U.S. patent application number 17/093440 was filed with the patent office on 2021-04-29 for methods and compositions for the treatment of melanoma.
The applicant listed for this patent is TAIGA BIOTECHNOLOGIES, INC.. Invention is credited to Gregory Alan BIRD, Yosef REFAELI, Brian C. TURNER.
Application Number | 20210121550 17/093440 |
Document ID | / |
Family ID | 1000005329130 |
Filed Date | 2021-04-29 |
![](/patent/app/20210121550/US20210121550A1-20210429\US20210121550A1-2021042)
United States Patent
Application |
20210121550 |
Kind Code |
A1 |
REFAELI; Yosef ; et
al. |
April 29, 2021 |
METHODS AND COMPOSITIONS FOR THE TREATMENT OF MELANOMA
Abstract
Provided herein are methods and compositions for the treatment
of melanoma using anti-tumor immune cells treated with a PTD-MYC
fusion protein (e.g., an HIV TAT-MYC fusion protein).
Inventors: |
REFAELI; Yosef; (Denver,
CO) ; TURNER; Brian C.; (Denver, CO) ; BIRD;
Gregory Alan; (Littleton, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TAIGA BIOTECHNOLOGIES, INC. |
Aurora |
CO |
US |
|
|
Family ID: |
1000005329130 |
Appl. No.: |
17/093440 |
Filed: |
November 9, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16184086 |
Nov 8, 2018 |
10864259 |
|
|
17093440 |
|
|
|
|
15668451 |
Aug 3, 2017 |
10149898 |
|
|
16184086 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/39 20130101;
A61K 39/0011 20130101; A61K 39/001152 20180801; A61K 2039/515
20130101; C12N 2510/00 20130101; C12N 5/0638 20130101; C12N
2501/606 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/39 20060101 A61K039/39; C12N 5/0783 20060101
C12N005/0783 |
Claims
1.-20. (canceled)
21. A method for preparing modified immune cells for melanoma
therapy, comprising contacting one or more immune cells in vitro
with a MYC fusion polypeptide, wherein the immune cells are from a
donor that has been exposed to one or more tumor antigens and
wherein the MYC fusion peptide comprises (i) a protein transduction
domain; (ii) a MYC polypeptide sequence and are reactive to a
tumor-specific antigen.
22. The method of claim 21, wherein the one or more modified immune
cells are derived from primary immune cells isolated from a subject
having melanoma.
23. The method of claim 21, further comprising expanding the
primary immune cells in vitro prior to contacting with the MYC
fusion peptide.
24. The method of claim 21, further comprising expanding the
primary immune cells following contacting with the MYC fusion
peptide.
25. The method of claim 21, wherein the cells are expanded using an
anti-CD3 antibody or irradiated allogenic feeder cells.
26. The method of claim 21, wherein the cells are expanded in the
presence of an exogenous cytokine.
27. The method of claim 26, wherein the cytokine is
interleukin-2.
28. The method of claim 21, wherein the MYC fusion peptide
translocates to the nucleus of the immune cell.
29. The method of claim 21, wherein the MYC fusion peptide exhibits
a biological activity of MYC.
30. The method of claim 21, wherein the MYC fusion peptide further
comprises one or more molecules that link the protein transduction
domain and the MYC polypeptide.
31. The method of claim 21, wherein the MYC fusion peptide
comprises a MYC fusion peptide with the following general
structure: protein transduction domain-X-MYC sequence, wherein
--X-- is molecule that links the protein transduction domain and
the MYC sequence.
32. The method of claim 21, wherein the protein transduction domain
sequence is a TAT protein transduction domain sequence.
33. The method of claim 32, wherein the TAT protein transduction
domain sequence is selected from the group consisting of TAT[48-57]
and TAT[57-48].
34. The method of claim 21, wherein the MYC fusion peptide
comprises SEQ ID NO: 1.
35. The method of claim 21, wherein the MYC fusion peptide is
acetylated.
36. The method of claim 21, wherein the one or more modified immune
cells have antitumor activity.
37. The method of claim 21, wherein the one or more modified immune
cells have antitumor activity against melanoma cells in the
subject.
38. The method of claim 21, wherein the one or more modified immune
cells comprise one or more anergic immune cells.
39. The method of claim 21, wherein the one or more immune cells
comprises one or more lymphocytes.
40. The method of claim 39, wherein the one or more lymphocytes
comprise a T cell, a B cell, an NK, or any combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is continuation of U.S. patent application
Ser. No. 16/184,086, filed on Nov. 8, 2018, which is a division of
U.S. patent application Ser. No. 15/668,451, filed on Aug. 3, 2017,
which is related to International Application No.
