 92875
module, will not only target the antigens to specific cells, but in addition give a response—
amplifying effect (adjuvant effect) by recruiting specific immune cells to the injection site. This
unique mechanism may be of great importance in a clinical g where patients can receive
the vaccine without any additional adjuvants since the vaccine itself gives the adjuvant effect.
The inventors of the present invention describes herein vaccine constructs where the
antigenic module consist of the E7 full length genetic sequence in fusion to the E6 full length
sequence originating from the HPV16 or HPV18 subtype. The advantage of this format is that
both E6 and E7 will be present in one construct and may thus be equally expressed in vivo.
Consequently, one vaccibody molecule consisting of a multi—antigenic unit may represent
equal levels of E6 and E7 for the immune system. The HPV16 E6 and E7 gene products are
oncogenic in their natural form. To neutralize their oncogenic properties, mutations at specific
sites may be introduced in the E6 and E7 genetic sequence.
The mutations, ing ons, may be introduced at specific sites, known to t the
oncogenic ties of E6 and E7, such as any one described in any of Dalal S et al., J Virol,
1996; ML'inger K et al., EMBO, 1989; wa S et al., Virology, 1995; Crook Tet al., Cell,
1991; er K et al., HPV Compendium Online, 1997
(http://www.stdgen.lanl.gov/COMPENDIUM_PDF/97PDF/3/E7.pdf); , M et al., J Virol,
2002; Nominé Y et a., Molecular Cell, 2006; Moody C et al., Nat Rev Cancer, 2010, Polakova
I et al., Vaccine, 2010; Xie Q, Virologica Sinica, 2011; Mesple‘de T et al., J Virol, 2012; US
102084 and US6306397, which references are hereby incorporated by reference.
Accordingly, in some s of the ion, the constructs according to the present
invention contain HPV16 E6, E7 or HPV16 E6/E7 chimeric constructs with one or more
ons in either of HPV16 E6, E7 or both at a position known to inhibit the oncogenic
properties as described in Dalal S et al., J Virol, 1996; Miinger K et al., EMBO, 1989;
Nakagawa S et al., Virology, 1995; Crook T et al., Cell, 1991; Miinger K et al., HPV
Compendium Online, 1997
(http://www.stdgen.lanl.gov/COMPENDIUM_PDF/97PDF/3/E7.pdf); Nguyen, M et al., J Virol,
2002; Nominé Y et a., Molecular Cell, 2006; Moody C et al., Nat Rev Cancer, 2010, Polakova
I et al., Vaccine, 2010; Xie Q, Virologica Sinica, 2011; Mesple‘de T et al., J Virol, 2012; US
102084 or US6306397. In other aspects of the invention, the constructs according to
the present invention contain HPV18 E6, E7 or HPV18 E6/E7 chimeric constructs with one or
more mutations in either of HPV18 E6, E7 or both at a position known to inhibit the
oncogenic properties as described in Dalal S et al., J Virol, 1996; Miinger K et al., EMBO,
1989; Nakagawa S et al., Virology, 1995; Crook T et al., Cell, 1991; Miinger K et al., HPV
Compendium Online, 1997
(http://www.stdgen.lanl.gov/COMPENDIUM_PDF/97PDF/3/E7.pdf); Moody C et al., Nat Rev
Cancer, 2010, US 2008/0102084 and US6306397.
WO 92875
There is a possibility that the vaccibody—moiety (targeting and dimerization modules) may
eradicate the oncogenic properties of E6 and E7 wildtype proteins in the final fusion protein.
Thus, in yet another aspect of the invention is the utilization of the wildtype full—length E6
and/or E7 sequences in the ody construction.
The invention describes several variant of Vaccibody HPV therapeutic DNA vaccines all based
on the overall format described in figure 1, the eutic vaccibody—HPV DNA vaccines
encodes genes that are naturally expressed in humans; the targeting module genes encode
the chemokine hMIP—ld, which binds to its cognate receptors, CCR1 and CCR5 sed on
the cell e of APCs. The zation module genes may encode hinge regions and
constant heavy chain 3, such as from human IgG3 which connects two vaccibody monomers
generating a homodimer molecule. Genes ng the vaccine module for the current
strategy consist of HPV, such as HPV16 and/or HPV18 E7 and E6 antigens, such as the full
length HPV16 E7 and E6 ns, optionally comprising one or more mutation to inhibit the
nic properties. Once administered in vivo by i.d. injection followed by electroporation,
dermal cells taking up the vaccine construct will express the vaccibody—HPV molecule. The in
vivo produced vaccibody es target to CCR1 and CCR5 expressed on the surface of APCs
in the skin, in particular DCs. The binding of the vaccibody molecule to its cognate ors
leads to internalization of the complex in the APC, degradation of the proteins into small
peptides that are loaded onto MHC molecules and presented to CD4+ and CD8+ T cells to
induce HPV16 E6 and E7 specific immune responses. Once stimulated and with help from
ted CD4+ T cells, CD8+ T cells will target and kill HPV16 E6 and E7 sing cells
(Figure 2). Such enhanced immune responses to a vaccine with a “built—in” adjuvant effect
may ially overcome tumor—escape (tumor immune surveillance) by breaking
immunological tolerance and efficiently kill malignant cells. The hMIP—ld targeting unit may
be connected through a dimerization motif, such as a hinge , to an antigenic unit,
wherein the later is in either the COOH—terminal or the NH2—terminal end. The t
invention not only relates to a DNA sequence coding for this recombinant protein, but also to
expression vectors comprising these DNA sequences, cell lines comprising said expression
vectors, to treatment of mammals preferentially by immunization by means of Vaccibody
DNA, Vaccibody RNA, or Vaccibody protein, and finally to pharmaceuticals and a kit
comprising the said molecules.
The dimerization motif in the proteins according to the present invention may be constructed
to include a hinge region and an immunoglobulin domain (e.g. CV3 domain), e.g.
carboxyterminal C domain (CH3 domain), or a sequence that is substantially identical to said C
domain. The hinge region may be Ig derived and contributes to the dimerization through the
formation of an interchain covalent bond(s), e.g. disulfide bridge(s). In addition, it functions as
a flexible spacer between the domains allowing the two targeting units to bind aneously
to two target molecules on APC expressed with variable distances. The immunoglobulin
domains contribute to homodimerization through non—covalent interactions, e.g. hydrophobic
interactions. In a preferred embodiment the CH3 domain is derived from IgG. These
dimerization motifs may be exchanged with other multimerization moieties (e.g. from other Ig
isotypes/subclasses). Preferably the dimerization motif is derived from native human proteins,
such as human IgG.
It is to be understood that the dimerization motif may have any orientation with t to
nic unit and ing unit. In one embodiment the antigenic unit is in the COOH—
terminal end of the dimerization motif with the targeting unit in the N—terminal end of the
dimerization motif. In another embodiment the antigenic unit is in the N—terminal end of the
dimerization motif with the targeting unit in the COOH—terminal end of the dimerization motif.
International application , which is hereby incorporated by nce
discloses nucleic acid sequences and vectors, which may be used ing to the present
invention.
The proteins according to the present ion include an antigenic unit derived from HPV,
such as HPV16 E7 and E6 antigens, such as the full length HPV16 E7 and E6 antigens, as well
as immunogenic fragments or variants thereof. The antigenic sequence should be of sufficient
length. The minimal length of such nic unit may be around 9 amino acids. Accordingly
in some embodiments, the antigenic unit derived from HPV comprises an amino acid
sequence of at least 9 amino acids corresponding to at least about 27 tides in a nucleic
acids sequence encoding such antigenic unit. Preferably the antigenic unit derived from HPV
is considerably longer, such as the full length HPV16 E7 and E6 antigens. Diversity arises
within a given HPV pe through limited nucleotide changes in the coding (at a frequency
of <2%) and non—coding (at a frequency of <5%) regions (Bernard, HU et al., IntJ Cancer,
2006). Such variants phylogenetically segregate based on their geographical origin and are
therefore labeled European, African, Asian, Asian—American and North American. Insertion of
such sequences in a Vaccibody format might lead to activation of both arms of the immune
response.
Immunization by means of Vaccibody protein, Vaccibody DNA, or Vaccibody RNA, the latter
two executed e.g. by intramuscular or intradermal injection with or without a following
electroporation, are all le methods according to the present ion.
As discussed above, the present invention relates to a e composition against cancer or
infectious diseases caused by HPV, the vaccine composition comprising an logically
ive amount of the nucleic acid encoding the molecule of the invention or degenerate
variants thereof. The vaccine may be able to trigger both a T—cell— and B—cell immune
response. The present invention also relates to a kit comprising Vaccibody DNA, RNA, or
protein for diagnostic, medical or scientific purposes.
The invention r relates to a method of preparing the inant molecule of the
invention comprising, transfecting the vector comprising the molecule of the invention into a
cell population; culturing the cell population; collecting recombinant protein expressed from
the cell population; and purifying the expressed protein.
The above bed nucleotide sequences may be inserted into a vector suited for gene
therapy, e.g. under the control of a specific promoter, and introduced into the cells. In some
embodiments the vector comprising said DNA sequence is a virus, e.g. an adenovirus, vaccinia
virus or an adeno—associated virus. In some embodiments a retroviruses is used as vector.
Examples of suitable retroviruses are e.g. MoMuLV or . For the purpose of gene
therapy, the DNA/RNA sequences according to the invention can also be orted to the
target cells in the form of colloidal dispersions. They comprise e.g. liposomes or lipoplexes.
The present invention encompasses the use of a targeting unit as well as an antigenic unit
having minimum degree of sequence identity or sequence homology with amino acid
ce(s) defined herein or with a polypeptide having the specific properties defined
herein. The present invention encompasses, in particular, the use of peptide variants or
e units to be used in the constructs according to the present invention having a degree
of ce identity with any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ
ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or
SEQ ID NO:34. Here, the term “variant” means an entity having a certain degree of sequence
identity with the subject amino acid sequences or the subject nucleotide sequences, where
the subject amino acid sequence preferably is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ
ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, or SEQ ID NO:34.
In one aspect, the variant or nt amino acid ce and/or nucleotide sequence
should provide and/or encode a polypeptide which retains the functional activity and/or
es the activity of a polypeptide of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ
ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or
SEQ ID NO:34.
In the present context, a variant sequence is taken to include an amino acid sequence which
may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98% or at least 99%, identical to the subject sequence. Typically, the variants used
according to the present invention will comprise the same active sites etc. as the subject
amino acid sequence. Although homology can also be considered in terms of similarity (i.e.
amino acid residues having similar chemical properties/functions), in the context of the
present invention it is red to express homology in terms of sequence identity.
