Note: Descriptions are shown in the official language in which they were submitted.
VACCINE COMBINATION AND METHOD FOR USING THE SAME
REFERENCE TO SEQUENCE LISTING
[0001] Accompanying this application is a sequence listing in an American
Standard Code
for Information Interchange (ASCII) text file named "211223-US86679-Sequence
Listing-
v1F.txt", created December 07, 2021, and having a size of 18,253 bytes.
[0002] [blank]
1
Date Recue/Date Received 2023-06-06
BACKGROUND
1. Field
[0003] The present disclosure generally relates to vaccine combinations,
particularly for a
human papillomavirus (HPV)-associated disease, and more specifically to
improving the vaccine
combination by the particular design of a priming vaccine and a boosting
vaccine.
2. Related Art
[0004] HPV is a common etiological agent in several human cancers,
including cervical, anal,
penile, vulvar, vaginal, and head and neck cancers. Current vaccines against
HPV, such as
Gardasil and Cervarix , have shown clinical efficacy in preventing HPV
infection, but they are
ineffective in treating patients with existing HPV infection or HPV-associated
cancers. As such,
development of therapeutic vaccines for patients infected with HPV or even
suffering from HPV-
associated diseases are highly demanded.
[0005] Deoxyribonucleic acid (DNA) vaccination is a technique for
protecting against
infection or treating disease by injection with a genetically engineered
plasmid containing a
DNA sequence encoding one or more antigens. DNA vaccines have theoretical
advantages over
conventional vaccines, including safety, speed, and predictability of
manufacture; temperature
stability; flexibility in design; and the ability to induce a wider range of
immune response types.
[0006] An effective vaccine usually requires more than one-time
immunization in the form of
prime-boost. Traditionally the same vaccines are given multiple times as
homologous boosts.
Some findings suggest that prime-boost can be done with different types of
vaccines containing
the same antigens. In many cases such heterologous prime-boost can be more
immunogenic than
homologous prime-boost (Curr Opin Immunol. 2009 Jun; 21(3): 346-351).
Heterologous prime-
boost may include administration of two different vectors or delivery systems
expressing the same
2
Date Recue/Date Received 2022-01-18
or overlapping antigenic inserts. It has been known that using certain vector
combinations could
increase both antibody and T cell immunity. However, while a priming vaccine
and a boosting
vaccine, which both can be either a fusion protein of antigens or a vector
encoding the same, may
have the same antigen arrangement in the fusion protein, the enhanced immunity
generated by the
prime-boost regimen may also include boosting an immune response against the
junction-
associated epitopes in the fusion protein of antigens and may lead to an
impaired immune response
against the target pathogen or other undesired effects.
[0007] Furthermore, before providing a newly developed DNA vaccine to the
clinics, the
potential to induce autoimmunity by vaccinating with novel sequences is a
great concern that
should be addressed. Such adverse reaction to vaccines may be viewed as a
result of the interaction
between susceptibility of the vaccinated subject and various vaccine
components. The significant
similarity between certain pathogenic elements contained in the vaccine and
specific human
proteins may lead to immune cross-reactivity, wherein the reaction of the
immune system towards
the pathogenic antigens may harm the similar human proteins (self-antigens),
essentially causing
autoimmune disease (Cell Mol Irnmunol. 2018 Jun;15(6):586-594). Therefore, in
addition to
elevating the immunogenicity of vaccines, it is essential as well to avoid
potential induction of
autoimmunity due to cross-reactivity when developing vaccines.
[0008] In view of the reasons mentioned above, there exists an unmet need
for providing a
vaccine for treating HPV-associated diseases with enhanced immunogenicity and
improved safety,
as reflected in a reduced risk to generate an immune response against the
junction-associated
epitopes potentially in the fusion protein expressed by or contained in the
vaccine and to induce
cross-reactivity against self-antigens.
BRIEF SUMMARY OF THE DISCLOSURE
[0009] In a first aspect of the present disclosure, a vaccine combination
may be provided. The
vaccine combination may include a first vaccine and a second vaccine. The
first vaccine may
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Date Recue/Date Received 2022-01-18
include a first fusion protein or a first polynucleotide encoding the first
fusion protein. The first
fusion protein, in an order from N to C terminus, may include: (i) an E7
protein of human
papillomavirus type 16 (HPV-16) or a functional variant thereof (ii) an E7
protein of human
papillomavirus type 18 (HPV-18) or a functional variant thereof; (iii) an E6
protein of HPV-16 or
a functional variant thereof; (iv) an E6 protein of HPV-18 or a functional
variant thereof; and (v)
a heat shock protein or a functional variant thereof The E7 protein of HPV-16
or a functional
variant thereof may comprise SEQ ID NO: 1 at a beginning of the protein and
SEQ ID NO: 2 at
an end of the protein. The E7 protein of HPV-18 or a functional variant
thereof may comprise SEQ
ID NO: 3 at a beginning of the protein and SEQ ID NO: 4 at an end of the
protein. The E6 protein
of HPV-16 or a functional variant thereof may comprise SEQ ID NO: 5 at a
beginning of the
protein and SEQ ID NO: 6 at an end of the protein. The E6 protein of HPV-18 or
a functional
variant thereof may comprise SEQ ID NO: 7 at a beginning of the protein and
SEQ ID NO: 8 at
an end of the protein. The second vaccine may include a second fusion protein
and a third fusion
protein, or a second polynucleotide encoding the second fusion protein and the
third fusion protein.
