U.S. patent application number 15/424473 was filed with the patent office on 2017-08-10 for therapeutic cancer vaccine containing tumor-associated neoantigens and immunostimulants in a delivery system.
The applicant listed for this patent is Xeme Biopharma Inc.. Invention is credited to Mircea C. Popescu, Richard J. Robb.
Application Number | 20170224796 15/424473 |
Document ID | / |
Family ID | 59498168 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170224796 |
Kind Code |
A1 |
Popescu; Mircea C. ; et
al. |
August 10, 2017 |
Therapeutic Cancer Vaccine Containing Tumor-Associated Neoantigens
and Immunostimulants in a Delivery System
Abstract
A therapeutic vaccine and a method of cancer treatment by
inducing humoral and cellular immune responses against malignant
cells is provided. The vaccine comprises a delivery system that
incorporates at least one peptide whose sequence encompasses a
genetic mutation associated with a malignancy (neoantigen), at
least one immunostimulant, and at least one type of lipid
molecule.
Inventors: |
Popescu; Mircea C.;
(Plainsboro, NJ) ; Robb; Richard J.;
(Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Xeme Biopharma Inc. |
Lombard |
IL |
US |
|
|
Family ID: |
59498168 |
Appl. No.: |
15/424473 |
Filed: |
February 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62292029 |
Feb 5, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
A61K 39/0011 20130101; A61K 2039/55533 20130101; C12Q 2600/106
20130101; C12Q 1/6886 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A therapeutic cancer vaccine comprising: at least one
neoantigen, wherein the neoantigen comprises a tumor-associated
genetic mutation peptide, and wherein the tumor-associated genetic
mutation peptide further includes an attachment element selected
from the group consisting of a transmembrane domain, a fatty acid
linker, and a tryptophan attachment element, wherein the attachment
element comprises a peptide sequence of 1 to 30 amino acids; at
least one immunostimulant; and at least one type of lipid
molecule.
2. The vaccine of claim 1, wherein the neoantigen is identified by
a genetic sequencing of RNA or DNA contained in a hematologic tumor
or a tumor-tissue sample obtained by needle biopsy or surgical
excision from one or more tumor sites of a patient.
3. The vaccine of claim 1, wherein the neoantigen is a fusion
peptide, wherein the fusion peptide comprises a plurality of
different tumor-associated genetic mutations.
4. The vaccine of claim 1, wherein the lipid molecule is selected
from the group consisting of phospholipids, glycolipids,
cholesterol, and derivatives of the lipid molecules.
5. The vaccine of claim 4, wherein the lipid molecule is a
saturated or an unsaturated phospholipid or glycolipid, or any
combination thereof.
6. The vaccine of claim 1, wherein the lipid molecule comprises
lipid bilayers, and wherein the attachment element integrates with
the lipid bilayers.
7. The vaccine of claim 1, wherein the fatty acid linker is
attached to the neoantigen by an extension of one or more amino
acids to the neoantigen sequence at its N or C-terminus.
8. The vaccine of claim 1, wherein the transmembrane domain
attachment element is at least 20 amino acids in length.
9. The vaccine of claim 1, wherein the transmembrane domain
attachment element is 30 to 70 amino acids in length.
10. The vaccine of claim 1, wherein the transmembrane domain
attachment element is TTEYQVAVAGIVFLLISVLLLSGLTWQRRQRK.
11. The vaccine of claim 1, wherein the tryptophan attachment
element comprises at least one tryptophan amino acid.
12. The vaccine of claim 11, wherein the tryptophan attachment
element is NRWIT.
13. The vaccine of claim 1, wherein the immunostimulant is a
cytokine selected from the group consisting of interleukin 2
(IL-2), GM-CSF, M-CSF, and interferon-gamma (IFN-.gamma.).
14. The vaccine according to claim 1, wherein the immunostimulant
is a Toll-like Receptor agonist and/or adjuvant selected from the
group consisting of monophosphoryl lipid A, lipid A, and muramyl
dipeptide (MDP) lipid conjugate and double stranded RNA.
15. The vaccine according to claim 1, wherein the immunostimulant
is a costimulatory membrane protein and/or cell adhesion protein
selected from the group consisting of CD80, CD86, and ICAM-1.
16. The vaccine according to claim 1, wherein the neoantigen is
selected based upon a predicted affinity for binding to the
patient's Major Histocompatibility Complex (MHC).
17. A method of treating cancer in a patient comprising
administering to the patient an effective amount of a therapeutic
cancer vaccine according to claim 1.
18. A method of cancer treatment by inducing humoral and cellular
immune responses against cancer cells in a patient comprising
genetically sequencing a tumor-tissue sample from a patient to
identify a plurality of neoantigens present in the tumor-tissue
sample, and wherein the neoantigens comprise tumor-associated
genetic mutation peptides; selecting at least one neoantigen based
upon a predicted affinity for binding to a Major Histocompatibility
Complex (MHC) of the patient, wherein MHC proteins on a surface of
antigen-presenting cells of the patient bind and present the
neoantigens to helper and effector T cells of an immune system of
the patient, wherein an immune response is directed to the cancer
cells in a patient; administering to the patient a therapeutically
effective amount of a vaccine comprising the neoantigen, wherein
the neoantigen comprises a tumor-associated genetic mutation
peptide, and wherein the tumor-associated genetic mutation peptide
further includes an attachment element selected from the group
consisting of a transmembrane domain, a fatty acid linker, and a
tryptophan attachment element, wherein the attachment element
comprises a peptide sequence of 1 to 30 amino acids; at least one
immunostimulant; and at least one type of lipid molecule.
19. The method according to claim 18, wherein the vaccine is
administered to the patient at a prescribed dose by intradermal,
subcutaneous, intramuscular, intranodal, or intra-tumoral
injection, or any combination thereof.