PCT/US2017/045336, filed on Aug. 3, 2017. Each of which is hereby
incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing, which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 6, 2020, is named 106417-0518 Sequence Listing.txt and is
21,978 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Adoptive cell transfer (ACT) is a form of immunotherapy that
involves the transfer of immune cells with antitumor activity into
patients. ACT typically involves isolation of lymphocytes with
antitumor activity from a patient, culturing the lymphocytes in
vitro to expand the population, and then infusing the lymphocytes
into the cancer-bearing host. Lymphocytes used for adoptive
transfer can either be derived from the stroma of resected tumors
(e.g., tumor infiltrating lymphocytes), from the lymphatics or
lymph nodes, or from the blood. In some cases, the isolated
lymphocytes are genetically engineered to express antitumor T cell
receptors (TCRs) or chimeric antigen receptors (CARs). The
lymphocytes used for infusion can be isolated from a donor
(allogeneic ACT), or from the cancer-bearing host (autologous
ACT).
SUMMARY OF THE INVENTION
[0004] Provided herein, in certain embodiments, are methods for
adoptive cell transfer for the treatment of melanoma. In some
embodiments, provided are methods for the treatment of melanoma in
a subject comprising administering a therapeutically effective
amount of immune cells having antitumor activity to the subject,
wherein the immune cells are contacted with a protein transduction
domain (PTD)-MYC fusion polypeptide prior to administration to the
subject. In some embodiments, the immune cells comprise one or more
lymphocytes. In some embodiments, the one or more lymphocytes
comprise T cells and/or B cells. In some embodiments, the one or
more lymphocytes comprise tumor-infiltrating lymphocytes. In some
embodiments, the melanoma is a metastatic melanoma. In some
embodiments, the melanoma is a superficial spreading melanoma, a
nodular melanoma, a lentigo maligna melanoma, or an acral melanoma.
In some embodiments, the immune cells are obtained from a donor
subject having melanoma. In some embodiments, donor subject and the
subject receiving the immune cells are the same (i.e., autologous
ACT). In some embodiments, donor subject and the subject receiving
the immune cells are different (i.e., allogeneic ACT).
[0005] In some embodiments, the PTD-MYC fusion polypeptide
comprises: (i) an HIV TAT protein transduction domain; and (ii) a
MYC polypeptide sequence. In some embodiments, the PTD-MYC fusion
polypeptide translocates to the nucleus of the immune cell. In some
embodiments, the PTD-MYC fusion polypeptide exhibits a biological
activity of MYC, such as the activation of MYC target genes. In
some embodiments, the fusion peptide comprises SEQ ID NO: 1.
[0006] Described herein, in certain embodiments are compositions
comprising (a) a MYC fusion peptide, comprising (i) a protein
transduction domain; (ii) a MYC polypeptide sequence; and (b) one
or more primary immune cells isolated from a donor subject that has
a melanoma tumor, wherein the one or more primary immune cells are
reactive against a melanoma-specific antigen. In some embodiments,
the MYC fusion peptide translocates to the nucleus of the one or
more primary immune cells. In some embodiments, the MYC fusion
peptide exhibits a biological activity of MYC. In some embodiments,
the MYC fusion peptide further comprises one or more molecules that
link the protein transduction domain and the MYC polypeptide. In
some embodiments, the MYC fusion peptide comprises a MYC fusion
peptide with the following general structure:
[0007] protein transduction domain-X-MYC sequence,
[0008] wherein --X-- is molecule that links the protein
transduction domain and the MYC sequence. In some embodiments, the
protein transduction domain sequence is a TAT protein transduction
domain sequence. In some embodiments, the TAT protein transduction
domain sequence is selected from the group consisting of TAT[48-57]
and TAT[57-48]. In some embodiments, the MYC fusion peptide
comprises SEQ ID NO: 1. In some embodiments, the MYC fusion peptide
is acetylated. In some embodiments, the one or more immune cells
have antitumor activity against melanoma cells. In some
embodiments, the one or more immune cells comprises one or more
lymphocytes. In some embodiments, the one or more lymphocytes
comprises a T cell, a B cell, an NK cell, or any combination
thereof. In some embodiments, the T cell is selected from the group
consisting of naive T cells, CD4+ T cells, CD8+ T cells, memory T
cells, activated T cells, anergic T cells, tolerant T cells,
chimeric T cells, and antigen-specific T cells. In some
embodiments, the B cells are selected from the group consisting of
naive B cells, plasma B cells, activated B cells, memory B cells,
anergic B cells, tolerant B cells, chimeric B cells, and
antigen-specific B cells. In some embodiments, the one or more
lymphocytes is a tumor-infiltrating lymphocyte, T-cell receptor
modified lymphocyte, or a chimeric antigen receptor modified
lymphocyte. In some embodiments, the tumor-infiltrating lymphocyte
has a CD8+CD25+ signature. In some embodiments, the
tumor-infiltrating lymphocyte has a CD4+CD25+ signature. In some
embodiments, the one or more immune cells comprises a detectable
moiety.