The second fusion protein may include an E6 protein of HPV-16 or a functional
variant thereof;
and an E7 protein of HPV-16 or a functional variant thereof The third fusion
protein may include
an E6 protein of HPV-18 or a functional variant thereof; and an E7 protein of
HPV-18 or a
functional variant thereof. Amino acid sequences of junction regions in the
first fusion protein may
be different from those in the second fusion protein and the third fusion
protein.
[0010] In some embodiments, the E7 protein of HPV-16 may comprise SEQ ID
NO: 9; the
E7 protein of HPV-18 may comprise SEQ ID NO: 10; the E6 protein of HPV-16 may
comprise
SEQ ID NO: 11; and the E6 protein of HPV-18 may comprise SEQ ID NO: 12.
[0011] In some embodiments, the E7 protein of HPV-16 may be encoded by SEQ
ID NO: 13;
the E7 protein of HPV-18 may be encoded by SEQ ID NO: 14; the E6 protein of
HPV-16 may be
encoded by SEQ ID NO: 15; and the E6 protein of HPV-18 may be encoded by SEQ
ID NO: 16.
[0012] In some embodiments, the first polynucleotide may include SEQ ID NO:
17.
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Date Recue/Date Received 2022-01-18
[0013] In some embodiments, the first polynucleotide may include SEQ ID NO:
18.
[0014] In some embodiments, the second polynucleotide may be contained
within a
recombinant virus.
[0015] In some embodiments, the recombinant virus may be TA-HPV.
[0016] In a second aspect of the present disclosure, a method for treating
an HPV-associated
disease in a subject in need thereof may be provided. The method may include
administering the
aforementioned vaccine combination to the subject. The first vaccine may be
administered as a
priming vaccine, and the second vaccine may be administered as a boosting
vaccine.
[0017] In some embodiments, the method may further include administering
the first vaccine
to the subject as a boosting vaccine after the administration of the first
vaccine as a priming vaccine
and prior to the administration of the second vaccine as a boosting vaccine.
[0018] In some embodiments, the first vaccine may comprise the first
polynucleotide. In some
embodiments, the first vaccine may be administered at a dose ranging from 10
micrograms per
subject to 20 milligrams per subject.
[0019] In some embodiments, the second vaccine may comprise TA-HPV. In some
embodiments, the second vaccine may be administered at a dose ranging from 1 x
104 plaque-
forming units (pfu) to 2 x 109 pfu.
[0020] In some embodiments, the method may further include administering a
chemotherapy,
radiotherapy, chemo-radiotherapy, cryotherapy, thermotherapy, targeted
therapy, cellular therapy,
gene therapy, or immunotherapy in combination with the administration of the
vaccine
combination.
[0021] In some embodiments, the chemotherapy, radiotherapy, chemo-
radiotherapy,
cryotherapy, thermotherapy, targeted therapy, cellular therapy, gene therapy,
or immunotherapy
may be administered prior to and/or simultaneously with the administration of
the first vaccine.
[0022] In some embodiments, the immunotherapy may comprise administering an
immune
checkpoint inhibitor to the subject.
Date Recue/Date Received 2022-01-18
[0023] In some embodiments, the immune checkpoint inhibitor may be an
immune modulator
targeting programmed cell death protein 1 (PD-1), programmed death-ligand 1
(PD-L1), cytotoxic
T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulator (ICOS), T-
cell
immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-
3) or T cell
immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM)
domain (TIGIT).
[0024] In some embodiments, the immune checkpoint inhibitor may be an anti-
PD-1 antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] So that the manner in which the above-recited features of the
present disclosure can be
understood in detail, a more particular description of the disclosure, briefly
summarized above,
may be had by reference to embodiments, some of which are illustrated in the
appended drawings.
It is to be noted, however, that the appended drawings illustrate only typical
embodiments of this
disclosure and are therefore not to be considered limiting of its scope, for
the disclosure may admit
to other equally effective embodiments.
[0026] FIG. lA is a schematic illustration showing a DNA construct encoding
the first fusion
protein of the first vaccine of the vaccine combination according to an
embodiment of the present
disclosure.
[0027] FIG. 1B is a schematic illustration showing a cassette for
expression of an HPV-16 E6-
E7 fusion protein and an HPV-18 E6-E7 fusion protein inserted into the genome
of a vaccinia virus
of the vaccine combination according to an embodiment of the present
disclosure.
[0028] FIG. 2A is a schematic illustration of the experimental design for
characterizing HPV
antigen-specific CD8+ T cell-mediated immune responses in mice vaccinated with
the vaccine
combination of the present disclosure in a prime and boost strategy (two DNA
doses and one
recombinant virus dose, DDV regimen) or a DNA vaccine alone in a prime and
boost strategy
(three DNA doses, DDD regimen), in accordance with an embodiment of the
present disclosure.