20. The method according to claim 18, wherein the patient receives
multiple vaccine injections at separate sites.
21. The method according to claim 18, wherein the patient receives
multiple vaccinations at prescribed time intervals.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application No. 62/292,029, filed Feb. 5, 2016, which is
hereby incorporated herein by reference in its entirety for all
purposes.
FIELD
[0002] The technology described herein relates to the
identification of tumor-associated antigens and proteins, and the
development of associated peptide vaccines for the therapeutic
treatment of cancer. Specifically, this disclosure relates to
cancer vaccines engineered from an individual patient's cancer that
include natural or modified neoantigens that stimulate the body's
humoral and cellular immune responses against the invading cancer
cells.
BACKGROUND
[0003] Efforts to treat patients with cancer utilizing the immune
system dates back to the 1890s. Cancer immunology and immunotherapy
has advanced since then, and researchers have gained a better
understanding of how the immune system identifies and attempts to
destroy cancer cells. Researchers have also gained a better
understanding of how cancers can undermine the immune system's
ability to identify and destroy the cancer cells and significant
progress has been made in the past decade in the treatment of
cancer. Targeted forms of chemotherapy and various types of passive
and active immunotherapy have improved clinical response rates,
delayed disease progression, and prolonged survival.
[0004] Nevertheless, additional and less-toxic therapeutic
modalities are needed to address the family of cancer diseases. In
particular, the individualized genetic make-up of cancer requires a
more patient-specific response. Therapeutic vaccination, for
example, aims to induce an active immune response to the patient's
tumor and is an approach that holds particular promise for
specificity and low toxicity, with the potential of long-term
disease-free survival by activating the host's anti-tumor immune
surveillance. Successful development of cancer vaccines, however,
has faced many challenges. For example, one of the main problems is
a lack of efficacy due to weak immunogenicity of the vaccine and
the difficulty of identifying tumor specific antigens common to all
patients within a particular indication. Another important reason
for the limited success of therapeutic cancer vaccines has been the
lack of recognition that cancer is a heterogeneous genetic disease
and that the magnitude of mutations found in a tumor demand an
immunologically critical approach to capturing the tumor antigenic
diversity in a vaccine. Vaccines made with only one or a few
tumor-associated antigens generally have been unsuccessful or
marginally successful in clinical studies.
[0005] To overcome some of these deficiencies, some vaccine
products have utilized whole tumor cells or tumor-cell extracts.
Another recent approach is to perform genetic sequencing of biopsy
material to identify the full range of genetic mutations (i.e.,
neoantigens), present in tumor-cell proteins. A subset of the
identified genetic mutations or neoantigens is then selected based
upon their predicted affinity for binding to the individual
patient's Major Histocompatibility Complex (MHC). A typical cancer
patient will have 100 to 150 genetic mutations, often consisting of
a single amino acid substitution. Of these 20-25 mutations will be
encoded within peptide segments that bind well to the patient's
particular MHC protein alleles.
[0006] MHC proteins on the surface of antigen-presenting cells
(APC) bind and present peptide antigens to the helper and effector
T cells of the immune system, thus directing an immune response to
the tumor. MHC Class I proteins typically present peptides of 8-11
amino acids in length while MHC Class II proteins present peptides
of 20-25 amino acids. Such neoantigen peptides can be utilized as
the tumor antigen component of a cancer vaccine. Two other
approaches that have been used to increase the efficacy of cancer
vaccines include the incorporation of tumor antigens into a
liposomal particle to enhance uptake by antigen presenting cells,
and the use of immunostimulating cytokines, such as interleukin 2
(IL-2) or GM-CSF, to more effectively induce cell-mediated and
humoral immunity. See U.S. Pat. No. 6,312,718, Vaccine for B-Cell
Malignancies, and U.S. Pat. No. 6,544,549, Multilamellar
Coalescence Vesicles (MLCV) Containing Biologically Active
Compounds, incorporated herein by reference in their entirety for
all purposes.
SUMMARY
[0007] This Summary provides an introduction to some general
concepts relating to this disclosure in a simplified form that are
further described below in the Detailed Description. This Summary
is not intended to identify key features or essential features of
the disclosure.
[0008] Embodiments of the present disclosure are directed to
therapeutic cancer vaccines and related methods of treatment that
combine neoantigen peptide sequences with immunostimulants in a
delivery system designed for efficient uptake by APC and activation
of effector immune responses in a cancer patient. Delivery systems
can include, for example, liposomes, systems made of cholesterol,
cholesterol hemisuccinate or alpha-tochoferol (e.g., vitamin E), or
other amphipathic molecules in which modified or synthesized
neoantigens can attach or insert. According to one aspect, the
therapeutic cancer vaccine includes at least one neoantigen, and
the neoantigen includes a tumor-associated genetic mutation
peptide. According to another aspect, the tumor-associated genetic
mutation peptide is engineered to further include an attachment
element. According to one aspect, the attachment element is a
transmembrane domain. According to another aspect, the attachment
element is a fatty acid linker. According to yet another aspect,
the attachment element is a tryptophan attachment element.
According to yet another aspect, the attachment element comprises a
peptide sequence of 1 to 30 amino acids. According to another
aspect, the therapeutic cancer vaccine also includes at least one
immunostimulant and at least one type of lipid molecule.