[0009] Described herein, in certain embodiments are methods for
treating a melanoma in a subject, comprising administering one or
more modified immune cells to the subject in need thereof, wherein
the one or more modified immune cells comprise a MYC fusion peptide
comprising (i) a protein transduction domain; (ii) a MYC
polypeptide sequence and are reactive to a tumor-specific antigen.
In some embodiments, the one or more modified immune cells are
derived from primary immune cells isolated from the subject. In
some embodiments, the one or more modified immune cells are derived
from primary immune cells isolated from a separate donor subject
having the same type of melanoma. In some embodiments, the one or
more modified immune cells are prepared by contacting the primary
immune cells in vitro with the MYC fusion peptide following
isolation. In some embodiments, the methods further comprise
expanding the primary immune cells in vitro prior to contacting
with the MYC fusion peptide. In some embodiments, the methods
further comprise expanding the primary immune cells following
contacting with the MYC fusion peptide. In some embodiments, the
cells are expanded using an anti-CD3 antibody. In some embodiments,
the cells are expanded using an irradiated allogenic feeder cells.
In some embodiments, the cells are expanded in the presence of an
exogenous cytokine. In some embodiments, the cytokine is
interleukin-2. In some embodiments, the MYC fusion peptide
translocates to the nucleus of the immune cell. In some
embodiments, the MYC fusion peptide exhibits a biological activity
of MYC. In some embodiments, the MYC fusion peptide further
comprises one or more molecules that link the protein transduction
domain and the MYC polypeptide. In some embodiments, the MYC fusion
peptide comprises a MYC fusion peptide with the following general
structure:
[0010] protein transduction domain-X-MYC sequence,
[0011] wherein --X-- is molecule that links the protein
transduction domain and the MYC sequence. In some embodiments, the
protein transduction domain sequence is a TAT protein transduction
domain sequence. In some embodiments, the TAT protein transduction
domain sequence is selected from the group consisting of TAT[48-57]
and TAT[57-48]. In some embodiments, the MYC fusion peptide
comprises SEQ ID NO: 1. In some embodiments, the MYC fusion peptide
is acetylated. In some embodiments, the one or more modified immune
cells have antitumor activity against melanoma cells in the
subject. In some embodiments, the one or more modified immune cells
have antitumor activity against melanoma cells in the subject. In
some embodiments, the one or more modified immune cells comprise
one or more anergic immune cells. In some embodiments, the one or
more immune cells comprises one or more lymphocytes. In some
embodiments, the one or more lymphocytes comprises a T cell, a B
cell, an NK, or any combination thereof. In some embodiments, the T
cell is selected from the group consisting of naive T cells, CD4+ T
cells, CD8+ T cells, memory T cells, activated T cells, anergic T
cells, tolerant T cells, chimeric T cells, and antigen-specific T
cells. In some embodiments, the B cells are selected from the group
consisting of naive B cells, plasma B cells, activated B cells,
memory B cells, anergic B cells, tolerant B cells, chimeric B
cells, and antigen-specific B cells. In some embodiments, the one
or more lymphocytes is a tumor-infiltrating lymphocyte, T-cell
receptor modified lymphocyte, or a chimeric antigen receptor
modified lymphocyte. In some embodiments, the lymphocyte has a
CD8+CD28-CD152- signature. In some embodiments, the lymphocyte has
a CD8+CD25+ signature. In some embodiments, the lymphocyte has a
CD4+CD25+ signature. In some embodiments, the methods further
comprise isolating the primary immune cells from the donor subject.
In some embodiments, the donor subject has melanoma. In some
embodiments, the one or more modified immune cells are administered
intravenously, intraperitoneally, subcutaneously, intramuscularly,
or intratumorally. In some embodiments, the methods further
comprise lymphodepleting the subject prior to administration of the
one or more modified immune cells. In some embodiments, the methods
further comprise administering a cytokine to the subject. In some
embodiments, the cytokine is administered prior to, during, or
subsequent to administration of the one or more modified immune
cells. In some embodiments, the cytokine is selected from a group
consisting of interferon .alpha., interferon .beta., interferon
.gamma., complement C5a, IL-2, TNFalpha, CD40L, IL12, IL-23, IL15,
IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3,
CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20,
CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26,
CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8,
CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1,
CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15,
CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9,
CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2. In some
embodiments, the melanoma is metastatic. In some embodiments, the
subject is a human or an animal. In some embodiments, the methods
further comprise administering an additional cancer therapy. In
some embodiments, the additional cancer therapy is selected from
among chemotherapy, radiation therapy, immunotherapy, monoclonal
antibodies, anti-cancer nucleic acids or proteins, anti-cancer
viruses or microorganisms, and any combinations thereof. In some
embodiments, the one or more modified immune cells comprises a
detectable moiety.