Sequence identity comparisons can be conducted by eye, or more y, with the aid of
readily ble ce comparison computer programs. These commercially ble
computer programs use complex comparison algorithms to align two or more sequences that
best reflect the ionary events that might have led to the difference(s) between the two
or more sequences. ore, these algorithms operate with a scoring system rewarding
alignment of identical or similar amino acids and penalising the insertion of gaps, gap
extensions and alignment of non—similar amino acids. The scoring system of the comparison
algorithms include:
i) assignment of a penalty score each time a gap is inserted (gap penalty score),
ii) assignment of a penalty score each time an ng gap is extended with an extra
position (extension penalty ,
iii) assignment of high scores upon alignment of identical amino acids, and
iv) assignment of variable scores upon alignment of non—identical amino acids.
Most alignment programs allow the gap penalties to be modified. However, it is preferred to
use the default values when using such software for sequence isons.
The scores given for ent of non—identical amino acids are assigned according to a
scoring matrix also called a substitution matrix. The scores ed in such substitution
matrices are reflecting the fact that the likelihood of one amino acid being substituted with
another during evolution varies and depends on the physical/chemical nature of the amino
acid to be substituted. For example, the hood of a polar amino acid being substituted
with another polar amino acid is higher compared to being substituted with a hydrophobic
amino acid. Therefore, the scoring matrix will assign the highest score for identical amino
acids, lower score for non—identical but similar amino acids and even lower score for non—
identical non—similar amino acids. The most frequently used scoring es are the PAM
matrices (Dayhoff et al. (1978), Jones et al. ), the BLOSUM matrices (Henikoff and
Henikoff (1992)) and the Gonnet matrix t et al. (1992)).
Suitable computer programs for carrying out such an alignment include, but are not limited
to, Vector NTI (Invitrogen Corp.) and the ClustalV, ClustalW and ClustalW2 programs
(Higgins DG & Sharp PM (1988), s et al. (1992), Thompson et al. (1994), Larkin et al.
(2007). A selection of different alignment tools is available from the ExPASy Proteomics
server at www.expasy.org. Another example of software that can perform sequence
alignment is BLAST (Basic Local Alignment Search Tool), which is available from the webpage
of National Center for Biotechnology Information which can currently be found at
http://www.ncbi.n|m.nih.gov/ and which was firstly described in Altschul et al. (1990) J. Mol.
Biol. 215; 403—410.
Once the software has produced an alignment, it is possible to calculate % similarity and %
sequence identity. The software typically does this as part of the sequence comparison and
generates a numerical .
In one embodiment, it is preferred to use the ClustalW software for performing sequence
ents. Preferably, alignment with lW is med with the following parameters
for pairwise alignment:
Substitution matrix: Gonnet 250
Ga- o-en -enaIt :
Ga- extension -ena|t :
Ga- end -ena|t :
ClustalW2 is for example made available on the internet by the European ormatics
Institute at the EMBL—EBI webpage www.ebi.ac.uk under tools — sequence analysis —
ClustalW2. Currently, the exact address of the ClustalW2 tool is
www.ebi.ac.uk/Tools/clustalw2.
In r embodiment, it is preferred to use the program Align X in Vector NTI rogen)
for performing sequence alignments. In one embodiment, Exp10 has been may be used with
default settings:
Gap opening penalty: 10
Gap extension penalty: 0.05
Gapseparation y range: 8
Score matrix: blosum62mt2
Thus, the present ion also encompasses the use of variants, fragments, and derivatives
of any amino acid sequence of a protein, polypeptide, motif or domain as defined herein,
ularly those of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34.
The sequences, particularly those of variants, fragments, and derivatives of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ
ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34, may also have deletions, insertions
or substitutions of amino acid residues which produce a silent change and result in a
functionally equivalent substance. Deliberate amino acid substitutions may be made on the
basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues as long as the secondary binding activity of the substance
is retained. For example, negatively charged amino acids include aspartic acid and glutamic
acid; positively charged amino acids include lysine and arginine; and amino acids with
uncharged polar head groups having similar hydrophilicity values include leucine, cine,
valine, e, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and
tyrosine.
The present invention also encompasses conservative substitution (substitution and
ement are both used herein to mean the interchange of an existing amino acid e,
with an alternative residue) that may occur i.e. or—like substitution such as basic for
basic, acidic for acidic, polar for polar etc. Non—conservative substitution may also occur i.e.
from one class of residue to another or alternatively involving the ion of unnatural
amino acids such as ornithine nafter referred to as Z), obutyric acid ornithine
nafter referred to as B), norleucine ornithine (hereinafter referred to as O),
pyriylalanine, thienylalanine, ylalanine and phenylglycine.
Conservative substitutions that may be made are, for example within the groups of basic
amino acids (Arginine, Lysine and Histidine), acidic amino acids (glutamic acid and aspartic
acid), aliphatic amino acids (Alanine, Valine, Leucine, Isoleucine), polar amino acids
(Glutamine, Asparagine, Serine, Threonine), aromatic amino acids (Phenylalanine,
Tryptophan and Tyrosine), hydroxyl amino acids (Serine, Threonine), large amino acids
(Phenylalanine and Tryptophan) and small amino acids (Glycine, Alanine).
2012/076404
Replacements may also be made by unnatural amino acids include; alpha* and alpha—
disubstituted* amino acids, N—alkyl amino acids*, lactic acid*, halide derivatives of natural
amino acids such as trifluorotyrosine*, p—Cl—phenylalanine*, p—Br—phenylalanine*, p—I—
phenylalanine*, L—allyl—glycine*, B—alanine*, L—oc—amino butyric acid*, L—y—amino butyric
acid*, L—oc—amino isobutyric acid*, L—e—amino caproic acid#, 7—amino heptanoic acid*, L—
nine sulfone‘“, eucine*, L—norvaline*, p—nitro—L—phenylalanine*, L—
hydroxyproline#, L—thioproline*, methyl derivatives of phenylalanine (Phe) such as 4—methyl—
Phe*, pentamethyl—Phe*, L—Phe (4—amino)#, L—Tyr (methyl)*, L—Phe (4—isopropyl)*, L—Tic
(1,2,3,4—tetrahydroisoquinoline—3—carboxyl acid)*, L—diaminopropionic acid # and L—Phe (4—
benzyl)*. The notation * has been utilised for the purpose of the sion above ing to
homologous or non—conservative substitution), to indicate the hydrophobic nature of the
derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #*
indicates am phipathic characteristics.
Variant amino acid ces may e suitable spacer groups that may be ed
between any two amino acid residues of the sequence including alkyl groups such as methyl,
ethyl or propyl groups in addition to amino acid spacers such as glycine or B—alanine es.
A further form of variation, involves the presence of one or more amino acid residues in
peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt,
“the peptoid form" is used to refer to variant amino acid residues wherein the oc—carbon
substituent group is on the residue’s nitrogen atom rather than the oc—carbon. Processes for
preparing peptides in the peptoid form are known in the art, for example Simon R] et al.
(1992), Horwe|| DC. (1995).
In one embodiment, the variant targeting unit used in the homodimeric protein according to
the t invention is variant having the sequence of amino acids at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%
amino acid sequence identity therewith.
In one aspect, preferably the protein or sequence used in the present invention is in a
purified form. The term “purified” means that a given component is present at a high level.
The component is desirably the predominant active component present in a composition.
A “variant” or “variants” refers to proteins, polypeptides, units, , domains or c
acids. The term “variant” may be used interchangeably with the term t.” Variants
include insertions, substitutions, transversions, truncations, and/or inversions at one or more
locations in the amino acid or nucleotide ce, respectively. The phrases “variant
polypeptide variant" and “variant enzyme" mean a polypeptide/protein that
, polypeptide ,
has an amino acid sequence that has been modified from the amino acid sequence of SEQ ID
NO: 1. The t polypeptides include a polypeptide having a certain percent, e.g., 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of sequence identity
with the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ
ID NO:34.
“Variant nucleic acids" can include sequences that are complementary to sequences that are
capable of izing to the nucleotide ces ted herein. For example, a variant
sequence is complementary to sequences capable of hybridizing under stringent conditions,
e.g., 50°C and 0.2X SSC (1X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), to the
nucleotide sequences presented herein. More particularly, the term variant encompasses
ces that are mentary to sequences that are capable of hybridizing under highly
stringent conditions, e.g., 65°C and 0.1X SSC, to the nucleotide sequences presented herein.
The melting point (Tm) of a variant nucleic acid may be about 1, 2, or 30C lower than the Tm
of the wild—type nucleic acid. The variant nucleic acids include a cleotide having a
certain percent, e.g., 80%, 85%, 90%, 95%, or 99%, of sequence identity with the nucleic
acid encoding SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ
ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34,
encoding the ric protein which can form the homodimeric protein according to
invention.
A specific category of mutations are the mutations in E6 and E7:
The E6 protein may be detoxified by rendering the p53 binding impossible. Five positions in
the full length HPV16 E6 protein are sites for mutations for inactivation of E6 functionality,
F47, L50, C63, C106 and 1128. Any amino acid substitution in these positions may lead to
inactivation of E6 and induces tumor suppression. Substitutions in any one of these positions
with any one different amino acid may potentially be utilized. Sites for potential mutations
are shown in SEQ ID NO:22.
In the E7 protein there are conserved regions associated with oncogenic properties (see
Phelps et al J. Virol. April 1992, vol. 66, no. 242; Gulliver et al J Virol. 1997,
; 71(8)) including an N—terminal Rb (retinoblastoma binding protein) binding—site motif
(LXCXE) and two conserved regions 3 (upstream and downstream) with a Zn— binding motif
. The preferred mutation sites in the motif are C24 and E26. Preferred sites in
the two CXXC motifs are C58, C61, C91 and C94. However, any mutations in these regions
can be envisaged to be substituted for the reduction of binding ons and thus abolish the
oncogenic effects of E7. Sites for potential mutations are shown in SEQ ID NO:23.
Signal peptide:
A signal e at the N—terminal end of the nascent polypeptide directs the molecule into
the ER before transport to into the Golgi complex. The signal peptide is cleaved off by signal
peptidase once it has served its purpose of targeting and importing the protein to the ER.
These signal peptides are generally between 15 and 30 amino acids, but can have more than
50 residues (Martoglio, B. et al., Trends in Cell y, 1998, Knappskog, S. et al., J
Biotechnol, 2007). The native signal peptide may be replaced by signal peptides from any
mammalian, prokaryotic or marine origin. Commonly used signal peptides are e.g. humanIL—
2 and human albumin due to their natural ability to secrete large amounts of protein. The
choice of signal e can have a considerable impact on the amount of sized and
secreted protein.
In some ments, the signal peptide used in the protein construct according to the
present invention is d from a chemokine protein, such as the signal sequence of
LD78beta.
In some embodiments the signal peptide is not derived from pLNOH2 (Bl—8 variable
immunoglobulin leader) disclosed in the international application with International
Application No: .
In some embodiments the signal peptide is not derived from an immunoglobulin gene.
The term “homodimeric protein" as used herein refers to a protein comprising two individual
identical strands of amino acids, or subunits held together as a single, dimeric protein by
hydrogen bonding, ionic (charged) interactions, actual covalent disulfide bonding, or some
combination of these interactions.