[0009] According to another aspect, a method of cancer treatment by
inducing humoral and cellular immune responses against cancer cells
in a patient is disclosed. According to one aspect, the method
requires the genetic sequencing a tumor-tissue sample from a
patient to identify a plurality of neoantigens present in the
tumor-tissue sample, and wherein the neoantigens include
tumor-associated genetic mutation peptides. According to one
aspect, at least one neoantigen is selected based upon a predicted
affinity for binding to a MHC of the patient, wherein MHC proteins
on a surface of antigen-presenting cells of the patient bind and
present the neoantigens to helper and effector T cells of an immune
system of the patient, and wherein an immune response is directed
to the cancer cells in a patient. According to another aspect, a
therapeutically effective amount of a vaccine including the
neoantigen is administered to the patient, wherein the neoantigen
includes a tumor-associated genetic mutation peptide, and wherein
the tumor-associated genetic mutation peptide further includes an
attachment element selected from the group consisting of a
transmembrane domain, a fatty acid linker, and a tryptophan
attachment element, wherein the attachment element comprises a
peptide sequence of 1 to 30 amino acids; at least one
immunostimulant; and at least one type of lipid molecule.
[0010] Further features and advantages of certain embodiments of
the present disclosure will become more fully apparent in the
following description of embodiments and drawings thereof, and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee. The foregoing and
other features and advantages of the present embodiments will be
more fully understood from the following detailed description of
illustrative embodiments taken in conjunction with the accompanying
drawings in which:
[0012] FIG. 1 schematically depicts a cancer vaccine incorporating
tumor neoantigen peptides within a multilamellar liposome structure
consisting of phospholipid bilayers. The expanded view of the
liposome schematically depicts the structure of DMPC (dimyristoyl
phosphatidy choline) composed of fatty acid chains and the glycerol
phosphatidylcholine headgroup.
[0013] FIG. 2 schematically depicts a cancer vaccine incorporating
tumor neoantigen peptides within a multilamellar liposome structure
consisting of phospholipid bilayers via a transmembrane domain. The
expanded view schematically depicts the transmembrane alpha helix
penetrating across the entire phospholipid bilayer.
[0014] FIG. 3 schematically depicts a cancer vaccine incorporating
tumor neoantigen peptides within a multilamellar liposome structure
consisting of phospholipid bilayers via a fatty acid linker. The
expanded view schematically depicts the fatty acid and neoantigen
peptide anchored to the phospholipid bilayer.
[0015] FIG. 4 schematically depicts a cancer vaccine incorporating
tumor neoantigen peptides within a multilamellar liposome structure
consisting of phospholipid bilayers via an attachment element
comprising a tryptophan (Trp-W) attachment element. The expanded
view schematically depicts the tryptophan attachment element and
neoantigen peptide anchored to the phospholipid bilayer.
DETAILED DESCRIPTION
[0016] In the following description of various examples of
therapeutic cancer vaccines and related methods of treatment of the
disclosure, reference is made to the accompanying drawings, which
form a part hereof, and in which are shown by way of illustration
various example structures and environments in which aspects of the
disclosure may be practiced. It is to be understood that other
structures and environments may be utilized and that structural and
functional modifications may be made from the specifically
described structures and methods without departing from the scope
of the present disclosure.
[0017] Aspects of the present disclosure are directed to a
therapeutic cancer vaccine that includes 1) at least one
neoantigen, wherein the neoantigen comprises a tumor-associated
genetic mutation peptide, and wherein the tumor-associated genetic
mutation peptide further includes an attachment element selected
from the group consisting of a transmembrane domain, a fatty acid
linker, and a tryptophan attachment element (or combinations
thereof), wherein the attachment element comprises a peptide
sequence of 1 to 30 amino acids; 2) at least one immunostimulant;
and 3) at least one type of lipid molecule. FIG. 1 shows such a
cancer vaccine that incorporates tumor-neoantigen peptides along
with the immunostimulant, IL-2, in a multilamellar liposome
structure consisting of phospholipid bilayers. In another exemplary
embodiment of the disclosure, the therapeutic cancer vaccine
includes a neoantigen that is identified by a genetic sequencing of
the RNA (or DNA) contained in a hematologic tumor or a solid
tumor-tissue sample obtained by needle biopsy, surgical excision,
or other suitable method from one or more tumor sites of a patient.
The genetic sequencing of a patient's tumor sample may be performed
by techniques readily known to one skilled in the art or by using
standard procedures, as described, for example, in U.S. Patent
Publication No. 2011/0293637, Composition and Methods of
Identifying Tumor Specific Neoantigens, incorporated herein by
reference in its entirety for all purposes. After the genetic
sequencing is completed, the identified peptide sequences
surrounding the cancer mutation are evaluated for their potential
binding affinity to the patient's Class I and Class II MHC
proteins. By using techniques readily known to one skilled in the
art, the neoantigen peptides with the highest binding affinity for
the patient's MHC proteins are selected for use in the vaccine. In
certain aspects, one or more of the identified neoantigen peptides
are engineered by using automated synthetic techniques, readily
known to those skilled in the art, and incorporated, with at least
one immunostimulant, into a liposome as depicted in FIG. 1.
[0018] In another aspect, the vaccine includes a neoantigen that is
a fusion peptide, wherein the fusion peptide comprises a plurality
of different tumor-associated genetic mutations. The fusion peptide
of the vaccine can be engineered to incorporate multiple peptide
sequences that contain different genetic cancer mutations and are
combined as one or more longer peptide sequences. Again, the
different neoantigens can be combined by any technique already
known to those skilled in the art. For example, peptides of up to
70 amino acids in length can be routinely synthesized by
solid-phase automated peptide synthesis instruments. This would
allow fusion of several MHC Class I neoantigen peptides (8-11 amino
acids in length). Moreover, multiple synthetic fusion peptides of
70 amino acids in length can be chemically ligated to engineer a
much longer fusion peptide. Folding and solubility may, however, be
a concern as the length is increased. In one aspect, the vaccine
includes a neoantigen that is selected based upon a predicted
affinity for binding to the patient's MHC. As discussed above, the
patient's neoantigen peptides that have been identified as having
the highest binding affinity to the MHC proteins can be combined
into one or more fusion peptides, or the various neoantigen
peptides can be incorporated into the vaccine individually. It is
believed that a synergistic or additive effect will come from
having multiple different neoantigen mutation peptides, regardless
of whether or not the peptides are individually incorporated into a
liposome vaccine or as one fusion peptide, that will elicit a more
effective immune response to the targeted cancer of the patient. As
shown in FIG. 1, the vaccine includes at least one fusion peptide,
together with at least one immunostimulant, into a liposome.