[0012] Also described herein, in certain embodiments are methods
for preparing modified immune cells for melanoma therapy,
comprising contacting one or more immune cells in vitro with a MYC
fusion polypeptide, wherein the immune cells are from a donor that
has been exposed to one or more tumor antigens and wherein the MYC
fusion peptide comprises (i) a protein transduction domain; (ii) a
MYC polypeptide sequence and are reactive to a tumor-specific
antigen. In some embodiments, the one or more modified immune cells
are derived from primary immune cells isolated from a subject
having melanoma. In some embodiments, the methods further comprise
expanding the primary immune cells in vitro prior to contacting
with the MYC fusion peptide. In some embodiments, the methods
further comprise expanding the primary immune cells following
contacting with the MYC fusion peptide. In some embodiments, the
cells are expanded using an anti-CD3 antibody. In some embodiments,
the cells are expanded using an irradiated allogenic feeder cells.
In some embodiments, the cells are expanded in the presence of an
exogenous cytokine. In some embodiments, the cytokine is
interleukin-2. In some embodiments, the MYC fusion peptide
translocates to the nucleus of the immune cell. In some
embodiments, the MYC fusion peptide exhibits a biological activity
of MYC. In some embodiments, the MYC fusion peptide further
comprises one or more molecules that link the protein transduction
domain and the MYC polypeptide. In some embodiments, the MYC fusion
peptide comprises a MYC fusion peptide with the following general
structure:
[0013] protein transduction domain-X-MYC sequence,
[0014] wherein --X-- is molecule that links the protein
transduction domain and the MYC sequence. In some embodiments, the
protein transduction domain sequence is a TAT protein transduction
domain sequence. In some embodiments, the TAT protein transduction
domain sequence is selected from the group consisting of TAT[48-57]
and TAT[57-48]. In some embodiments, the MYC fusion peptide
comprises SEQ ID NO: 1. In some embodiments, the MYC fusion peptide
is acetylated. In some embodiments, the one or more modified immune
cells have antitumor activity. In some embodiments, the one or more
modified immune cells have antitumor activity against melanoma
cells in the subject. In some embodiments, the one or more modified
immune cells comprise one or more anergic immune cells. In some
embodiments, the one or more immune cells comprises one or more
lymphocytes. In some embodiments, the one or more lymphocytes
comprises a T cell, a B cell, an NK, or any combination thereof. In
some embodiments, the T cell is selected from the group consisting
of naive T cells, CD4+ T cells, CD8+ T cells, memory T cells,
activated T cells, anergic T cells, tolerant T cells, chimeric T
cells, and antigen-specific T cells. In some embodiments, the B
cells are selected from the group consisting of naive B cells,
plasma B cells, activated B cells, memory B cells, anergic B cells,
tolerant B cells, chimeric B cells, and antigen-specific B cells.
In some embodiments, the one or more lymphocytes is a
tumor-infiltrating lymphocyte, T-cell receptor modified lymphocyte,
or a chimeric antigen receptor modified lymphocyte. In some
embodiments, the lymphocyte has a CD8+CD28-CD152- signature. In
some embodiments, the lymphocyte has a CD8+CD25+ signature. In some
embodiments, the lymphocyte has a CD4+CD25+ signature.
[0015] Also described herein, in certain embodiments, are
compositions comprising: (a) one or more isolated primary immune
cells that have been exposed to a melanoma cell line; and (b) a MYC
fusion peptide, comprising (i) a protein transduction domain; (ii)
a MYC polypeptide sequence; wherein the one or more primary immune
cells are reactive against a melanoma-specific antigen.
[0016] Also described herein, in certain embodiments, are any of
the aforementioned compositions for use in treating a melanoma.
Also described herein, in certain embodiments, are any of the
aforementioned compositions for use in the manufacture of a
medicament for use in treating a melanoma.
[0017] Also described herein, in certain embodiments, are methods
for increasing the efficacy of adoptive cell therapy or T-cell
therapy in a subject comprising administering any of the
aforementioned compositions.
[0018] Also described herein, in certain embodiments, are
tumor-infiltrating lymphocytes comprising a MYC fusion peptide,
comprising (i) a protein transduction domain; (ii) a MYC
polypeptide sequence. In some embodiments, the tumor-infiltrating
lymphocytes are derived from primary tumor-infiltrating lymphocytes
isolated from a subject that has cancer (e.g., melanoma).
[0019] Also described herein, in certain embodiments, are
lymphocytes comprising a chimeric antigen receptor and a MYC fusion
peptide, comprising (i) a protein transduction domain; (ii) a MYC
polypeptide sequence. In some embodiments, the lymphocytes are
derived from primary lymphocytes isolated from a subject that has
cancer (e.g., melanoma).