[0029] FIG. 2B is a bar chart showing the percentage of HPV-16 E7-specific
CD8+ T cells/
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Date Recue/Date Received 2022-01-18
total CD8+ cells in peripheral blood mononuclear cells (PBMCs) collected from
naïve mice or
mice treated with the vaccine combination of the present disclosure (DDV
regimen) or the DNA
vaccine alone (DDD regimen) using HPV-16 E7 (aa 49-57) peptide-loaded tetramer
staining, using
a two-tailed Student's t-test, in accordance with an embodiment of the present
disclosure.
[0030] FIG. 2C is a bar chart of the number of HPV-16 E7-specific IFN-7+
CD8+ T cells/3 x
105 splenocytes from mice vaccinated with the vaccine combination of the
present disclosure
(DDV regimen) or the DNA vaccine alone (DDD regimen) after stimulation with
either HPV-16
E6 (aa 50-57), peptide HPV-16 E7 (aa 49-57) peptide or HPV-18 E6 (aa 67-75)
peptide, in
accordance with an embodiment of the present disclosure.
[0031] FIG. 3A is a schematic illustration of the experimental design for
characterizing the
HPV antigen-specific immune response and antitumor effects in TC-1 tumor-
bearing mice treated
with the vaccine combination of the present disclosure in a prime and boost
strategy with or
without anti-PD-1 antibody, in accordance with an embodiment of the present
disclosure.
[0032] FIG. 3B is a bar chart showing the percentage of HPV-16 E7-specific
CD8+ T cells/
total CD 8+ T cells in peripheral blood mononuclear cells (PBMCs) prepared
from untreated mice,
mice treated with anti-PD-1 antibody alone, mice treated with the vaccine
combination of the
present disclosure (DDV), or mice treated with the vaccine combination of the
present disclosure
in combination with anti-PD-1 antibody (Anti-PD-1+DDV), using HPV-16 E7 (aa 49-
57) peptide-
loaded tetramer staining, using a two-tailed Student's t-test, in accordance
with an embodiment of
the present disclosure.
[0033] FIG. 3C is a curve chart showing TC-1 tumor volumes in the untreated
mice, the mice
treated with anti-PD-1 antibody alone, the mice treated with the vaccine
combination of the present
disclosure (DDV), or the mice treated with the vaccine combination of the
present disclosure in
combination with anti-PD-1 antibody (Anti-PD-1+DDV), in accordance with an
embodiment of
the present disclosure.
[0034] FIG. 3D is a Kaplan-Meier survival curve showing the probability of
survival in the
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Date Recue/Date Received 2022-01-18
untreated mice, the mice treated with anti-PD-1 antibody alone, the mice
treated with the vaccine
combination of the present disclosure (DDV), or the mice treated with the
vaccine combination of
the present disclosure in combination with anti-PD-1 antibody (Anti-PD-1+DDV),
in accordance
with an embodiment of the present disclosure.
[0035] In accordance with common practice, the various described features
are not drawn to
scale and are drawn to emphasize features relevant to the present disclosure.
Like reference
characters denote like elements throughout the figures and text.
DETAILED DESCRIPTION
[0036] The present disclosure will now be described more fully hereinafter
with reference to
the accompanying drawings, in which exemplary embodiments of the disclosure
are shown. This
disclosure may, however, be embodied in many different forms and should not be
construed as
limited to the exemplary embodiments set forth herein. Rather, these exemplary
embodiments are
provided so that this disclosure will be thorough and complete, and will fully
convey the scope of
the disclosure to those skilled in the art. Like reference numerals refer to
like elements throughout.
[0037] The terminology used herein is for the purpose of describing
particular exemplary
embodiments only and is not intended to be limiting of the disclosure. As used
herein, the singular
forms "a", "an", and "the" are intended to include the plural forms as well,
unless the context
clearly indicates otherwise. It will be further understood that the terms
"comprises" and/or
"comprising," or "includes" and/or "including" or "has" and/or "having", when
used herein,
specify the presence of stated features, regions, integers, steps, operations,
elements, and/or
components, but do not preclude the presence or addition of one or more other
features, regions,
integers, steps, operations, elements, components, and/or groups thereof.
[0038] The term "functional variant", as used herein, refers to a protein
(i) having at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acid
sequence to
the wild-type protein; and (ii) retaining the immunogenicity of the wild-type
protein, for example,
8
Date Recue/Date Received 2022-01-18
the ability to induce protein-specific CD8+ T cells.
[0039] The term "in combination with", as used herein, includes the
administration of two or
more vaccines or therapeutic agents simultaneously, or sequentially in any
order within no specific
time limits, unless otherwise indicated, in the course of treating the same
disease in the same
patient.
[0040] Unless otherwise defined, all terms (including technical and
scientific telins) used
herein have the same meaning as commonly understood by one of ordinary skill
in the art to which
this disclosure belongs. It will be further understood that terms, such as
those defined in commonly
used dictionaries, should be interpreted as having a meaning that is
consistent with their meaning
in the context of the relevant art and the present disclosure, and will not be
interpreted in an
idealized or overly formal sense unless expressly so defined herein.