[0019] In yet another aspect, the vaccine includes a lipid molecule
that is selected from the group consisting of phospholipids,
glycolipids, cholesterol, and derivatives of the lipid molecules.
In still yet other aspects, the lipid molecule is a saturated or an
unsaturated phospholipid or glycolipid, or any combination of such
molecules. In other aspects, the lipid molecule can include 1,2
dimyristoylphosphatidyl choline, 1,2 dipalmitoylphosphatidyl
choline, 1,2 dimyristoylphosphatidyl glycerol, cholesterol,
cholesterol hemisuccinate and other derivatives of the above. The
lipid molecule further includes lipid bilayers. In certain aspects,
the neoantigen is engineered to include the tumor-associated
mutation peptide that also includes an attachment element, and the
attachment element is a transmembrane domain attachment element, a
fatty acid linker, a tryptophan attachment element, a combination
thereof, or other attachment elements that integrate or anchor
themselves, along with the neoantigen, into or onto the lipid
bilayers as shown in FIG. 2. The transmembrane domain attachment
element includes a peptide sequence of 20 to 30 amino acids.
Transmembrane domains generally consist of alpha helical peptide
segments that span the entire lipid bilayer and typically consist
of a minimum of 20-25 amino acids. In other aspects, the
transmembrane domain includes peptide sequences of 25-30, 30-35, or
35-40 amino acids. In still other aspects, the transmembrane domain
includes peptide sequences of 25, 30, 35, 40, and 45 amino acids.
In other aspects, the transmembrane domain includes peptide
sequences of at least 20 amino acids in length. In yet other
aspects, the transmembrane domain includes peptide sequences of 30
to 70 amino acids in length. In other aspects, the transmembrane
domain may include additional spacer amino acids or may include
charged amino acids to anchor the transmembrane domain attachment
element on the opposite side of the lipid bilayer. In still other
aspects, the neoantigen is engineered to include the
tumor-associated mutation peptide that also includes an attachment
element. Attachment elements do not generally span the entire
phospholipid bilayer if the attachment element consists of 20 or
less amino acids.
[0020] Other aspects of the present disclosure are directed to a
therapeutic cancer vaccine that includes 1) at least one
neoantigen, wherein the neoantigen includes a tumor-associated
genetic mutation peptide, and wherein the tumor-associated genetic
mutation peptide further includes an attachment element selected
from the group consisting of a transmembrane domain, a fatty acid
linker, and a tryptophan attachment element, wherein the attachment
element comprises a peptide sequence of 1 to 30 amino acids; 2) at
least one immunostimulant; and 3) at least one type of lipid
molecule. In another aspect, the transmembrane domain attachment
element is extended at its amino terminal end with at least one
amino acid. The amino terminal end of the transmembrane domain will
link to the neoantigen peptide and may have additional "spacer"
amino acids in between. In yet another aspect, the transmembrane
domain attachment element is extended at a carboxy terminal end
with at least one amino acid. In yet another aspect, the
transmembrane domain attachment element is:
NH.sup.2-TTEYQVAVAGIVFLLISVLLLSGLTWQRRQRK-COOH (SEQ ID NO:1).
According to one aspect, the peptide containing the genetic
mutation is preceded during automated synthesis by a hydrophobic
peptide sequence of 20 to 30 amino acids. In one aspect, the
synthesized peptide includes 1-50 amino acids. In another aspect,
the synthesized peptide includes 1-70 amino acids. In one aspect,
the engineered peptide acts as a membrane attachment element or
transmembrane anchoring element for incorporation into the lipid
bilayers of the liposomes comprising the vaccine. According to one
aspect, the engineered neoantigen can have the following general
structure:
[0021] [NH.sub.2-Neoantigen Sequence-Attachment Element-COOH]
[0022] In yet another embodiment, the vaccine includes a neoantigen
that further includes a tumor-associated genetic mutation peptide,
wherein the tumor-associated genetic mutation peptide further
includes an attachment element, wherein the attachment element is a
fatty acid linker, and the fatty acid linker is attached to the
neoantigen through an epsilon amino group, and the epsilon amino
group is part of a lysine residue. For example, the neoantigen
peptide can be extended at its carboxy terminus with a lysine
residue to which a fatty acid is attached through the lysine
epsilon amino group. The fatty acid integrates with the lipid
bilayers as shown in FIG. 3. It is noted that the fatty acid
conjugation to the neoantigen by an epsilon amino group is a single
example of chemically linking fatty acids to peptides. Accordingly,
one skilled in the art would understand that there are other
mechanisms to provide such a linkage. For example, peptides with
extensions of one or more amino acids to the neoantigen sequence at
its N or C-terminus may be conjugated to a fatty acid. Further,
more than one fatty acid may be conjugated to the extension of the
neoantigen sequence.
[0023] In another embodiment, wherein the vaccine further includes
a neoantigen, wherein the neoantigen further includes a
tumor-associated genetic mutation peptide, and wherein the
tumor-associated genetic mutation peptide further includes an
attachment element, wherein the attachment element is a tryptophan
attachment element, and the tryptophan attachment element includes
at least one tryptophan amino acid as shown in FIG. 4. According to
one aspect, the amino acid extension contains a tryptophan, two or
more tryptophans, or is entirely composed of tryptophan. In one
aspect, the tryptophan attachment element is NRWIT (SEQ ID NO:2).