[0020] Also described herein, in certain embodiments, are methods
for preparing a composition for adoptive cell therapy comprising
contacting one or more primary immune cells with MYC fusion
peptide, comprising (i) a protein transduction domain; (ii) a MYC
polypeptide sequence, wherein one or more primary immune cells are
isolated from a patient having melanoma, and wherein one or more
primary immune cells are reactive to a melanoma-specific
antigen.
[0021] Also provided are kits comprising the MYC-fusion
polypeptides and/or MYC-fusion polypeptide-modified immune cells
provided herein for use in treating a melanoma. In some
embodiments, the kit comprises one for more reagents for the
detection of the administered MYC-fusion polypeptides and/or
MYC-fusion polypeptide-modified immune cells. In some embodiments,
the kit comprises cells for treatment with a MYC-fusion polypeptide
provided herein, for example, hematopoietic stem cells, donor
leukocytes, T cells, or NK cells. In some embodiments, the kit
comprises associated instructions for using the MYC-fusion
polypeptides and/or MYC-fusion polypeptide-modified immune
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 illustrates results for survival of melanoma
tumor-bearing mice following infusion of lymphocytes from
tumor-bearing donor mice treated with TAT-MYC for 1 hour. Mice were
treated with TAT-MYC lymphocytes, lymph cells treated with a
control protein or left untreated. Day of death recorded with day
of treatment as Day 0.
[0023] FIG. 2 illustrates results for survival of melanoma
tumor-bearing mice following infusion of lymphocytes from
tumor-bearing donor mice treated with TAT-MYC (repeat of experiment
shown in FIG. 1). Mice were treated with TAT-MYC lymphocytes, lymph
cells treated with a control protein or left untreated. Day of
death recorded with day of treatment as Day 0.
[0024] FIG. 3 illustrates results for survival of melanoma
tumor-bearing mice following infusion of different amounts of
lymphocytes from tumor-bearing donor mice treated with TAT-MYC.
Mice were treated with TAT-MYC lymphocytes, lymph cells treated
with a control protein or left untreated. Day of death recorded
with day of treatment as Day 0.
[0025] FIG. 4 illustrates results for survival of melanoma
tumor-bearing mice following infusion of different amounts of
lymphocytes from tumor-bearing donor mice treated with TAT-MYC.
Mice were treated with TAT-MYC lymphocytes, lymph cells treated
with a control protein or left untreated. Day of death recorded
with day of treatment as Day 0.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present disclosure is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
disclosure. All the various embodiments of the present disclosure
will not be described herein. Many modifications and variations of
the disclosure can be made without departing from its spirit and
scope, as will be apparent to those skilled in the art.
Functionally equivalent methods and apparatuses within the scope of
the disclosure, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing
descriptions. Such modifications and variations are intended to
fall within the scope of the appended claims. The present
disclosure is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
[0027] It is to be understood that the present disclosure is not
limited to particular uses, methods, reagents, compounds,
compositions or biological systems, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting.
[0028] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0029] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 cells
refers to groups having 1, 2, or 3 cells. Similarly, a group having
1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so
forth.
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
I. Definitions
[0031] The terminology used herein is for the purpose of describing
particular 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.
[0032] As used herein, the term "about" means that a value can vary
+/-20%, +/-15%, +/-10% or +/-5% and remain within the scope of the
present disclosure. For example, "a concentration of about 200
IU/mL" encompasses a concentration between 160 IU/mL and 240
IU/mL.
[0033] As used herein, the term "administration" of an agent to a
subject includes any route of introducing or delivering the agent
to a subject to perform its intended function. Administration can
be carried out by any suitable route, including intravenously,
intramuscularly, intraperitoneally, or subcutaneously.
Administration includes self-administration and the administration
by another.
[0034] The term "amino acid" refers to naturally occurring and
non-naturally occurring amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally encoded amino acids are
the 20 common amino acids (alanine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine
and selenocysteine. Amino acid analogs refers to agents that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, such as, homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (such as, norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. In some embodiments, amino acids
forming a polypeptide are in the D form. In some embodiments, the
amino acids forming a polypeptide are in the L form. In some
embodiments, a first plurality of amino acids forming a polypeptide
are in the D form and a second plurality are in the L form.
[0035] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
code.
[0036] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to naturally occurring amino acid
polymers as well as amino acid polymers in which one or more amino
acid residues is a non-naturally occurring amino acid, e.g., an
amino acid analog. The terms encompass amino acid chains of any
length, including full length proteins, wherein the amino acid
residues are linked by covalent peptide bonds.