[0041] A vaccine combination of the present disclosure is provided for a
subject having a
human papillomavirus (HPV)-associated disease. The subject having an HPV-
associated disease
may be a mammal (e.g., human, mice, etc.) suffering from, but not limited to,
warts, papilloma,
intraepithelial neoplasia, penile cancer, vaginal cancer, vulva cancer, anal
cancer, oropharyngeal
cancer, non-melanoma skin cancer, conjunctival cancer, or cervical cancer.
[0042] The vaccine combination may include a first vaccine and a second
vaccine. The first
vaccine includes a first fusion protein or a first polynucleotide that is
designed to encode the first
fusion protein. The first fusion protein includes a plurality of HPV antigens
and a heat shock
protein having a specific arrangement and thereby encompasses specific
junction regions between
the HPV antigens and between the HPV antigen and the heat shock protein. The
second vaccine
includes a second fusion protein and a third fusion protein, or a second
polynucleotide that is
designed to encode the second fusion protein and the third fusion protein. The
second fusion
protein includes two HPV antigens having a specific arrangement and thereby
encompasses a
specific junction region between the HPV antigens. The third fusion protein
also includes two HPV
antigens having a specific arrangement and thereby encompasses a specific
junction region
9
Date Recue/Date Received 2022-01-18
between the HPV antigens. The amino acid sequences of the junction regions in
the first fusion
protein are different from those in the second fusion protein and the third
fusion protein.
[0043] Specifically, the specific arrangement of HPV antigens of the first
fusion protein, the
second fusion protein and the third fusion protein are described as follows:
[0044] The first fusion protein, in an order from N to C terminus, may
include (i) an E7 protein
of human papillomavirus type 16 (HPV-16) or a functional variant thereof,
wherein the E7 protein
of HPV-16 or a functional variant thereof comprises SEQ ID NO: 1 at the
beginning of the protein
and SEQ ID NO: 2 at the end of the protein; (ii) an E7 protein of human
papillomavirus type 18
(HPV-18) or a functional variant thereof, wherein the E7 protein of HPV-18 or
a functional variant
thereof comprises SEQ ID NO: 3 at the beginning of the protein and SEQ ID NO:
4 at the end of
the protein; (iii) an E6 protein of HPV-16 or a functional variant thereof,
wherein the E6 protein
of HPV-16 or a functional variant thereof comprises SEQ ID NO: 5 at the
beginning of the protein
and SEQ ID NO: 6 at the end of the protein; (iv) an E6 protein of HPV-18 or a
functional variant
thereof, wherein the E6 protein of HPV-18 or a functional variant thereof
comprises SEQ ID NO:
7 at the beginning of the protein and SEQ ID NO: 8 at the end of the protein;
and (v) a heat shock
protein or a functional variant thereof. Thereby, junctions are formed between
the adjacent HPV
antigens (e.g., E7 protein of HPV-16 and E7 protein HPV-18; E7 protein HPV-18
and E6 protein
of HPV-16; and E6 protein of HPV-16 and E6 protein HPV-18) and between the E6
protein of
HPV-18 and the heat shock protein in the first fusion protein.
[0045] The second fusion protein, in an order from N to C terminus, may
include an E6 protein
of HPV-16 or a functional variant thereof; and an E7 protein of HPV-16 or a
functional variant
thereof. The third fusion protein, in an order from N to C terminus, may
include an E6 protein of
HPV-18 or a functional variant thereof; and an E7 protein of HPV-18 or a
functional variant thereof
Thereby, a junction is formed between the adjacent HPV antigens (i.e., E6
protein of HPV-16 and
E7 protein of HPV-16; or E6 protein of HPV-18 and E7 protein of HPV-18) in the
second fusion
protein or the third fusion protein.
Date Recue/Date Received 2022-01-18
[0046] Due to the aforementioned arrangement of the first fusion protein,
the second fusion
protein and the third fusion protein, the amino acid sequences of the junction
regions in the first
fusion protein are different from those in the second fusion protein and the
third fusion protein.
[0047] Examples of the first polynucleotide of the first vaccine and the
second polynucleotide
of the second vaccine according to the vaccine combination are illustrated in
FIG. I A and FIG. 1B,
respectively.
[0048] FIG. IA illustrates an exemplary first polynucleotide (denoted as
PBI-11) that encodes
the first fusion protein. The first polynucleotide includes a fusion gene
encoding the first fusion
protein, which in an order from N to C terminus comprises: a signal peptide
(denoted as -S"), an
E7 protein of HPV-16 (denoted as E7(16)), an E7 protein of HPV-18 (denoted as
E708)), an E6
protein of HPV-16 (denoted as E6(16)), an E6 protein of HPV-18 (denoted as
E6(18)), and a 70
kilodalton heat shock protein (HSP70).
[0049] The first fusion protein thereby has junction regions formed between
the adjacent HPV
antigens (e.g., E7 protein HPV-16 and E7 protein HPV-18, E7 protein HPV-18 and
E6 protein of
HPV-16. E6 protein of HPV-16 and E6 protein of HPV-18).