The term “dimerization motif", as used herein, refers to the sequence of amino acids between
the antigenic unit and the targeting unit comprising the hinge region and the optional second
domain that may bute to the dimerization. This second domain may be an
immunoglobulin domain, and optionally the hinge region and the second domain are
connected through a . ingly the dimerization motif serves to t the antigenic
unit and the targeting unit, but also contain the hinge region that facilitates the dimerization
of the two monomeric proteins into a homodimeric protein ing to the invention.
The term “targeting unit" as used herein refers to a unit that delivers the protein with its
antigen to mouse or human APC for MHC class II—restricted presentation to CD4+ T cells or for
providing cross presentation to CD8+ T cells by MHC class I restriction. The targeting unit
used in the constructs according to the present invention is derived from or identical to mature
LD78—beta.
The term “antigenic unit" as used herein refers to any molecule, such as a e which is
able to be specifically recognized by an antibody or other component of the immune system,
such as a surface receptor on T—cells. Included within this tion are also immunogens that
are able to induce an immune response. The terms “epitope” or “antigenic epitope" is used to
refer to a distinct molecular surface, such as a lar surface provided by a short peptide
sequence within an antigenic unit. In some embodiments the antigenic unit comprises two ore
more antigenic epitopes. The antigenic unit used in the constructs according to the present
invention is derived from or cal to the early gene products E6 and E7 from HPV, such as
from HPV16 or HPV18.
The term “hinge region" refers to a peptide sequence of the homodimeric protein that
facilitates the dimerization, such as through the formation of an interchain covalent bond(s),
e.g. disulfide bridge(s). The hinge region may be Ig d, such as hinge exons h1+h4 of an
Ig, such as IgG3.
Specific embodiments of the invention:
As described above, the present invention relates to a homodimeric protein of two identical
amino acid chains, each amino acid chain comprising (1) a signal peptide, (2) a targeting
unit, (3) a dimerization motif, and (4) an nic unit, said targeting unit comprising an
amino acid sequence having at least 80 % sequence identity to the amino acid sequence 24—
93 of SEQ ID NO:1, and an antigenic unit sing an amino acid sequence of human
papillomavirus (HPV), such as an antigenic unit sing an amino acid sequence of HPV16
and/or HPV18, such as an antigenic unit derived from early proteins E6 and/or E7 of HPV16
and/or HPV18. In some embodiments according to the present invention, the targeting unit,
dimerization motif and antigenic unit in the amino acid chain are in the N—terminal to C—
al order of targeting unit, dimerization motif and antigenic unit.
In some embodiments, the antigenic unit used in the constructs according to the t
invention is derived from HPV16, such as from early proteins E6 and/or E7.
In some embodiments, the antigenic unit used in the constructs according to the present
invention is derived from E6 of HPV16.
In some embodiments, the antigenic unit used in the constructs according to the present
invention is derived from E7 of HPV16.
In some embodiments, the antigenic unit used in the constructs according to the present
invention is derived from HPV18, such as from early proteins E6 and/or E7.
In some embodiments, the antigenic unit used in the constructs according to the present
invention is derived from E6 of HPV18.
In some embodiments, the antigenic unit used in the constructs according to the t
ion is derived from E7 of HPV18.
In some ments ing to the present invention, the signal peptide consists of an
amino acid sequence having at least 80 % sequence identity to the amino acid sequence 1—
23 of SEQ ID NO:1.
In some embodiments according to the present invention, the signal peptide consists of an
amino acid sequence having at least 85%, such as at least 86%, such as at least 87%, such
as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as
at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at
least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%
sequence ty to the amino acid sequence 1—23 of SEQ ID NO:1.
In some embodiments according to the present invention, the targeting unit consists of an
amino acid sequence having at least 85%, such as at least 86%, such as at least 87%, such
as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as
at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at
least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence
identity to the amino acid ce 24—93 of SEQ ID NO:1.
In some embodiments according to the present invention, the dimerization motif comprises a
hinge region and optionally another domain that facilitate dimerization, such as an
globulin domain, optionally connected through a linker.
In some embodiments according to the present invention, the hinge region is Ig derived,
such as derived from IgG3.
In some ments according to the t invention, the hinge region has the ability to
form one, two, or several covalent bonds. In some embodiments ing to the present
invention, the covalent bond is a disulphide bridge.
In some embodiments ing to the present invention, the immunoglobulin domain of the
dimerization motif is a carboxyterminal C domain, or a sequence that is substantially identical
to the C domain or a variant thereof.
In some embodiments according to the t invention, the carboxyterminal C domain is
derived from IgG.
In some embodiments according to the present invention, the immunoglobulin domain of the
dimerization motif has the ability to homodimerize.
In some embodiments ing to the present invention, the immunoglobulin domain has
the ability to homodimerize via noncovalent interactions. In some embodiments according to
the present invention, the noncovalent interactions are hydrophobic interactions.
In some embodiments according to the present invention, the dimerization domain does not
se the CH2 domain.
In some embodiments according to the present invention, the zation motif consists of
hinge exons hi and h4 connected through a linker to a CH3 domain of human IgG3.
In some embodiments according to the present invention, the dimerization motif consist of
an amino acid sequence having at least 80 % sequence ty to the amino acid sequence
94-237 of SEQ ID NO:3.
In some embodiments according to the present invention, the linker is a G3SZG3SG linker.
In some embodiments according to the present invention, the antigenic unit and the
dimerization motif is ted through a linker, such as a GLGGL linker or a GLSGL linker.
In some embodiments according to the present invention, the targeting unit consists of
amino acids 24—93 of SEQ ID NO:1, or a t thereof.
In some embodiments according to the present invention, the homodimeric protein have
increased affinity for any one chemokine receptor ed from CCR1, CCR3 and CCR5 as
compared to the affinity of the same homodimeric protein with the targeting unit consisting
of amino acids 24—93 of SEQ ID NO:1, or a variant f.
In some embodiments according to the t ion, the antigenic unit comprises an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to the amino acid sequence 243—293 of SEQ ID NO:3.
In some embodiments according to the present invention, the antigenic unit consists of an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to the amino acid sequence 243—293 of SEQ ID NO:3.
In some embodiments ing to the present invention, the antigenic unit ses one or
more amino acid substitutions at a position selected from the list consisting of F47, L50, C63,
C106 and 1128 of SEQ ID NO:22, or a deletion involving one or more amino acid selected
from the list consisting of Y43—L50 of SEQ ID NO:22.
In some embodiments ing to the present ion, the antigenic unit comprises not
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 amino acid substitutions and/or
deletions relative to SEQ ID NO:22.
In some embodiments according to the present ion, the antigenic unit comprises the
amino acid sequence 243—293 of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9,
or a variant or antigenic fragment thereof.
In some embodiments according to the present invention, the antigenic unit consists of the
amino acid sequence 243—293 of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9,
or a variant or antigenic fragment thereof.
2012/076404
In some embodiments according to the present invention, the antigenic unit comprises an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to the amino acid sequence 243—340 of SEQ ID NO:11.
In some ments according to the present invention, the antigenic unit consists of an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence ty to the amino acid sequence 243—340 of SEQ ID NO:11.
In some embodiments ing to the present invention, the antigenic unit comprises one or
more amino acid substitutions at a position selected from the list consisting of C24, E26, C58,
C61, C91, and C94 of SEQ ID NO:23, or a deletion involving one or more amino acid selected
from the list consisting of L22—E26 and/or C58—C61 and/or C91—S95 of SEQ ID NO:23.
In some embodiments according to the present invention, the antigenic unit ses not
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 amino acid substitutions and/or
deletions relative to SEQ ID NO:23.
In some embodiments according to the present invention, the antigenic unit comprises the
amino acid sequence 0 of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID
NO:17, or a variant or antigenic fragment thereof.
In some embodiments according to the present invention, the antigenic unit consists of the
amino acid sequence 243—340 of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID
NO:17, or a variant or antigenic fragment thereof.
In some embodiments according to the t invention, the antigenic unit comprises an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to the amino acid sequence 243—501 of SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:32, or SEQ ID NO:34.
In some embodiments ing to the present invention, the antigenic unit consists of an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to the amino acid sequence 243—501 of SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:32, or SEQ ID NO:34.
In some embodiments according to the present invention, the antigenic unit comprising an
amino acid sequence of human omavirus 16 (HPV16) derived from both early proteins
E6 and E7.
In some embodiments according to the t invention, the antigenic unit comprising an
amino acid sequence of human papi||omavirus 18 ) derived from both early proteins
E6 and E7.
In some embodiments according to the present invention, the antigenic unit comprises one or
more amino acid tutions at a position selected from the list consisting of F47, LSOG,
C63, C106,1128T of SEQ ID NO:22 and C24, E26, C58, C61, C91, C94 of SEQ ID NO:23.
In some embodiments ing to the present invention, the antigenic unit comprises not
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 amino acid substitutions and/or
deletions relative to SEQ ID NO:22 and SEQ ID NO:23.
In some embodiments according to the present invention, the antigenic unit ts of the
amino acid sequence 243—501 of SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:32, or SEQ ID
NO:34, or a t or antigenic fragment thereof.
In some embodiments according to the present invention, the amino acid chain consists of an
amino acid sequence selected from the list consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, SEQ ID NO:32, and SEQ ID NO:34, or a variant or antigenic fragment
thereof.
In some ments according to the present invention, the nic unit comprises an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to any one amino acid sequence ed from SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
In some embodiments ing to the present invention, the antigenic unit consist of an
amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such
as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as
at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at
least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at
least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at
least 99% sequence identity to any one amino acid sequence selected from SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
In some embodiments the homodimeric protein according to the present invention, is in its
mature form without any signal peptide sequence.
In some embodiments the nucleic acid molecule according to the present invention is human
codon optimized.
It is to be understood that a human codon optimized nucleic acid molecule according to the
present invention comprises one or more nucleic acid substitution as compared to the wild
type sequence, which substitution provides for a codon with higher frequency of usage in
human coding regions. ncy of codon usage in homo sapiens can be found at
http ://biowiki.edu—wiki.org/en/codon_table
In some ments the nucleic acid molecule according to the t invention is
comprising any one of nucleotide sequences selected from the list consisting of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:31 and SEQ ID NO:33, or a
variant thereof.
In some embodiments the nucleic acid molecule ing to the present invention is
comprised by a vector.
In some ments the nucleic acid molecule according to the present invention is
formulated for administration to a patient to induce tion of the homodimeric n in
said patient.
In some embodiments the vaccine according to the present ion further comprises a
pharmaceutically acceptable carrier and/or adjuvant.
In some ments, the method of treating or preventing a HPV induced disease or
condition, such as a cancer or an infectious disease caused by HPV in a patient according to
the present invention comprises administering to the patient in need thereof of a nucleic acid
molecule, such as a DNA, ing to the t invention with a subsequent step of
electroporation. In some embodiments the administration is performed intra dermal or intra
muscular.
EXAMPLE 1
Construction and expression of the vaccines.