[0037] As used herein, a "control" is an alternative sample used in
an experiment for comparison purpose. A control can be "positive"
or "negative." For example, where the purpose of the experiment is
to determine a correlation of the efficacy of a therapeutic agent
for the treatment for a particular type of disease, a positive
control (a composition known to exhibit the desired therapeutic
effect) and a negative control (a subject or a sample that does not
receive the therapy or receives a placebo) are typically
employed.
[0038] As used herein, the term "effective amount" or
"therapeutically effective amount" refers to a quantity of an agent
sufficient to achieve a desired therapeutic effect. In the context
of therapeutic applications, the amount of a therapeutic peptide
administered to the subject can depend on the type and severity of
the infection and on the characteristics of the individual, such as
general health, age, sex, body weight and tolerance to drugs. It
can also depend on the degree, severity and type of disease. The
skilled artisan will be able to determine appropriate dosages
depending on these and other factors.
[0039] As used herein, the term "expression" refers to the process
by which polynucleotides are transcribed into mRNA and/or the
process by which the transcribed mRNA is subsequently being
translated into peptides, polypeptides, or proteins. If the
polynucleotide is derived from genomic DNA, expression can include
splicing of the mRNA in a eukaryotic cell. The expression level of
a gene can be determined by measuring the amount of mRNA or protein
in a cell or tissue sample. In one aspect, the expression level of
a gene from one sample can be directly compared to the expression
level of that gene from a control or reference sample. In another
aspect, the expression level of a gene from one sample can be
directly compared to the expression level of that gene from the
same sample following administration of the compositions disclosed
herein. The term "expression" also refers to one or more of the
following events: (1) production of an RNA template from a DNA
sequence (e.g., by transcription) within a cell; (2) processing of
an RNA transcript (e.g., by splicing, editing, 5' cap formation,
and/or 3' end formation) within a cell; (3) translation of an RNA
sequence into a polypeptide or protein within a cell; (4)
post-translational modification of a polypeptide or protein within
a cell; (5) presentation of a polypeptide or protein on the cell
surface; and (6) secretion or presentation or release of a
polypeptide or protein from a cell.
[0040] The term "linker" refers to synthetic sequences (e.g., amino
acid sequences) that connect or link two sequences, e.g., that link
two polypeptide domains. In some embodiments, the linker contains
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of amino acid sequences.
[0041] The terms "lyophilized," "lyophilization" and the like as
used herein refer to a process by which the material (e.g.,
nanoparticles) to be dried is first frozen and then the ice or
frozen solvent is removed by sublimation in a vacuum environment.
An excipient can be included in pre-lyophilized formulations to
enhance stability of the lyophilized product upon storage. The
lyophilized sample can further contain additional excipients.
[0042] As used herein the term immune cell refers to any cell that
plays a role in the immune response. Immune cells are of
hematopoietic origin, and include lymphocytes, such as B cells and
T cells; natural killer cells; myeloid cells, such as monocytes,
macrophages, dendritic cells, eosinophils, neutrophils, mast cells,
basophils, and granulocytes.
[0043] The term "lymphocyte" refers to all immature, mature,
undifferentiated and differentiated white lymphocyte populations
including tissue specific and specialized varieties. It
encompasses, by way of non-limiting example, B cells, T cells, NKT
cells, and NK cells. In some embodiments, lymphocytes include all B
cell lineages including pre-B cells, progenitor B cells, early
pro-B cells, late pro-B cells, large pre-B cells, small pre-B
cells, immature B cells, mature B cells, plasma B cells, memory B
cells, B-1 cells, B-2 cells and anergic AN1/T3 cell
populations.
[0044] As used herein, the term T-cell includes naive T cells, CD4+
T cells, CD8+ T cells, memory T cells, activated T cells, anergic T
cells, tolerant T cells, chimeric T cells, and antigen-specific T
cells.
[0045] The term "B cell" or "B cells" refers to, by way of
non-limiting example, a pre-B cell, progenitor B cell, early pro-B
cell, late pro-B cell, large pre-B cell, small pre-B cell, immature
B cell, mature B cell, naive B cells, plasma B cells, activated B
cells, anergic B cells, tolerant B cells, chimeric B cells,
antigen-specific B cells, memory B cell, B-1 cell, B-2 cells and
anergic AN1/T3 cell populations. In some embodiments, the term B
cell includes a B cell that expresses an immunoglobulin heavy chain
and/or light chain on its cells surface. In some embodiments, the
term B cell includes a B cell that expresses and secretes an
immunoglobulin heavy chain and/or light chain. In some embodiments,
the term B cell includes a cell that binds an antigen on its
cell-surface. In some embodiments disclosed herein, B cells or
AN1/T3 cells are utilized in the processes described. In certain
embodiments, such cells are optionally substituted with any animal
cell suitable for expressing, capable of expressing (e.g.,
inducible expression), or capable of being differentiated into a
cell suitable for expressing an antibody including, e.g., a
hematopoietic stem cell, a naive B cell, a B cell, a pre-B cell, a
progenitor B cell, an early Pro-B cell, a late pro-B cell, a large
pre-B cell, a small pre-B cell, an immature B cell, a mature B
cell, a plasma B cell, a memory B cell, a B-1 cell, a B-2 cell, an
anergic B cell, or an anergic AN1/T3 cell.