In another aspect, the membrane attachment element is any sequence
of amino acids having an affinity for lipid membranes. According to
another aspect, the amino acid attachment element, or transmembrane
attachment or anchoring element is added to synthesis of the
neoantigen peptide at the amino and/or carboxy terminal end and/or
at any position within the neoantigen peptide sequence. In another
embodiment, the membrane attachment element is a synthetic polymer
able to be attached during or after synthesis of the neoantigen
peptide and added at the amino and/or carboxy terminal end and/or
at any position within the neoantigen peptide sequence.
[0024] In yet another embodiment, the therapeutic cancer vaccine
that includes 1) at least one neoantigen, wherein the neoantigen
includes a tumor-associated genetic mutation peptide, and wherein
the tumor-associated genetic mutation peptide further includes a
transmembrane domain attachment element, wherein the transmembrane
domain attachment element includes a peptide sequence of 20 to 30
amino acids; 2) at least one immunostimulant; 3) and at least one
type of lipid molecule; and the vaccine further includes either 4)
a second antigen that includes at least one second tumor-associated
genetic mutation peptide, wherein the second tumor-associated
genetic mutation peptide further includes an attachment element,
wherein the attachment element is a fatty acid linker, and wherein
the fatty acid linker includes one or more amino acids chemically
modified to include at least one hydrophobic peptide that
integrates with the lipid bilayers; or the vaccine further includes
5) a third neoantigen, wherein the third neoantigen includes a
tumor-associated genetic mutation peptide, and wherein the
tumor-associated genetic mutation peptide further includes an
attachment element, wherein the attachment element is a tryptophan
attachment element, and the tryptophan attachment element includes
at least one tryptophan amino acid. In yet another embodiment, the
vaccine includes at least one of both the second and the third
neoantigens described above.
[0025] In another aspect, the peptide containing the genetic
mutation is synthesized without altering the basic tumor neoantigen
sequence, using one or more amino acids that are chemically
modified with the addition of one or more hydrophobic entities
known in the art to interact with and/or integrate into lipid
membranes. In yet another aspect, the chemical modification
consists of one or more fatty acid or phospholipid molecules to
improve the integration of the peptide into the lipid bilayers of
the liposomes comprising the vaccine. In another aspect, the
chemical modification includes any chemical having an affinity for
lipid membranes. In still another aspect, the amino and/or carboxy
terminal ends of the neoantigen peptide are extended with one or
more amino acids, at least one of which has a chemical modification
known to interact with lipid membranes. In other aspects, the
chemical modification is added after synthesis of the neoantigen
peptide. In other aspects, the chemical modification is added at
the same time as the synthesis of the neoantigen peptide.
[0026] In another embodiment, the present disclosure is directed to
a therapeutic cancer vaccine that includes at least one neoantigen,
wherein the neoantigen includes a tumor-associated genetic mutation
peptide, and wherein the tumor-associated genetic mutation peptide
further includes an attachment element selected from the group
consisting of a transmembrane domain, a fatty acid linker, and a
tryptophan attachment element, wherein the attachment element
comprises a peptide sequence of 1 to 30 amino acids; at least one
immunostimulant; and at least one type of lipid molecule. In one
aspect, the immunostimulant includes one or more cytokines, such as
interleukin 2 (IL-2), GM-CSF, M-CSF, and interferon-gamma
(IFN-.gamma.), one or more Toll-like Receptor agonists and/or
adjuvants, such as monophosphoryl lipid A, lipid A, muramyl
dipeptide (MDP) lipid conjugate and double stranded RNA, or one or
more costimulatory membrane proteins and/or cell adhesion proteins,
such CD80, CD86 and ICAM-1, or any combination of the above. In one
aspect, the vaccine includes an immunostimulant that is a cytokine
selected from the group consisting of interleukin 2 (IL-2), GM-CSF,
M-CSF, and interferon-gamma (IFN-.gamma.). In another aspect, the
vaccine includes an immunostimulant that is a Toll-like Receptor
agonist and/or adjuvant selected from the group consisting of
monophosphoryl lipid A, lipid A, and muramyl dipeptide (MDP) lipid
conjugate and double stranded RNA. In yet another aspect, the
vaccine includes an immunostimulant that is a costimulatory
membrane protein and/or cell adhesion protein selected from the
group consisting of CD80, CD86, and ICAM-1.
[0027] Other aspects of the disclosure relate to a method of
treating cancer in a patient by administering to the patient an
effective amount of a therapeutic cancer vaccine that includes at
least one neoantigen, wherein the neoantigen includes a
tumor-associated genetic mutation peptide, and wherein the
tumor-associated genetic mutation peptide further includes 1) an
attachment element selected from the group consisting of a
transmembrane domain, a fatty acid linker, and a tryptophan
attachment element, wherein the attachment element comprises a
peptide sequence of 1 to 30 amino acids; 2) at least one
immunostimulant; 3) and at least one type of lipid molecule.
[0028] The vaccine is manufactured by aseptic techniques by any
method for creating multilamellar liposomes that is readily known
to one skilled in the art. For example, the vaccine can be
manufactured by combining sterilized solutions of neoantigen
peptides, immunostimulants, and lipids, and subjecting the
combination to repeated cycles of freezing, thawing, and sonication
within a sterilized reaction vessel. The vaccine can also be made
by adding a sterilized solution of neoantigen peptides and a
sterilized solution of immunostimulants, sequentially or
simultaneously, to a sterilized solution of lipids in the form of
small unilamellar vesicles, all within a sterilized reaction
vessel. The reaction vessel is incubated at a prescribed
temperature for a prescribed length of time during which
multi-lamellar liposomes form incorporating the peptides and
immunostimulants within and between the lipid bilayers. Once
manufactured, the vaccine is aseptically divided into sterilized
vials which are sealed and stored for use.