Gene sequences were designed according to the following structure: 1: native leader
sequence for human LD78 b, 2: full length LD78b sequence. 3: Human hinge—region 1 from
IgG3. 4: Human hinge region 4 from IgG3. 5: Glycine— Serine linker. 6: Human CH3 domain
from IgG3. 7: Glycine—Leucine linker. 8: wildtype and mutant Human papilloma virus
oncogenes E6, E7 and fusion proteins of both E6 and E7 divided by a Glycine— Serine linker.
The constructs are designated according to their E6 and or E7 ition as follows:
VBlOOl: Vaccibody—E6 wild type;
VBlOOS: Vaccibody—E7 wild type;
The mutants are designated according to the amino acid position in the corresponding native
E6 or E7 sequence.
VBlOOZ: Vaccibody—E6 C63R;
VBlOO3: ody—E6 C106R;
v31004: Vaccibody—E6 F47R, C63R, C106R;
VBlOO6: ody—E7 C24G, E26G;
VBlOO7: Vaccibody—E7 C24G, E26G, C58G, C61G;
VBlOOS: Vaccibody—E7 C24G, E26G, C91G, C94G;
v31009: Vaccibody— E7 C24G, E26G/ E6 F47R, C63R, C106R;
VBlOl6: Vaccibody— E7 C24G, E26G/ E6 C63R, C106R;
VBlOZO: Vaccibody— E7 C24G, E26G/ E6 F47R, C63R, C106R human codon optimized
VBlOZl: Vaccibody— E7 C24G, E26G/ E6 F47R, LSOG, C106R, 1128T human codon optimized
l vaccines composed of only the antigens were included:
Control 1: E7 C24G, E26G/ E6 F47R, C63R, C106R;
Control 2: E7 C24G, E26G/ E6 C63R, C106R
All gene sequences were ordered from Aldevron (Fargo ND, USA) or ns MWG GmbH and
cloned into the expression vector pUMVC4a.
All constructs were transfected in to 293E cells and verified expression of intact vaccibody
proteins were performed by dot blot and ELISA (data not shown). All amino acid sequences
except for Controls 1 and 2 are shown as SEQ IDs.
EXAMPLE 2.
Immune response s
VB 1009,VBlOl6, VBlOZO and VBlOZl were selected as vaccine candidates with their
corresponding controls 1 and 2 respectively. As a negative control empty pUMVC4a vector
was ed.
, 12.5 and 1.4 pg plasmid DNA of each candidate was injected intradermal in the lower
back of C57Bl/6 mice followed by electroporation, Dermavax, Cellectis , France). 7 days
later the mice were boosted with similar amounts of vaccines and control plasmids. At day 21
the mice were killed and s were harvested.
The T cell responses were calculated by ELISPOT. (Figures 3 a, b and c)
EXAMPLE 3.
eutic effect
VBlOl6, VBlOZO and VBlOZl with the corresponding controls 1 and 2 were selected as the
e candidate for therapeutic vaccine studies.
5x104 or 5x105 TC—1 cells (Johns Hopkins University, Baltimore, USA, Lin KY et a/., Cancer
Res, 1996) were injected in the neck or thigh region of C57Bl/6 mice. After days 3 and 10 or
day 3,7 and 10, the mice were vaccinated with 2pg, 10pg, 12.5 pg or 20pg of plasmid DNA
followed by electroporation, Dermavax, Cellectis France. Tumor size were measured two to
three times a week up until day 49 after TC—1 cell injection (Figure 4, 5 and 6)
EXAMPLE 4.
A therapeutic DNA vaccine to be used may be prepared by GMP manufacturing of the plasmid
vaccine according to regulatory authorities’ guidelines, including GMP cell banking, GMP
cturing of drug substance and drug product, ICH stability studies and Fill & Finish of
the DNA vaccine. The DNA vaccine may be formulated by dissolving in a saline solution, such
as 10nM Tris, 1mM EDTA at a concentration of 2—5 mg/ml. The vaccine may be administered
either intra—dermal or muscular with or without following oporation.
SEQUENCES:
C—C motif chemokine 3—like 1 precursor including signal peptide (aa 1—23 in bold) and mature
peptide (LD78—beta), aa 24—93 (SEQ ID NO:1):
MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQN FIADYFETSSQCSKPSVIFLTKR
G RQVCADPSEEWVQKYVSD LELSA
The specific DNA and corresponding amino acid ces of vaccibody HPV constructs:
E6 g E7 single constructs:
For the purpose of illustration only, the different s of the constructs are separated by
an |"with the domains in the ing order: Signal peptide | human MIP-lCl | Hinge h1 | Hinge
h4 | Gly—Ser Linker or Gly—Leu | hCH3 IgG3 |Gly—Ser Linker or Gly—Leu linker| wildtype or
mutant full length E6 g E7. Amino acids or nucleotides in bold illustrates sites of mutations.
DNA sequence of VBlOOl (SEQ ID NO:2):
ATGCAGGTCTCCAcr‘Gcr‘GCCCTTGCCG"CCTCCTCr‘GCACCATGGCTC"CTGCAACCAGGF‘CCF‘CTC" | C'T
GCTGCTGACACGCCGACCGCCTGC"GCT"CAGC"ACACC"CCCGACAGA'TCCACAGAAT'T‘CA"AGC"GACTACT""G
AGACGAGCAGCCAG'CC"CCAAGCCCAG"GTCA"C""CC"AACCAAGAGAGGCCGGCAGG"CTG"GC"GACCCCAG"GA
GGTCCAGAAAF‘ACGTCAGF‘GACCTGGAGCF‘GAGF‘GCC | GAGCTCAAAACCCCAC'T‘GG'CACACAAC"CACAC
A | GAGCCCAAA"CTr‘Gr‘GACACACCTCCCCCG"GCCCAAGG"GCCCA | GGCGGTGGAAGCAGCGGAGG"GGAAGTGGA |
GGACAGCCCCGAGAACCACAGG"G"ACACCC"GCcccCA"CCCGGGAGGAGATGACCAAGAACCAGG"CAGCC"GACCT
GCCTGG"CAAAGGC""CTACCCCAGCGACATCGCCGTGGAG"GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCC"CCCA"GC"GGAC"CCGACGGCTCCW“C""CCTCTACAGCAAGCTCACCGr‘GGACAAGAGCAGGr‘GGCAGCAG
GGGAACATCTF‘C"CAF‘GCF‘CCGF‘GAF‘GCATGAGGC"‘CF‘GCACAACCGCF‘TCACGCAGAAGAGCCF‘CTCCC"GTC"CCGG
G"AAA| GGCCTCGGTGGCU‘G AF‘GF‘TF‘CAGGACCCACAGGAGCGACCCAGAAAG""ACCACAGF‘TAF‘GCACAGAGCF‘G
CAAACAAC"A"ACA"GA"A"AA"A""AGAA"G"G"G"ACTGCAAGCAACAGT"AC"GCGACG"GAGG"A"A"GAC"T“"G
CW...“CGGGAFTHAHGCA..AGHAHAHAGAGAHGGGAAHCCA..A..GC“GHAHGHGAHAAAHGHHTAAAGTHWAW.CHAA
AAF‘F‘AGTGAGF‘AF‘AGACAF‘F‘A""G""‘AF‘AGF‘""‘GF‘AF‘GGAACAACA"TAGAACAGCAATACAACAAACCGF‘F‘GF‘GF‘GA"
""G""AA""AGG"G"A""AAC"G"CAAAAGCCAC"G"G"CCF‘GAAGAAAAGCAAAGACA"C"GGACAAAAAGCAAAGA"
40 r“CCAF‘AAF‘ATAAGGGG"CGGTGGACCGGTCGAF‘GF‘AF‘G"CTF‘GF‘TGCAGA"CATCAAGAACACGTAGAGAAACCCAGC"
G"AA
Protein sequence of VBlOOl (Homodimeric construct ing to the invention with E6, SEQ
ID NO:3): Amino acid sequence 393 amino acids.
QVSTAA IAVT. ICTMAT.CNQV IS IAPTIAADTPTACCFSYTSRQIPQNFIAD
QCS (PSVIFDTKRGRQVCADPS * *WVQKYVSD .4 ISAI * IKTP IG
DTTHT I EPKSCDTPPPCPRCP I GGGSSGGGSG GQPRTPQVYTTIPPS Q* * TK
\IQVSLTCuV <GFYPSDIAV* W * SSGQP * VVYNFTTPPM.J:DS:DGS FF IYS < .
TVDKS QWQQGVIFSCSVMHTA Q (S :8 ISPGK G IGG 'I FQDPQER
PRUIPQ uCT': IQTTI-IDII QQ . |QR-T‘VY:DFAFQ:D.JCIVYR:DG\I
PYAVCD (CL (FYSKISEYR-IYCYSTIYGTT F‘QQYN (P ICD . IIRCINCQ<
PTICP * * (QR-I 0pa.pa.)0i QFHNI QGRWTGRC SCCQSS QT QRTTQ .*
DNA sequence of VBlOOZ (SEQ ID NO:4):
ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT I GCACCACTT
GCTGCTGACACGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAGCTGACTACTTTG
AGACGAGCAGCCAGTGCTCCAAGCCCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGA
GGAGTGGGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCC I GAGCTCAAAACCCCACTTGGTGACACAACTCACAC
A I GAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA I GGCGGTGGAAGCAGCGGAGGTGGAAGTGGA I
GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAA I GGCCTCGGTGGCCTG ATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTG
CAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTG
Cmmm.—1CGGGANT“AHGCAnAGHAmAnAGAGAHGGGAAHCCAmAmGCmeACG_AGAmAAATGT.—1TAAAGTr1r1.—1Am.—1CmAA
AATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGAT
TTGTTAATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGAT
TCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCT
GTAA
Protein sequence of VBlOOZ (Homodimeric construct according to the invention, SEQ ID
NO:5): Amino acid sequence, 393 amino acids.
QVSTAA IAVT. ICTMAT.CNQV IS ADTPTACCFSYTSRQIPQNFIAD
YFETSSQCS (PSVIFDTKRGRQVCADPS * *WVQKYVSD .4 ISAI * IKTP IG
DTTHT I EPKSCDTPPPCPRCP I GGGSSGGGSG GQPR'I‘PQVYTTIPPS Q* * TK
\IQVSLTCuV <GFYPSDIAV* W * SSGQP * \I\IYNTTPPM_JDSDGS FF IYS ( .