[0046] As used herein "adoptive cell therapeutic composition"
refers to any composition comprising cells suitable for adoptive
cell transfer. In exemplary embodiments, the adoptive cell
therapeutic composition comprises a cell type selected from a group
consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e.
heterologous T-cell receptor) modified lymphocytes and CAR (i.e.
chimeric antigen receptor) modified lymphocytes. In another
embodiment, the adoptive cell therapeutic composition comprises a
cell type selected from a group consisting of T-cells, CD8+ cells,
CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and
peripheral blood mononuclear cells. In another embodiment, TILs,
T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells,
regulatory T-cells or peripheral blood mononuclear cells form the
adoptive cell therapeutic composition. In one embodiment, the
adoptive cell therapeutic composition comprises T cells.
[0047] As used herein "tumor-infiltrating lymphocytes" or TILs
refer to white blood cells that have left the bloodstream and
migrated into a tumor.
[0048] The terms "MYC" and "MYC gene" are synonyms. They refer to a
nucleic acid sequence that encodes a MYC polypeptide. A MYC gene
comprises a nucleotide sequence of at least 120 nucleotides that is
at least 60% to 100% identical or homologous, e.g., at least 60,
65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%,
96%, 97%, 98%, or any other percent from about 70% to about 100%
identical to sequences of NCBI Accession Number NM-002467. In some
embodiments, the MYC gene is a proto-oncogene. In certain
instances, a MYC gene is found on chromosome 8, at 8q24.21. In
certain instances, a MYC gene begins at 128,816,862 bp from pter
and ends at 128,822,856 bp from pter. In certain instances, a MYC
gene is about 6 kb. In certain instances, a MYC gene encodes at
least eight separate mRNA sequences--5 alternatively spliced
variants and 3 unspliced variants.
[0049] The terms "MYC protein," "MYC polypeptide," and "MYC
sequence" are synonyms and refer to the polymer of amino acid
residues disclosed in NCBI Accession Number
UniProtKB/Swiss-Prot:P01106.1 (MYC isoform 1) or NP_002458.2
(UniProtKB/Swiss-Prot:P01106.2; MYC isoform 2), and functional
homologs, analogs or fragments thereof. The sequence of or
UniProtKB/Swiss-Prot:P01106.1 is:
TABLE-US-00001 (SEQ ID NO: 2)
MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDI
WKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQ
LEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSE
KLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFP
YPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLREETP
PTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSP
LVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCT
SPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPK
VVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKIAKLEQLRNSCA
The sequence of NP_002458.2 (UniProtKB/Swiss-Prot:P01106.2) is:
TABLE-US-00002 (SEQ ID NO: 11)
MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQ
QQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLR
GDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQD
CMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDL
SAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESS
PQGSPEPLVLREETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSES
GSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLD
SVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFAL
RDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQ
LKIAKLEQLRNSCA
[0050] In some embodiments, the MYC polypeptide is a complete MYC
polypeptide sequence. In some embodiments, the MYC polypeptide is a
partial MYC polypeptide sequence. In some embodiments, the MYC
polypeptide comprises at least 400 consecutive amino acids of SEQ
ID NO: 2 OR 11. In some embodiments, the MYC polypeptide comprises
at least 400 consecutive amino acids of SEQ ID NO: 2 OR 11 and
retains at least one MYC activity. In some embodiments, the MYC
polypeptide comprises at least 400, at least 410, at least 420, at
least 430, or at least 450 consecutive amino acids of SEQ ID NO: 2
OR 11. In some embodiments, the MYC polypeptide comprises at least
400, at least 410, at least 420, at least 430, or at least 450
consecutive amino acids of SEQ ID NO: 2 OR 11 and retains at least
one MYC activity. In some embodiments, the MYC polypeptide is
c-MYC. In some embodiments, the MYC polypeptide sequence comprises
the sequence shown below:
TABLE-US-00003 (SEQ ID NO: 3)
MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQ
QQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLR
GDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQD
CMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDL
SAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESS
PQGSPEPLVLREETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSES
GSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLD
SVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFAL
RDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQ LKHKLEQLR
[0051] In some embodiments, the MYC polypeptide sequence comprises
the sequence shown below:
TABLE-US-00004 (SEQ ID NO: 4)
PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIW
KKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQL
EMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEK
LASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPY
PLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLREETPP
TTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPL
VLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTS
PRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKV
VILKKATAYILSVQAEEQKLISEEDLLRKRREQLKIAKLEQLR.