[0029] Other aspects of the disclosure relate to a method of cancer
treatment by inducing humoral and cellular immune responses against
cancer cells in a patient that includes the steps of genetically
sequencing a tumor-tissue sample from a patient to identify a
plurality of neoantigens present in the tumor-tissue sample, and
wherein the neoantigens include tumor-associated genetic mutation
peptides. At least one neoantigen is selected based upon a
predicted affinity for binding to the MHC of the patient, wherein
the corresponding MHC proteins on the surface of the
antigen-presenting cells of the patient bind and present the
neoantigens to helper and effector T cells of the patient's immune
system. Resultantly, the patient's immune system then directs an
effective immune response to the cancer cells in a patient. As
discussed above, to identify those neoantigens that have the
highest affinity for binding to the particular patient's MHC, a
genetic sequencing (i.e., nucleic acid) is performed on the biopsy
material to identify the full range of genetic mutations (i.e.,
neoantigens), present in the proteins associated with the biopsy
material. Again, the genetic sequencing of a patient's cancer
sample may be performed by techniques readily known to one skilled
in the art or by using standard procedures, as described above.
[0030] According to one aspect, the patient is then administered a
therapeutically effective amount of a vaccine that includes the
neoantigen peptides identified as those with the highest binding
affinity for the patient's MHC proteins. According to another
aspect, the neoantigen, which includes a tumor-associated genetic
mutation peptide, further includes an attachment element selected
from the group consisting of a transmembrane domain, a fatty acid
linker, and a tryptophan attachment element (or combinations
thereof), wherein the attachment element comprises a peptide
sequence of 1 to 30 amino acids; at least one immunostimulant; and
at least one type of lipid molecule.
[0031] Other aspects of the disclosure relate to method of cancer
treatment by inducing humoral and cellular immune responses against
cancer cells in a patient that includes genetically sequencing a
tumor-tissue sample from a patient to identify a plurality of
neoantigens present in the tumor-tissue sample, and wherein the
neoantigens comprise tumor-associated genetic mutation peptides,
then selecting at least one neoantigen based upon a predicted
affinity for binding to a Major Histocompatibility Complex (MHC) of
the patient, wherein MHC proteins on a surface of
antigen-presenting cells of the patient bind and present the
neoantigens to helper and effector T cells of an immune system of
the patient, wherein an immune response is directed to the cancer
cells in a patient, subsequently administering to the patient a
therapeutically effective amount of a vaccine that includes the
neoantigen, wherein the neoantigen includes a tumor-associated
genetic mutation peptide, and wherein the tumor-associated genetic
mutation peptide further includes an attachment element selected
from the group consisting of a transmembrane domain, a fatty acid
linker, and a tryptophan attachment element, wherein the attachment
element comprises a peptide sequence of 1 to 30 amino acids; at
least one immunostimulant; and at least one type of lipid molecule.
According to other aspects, the method of cancer treatment by
inducing humoral and cellular immune responses against cancer cells
in a patient may include any of the vaccines, components of the
vaccines, or combinations of the vaccines described above.
[0032] According to another aspect, the method of cancer treatment
by inducing humoral and cellular immune responses against cancer
cells in a patient may include administering the vaccine to the
patient at a prescribed dose by intradermal, subcutaneous,
intramuscular, intranodal, or intra-tumoral injection, or any
combination thereof. According to another aspect, the patient
receives multiple vaccine injections at separate sites or the
patient may receive multiple vaccine injections at the same site.
According to yet another aspect, the patient receives multiple
vaccinations at prescribed time intervals. According to other
aspects, the time intervals may include time intervals such as
every 1, 2, 3, or 4 weeks or every 2 to 4 weeks.
[0033] An effective therapeutic vaccine and related method of
treatment by inducing humoral and cellular immune responses against
malignant cells is described in this disclosure. The vaccine
comprises a delivery system that incorporates at least one peptide
whose sequence encompasses a patient-specific genetic mutation
associated with malignancy (neoantigen), at least one
immunostimulant, and at least one type of lipid molecule. Such a
combination provides a novel and more potent vaccine formulation
for treating cancer. The disclosed vaccine and related method
provide a synergistic effect that induces a more effective immune
response, uniquely tailored for an individual patient's tumor
cells, directed against the patient's malignant cells.
[0034] As used herein, the terms "protein" and "polypeptide" and
"peptide" are used interchangeably herein to designate a series of
amino acid residues, connected to each other by peptide bonds
between the alpha-amino and carboxy groups of adjacent residues.
The terms "protein," "peptide," and "polypeptide" refer to a
polymer of amino acids, including modified amino acids (e.g.,
phosphorylated, glycated, glycosylated, etc.) and amino acid
analogs, regardless of its size or function. "Protein" and
"polypeptide" are often used in reference to relatively large
polypeptides, whereas the term "peptide" is often used in reference
to small polypeptides, but usage of these terms in the art
overlaps. The terms "protein" and "peptide" are used
interchangeably herein when referring to a gene product and
fragments thereof. Thus, exemplary polypeptides, peptides, or
proteins include gene products, naturally occurring proteins,
homologs, orthologs, paralogs, fragments and other equivalents,
variants, fragments, and analogs of the foregoing.
[0035] An "antigen" is a substance that upon introduction into a
vertebrate animal stimulates the production of antibodies or
cell-mediated immune responses. A "tumor-associated antigen" is a
molecule produced by or associated with malignant cells, but is not
normally expressed, or expressed at very low levels, by a
non-malignant cell. A "neoantigen" is class of tumor antigens that
arises from tumor-specific mutations in an expressed protein.