[0050] In some embodiments, the E7 protein of HPV-16 may comprise SEQ ID
NO: 9, the E7
protein of HPV-18 may comprise SEQ ID NO: 10, the E6 protein of HPV-16 may
comprise SEQ
ID NO: 11, and the E6 protein of HPV-18 may comprise SEQ ID NO: 12.
[0051] In some embodiments, the E7 protein of HPV-16 may be encoded by SEQ
ID NO: 13,
the E7 protein of HPV-18 may be encoded by SEQ ID NO: 14, the E6 protein of
HPV-16 may be
encoded by SEQ ID NO: 15, and the E6 protein of HPV-18 may be encoded by SEQ
ID NO: 16.
[0052] In some embodiments, pBI-11 may be used as the first vaccine of the
instant vaccine
combination. pBI-11, which has a nucleotide sequence as set forth in SEQ ID
NO: 17, is a DNA
construct including a fusion gene which includes: a subsequence encoding a
signal peptide; an
optimized HPV subsequence (FIG. 1A, fragments 101) encoding an E7 protein of
HPV-16 as set
forth in SEQ ID NO: 9, an E7 protein of HPV-18 as set forth in SEQ ID NO: 10,
an E6 protein of
11
Date Recue/Date Received 2022-01-18
HPV-16 as set forth in SEQ ID NO: 11, and an E6 protein of HPV-18 as set forth
in SEQ ID NO:
12; and a subsequence encoding an HSP70.
[0053] In some embodiments, alternatively and/or additively, pBI-12 may be
used as the first
vaccine of the instant vaccine combination. pBI-12, which has a nucleotide
sequence as set forth
in SEQ ID NO: 18, is a DNA construct comprising a fusion gene which includes:
an optimized
signal sequence encoding a signal peptide; an optimized HPV subsequence
encoding an E7 protein
of HPV-16 as set forth in SEQ ID NO: 9, an E7 protein of HPV-18 as set forth
in SEQ ID NO: 10,
an E6 protein of HPV-16 as set forth in SEQ NO: 11, and an E6 protein of HPV-
18 as set forth
in SEQ ID NO: 12; and a subsequence encoding an HSP70.
[0054] In some embodiments, the first vaccine may comprise the first
polynucleotide, and the
first vaccine may be administered at a dose ranging from 10 micrograms per
subject to 20
milligrams per subject.
[0055] FIG. 1B illustrates an exemplary second polynucleotide that encodes
the second fusion
protein and the third fusion protein. As shown in FIG. 1B, a cassette for
expression of the second
fusion protein and the third fusion protein is contained within the vaccinia
virus genome (denoted
as TA-HPV). The second polynucleotide includes a first open reading frame for
encoding an E6
and an E7 protein of HPV-16 (denoted as HPV16 E67 in FIG. 1B); and a second
open reading
frame for encoding an E6 and an E7 protein of HPV-18 (denoted as HPV18 E67 in
FIG. 1B).
[0056] In some embodiments, the second polynucleotide may be contained in a
recombinant
virus.
[0057] In some embodiments, the recombinant virus which contains the second
polynucleotide may be, but not limited to TA-HPV. The TA-HPV may be
administered at a dose
ranging from 1 x 104 pfu to 2 x 109 pfu. The TA-HPV may be administered
preferably at a dose
ranging from 2 x 104 pfu to 5 x 107 pfu.
[0058] In some embodiments, the vaccine combination of the present
disclosure may be
administered to a subject for treating an HPV-associated disease.
12
Date Recue/Date Received 2022-01-18
[0059] In some embodiments, the first vaccine of the vaccine combination
may be
administered as a priming vaccine and the second vaccine of the vaccine
combination may be
administered subsequently as a boosting vaccine, so as to provide a
combination therapy being
used in a heterologous prime-boost regimen to enhance a subject's immune
responses against HPV.
For example, the first vaccine may comprise the first polynucleotide and be
administered at a dose
ranging from 10 micrograms per subject to 20 milligrams per subject, and the
second vaccine may
comprise TA-HPV and be administered at a dose ranging from 1 x 104 pfu to 2 x
109 pfu.
[0060] In some embodiments, the first vaccine of the vaccine combination
may be
administered as a priming vaccine and be administered subsequently as a
boosting vaccine, and
after the administration of the first vaccine as the boosting vaccine, the
second vaccine of the
vaccine combination may be administered subsequently also as a boosting
vaccine, so as to provide
a combination therapy being used in a heterologous prime-boost regimen to
enhance a subject's
immune responses against HPV.
[0061] In some embodiments, the vaccine combination may further include an
immune
checkpoint inhibitor. The immune checkpoint inhibitor may be administered
simultaneously
and/or sequentially in any order with the administration of the first vaccine
and/or the second
vaccine. For example, the immune checkpoint inhibitor may be administered
prior to and/or
simultaneously with the administration of the first vaccine.
[0062] In some embodiments, the immune checkpoint inhibitor may be an
immune modulator
that targets programmed cell death protein 1 (PD-1), programmed death-ligand 1
(PD-L1),
cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulator
(ICOS). T-cell
immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-
3) or T cell
immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM)
domain (TIGIT).