TVDKS QWQQGVIFSCSVMH'I‘A IHNRFTQ (S :8 ISPGKIG IGG 'I FQDPQER
PR (TIPQ uCT'I‘ IQTTI {DII TCVYCKQQ . RR'I‘VYDFAF IVYR:DG\I
PYAVED (CL (FYSKISEYR-IYCYSTIYGTT F‘QQYN (P ICD . IIRCINCQ (
PTICP * * (QR-I DKKQ QFHNI QGRWTGRC SCC QSS QT QRTTQ .*
DNA sequence of VB 1003 (SEQ ID NO:6):
ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT I GCACCACTT
GCTGCTGACACGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAGCTGACTACTTTG
AGACGAGCAGCCAGTGCTCCAAGCCCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGA
GGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCC I GAGCTCAAAACCCCACTTGGTGACACAACTCACAC
A I GAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA I GGCGGTGGAAGCAGCGGAGGTGGAAGTGGA I
GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAA I GGCCTCGGTGGCCTG ATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTG
CAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTG
1CGGGANT“AHGCAnAGHAmAnAGAGAHGGGAATCCAnAmGCmeATGTGAnAAATGT.—1TAAAGTHr1.—1Am.—1CmAA
AATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGAT
ATTAGGTGTATTAACCG_ACAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGAT
TCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCT
GTAA
Protein sequence of VBlOO3 (Homodimeric construct according to the invention, SEQ ID
NO:7): Amino acid sequence, 393 amino acids.
QVSTAA IAVT. ICTMAT.CNQV IS IAPTIAADTPTACCFSYTSRQIPQNFIAD
YFETSSQCS (PSVIFDTKRGRQVCADPS * *WVQKYVSD .4 ISAI * IKTP IG
DTTH"1 I EPKSCDTPPPCPRCP I GGGSSGGGSG GQPR'I‘PQVYTTIPPS Q* * TK
\IQVSLTCuV (GFYPSDIAV* W * SSGQP * \T\IYNTTPPM_JDSDGS FF IYS ( .
TVDKS QWQQGVIFSCSVMH'I‘A IHNRFTQ (S :8 ISPGK G IGG 'I FQDPQER
PRUIPQ uCT'I‘ IQTTI-IDII 'I‘CVYCKQQ . uQR'I‘VYDFAF{DDCIVYRDGV
PYAVCD (CL (FYSKISEYR-IYCYSTIYGTT F‘QQYN (P ICD . IRCINEQ<
PTICP * * (QR-I 0pa.pa.)0i QFHNI QGRWTGRC SCCQSS QT QRTTQ .*
DNA ce of VBlOO4 (SEQ ID NO:8):
ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCTIGCACCACTT
GCTGCTGACACGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATT"CATAGCTGACTACTTTG
AGACGAGCAGCCAGTGC"CCAAGCCCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGA
GGAGTGGGTCCAGAAATACGTCAG"GACCTGGAGCTGAGTGCC I GAGCTCAAAACCCCAC""""GG"“GACACAAC"1CACAC
ZXI (EZXCECE(ECEZXZXZXF‘(:TF'"(}'“(}ZX(:ZX(:ZX(3(EUTCECE(3(3(3C3'"C3(3(:(31XZX(3C3"C3(3(3(:ZX ICECE(3C3C31?(}(}ZXZXC}(:ZXC}(3(3(EZX(}(}'"(}(}ZXZX(}TF(}(}ZXI
GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
TCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACATCTTC"CATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAAIGGCCTCGGTGGCCTG ATGTT"CAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTG
CAAACAAC""ATACATGATATAATA"1"AGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACT"1"G
CmECGGGANT“AHGCAmAGHAmAmAGAGAHGGGAAHCCAmAmGCmeACG_AGAmAAATG".—1TAAAGTr1r1.—1Am.—1CmAA
AAT""AGTGAG""ATAGACATTA"1""G"1"“ATAGT"1""GTATGGAACAACA"1TAGAACAGCAATACAACAAACCG"1"“GTGTGA"1
T""G"1""AA"1"“AGGTGTA"1"AACCG_ACAAAAGCCAC""G""GTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGA"1
"1CCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGC"1
GTAA
Protein sequence of VBlOO4 imeric construct according to the invention, SEQ ID
NO:9): Amino acid ce, 393 amino acids.
QVSTAAIAV.LCTMALCNQVISAPIAADTPTACCFSYTSQQIPQVFIAD
YFETSSQCS(PSVIFDTKRGRQVCADPS**WVQKYVSDu*.SA4L<TP C)
DTTHTEP(SCDTPPPCPRCPGGGSSGGGSGGQPRTPQVYTIPPSR**
CDV<GFYPSDIAV*W*SSGQP*VVYNTTPPMDDSDGSFFIYS Ara A
TVDKSQWQ GVIFSCSVMHTAIHNRFTQ<SISISPGKGIGGIMFQDPQ Lu (u
PR<LPQLCTTIQTTI{DII :CVYCKQQIIQRTVYDFARQDJCIVYQ UC)A
PYAVEDKCL{FYSKISEYRiYCYSLYGTTITQQYN<PICDIIIRCIVEQ<
PLCP**KQR{ DKKQQFHNIQGRWTGRC SCCQSSQTQQETQL*
DNA sequence of VBlOOS (SEQ ID NO:10):
ATGCAGGTCTCCACTGCTGCCCTTGCCG"1CCTCCTCTGCACCATGGCTC"1CTGCAACCAGG"1CCTCTC"1 I GCACCACTT
GCTGCTGACACGCCGACCGCCTGCTGCT"1CAGCTACACCTCCCGACAGATTCCACAGAATT"1CATAGCTGACTACTTTG
AGACGAGCAGCCAGTGC"1CCAAGCCCAGTGTCA"1C"""1CCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGA
GGAGTGGGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCC I GAGCTCAAAACCCCAC""""GG""GACACAAC"1CACAC
A I GAGCCCAAA"1CTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA I GGCGGTGGAAGCAGCGGAGGTGGAAGTGGA I
GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG"1CAGCCTGACCT
GCCTGG"1CAAAGGCT"1CTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCTCCCATGCTGGAC"1CCGACGGCTCCT"1CTTCCTCTACAGCAAGC"1CACCGTGGACAAGAGCAGGTGGCAGCAG
A"1CTTC"1CATGCTCCGTGATGCATGAGGCTC"GCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAA GGCCTCGGTGGCC"1G I ATGCATGGAGATACACCTACAT"1GCATGAATATATGTTAGA"1T"GCAACCAGAGACA
ACTGA"1C"1CTAC"GTTATGAGCAA"1"AAATGACAGCTCAGAGGAGGAGGATGAAATAGATGG"1CCAGCTGGACAAGCAG
ACAGAGCCCAT""ACAATA"1""GTAACC"1"1TTGTTGCAAGTGTGAC"1C""ACGCT"1CGG"1"GTGCGTACAAAGCAC
ACACGTAGACAT"1CGTACTTTGGAAGACCTGT"AATGGGCACACTAGGAATTGTGTGCCCCA"1CTGTTC"1CAGAAACCA
WO 92875
Protein sequence of VBlOOS (Homodimeric construct according to the invention with E7, SEQ
ID NO:11):Anuno acm sequence,340 anuno adds.
QVS"AA .AVT. .CTMAT .CNQV AADTP’1ACCFSYTSRQI PQNFIAD
YFETSSQCS (PSVIE4T <RGRQVCADPS* * SD .4 .SAI * .KTP
DTT-I"1 "‘LPKSCDTPPPCPRCP IGGGSSGGGSG GQPRTPQVYTT .PPS
\TQVS ITC .V DIAV*W * SSGQP *VVYN"TPPMD,S:
TVD<S QWQQGNIFSCSV H'3A -IN QFTQ {S ASLSPGKI
-I':‘Y .3 QPTTTDLYCYTQ .Ni 88* * *3 *IiDGPAGQAEP
C {C38 ".4 QLCVQS THV:DIRTTEiJU-3T. . GT .GIVCPICSQ
DNAsequenceofVBlOO6(SEQIE)NO:12y
ATGCAGGTCTCCACTGCTGCCCTTGCCG"‘CCTCCTC""GCACCATGGCTC"1CTGCAACCAGGTCCTCTC"1 I GCACCACTT
GACACGCCGACCGCCTGCTGCT"1CAGCTACACCTCCCGACAGA"1TCCACAGAATTTCATAGCTGACTACTTTG
AGACGAGCAGCCAGTGC"1CCAAGCCCAG"1GTCATCT”CCTAACCAAGAGAGGCCGGCAGG"CTGTGCTGACCCCAGTGA
GGAGTGGGTCCAGAAA"“ACGTCAG’1GACCTGGAGCTGAGTGCC I GAGCTCAAAACCCCACTTGGTGACACAAC"CACAC
AIGAGCCCAAA’1CTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA I GGCGGTGGAAGCAGCGGAGGTGGAAGTGGAI
GGACAGCCCCGAGAACCACAGGTGTACACCC""GCCCCCA’1CCCGGGAGGAGATGACCAAGAACCAGG"1CAGCCTGACCT
GCCTGG"CAAAGGC""""CTACCCCAGCGACATCGCCGTGGAG"1GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCTCCCA""GCTGGAC'‘CCGACGGCTCC""CT"CCTCTACAGCAAGC"" CACCG'1GGACAAGAGCAGGTGGCAGCAG
GGGAACATCTTCTCA""GCTCCGTGATGCA'‘GAGGC"C"GCACAACCGC"‘TCACGCAGAAGAGCC"1CTCCCTGTCTCCGG
GTAAA GGCCTCGGTGGCC'“GIATGCA"GGAGA"ACACCTACAT"GCA"GAA"ATA"G""AGA"""1GCAACCAGAGACA
ACTGATC"CTACGGATATGGACAATTAAA"‘GACAGCTCAGAGGAGGAGGA"GAAA"AGA"GG"CCAGCTGGACAAGCAG
AACCGGACAGAGCCCATTACAA"1A““GHAACCHHTHGHTGCAAGHGTGACHCHACGCTHCGGHHGF1GCGTACAAAGCAC
ACACGTAGACATTCGTACT"‘TGGAAGACC"GT"AA"GGGCACAC"AGGAA"TGTG"GCCCCA"C"GTTC"CAGAAACCA
TAA
Protein sequence of VBlOO6 (Homodimeric construct according to the invention, SEQ ID
|VO:13):Anfino acm sequence,340 annno acMs.
QVS"AA .AV .T .CTMAT .CNQV .SAP .AADTPTACCFSYTS QQI PQVFIAD
YFETSSQCS (PSVIEJTKRGRQVCADPS * * SD .4 .SA* T. ("PEG
DTTHTEP (S C,3.—1PPPCPRCPGGGS SGGGSGGQPR'I‘PQVYT .PPSR* 4Mr1<
\IQVS ITC V DIAV* W * SSGQP * TP PMD,DS:DGS FF .YSK .