[0036] Proteins or molecules of the "major histocompatibility
complex (MHC)" are proteins capable of binding peptides that result
from the proteolytic cleavage of protein antigens and representing
potential T-cell epitopes, transporting them to the cell surface
and presenting them there to specific cells, in particular
cytotoxic T-lymphocytes or T-helper cells. The MHC of an
individual's genome comprises the genetic region whose gene
products expressed on the cell surface are important for binding
and presenting endogenous and/or foreign antigens for regulating
immune response. The major histocompatibility complex is classified
into two gene groups coding for different proteins, namely
molecules of MHC class I and molecules of MHC class II. The
molecules of the two MHC classes are specialized for different
antigen sources. The molecules of MHC class I present endogenously
synthesized antigens, for example viral proteins and tumor
antigens.
[0037] A "lipid" is any of a group of biochemicals which is
variably soluble in organic solvents, such as alcohol. Examples of
lipids include phospholipids, fats, waxes, and sterols, such as
cholesterol. A "liposome" is a microscopic vesicle that consists of
one or more lipid bilayers surrounding an aqueous compartment.
[0038] A "vaccine" is a material that is administered to a
vertebrate host to immunize the host against the same material.
Typically, a vaccine comprises material associated with a disease
state, such as viral infection, bacterial infection, and various
malignancies. A "therapeutic vaccine" is a vaccine administered to
a vertebrate host which already has the disease being targeted and
is designed to induce an immune response that causes disease
regression, delayed disease progression, prolonged disease-free
survival and/or overall survival.
[0039] An "immunostimulant" is any substance that stimulates the
immune system by inducing activation or increasing activity of any
of the immune system's components.
[0040] An "amino acid sequence" may be determined directly for a
protein or peptide, or inferred from the corresponding nucleic acid
sequence.
[0041] A "nucleic acid" or "nucleic acid sequence" may be any
molecule, preferably a polymeric molecule, incorporating units of
ribonucleic acid, deoxyribonucleic acid or an analog thereof. The
nucleic acid can be either single-stranded or double-stranded. A
single-stranded nucleic acid can be one nucleic acid strand of a
denatured double-stranded DNA. Alternatively, it can be a
single-stranded nucleic acid not derived from any double-stranded
DNA. In one aspect, the nucleic acid can be DNA. In another aspect,
the nucleic acid can be RNA. Suitable nucleic acid molecules are
DNA, including genomic DNA or cDNA. Other suitable nucleic acid
molecules are RNA, including mRNA.
[0042] Definitions of common terms in cell biology and molecular
biology can be found in The Encyclopedia of Molecular Biology,
published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);
Benjamin Lewin, Genes X, published by Jones & Bartlett
Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8) and Current Protocols in Protein Sciences 2009,
Wiley Intersciences, Coligan et al., eds.
[0043] Unless otherwise stated, the present disclosure is performed
using standard procedures, as described, for example in Sambrook et
al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001);
Davis et al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1995); or Methods in Enzymology:
Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A.
R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); and
Current Protocols in Protein Science (CPPS) (John E. Coligan, et.
al., ed., John Wiley and Sons, Inc.), which are all incorporated by
reference herein in their entireties.
[0044] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While specific embodiments of, and examples for,
the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize. For example, while method steps or functions are
presented in a given order, alternative embodiments may perform
functions in a different order, or functions may be performed
substantially concurrently. The teachings of the disclosure
provided herein can be applied to other procedures or methods as
appropriate. The various embodiments described herein can be
combined to provide further embodiments. Aspects of the disclosure
can be modified, if necessary, to employ the compositions,
functions and concepts of the above references and application to
provide yet further embodiments of the disclosure. Moreover, due to
biological functional equivalency considerations, some changes can
be made in protein structure without affecting the biological or
chemical action in kind or amount. These and other changes can be
made to the disclosure in light of the detailed description. All
such modifications are intended to be included within the scope of
the appended claims.
[0045] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
[0046] The following examples are set forth as being representative
of the present disclosure. These examples are not to be construed
as limiting the scope of the present disclosure as these and other
equivalent embodiments will be apparent in view of the present
disclosure, figures and accompanying claims.
Example I
Transmembrane Domain Attachment Element
[0047] Using a solid phase automated peptide synthesizer,
manufacture the peptide corresponding to the neoantigen amino acid
sequence together with a carboxy-terminal extension corresponding
to a transmembrane domain (TMID). Begin the synthesis at the
carboxy-terminal end of the TMID and proceed to the amino terminus,
followed by the sequence corresponding to the neoantigen peptide.
An example of a transmembrane domain is:
TABLE-US-00001 Example Transmembrane Domain (SEQ ID NO: 1)
NH.sub.2-Neoantigen Sequence-TTEYQVAVAGIVFLLISVLLLSGLTW
QRRQRK-COOH
[0048] When synthesis is complete, chemically remove the peptide
from the solid phase resin and lyopholize. Reconstitute the peptide
at a concentration of 1 to 10 mg/mL in an appropriate buffer, such
as 0.9% saline containing 0.5% diheptanoyl phosphatidylcholine,
DHPC.
[0049] Prepare small unilamellar vesicles (SUV) of a phospholipid,
such as dimyristoyl phosphatidylcholine (DMPC), or a mixture of
lipids (phospholipids, cholesterol, etc.) by high pressure
homogenization. Set aside at 37.degree. C. until used.
[0050] Prepare a solution of immune stimulant or mixture of immune
stimulants in 0.9% saline. For this example, the immune stimulant
is recombinant human Interleukin 2 (IL-2) and the concentration of
the solution is 40.times.10.sup.6 IU/mL.