For instance, the immune checkpoint may be an antibody targeting PD-1 (also
called "anti-PD-1
antibody").
[0063] In some embodiments, the first vaccine and the second vaccine of the
vaccine
13
Date Recue/Date Received 2022-01-18
combination may further include an ingredient, such as an adjuvant, to create
a stronger immune
response in the subject receiving the vaccine.
Example 1: Design and synthesis of the vaccine combination
[0064] A pBI-I DNA construct has been described previously as pNGVL4a-
SigE7(detox)HSP70 (C. Trimple et al. Vaccine 2003, 21:4036-4042). In one
example, the pBI-11
DNA construct was derived by Gibson assembly of a synthesized DNA fragment
encoding a fusion
protein of a signal peptide, an E7 protein of HPV-16, an E7 protein of HPV-18,
an E6 protein of
HPV-16, an E6 protein of HPV-18, and a 5' portion of HSP70 (up to the Tthl I
1I site), flanked 5'
by anEcoRI and Kozak site and 3' with a Tth111I site. The synthesized DNA
fragment was cloned
into the pBI-1 to replace the fragment between EcoRI and Tth111I in frame with
HSP70. The
expressing genes for the E7 protein of HPV-16, the E7 protein of HPV-18, the
E6 protein of HPV-
16, and the E6 protein of HPV-18 in pBI-11 were codon-optimized and encoded by
SEQ ID NO:
1-4 respectively. In another example, the pBI-12 DNA construct also included a
synthesized DNA
fragment encoding a fusion protein of the signal peptide, an E7 protein of HPV-
16, an E7 protein
of HPV-18, an E6 protein of HPV-16, an E6 protein of HPV-18, and a HSP70. The
expressing
genes for the signal peptide, HPV-I6 E7, HPV-I8 E7, HPV-I6 E6, and HPV-18 E6
in pBI-I2 were
codon-optimized. The synthesized DNA fragment of pBI-12 was cloned into the
pBI-1 to replace
the fragment between EcoRI and Tth111I in frame with HSP70.
[0065] The genes in the synthesized DNA fragment of each DNA construct that
have been
either optimized for gene expression (FIG. 1A, fragments 101) or based on
native papillomaviral
sequences (FIG. IA, fragments 103) are shown in FIG. 1A.
[0066] TA-HPV is a recombinant vaccinia viral vaccine expressing oncogenes
E6 and E7 of
HPV types 16 and 18. The HPV-16 and HPV-18 oncogenes E6 and E7 may be inserted
in a head-
to-head orientation under the control of the p7.5 and H6 promoters (denoted as
P7.5 and FI6,
respectively, in FIG. 1B) at a neutral site in the vaccinia virus Wyeth strain
genome (L. K.
14
Date Recue/Date Received 2022-01-18
Borysiewicz et al., Lancet., 1996 Jun 1;347(9014):1523-7). For both the HPV-16
and HPV-18
genes, the E6 termination codon may be altered to create an E6/E7 fused open
reading frame and
defined mutation introduced to inactivate the Rb-binding site in E7.
Example 2: The arrangement of the HPV antigens in the fusion protein encoded
in the
vaccine combination does not include peptides with sequences common to host
proteins that
can induce cross-reactive immunity against self-antigens.
[0067] To identify vaccine epitopes that might induce cross-reactivity against
self-antigens, the
sequence of the first fusion protein encoded by the first vaccine to those of
human proteins were
compared. Considering that peptide antigens are presented as fragments of
either 8-11 amino acids
on MHC class Ito CD8 T cells, or 12-20 amino acids on MHC class II to CD4 T
cells, a search
for all 8-mers generated from the HPV16/18 E6/E7 peptides plus the junctional
regions in pBI-11
encoded fusion protein against human protein sequences in UniProtTm, which
contains the Swiss-
ProtTM and TrEMBLI'm databases, was carried out.
[0068] Specifically, in order to search for potential novel peptides that may
be identical to
endogenous peptides, all linear sequences of 5-8 amino acids in length (5-mers
to 8-mers) from
amino acid 24-550 of pBI-11 were generated. The linear sequences span the last
7 amino acids of
the signal peptide, HPV-16 E7, HPV-18 E7, HPV-18 E6, HPV-16 E6, and the first
11 amino acids
of HSP70. In total, 520 8-mers, 521 7-mers, 522 6-mers, and 523 5-mers were
generated.
[0069] Furthermore, the sequences were submitted to the UniProtTm protein
database
(https://www.uniprot.org/) in groups of 80-100 sequences using the Peptide
Search Tool provided
at the website and searched for exact matches against the Swiss-ProtTm and
TrEMBLI'm databases
filtered for human proteins. The results revealed no exact match between the
pBI-11 encoded
fusion protein and endogenous human peptide sequences that are at least 8
amino acids in length.
[0070] An alternative searching approach was also conducted. The Immune
Epitope Database
(https://www.iedb.org/) was searched by using the same region of the pBI-11
protein sequence.