TVDKS QWQQGVI FS CSVMH'3A .HN QFTQ {S .ST.SPGKG .GGT .M {GiDTP'TJ
-I' .QPTT"*onygygo .NDSS * * *3 *Ii3GPAGQAEP:D QAHYNIVTFC
C {CDSTD QLCVQSTHVDI RTT F‘DT. . GT .GIVCP I CSQ <P*
DNAsequenceofVBlOO7(SEQIE)NO:14y
ATGCAGGTCTCCACTGCTGCCCTTGCCG"‘CCTCCTC""GCACCATGGCTC"1CTGCAACCAGGTCCTCTC"1 I GCACCACTT
GCTGCTGACACGCCGACCGCCTGCTGCT"1CAGCTACACCTCCCGACAGA"1TCCACAGAATTTCATAGCTGACTACTTTG
AGACGAGCAGCCAGTGC"1CCAAGCCCAG"1GTCATCT”CCTAACCAAGAGAGGCCGGCAGG"CTGTGCTGACCCCAGTGA
40 GGAGTGGGTCCAGAAA"“ACGTCAG’1GACCTGGAGCTGAGTGCC I GAGCTCAAAACCCCACTTGGTGACACAAC"CACAC
AIGAGCCCAAA’1CTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA I GGCGGTGGAAGCAGCGGAGGTGGAAGTGGAI
GGACAGCCCCGAGAACCACAGG"G"‘ACACCC""GCCCCCA’1CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG"1GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCTCCCA""GCTGGAC'‘CCGACGGCTCC""CT"CCTCTACAGCAAGC"1CACCGTGGACAAGAGCAGGTGGCAGCAG
45 GGGAACA"C""TCTCA""GCTCCGTGATGCA'"GAGGC'‘CTGCACAACCGC"‘TCACGCAGAAGAGCC"1CTCCCTGTCTCCGG
GTAAA GGCCTCGGTGGCC"‘GIATGCA’‘GGAGA"ACACCTACAT'1GCAHGAAHATAHGHHAGAHr1.—1GCAACCAGAGACA
ACTGATC"CTACGGATATGGACAATTAAA"‘GACAGCTCAGAGGAGGAGGA"GAAA’1AGATGGTCCAGCTGGACAAGCAG
AACCGGACAGAGCCCAT"‘ACAA"1Ar1r1Gr1AACCr""ngéTGCAAeggéeAc"‘C"ACGCT"CGG""G"1GCGTACAAAGCAC
ACACGTAGACATTCGTACT"1TGGAAGACC"‘GT"1AATGGGCACAC"‘AGGAATTGTG"1GCCCCATCTGTTCTCAGAAACCA
50 TAA
Protein sequence of VBlOO7 (Homodimeric construct according to the invention, SEQ ID
|VO:15):Anfino acm sequence,340 annno acMs.
ALAVLLCTMALCNQVLSAPLAA:DTPTACCFSYTSRQI PQNFIAD
YFETSSQCS (PSVIF.4TKRGRQVCAiDPS * WVQKYVSD .4 .SA* T.
DTT-ITEPKS CD"1PPPCPRCPGGGSSGGGSGGQPRF‘PQVYT .PPSR
\TQVS_I1CLV (GFYPS:DIAV *SSGQP 4 TPPMJDSDGS FF
TVD (S QWQQGVI FS CSVMH' QFTQ .S T .S PGKG Vl -IG:
-I':‘Y Q .QPFT" onygygo 3GPAGQAEPD VTFE
C (GDS "1.4RLCVQS THV:DIRTTEuLJ. .GIVCPI CSQ (P*
DNAsequenceofVBlOOS(SEQIE)NO:16y
ATGCAGGTCTCCAC'1GC'1GCCCTTGCCG'1CCTCCTC"1GCACCATGGCTC"1 CCAGG"1CC"1CTC"‘IGCACCAC"T
GCTGCTGACACGCCGACCGCCTGC'1GCT"1CAGC'11ACACCT1CCCGACAGA"1TCCACAGAATF1'11CA"1AGC"1GACTACT""G
AGACGAGCAGCCAG"1GC"1CCAAGCCCAG"1GTCA"1 C"1"1 C C'11AACCAAGAGAGGCCGGCAGG"1CTG"1GC"‘GACCCCAG’1GA
GGAGTGGGTCCAGAAA’1ACGTCAG"1GACCTGGAGC'1GAGT1GCC I GAGCTCAAAACCCCACF1'11GG'1‘GACACAAC"CACAC
CCAAA’1CT" 1GACACACCTCCCCCG"1GCCCAAGG"1GCCCA I GGAAGCAGCGGAGG"‘GGAAGTGGAI
GGACAGCCCCGAGAACCACAGG' 1ACACCC"1GCCCCCA"1C CCGGGAGGAGATGACCAAGAACCAGG"1CAGCC"GACCT
GCCTGG"CAAAGGC' 1CTACCCCAGCGACATCGCCGTGGAG"‘GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCC"CCCA’1GC'1GGAC'1CCGACGGCTCC" .CT.1CCTCTACAGCAAGC"1CACCG"GGACAAGAGCAGG"1GGCAGCAG
GGGAACA"C"TCTCA’1GCTCCG".GA.1GCA"1GAGGC'1CT1GCACAACCGC"‘TCACGCAGAAGAGCC"CTCCC'1GTCTCCGG
GTAAA GGTGGCC‘GIATGCA’1GGAGA"1ACACCTACAT"1GCA'1GAA'1ATA" .HAGAHF 1GCAACCAGAGACA
ACTGA”C"C"AC§§§' ‘GG_ACAA" 1AAA"1GACAGCTCAGAGGAGGAGGA"1GAAATAGA"GG"CCAGCTGGACAAGCAG
AACCGGACAGAGCCCAT"“ACAA’ 1AACC"1"T"G"TGCAAG'1GTGAC" 1ACGCT"1CGG'11'11G"1GCG"1ACAAAGCAC
ACACG"AGACATTCG'“ACT"‘TGGAAGACC’1GT"1AA"‘GGGCACAC’1AGGAA"1TGTGGGACCCA"1CGGATC"1CAGAAACCA
Protein sequence of VBlOOS (Homodimeric construct according to the invention, SEQ ID
|VO:17):Anfino acm sequence,340 annno acMs.
QVS'1AA .AV AT.CNQV .SAP .AADTPTACCFSYTS QQI PQVFIA:
YFETS SQCS (PSVI F.4TKRGRQVCADPS * WVQKYVSD .4 .SA* T. 1PT.G
DTT {'13 P (S PCPRCPGGGSSGGGSGGQPRF‘PQVYT .PPSR «Mr
\TQVS ."1C .V (GFYPSDIAV * SSGQP * \IVYNTTPPM.J:DS:DGS FF .YSK
TVD (S QWQQGVI FS CSVMH' . QFTQ .ST.SPGKG .GGT.1Vl-IGD"
-I':‘Y Q .QP'TT'1DT IYEYEQ D 1 DGPAGQAEPDQAHYNIVTFC
C (CDS "1.4 QLCVQS THVD I RTT (3‘, .GIV§P1§5o<P*
Constructs with E6 and E7:
For the purpose of illustration only, the different domains of the constructs are separated by
an “|" with the domains in the following order: Signal peptide | human MIP-lCl | Hinge
h1 | Hinge h4 | Gly—Ser Linker or Gly—Leu linker| hCH3 IgG3 |Gly—Ser Linker or u
linker| E7 | r Linker or Gly—Leu linker| E6 mutant. Amino acids or nucleotides in
bold illustrates sites of mutations.
4o DNAsequenceofVBlOO9(SEQIE)NO:18y
ATGCAGGTCTCCAC"1GC'1GCCCTTGCCG'1CCTCCTC"1GCACCATGGCTC"1CTGCAACCAGG'11CC"1CTC"1|GCACCAC"T
GCTGCTGACACGCCGACCGCCTGC"1GCT"1CAGC"1ACACC'11CCCGACAGA"1TCCACAGAAT"1"1CA"1AGC"1GACTACT""G
AGACGAGCAGCCAG"1GC'11CCAAGCCCAG"1GTCA"1C"1"1CC'11AACCAAGAGAGGCCGGCAGG'1CTG"1GC"1GACCCCAG"GA
GGAGTGGGTCCAGAAA"1ACGTCAG"1GACCTGGAGCT1GAGT1GCC I GAGCTCAAAACCCCAC'1'1GG"‘GACACAAC"CACAC
45 AIGAGCCCAAA’1CT" 1GACACACCTCCCCCG"1GCCCAAGG"1GCCCA I GGCGGTGGAAGCAGCGGAGG"‘GGAAGTGGAI
GGACAGCCCCGAGAACCACAGG" 1ACACCC"1GCCCCCA"1C CCGGGAGGAGATGACCAAGAACCAGG"1CAGCC"GACCT
GCCTGGT1CAAAGGC" 1CTACCCCAGCGACATCGCCGTGGAG"‘GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCr1CCCA".Gcr1GGAC"1CCGACGGCTCC" .CT.1CCTCTACAGCAAGC"1CACCG"GGACAAGAGCAGG"1GGCAGCAG
GGGAACA"C"TCTCA’1GCTCCG".GA.1GCA"‘GAGGC"C"GCACAACCGC"‘TCACGCAGAAGAGCC"CTCCC'1GTCTCCGG
50 GTAAAIGGCCTCGGTGGCC‘GIATGCA’‘GGAGA’1ACACCTACAT"1GCA"1GAA'11ATA" .HAGAHF 1GCAACCAGAGACA
ACTGATC"C"AC§§§' ‘GG_ACAA" "AAA"1GACAGCTCAGAGGAGGAGGA"1GAAATAGA'11GG"1 CCAGCTGGACAAGCAG
AACCGGACAGAGCCCATF1ACAA" 1AACCT"1T'11G'11TGCAAG"1GTGAC"1C"1ACGCT"1CGG"-n—-.Gr1GCGTACAAAGCAC
ACACGTAGACA'—1TCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCA
GGCGGTGGAAGCAGCGGAGGTGGAAG"1GGA ATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATG
CACAGAGCTGCAAACAACTA"‘ACATGA"4AmAAr4Am.4AGAAr4Gr4GTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTA
rfiArfiGACrfirfirfiGC""CGACGGGAT'4.4AHGCAnAGr4AmAnAGAGAr4GGGAATCCATATGCTGTACGAGATAAATGTTTAAAGT
CTAAAATTAGTGAG’1A""AGACATHANqumHAHAGHr4r4GTATGGAACAACATTAGAACAGCAATACAACAAACC
GmmeGmGAmmr“GTTAA"TAGGTGTATTAACCGACAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAA
AAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAG
AAACCCAGCTG"1AA
Protein sequence of VBlOO9 (Homodimeric construct according to the invention, SEQ ID
|VO:19):Anfino acm sequence,501 anflno acMs.
QVSTAA IAVT . .CTMAT .C\IQV .S I APT .AADTPTACCFSYTSRQI PQNFIAD
QCS (PSVI F.4T< QGRQVCADPS * * WVQKYVSD fl .SA |«.KTP.G
DTT-IT EPKS CDTPPPCP QCP |GGGSSGGGSG GQPRTPQVYTT.PPSQ* * TK
\IQVS ITC .V (GFYPSDIAV *W 4 SSGQP * \T\TYNTTP PM. sacs FF.YS<.