[0051] The final product is assembled under aseptic conditions in
an ISO 5 Biological Safety Cabinet (BCC), preferably within an ISO
7 Clean Room. For this example, the reaction vessel consists of a
20 mL glass vial with a magnetic stir bar, both of which have been
sterilized by dry heat sterilization. The reaction vessel is placed
on a magnetic stirrer containing a heating element and maintained
at 25.degree. C. during the preparation of the vaccine. During the
addition of the components to the reaction vessel, the liquid
contents within the vessel are mixed continuously using the stir
bar. Using a sterile disposable polypropylene syringe and a sterile
0.2 .mu.m filter, 6.4 mL of the DMPC SUV is filtered directly into
the glass reactor vessel. Using a separate syringe and 0.2 .mu.m
filter, 6.4 mL of neoantigen solution (1 to 10 mg/mL protein) is
filtered into the reaction vessel. With a third syringe and 0.2
.mu.m filter, 2.0 mL of diluted IL-2 (80.times.10.sup.6 IU total)
is filtered into the reaction vessel. The volume of the reaction
mixture is 14.8 mL. Once all the components have been added and
mixed, the reaction vessel is sealed with a sterile rubber septum
and metal ring crimp. It is then transferred to an incubator at
19.+-.2.degree. C. for 2 to 24 hrs.
[0052] After the incubation period is complete, the reaction vessel
is moved to an ISO 5 BSC where it is opened and 5.2 mL of sterile
0.9% saline is added to bring the total volume to 20 mL. The
contents are mixed briefly on a magnetic stirrer to assure
homogeneity and the product is then subdivided for final use.
[0053] Measure incorporation of the neoantigen-TMD peptide and the
immune stimulant IL-2 active components as follows:
[0054] For the Total content of the component in the final vaccine
product, dissolve the liposomes by combining a sample of vaccine
with an equal volume of 5% (w/v) of an appropriate detergent, such
as Igepal CA-630. Measure the content of IL-2 using a commercial
capture ELISA assay. Measure the content of neoantigen(s)-TMD by
running a sample on reverse phase HPLC and quantitating by using
standard curves derived with the individual purified neoantigen-TMD
peptides. For the Free content of the components, dilute a sample
of vaccine with an equal volume of 0.9% saline and centrifuge at
20,000 rcf for 15 min at 4.degree. C. Remove the supernatant
containing non-incorporated material. Assay for Free IL-2 using the
commercial capture ELISA assay and for Free neoantigen(s) using
reverse phase HPLC quantitated by standard curves.
Example II
Tryptophan Attachment Element
[0055] Using a solid phase automated peptide synthesizer,
manufacture peptide corresponding to the neoantigen amino acid
sequence together with a carboxy-terminal attachment element
containing one or more tryptophans (Trp; W). Begin the synthesis at
the carboxy-terminal end of the attachment element and proceed to
the amino terminus, followed by the sequence corresponding to the
neoantigen peptide. An example of a TRP-containing attachment
element is:
TABLE-US-00002 Example Tryptophan Attachment Element (SEQ ID NO: 2)
NH.sub.2-Neoantigen Sequence-NRWIT-COOH
[0056] When synthesis is complete, chemically remove the peptide
from the solid phase resin and lyopholize. Reconstitute the peptide
at a concentration of 1 to 10 mg/mL in an appropriate buffer, such
as 0.9% saline.
[0057] Prepare small unilamellar vesicles (SUV) of a phospholipid,
such as dimyristoyl phosphatidylcholine (DMPC), or a mixture of
lipids (phospholipids, cholesterol, etc.) by high pressure
homogenization. Set aside at 37.degree. C. until used.
[0058] Prepare a solution immune stimulant or mixture of immune
stimulants in 0.9% saline. For this example, the immune stimulant
is recombinant human Interleukin 2 (IL-2) and the concentration of
the solution is 40.times.10.sup.6 IU/mL.
[0059] The final product is assembled under aseptic conditions in
an ISO 5 Biological Safety Cabinet (BCC), preferably within an ISO
7 Clean Room. For this example, the reaction vessel consists of a
20 mL glass vial with a magnetic stir bar, both of which have been
sterilized by dry heat sterilization. The reaction vessel is placed
on a magnetic stirrer containing a heating element and maintained
at 25.degree. C. during the preparation of the vaccine. During the
addition of the components to the reaction vessel, the liquid
contents within the vessel are mixed continuously using the stir
bar. Using a sterile disposable polypropylene syringe and a sterile
0.2 .mu.m filter, 6.4 mL of the DMPC SUV is filtered directly into
the glass reactor vessel. Using a separate syringe and 0.2 .mu.m
filter, 6.4 mL of neoantigen solution (1 to 10 mg/mL protein) is
filtered into the reaction vessel. With a third syringe and 0.2
.mu.m filter, 2.0 mL of diluted IL-2 (80.times.10.sup.6 IU total)
is filtered into the reaction vessel. The volume of the reaction
mixture is 14.8 mL. Once all components have been added and mixed,
the reaction vessel is sealed with a sterile rubber septum and
metal ring crimp. It is then transferred to an incubator at
19.+-.2.degree. C. for 2 to 24 hrs.
[0060] After the incubation period is complete, the reaction vessel
is moved to an ISO 5 BSC where it is opened and 5.2 mL of sterile
0.9% saline is added to bring the total volume to 20 mL. The
contents are mixed briefly on a magnetic stirrer to assure
homogeneity and the product is then subdivided for final use.
Measure the incorporation of the neoantigen peptide(s) and IL-2 as
in Example I above.
Sequence CWU 1
1
2132PRTArtificialtransmembrane domain attachment element 1Thr Thr
Glu Tyr Gln Val Ala Val Ala Gly Ile Val Phe Leu Leu Ile 1 5 10 15
Ser Val Leu Leu Leu Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys 20
25 30 25PRTArtificialtryptophan attachment element 2Asn Arg Trp Ile
Thr 1 5
* * * * *