Date Recue/Date Received 2022-01-18
"Substring" for Linear Epitope to obtain any epitope sequences that are mapped
to the pBI-10.1
sequence (disclosed in U.S. Patent Application No. 17/534,256, filed on
November 23, 2021) were
selected. The search identified a B cell epitope derived from HPV-16 E7 in pBI-
11 (see Table 1
below). This epitope is also present in pBI-1, which has been tested for its
safety in human subjects.
Taken together, these results indicate that the arrangement of the fusion
protein encoded by the
first vaccine of the instant application (e.g., pBI-11 or pBI-12) is unlikely
to generate peptides that
could induce cross-reactive T cell immunity against self-antigens.
[0071] Table 1. Peptide epitope from pBI-11 protein identified in the
Immune Epitope
Database
HPV Protein Epitope' 'Human Protein Uniprot ID
HPV16 E7 RTLED Glutamate decarboxylase 2 (GAD2) A0A3B3IU09
[0072] In addition, pBI-11 encodes a fusion protein of the signal peptide, HPV-
16 E7, HPV-18
E7, HPV-16 E6, and HPV-18 E6 (E7(16)/E7(18)/E6(16)/E6(l8), see FIG. 1A),
arranged in an order
different from that in TA-HPV (see FIG. 1B). Such arrangement can further
avoid boosting
immune response against junction-associated epitopes potentially contained
within the fusion
protein encoded by pBI-11.
[0073] As a result, the vaccine combination of the instant application reduces
the risk to induce
cross-reactive immune response against self-antigens and the possibility to
generate immunity
against junction-associated epitopes in the fusion protein comprised within or
encoded by the
vaccine, and thus has an improved safety.
Example 3: HPV antigen-specific CD8+ T cell-mediated immune responses
generated by the
pBI-11 vaccine can be further enhanced by boost with TA-HPV vaccinia virus
vaccine.
[0074] In vivo T cell activation assays were performed to compare immune
response in mice
vaccinated with either pBI-11 alone, or pBI-11 in combination with TA-HPV.
[0075] FIG. 2A is a schematic illustration of the experiment design. 6- to
8-week-old female
C57BL/6 mice purchased from Taconic Biosciences (Germantown, NY) were
vaccinated pBI-11
16
Date Recue/Date Received 2022-01-18
(25 mg/50 ml/mouse) through intramuscular (I.M.) injection. The mice were
divided into a control
group (naive group), a DDD group and a DDV group. Each mouse in each group was
boosted with
the same regimen 7 days later. One week after the second vaccination, the DDD
group of the mice
(denoted as DDD in FIG.2A) was vaccinated with pBI-11 (25 mg/50 ml/mouse)
through I.M.
injection. The DDV group of mice (denoted as DDV in FIG. 2A) was vaccinated
with TA-HPV (1
x 106 pfu/50 ml/mouse) through I.M. injection. Six days after the last
vaccination, peripheral blood
was collected from the vaccinated or naive mice for HPV-16 E7 tetramer
staining. Fourteen days
after the last vaccination, splenocytes were prepared from the vaccinated mice
and stimulated with
either HPV-16 E6 (aa 50 to 57), HPV-16 E7 (aa 49 to 57), or HPV-18 E6 (aa 67
to 75) peptide in
the presence of GolgiPlug rim (BD Pharmingen, San Diego, CA). It is known that
the presentation
of epitope HPV-16 E6 (aa 50 to 57) is suppressed by the immunodominant epitope
of HPV-16 E7
(aa 49 to 57). Intracellular IFN-y cytokine staining assay was performed to
detect antigen-specific
CD8+ T cells. The cells were acquired with a FACSCaliburTm flow cytometer, and
data were
analyzed with CellQuest Pro software (BD Biosciences, Mountain View, CA).
[0076] As shown in FIG. 2B, mice vaccinated with the DDV regimen had
significantly higher
percentages of E7-specific CD8+ T cells than mice vaccinated with pBI-11 alone
(DDD) or naive
mice.
[0077] Furthermore, FIG. 2C shows that mice in a prime-vaccinia boost (DDV)
had
significantly higher numbers of HPV-16 E7-specific T cells (P = 0.0428) and
higher HPV-18 E6-
specific T cells (P = 0.2116) than those that only received pBI-11. In fact,
HPV16 E7-specific
CD8+ T cell-mediated immune responses in mice vaccinated with TA-HPV alone was
previously
characterized (Virology 2018 Dec; 525:205-215). It is found that mice
vaccinated with TA-HPV
alone did not generate appreciable HPV16 E7-specific CD8+ T cell-mediated
immune responses
(urology 2018 Dec; 525:205-215). However, our result indicates that TA-HPV
booster vaccination
is capable of simultaneously enhancing HPV-16 and HPV-18 antigen-specific CD8
T cell immune
responses generated after priming with pBI-11 DNA vaccine.
17
Date Recue/Date Received 2022-01-18
[0078] Based on the above results, the heterologous prime-boost vaccination
using the first
vaccine (e.g., pBI-11) in combination with the second vaccine (e.g., TA-HPV)
according to the
vaccine combination of the present disclosure induces an elevated HPV antigen-
specific CD8+ T
cell-mediated immune responses compared to administration with the first
vaccine (e.g., pBI-11)
alone.