TVD<S QWQQGNI FS CSV {TA .-IN QFTQ S PGK | LGGL | {GDTPTL
{TY .i3 .QPTTTD .YEYGQ .Ni388444 D «IDGPAGQAE P:U QAHYVIVTFC
C (CDST.4 THVDI zanDT. GT .GIVCP I CSQ P |GGGSSGGGSG|
FQDPQ':‘QP QKT.PQT.CT':' .QTTIHDI If:‘CVYCKQQT. . aTVYDFARzl4
CIVYQ DGVPYAVE:DKC.J {FYS (ISEYE {YCYST .YGTT .' UNAQQYN .
IQCIVEQ (P ICP * 4KQ Q -1 .DK {QRF {\II QGRWTGRCMS cczss QTRRETQ
DNAsequenceofVBlOl6(SEQIE)NO:20y
GTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT I GCACCACTT
GCTGCTGACACGCCGACCGCCTGC"1GCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAGCTGACTACTTTG
AGACGAGCAGCCAGTGCTCCAAGCCCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGA
GGAGTGGGTCCAGAAA"‘ACGTCAG""GACCTGGAGC'‘GAGTGCC |GAGCTCAAAACCCCACTTGGTGACACAACTCACAC
A|GAGCCCAAA’‘CTTGTGACACACCTCCCCCG"“GCCCAAGGTGCCCA|GGCGGTGGAAGCAGCGGAGGTGGAAGTGGA|
GGACAGCCCCGAGAACCACAGGTGTACACCC"“GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT
GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCCTCCCA'—1GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACATCTTCTCA"1GCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAA GGCCTCGGTGGCCTG |ATGCA"‘GGAGA"1ACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACA
ACTGATCTCTACGGATATGGACAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAG
AACCGGACAGAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCAC
ACACGTAGACA'—4—4CGHACHHHGGAAGAC C'—1GTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCA
GGCGGTGGAAGCAGCGGAGGTGGAAG"1GGA ATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATG
GCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTA
40 rfiArfiGACrfirfirfiGC"1r1r1r1CGGGAr1r4—4Ar4GCAmAGTAmAmAGAGAHGGGAATCCATATGCTGTACGAGATAAATGTTTAAAGT
.—-..—-..—-.A.—-.TCmAAAAmrqAGTGAGmAr‘AGACAT“Am“Gmr4Ar4AGr4r4r4GTATGGAACAACATTAGAACAGCAATACAACAAACC
GmmeGmGAmmr“GTTAA"TAGGTGTATTAACCGACAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAA
AAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAG
AAACCCAGCTG"1AA
Protein sequence of VBlOl6 (Homodimeric construct according to the invention, SEQ ID
|VO:21):Anfino acm sequence,501 anflno acMs
QVSTAA .AV .T .CTMAT .C\TQV .SAP .AA:DTPTACCFSYTS QQI PQVFIAD
YFETS SQCS (PSVI F.4T< QGRQVCADPS * * WVQKYVSD fl .SA 4T.(TPT.G
50 DTT-ITEP (S C: S SGGGSGGQPR':‘PQVYT .PP SR* *MT
\IQVS .TC DIAV*W 4 SSGQP * \IWYNTTPPM;DSDGS
{TA _ -IN QFTQ (S .ST.SPGKG
.NiU 88* * *3 4 IDGPAGQA:1
T. GT .GIVCPICSQ
55 Li .QTTIHDI If:‘CVYCKQQT.
.J4AFYS (ISEYE {YCYST .YGTT .'
.DK {QRF {\II QGRWTGRCMS CC
SEQ ID NO:22:
>tr|Q77816|Q77816_HPV16 E6 protein OS=Human papillomavirus type 16 GN=E6 PE=4
SV=1; (Underlined amino acids denotes amino acids that may be deleted; Potential amino
acids that may be mutated are highlighted)
MFQDPQERPRKLPQLCTELQ‘I‘I’IHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYS
HYCYSLYG'I‘I'LEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCR
SSRTRRETQL
SEQ ID NO:23:
>sp|P03129|VE7_HPV16 Protein E7 OS=Human papillomavirus type 16 GN=E7 PE=1 SV=1;
(Underlined amino acids denotes amino acids that may be deleted; Potential amino acids that
may be mutated are highlighted)
MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQ
RTLEDLLMGTLGIVCPICSQKP
SEQ ID NO:24:
>sp|P06463|VE6_HPV18 Protein E6 OS=Human papillomavirus type 18 GN=E6 PE=1 SV=1
MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSI
PHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRH
LNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV
SEQ ID NO:25:
>sp|P06788|VE7_HPV18 Protein E7 OS=Human papillomavirus type 18 GN=E7 PE=3 SV=2
MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHT
MLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWCASQQ
SEQ ID NO:26:
Hinge regions (IgG3 UH hinge), 12 amino acids: ELKTPLGD'I‘I'HT
SEQ ID NO:27:
Hinge region (IgG3, MH hinge, 15 amino acids): TPPPCPRCP
SEQ ID NO:28:
Gly—Ser Linker: GGGSSGGGSG
SEQ ID NO:29: hCH3 IgG3:
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTI'PPMLDSDGSFFLYSKL
WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQ ID NO:30: Linker: GLGGL
SEQ ID NO:31: DNA sequence of VBlOZO:
45 ATGCAGGTCTCCAC"GC"GCCCTTGCCG"CCTCCTC"GCACCATGGCTC"CTGCAACCAGG"CC"CTC"IGCACCAC"T
GCTGCTGACACGCCGACCGCCTGC"GCT"CAGC"ACACC"CCCGACAGA"TCCACAGAAT""CA"AGC"GACTACT""G
GCAGCCAG"GC"CCAAGCCCAG"GTCA"C""CC"AACCAAGAGAGGCCGGCAGG"CTG"GC"GACCCCAG"GA
GGAGTGGGTCCAGAAA"ACGTCAG'1GACCTGGAGC'1GAG'1GCC I GAGCTCAAAACCCCAC"1"1GG""GACACAAC"1CACAC
[XI CE(ECEZXZXZX'1(:TF""(}""(}ZX(:ZX(EZX(3(EUTCECECECE(3C3""C3(3(ECEZXZX(}C3'"C3(3(3(:ZX IC3C3C:C3C31T(E(EZXZXC3(:ZXC}(3(3(EZX(}(}""(}(}Z%]X(}TF(}(}ZXI
50 GGACAGCCCCGAGAACCACAGG"G"ACACCC"GCCCCCA"CCCGGGAGGAGATGACCAAGAACCAGG"CAGCC"GACCT
"CAAAGGC""CTACCCCAGCGACATCGCCGTGGAG"GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
CACGCC"CCCA"GC"GGACTCCGACGGC"CC""C""CCTCTACAGCAAGCTCACCG"GGACAAGAGCAGG"GGCAGCAG
GGGAACATCT"C"CA"GC"CCG"GA"GCATGAGGC"C"GCACAACCGCT"CACGCAGAAGAGCC"C"CCC"G"CTCCGG
G"AAA|GGCCTCGGTGGCCTG/ATGCA"GGCGA"ACCCCAACAC"CCA"GAGTACATGCTGGACC""CAGCCCGAGAC
55 "ACGGA"C"G"A"GGC"A"GGGCAG""GAA"GAC"CA"C"GAGGAGGAGGACGAAA"AGACGGCCCAGCTGG"CAAGCC
GAACCGGA"AGAGCCCAC"ACAACA""G"GACC""""GC"G"AAG"G"GACAGCAC"C"GAGAC"G"G"G"TCAGTCCA
C"CATG"CGACA”ACGCACAT"GGAGGA"C"CC"GA"GGGAACAC"GGGAATTGTG"G"CCCA"CTG"”CCCAAAAGCC
"1/GGAGGTGGAAGCAG""GGAGGCGG""""CAGGC/A""G""""CCAAGATCC""CAAGAACG""CCTCGTAAGC""GCCACAGCTG"1
G""ACCGAGCTTCAGACCACCA”TCAC&ACA""CPJ’"CC""GGAG""GCG""C""ATTGCAAACAGCAGC"CC"1""AGAAGGGAAG"1
G"ACGATTTTGCACGGAGGGACC"CTGCATCGTGTA"CGGGACGGCAATCCC"ATGCGGTACGGGA"AAATGCCTGAAG
T"C"ACAGCAAAATC"CCGAGTACCGGCACTACTGC"ACTCTC"C"A"GGGACGACTCTGGAACAGCAGTACAACAAGC
CCT"GTGCGA”C"GC"GATTCGC"GCATTAA"CGCCAGAAACC"C"G"GCCCAGAAGAGAAGCAAAGACACCTGGACAA
GAAACAGCGA""CCACAACATCCGAGGGAGA"GGACAGGGAGG"G"A"GAGC"GCTGTCGGAGTTC"AGGACAAGGCGC
GAAACCCAGC"""""GA
SEQ ID NO:32: Protein sequence of VBlOZO (Homodimeric construct according to the
invenUon Annno acm ce,501 annno adds:
QVS'1AA IAVT. .C\IQV IS IAPTIMiDTPr‘ACCFSYTSRQIPQNFIAD
YFETSSQCS {PSVIF_4T<QGRQVCADPS * *WVQKYVSD .4 ISAI * IKTP IG
DTT-I"1 EPKSCDTPPPCP QCP I GGGSSGGGSG GQPR'TPQVYTTIPPS Q* * TK
\TQVS 51C IV <GFYPSDIAV* W * SSGQP * VVYNr‘TPPMJ SDGSFF.YS<.
TVD (S WQQGNIFSCSVQ {TA u-IN QFTQ (S.JSLSPGK| LGGL| L
-I':‘Y .i3 IQP'TTTD IYEYEQ uNDSSd * *D* IDGPAGQAEP:U QAHYVIVTFC
C(CDS'TJQLCVQSTHVDI QTTF‘DT. . GT IGIVCPICSQ AP|GGGSSGGGSG|
FQDPQ': QP QKTE’QTICT'iJ IQTTIHDII F‘CVYCKQQT. . N zrvyoFAgzou
CIVY Q:DG\IPYAVE:DKC.J AFYS {ISEY Q-IYCYSTXGTT .'u LCD..
I QCIVEQ (P C]? * * KQQ A OK {QRF {\II QGRWTGRCMSCCQSSQTRRETQ
*
SEQ ID NO:33: DNA ce of VBlOZl:
ATGCAGGTCTCCAC"GC"GCCCTTGCCG"CCTCCTC"GCACCATGGCTC"CTGCAACCAGG"CC"CTC"IGCACCAC"T
GCTGCTGACACGCCGACCGCCTGC"GCT"CAGC"ACACC"CCCGACAGA"TCCACAGAAT""CA"AGC"GACTACT""G
AGACGAGCAGCCAG"GC"CCAAGCCCAG"GTCA"C""CC"AACCAAGAGAGGCCGGCAGG"CTG"GC"GACCCCAG"GA
GGAGTGGGTCCAGAAA"ACGTCAG"GACCTGGAGC"GAG"GCC|GAGCTCAAAACCCCAC"”GG"GACACAAC"CACAC
AIGAGCCCAAA"CT"G"GACACACCTCCCCCG"GCCCAAGG"GCCCA|GGCGGTGGAAGCAGCGGAGG"GGAAGTGGA|
GGACAGCCCCGAGAACCACAGG"G"ACACCC"GCCCCCA"CCCGGGAGGAGATGACCAAGAACCAGG"CAGCC"GACCT
GCCTGG"CAAAGGC""CTACCCCAGCGACATCGCCGTGGAG"GGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACAC
"CCCA"GC"GGACTCCGACGGCTCC""CT"CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGG"GGCAGCAG
GGGAACATCTTCTCA"GCTCCG"GATGCATGAGGC"CTGCACAACCGCTTCACGCAGAAGAGCCTCTCCC"GTCTCCGG
GTAAMGGCCTCGGTGGCCHEATGCATGGTGACACACCAACCCTGCACGAATACATGCTCGATCTGCAGCCAGAG
ACTACCGACCTTTACGGCTATGGGCAGTTGAACGACAGCTCTGAGGAGGAGGACGAGATCGATGGTCCTGCTGGA
CAAGCAGAACCAGACAGAGCCCACTACAACATCGTAACCTTTTGCTGCAAGTGTGACAGTACCCTTCGTTTGTGCG
TTCAGAGCACGCATGTCGACATTCGGACACTGGAGGATCTGCTCATGGGGACTCTGGGGATTGTGTGTCCTATTTG
CAGCCAGAAACQNGGCGGAGGATCTTCAGGAGGCGGGAGTGGCMJGTTCCAAGACCCTCAGGAACGCCCTCGG
AAACTGCCCCAATTGTGTACTGAGCTCCAGACAACGATACACGACATAATCCTGGAGTGCGTGTATTGCAAGCAGC
AGCTTCTGAGGAGGGAAGTGTACGATTTTGCCAGGAGAGATGGCTGCATTGTCTACCGAGATGGCAATCCCTATG
4o CGGTGTGTGATAAGTGTCTGAAGTTCTATTCCAAAATCAGCGAATATCGGCATTATTGCTACTCACTGTACGGAACT
ACCCTCGAACAGCAGTACAACAAACCGCTCTGTGATCTGCTGATCAGATGCATCAATCGGCAGAAACCCCTTTGTC
CCGAAGAGAAGCAAAGACACCTGGACAAGAAGCAGAGGTTCCACAATACCCGAGGTCGTTGGACTGGGCGCTGC
TGTTGTCGCTCCTCTCGCACAAGGAGAGAGACACAACTGTGA
45 SEQ ID NO:34: Protein sequence of VBlOZl (Homodimeric construct according to the
invenUon.Anfino acm sequence,501 annno adds:
QVS"AA.AVL.CTMALCVQV.S|APEAADTP"ACCFSYTSRQIPQNFIAD
YFETSSQCS<PSVIFuT<QGRQVCAQPSd*WVQKYVSD.*ISA|*.KTP.G
50 DTTI" EPKSCDTPPPCPQCP|GGGSSGGGSG GQPRTPQVYTLPPSQ44 TK
VQVS."C.V<GFYPSDIAV*W*SSGQPdVVYN"TPPMu SDGSFF.YS<.
TVD<S WQQGNIFSCSV {TAIINQFTQ<SDSLSPGK| LGGL| {GDTPTL
.QPTTTDIYGYGQINDSS***D*IDGPAGQAE@U VTFC
C<cos"uzLCVQSTHVDIzTL:3L. GT.GIVCPICSQ<P|GGGSSGGGSG|
55 "11 IO U "U IO U (U "U (Ux—i' ' "U 10 _. OH 1J'.QTTIHDIIITCVYCKQQLIQQTVYDFAEQDE
SEYQIYCYSEYGTTITQQYN<PLCDJL
A .DK<QRF{V QGRWTGRCMSCCQSSQTRRETQ
Claims (33)
1.
A nucleic acid molecule encoding an amino acid chain comprising (1) a signal peptide, (2) a targeting unit, (3) a dimerization motif, and (4) an antigenic unit, said 5 targeting unit comprising an amino acid sequence having at least 80 % sequence identity to the amino acid sequence 24-93 of SEQ ID NO:1, and an antigenic unit comprising an amino acid sequence of human papillomavirus (HPV) HPV16 and/or HPV18, preferably an antigenic unit derived from early proteins E6 and/or E7 of HPV16 and/or HPV18, which amino acid chain is able to form a homodimeric protein. 10 2. The nucleic acid molecule according to claim 1, wherein said targeting unit, dimerization motif and antigenic unit in said amino acid chain are in the N-terminal to C-terminal order of targeting unit, dimerization motif and antigenic unit.
3. The amino acid chain according to either one of claims 1 or 2, wherein said signal peptide ts of an amino acid ce having at least 80 % sequence 15 identity to the amino acid sequence 1-23 of SEQ ID NO:1.
4. The nucleic acid molecule according to any one of claims 1-3, wherein the zation motif comprises a hinge region and optionally an immunoglobulin domain, optionally connected h a linker.
5. The nucleic acid le ing to claim 4, comprising an immunoglobulin 20 domain which is a carboxyterminal C domain, or a sequence that is substantially identical to said C domain or a variant thereof.
6. The nucleic acid molecule according to any one of claims 1-5, wherein the dimerization motif consists of hinge exons h1 and h4 connected through a linker to a CH3 domain of human IgG3. 25
7. The nucleic acid le according to any one of claims 1-6, wherein the dimerization motif consists of an amino acid ce having at least 80 % sequence identity to the amino acid sequence 94-237 of SEQ ID NO:3.
8. The nucleic acid molecule according to any one of claims 1-9, wherein said targeting unit ts of amino acids 24-93 of SEQ ID NO:1, or a variant thereof.
9. The nucleic acid molecule according to any one of claims 1-8, wherein said nic unit comprises an amino acid ce having at least 80%sequence identity to the amino acid sequence 243-293 of SEQ ID NO:3 or to the amino acid sequence 243-340 of SEQ ID NO:11. 5 10. The nucleic acid molecule according to claim 9, wherein said antigenic unit ses one or more amino acid substitutions at a position selected from the list consisting of F47, L50, C63, C106 and I128 of SEQ ID NO:22, or a deletion involving one or more amino acid selected from the list consisting of Y43-L50 of SEQ ID NO:22 or wherein said antigenic unit comprises one or more amino acid substitutions at a
10 position selected from the list consisting of C24, E26, C58, C61, C91, and C94 of SEQ ID NO:23, or a deletion involving one or more amino acid selected from the list ting of L22-E26 and/or C58-C61 and/or C91-S95 of SEQ ID NO:23.
11. The nucleic acid molecule according to any one of claims 1-10, wherein said antigenic unit comprises an amino acid sequence of human papillomavirus 16 (HPV16) 15 derived from both early proteins E6 and E7
12. The nucleic acid molecule according to any one of claims 1-11, wherein said antigenic unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence 1 of SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:32, or SEQ ID NO:34. 20
13. The nucleic acid molecule according to claim 12, n said antigenic unit comprises one or more amino acid substitutions at a position selected from the list consisting of F47, L50, C63, C106 and I128 of SEQ ID NO:22 and C24, E26, C58, C61, C91, C94 of SEQ ID NO:23.
14. The nucleic acid molecule ing to any one of claims 12-13, wherein said 25 antigenic unit consists of the amino acid sequence 1 of SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:32, or SEQ ID NO:34, or a variant or antigenic fragment thereof.
15. The c acid molecule according to any one of claims 1-14, wherein said amino acid chain consists of an amino acid sequence selected from the list consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:32, and SEQ ID NO:34, or a variant or nic nt thereof.
16. The nucleic acid molecule according to any one of claims 1-15, wherein said amino acid chain consists of an amino acid sequence selected from the list consisting 5 of SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:32, and SEQ ID NO:34, or a variant or antigenic fragment thereof.
17. The nucleic acid molecule according to claim 1, wherein said amino acid chain consists of SEQ ID NO:32, or a variant or antigenic fragment thereof.
18. The nucleic acid molecule according to any one of claims 1-17, which nucleic 10 acid molecule is human codon optimized
19. The nucleic acid molecule according to any one of claims 1-18, which is a DNA or RNA, preferably a DNA.
20. The nucleic acid molecule ing to any one of claims 1-19 comprising a vector. 15
21. The c acid molecule according to any one of claims 1-20 formulated for administration to a patient to induce production of the dimeric protein in said patient.
22. A homodimeric protein of two amino acid chains encoded by a nucleic acid molecule according to any one of claims 1-19.
23. The homodimeric n according to claim 22, in its mature form without any 20 signal peptide sequence.
24. The homodimeric protein according to either one of claims 22 or 23, or the c acid molecule according to any one of claims 1-21, for use as a medicament.
25. A pharmaceutical composition comprising the homodimeric protein according to either one of claims 22 or 23, or the nucleic acid le according to any one of 25 claims 1-21.
26. A host cell comprising the nucleic acid molecule according to any one of claims 1-21, wherein the host cell is not a transformed host cell within a human body.
27. A method for preparing a homodimeric protein according to either one of claims 22 or 23, the method comprising a) transfecting the nucleic acid molecule ing to any one of claims 1-21 into a cell population; b) culturing the cell population; and c) collecting and ing the homodimeric protein expressed from the cell 10 population.
28. A method for preparing a vaccine comprising an immunologically effective amount of a nucleic acid molecule according to any one of claims 1-21, the method comprising a) preparing a nucleic acid molecule ing to any one of claims 1-21; 15 and b) dissolving the nucleic acid molecule obtained under step a) in a pharmaceutically acceptable carrier, diluent, or buffer.
29. A vaccine against HPV comprising an immunologically effective amount of a dimeric n according to either one of claims 22 or 23, or a c acid molecule 20 according to any one of claims 1-21, and a pharmaceutically acceptable r and/or adjuvant.
30. The vaccine according to claim 29 comprising an immunologically effective amount of a nucleic acid molecule according to any one of claims 1-21, preferably a DNA. 25
31. A meric protein according to either one of claims 22 or 23 or a nucleic acid le according to any one of claims 1-21, for use in the treatment or prevention of a HPV-induced disease or condition, such as a cancer or an infectious disease caused by HPV.
32. The homodimeric protein or the nucleic acid molecule according to claim 31, wherein the nucleic acid le, preferably a DNA, is formulated for administration to the patient through electroporation.
33. The meric protein or the nucleic acid molecule according to either one of 5 claims 31 or 32, formulated for intradermal or intramuscular administration.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161578542P | 2011-12-21 | 2011-12-21 | |
US61/578,542 | 2011-12-21 | ||
PCT/EP2012/076404 WO2013092875A1 (en) | 2011-12-21 | 2012-12-20 | Vaccines against hpv |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ626124A NZ626124A (en) | 2016-07-29 |
NZ626124B2 true NZ626124B2 (en) | 2016-11-01 |
Family
ID=
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