Example 4: The vaccine combination of the present disclosure can be combined
with anti-
PD-1 immune checkpoint blockade to improve therapeutic antitumor response.
[0079] The ability of the vaccine combination of the present disclosure in
combination with
an anti-PD-1 immune checkpoint inhibitor to generate therapeutic antitumor
effects against HPV-
associated diseases were examined in the HPV-16 E6/E7+ TC-1 tumor model.
[0080] FIG. 3A is a schematic illustration of the experiment design. 6- to
8-week-old female
C57BL/6 mice were injected with 2 x 105 of TC-1 tumor cells subcutaneously on
day 0. On day 3,
the mice were divided into 4 groups (untreated group, anti-PD-1 group. DDV
group and anti-PD-
1+DDV group). The mice in the anti-PD-1 group were injected with purified anti-
mouse PD-1
monoclonal antibody (MAb; clone 29F.1Al2, 200mg/mouse) via intraperitoneal
injection. The
treatment was repeated every other day. The mice in the DDV group were
vaccinated with pBI-11
(25mg/ 50m1/mouse) through LM. injection and boosted once 3 days later, and
were further
boosted with TA-HPV vaccinia virus 3 days later through skin scarification.
The mice in the anti-
PD-1+DDV group were treated with both anti-mouse PD-1 MAb and pBI-11 DNA
vaccine prime
followed by TA-HPV vaccinia virus boost. On day 27, PBMCs were collected for
the
characterization of HPV-16 E7-specific CD8 T cell-mediated immune responses
using HPV-16
E7 peptide (aa 49 to 57)-loaded tetramer staining.
[0081] In one example, schematic illustration of the experiment in FIG. 3A
shows that the
immune checkpoint inhibitor (e.g., anti-PD-1 antibody) was administered prior
to heterologous
prime-boost vaccination (e.g., DDV). However, in other examples, additively
and/or alternatively,
18
Date Recue/Date Received 2022-01-18
the immune checkpoint inhibitor may be administered during the course of
heterologous prime-
boost vaccination.
[0082] For tetramer staining, mouse PBMCs were stained with purified anti-
mouse CD16/32
first and then stained with FITC-conjugated anti-mouse CD8a and PE-conjugated
HPV-16 E7 (an
49-57) peptide-loaded H-2Db tetramer at 4 C for 1 hour. After washing, the
cells were stained with
7-AAD. The cells were acquired with the FACSCaliburTM flow cytometer and
analyzed with
CellQuest Pro software (BD Biosciences, Mountain View, CA). The results of
tetramer staining
are shown in FIG. 3B.
[0083] The growth of the tumor was monitored twice a week by palpation and
digital caliper
measurement. Tumor volume was calculated using the formula [largest diameter x
(perpendicular
diameter)2] x 3.14/6 and is shown in FIG. 3C. The survival rate of the tumor-
bearing mice was
recorded, as illustrated in FIG. 3D, where both natural death and a tumor
diameter greater than
2 cm leading to death were counted as death.
[0084] As shown in Fig. 3B, mice receiving DDV regimen, either alone or with
anti-PD-1
antibody, displayed HPV-16 E7-specific CD8+T cell-mediated immune responses,
whereas in the
absence of vaccination with anti-PD-1 antibody, treatment of anti-PD-1
antibody did not elicit a
detectable HPV-16 E7-specific CD8+ T cell response.
[0085] Furthermore, FIG. 3C demonstrated that addition of anti-PD-1 antibody
treatment to the
DDV regimen significantly enhanced the therapeutic antitumor effects. This
suggests synergy of
vaccination and anti-PD-1 antibody treatment and that the latter is not
effective without a prior
immune response. Additionally, FIG. 3D shows that the combinational treatment
(anti-PD-1+DDV)
translated into significantly (P = 0.0073 when compared to DDV, and P = 0.0002
when compared
to anti-PD-1) better survival of the tumor-bearing mice.
[0086] In conclusion, the vaccine combination of the present disclosure
including a first vaccine
which comprises a first fusion protein with an indicated arrangement of HPV
antigens, or a first
polynucleotide encoding the same; and a second vaccine which comprises a
second fusion protein
19
Date Recue/Date Received 2022-01-18
and a third fusion protein both with an arrangement of HPV antigens different
from that in the first
fusion protein, or a second polynucleotide encoding the second fusion protein
and the third fusion
protein, not only reduces the risk to induce cross-reactive immunity against
self-antigens and to
boost an immune response against junction-associated epitopes in the fusion
protein contained in
or encoded by the vaccine, but also exhibits a significant HPV antigen-
specific immune response.
Moreover, the vaccine combination of the present disclosure in combination
with an immune
checkpoint inhibitor (e.g., anti-PD-1 antibody) elicits a stronger HPV antigen-
specific antitumor
response in vivo and further translates into more potent antitumor efficacy.
[0087] Those
skilled in the art will readily observe that numerous modifications and
alterations
of the device and method may be made while retaining the teachings of the
disclosure. Accordingly,
the above disclosure should be construed as limited only by the metes and
bounds of the appended
claims.
Date Recue/Date Received 2022-01-18