[0052] In some embodiments, a MYC polypeptide comprises an amino
acid sequence that is at least 40% to 100% identical, e.g., at
least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%,
99%, or any other percent from about 40% to about 100% identical to
the sequence of NCBI Accession Number NP002458.2 or
UniProtKB/Swiss-Prot Accession Number P01106.1. In some
embodiments, MYC polypeptide refers to a polymer of 439 amino
acids, a MYC polypeptide that has not undergone any
post-translational modifications. In some embodiments, MYC
polypeptide refers to a polymer of 439 amino acids that has
undergone post-translational modifications. In some embodiments,
the MYC polypeptide is 48,804 kDa. In some embodiments, the MYC
polypeptide contains a basic Helix-Loop-Helix Leucine Zipper
(bHLH/LZ) domain. In some embodiments, the bHLH/LZ domain comprises
the sequence of:
ELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKH
KLEQLR (SEQ ID NO: 5). In some embodiments, the MYC polypeptide is
a transcription factor (e.g., Transcription Factor 64). In some
embodiments, the MYC polypeptide contains an E-box DNA binding
domain. In some embodiments, the MYC polypeptide binds to a
sequence comprising CACGTG. In some embodiments, the MYC
polypeptide promotes one or more of cell survival and/or
proliferation. In some embodiments, a MYC polypeptide includes one
or more of those described above, and includes one or more
post-translational modifications (e.g., acetylation). In some
embodiments, the MYC polypeptides comprise one or more additional
amino acid residues at the N-terminus or C-terminus of the
polypeptide. In some embodiments, the MYC polypeptides are fusion
proteins. In some embodiments, the MYC polypeptides are linked to
one or more additional peptides at the N-terminus or C-terminus of
the polypeptide.
[0053] Proteins suitable for use in the methods described herein
also includes functional variants, including proteins having
between 1 to 15 amino acid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, deletions,
or additions, compared to the amino acid sequence of any protein
described herein. In other embodiments, the altered amino acid
sequence is at least 75% identical, e.g., 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino
acid sequence of any protein inhibitor described herein. Such
sequence-variant proteins are suitable for the methods described
herein as long as the altered amino acid sequence retains
sufficient biological activity to be functional in the compositions
and methods described herein. Where amino acid substitutions are
made, the substitutions can be conservative amino acid
substitutions. Among the common, naturally occurring amino acids,
for example, a "conservative amino acid substitution" is
illustrated by a substitution among amino acids within each of the
following groups: (1) glycine, alanine, valine, leucine, and
isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine
and threonine, (4) aspartate and glutamate, (5) glutamine and
asparagine, and (6) lysine, arginine and histidine. The BLOSUM62
table is an amino acid substitution matrix derived from about 2,000
local multiple alignments of protein sequence segments,
representing highly conserved regions of more than 500 groups of
related proteins (Henikoff et al., (1992), Proc. Natl Acad. Sci.
USA, 89:10915-10919). Accordingly, the BLOSUM62 substitution
frequencies are used to define conservative amino acid
substitutions that, in some embodiments, are introduced into the
amino acid sequences described or disclosed herein. Although it is
possible to design amino acid substitutions based solely upon
chemical properties (as discussed above), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3).
[0054] The phrases "E-box sequence" and "enhancer box sequence" are
used interchangeably herein and mean the nucleotide sequence
CANNTG, wherein N is any nucleotide. In certain instances, the
E-box sequence comprises CACGTG. In certain instances, the basic
helix-loop-helix domain of a transcription factor encoded by MYC
binds to the E-box sequence. In certain instances the E-box
sequence is located upstream of a gene (e.g., p21, Bc1-2, or
ornithine decarboxylase). In certain instances, the MYC polypeptide
contains an E-box DNA binding domain. In certain instances, the
E-box DNA binding domain comprises the sequence of KRRTHNVLERQRRN
(SEQ ID NO: 6). In certain instances, the binding of the
transcription factor encoded by MYC to the E-box sequence, allows
RNA polymerase to transcribe the gene downstream of the E-box
sequence.
[0055] The term "MYC activity" or "MYC biological activity" or
"biologically active MYC" includes one or more of enhancing or
inducing cell survival, cell proliferation, and/or antibody
production. By way of example and not by way of limitation, MYC
activity includes enhancement of expansion of anti-CD3 and
anti-CD28 activated T-cells and/or increased proliferation of
long-term self-renewing hematopoietic stem cells. MYC activity also
includes entry into the nucleus of a cell, binding to a nucleic
acid sequence (e.g., binding an E-box sequence), and/or inducing
expression of MYC target genes.
[0056] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to an animal, typically
a mammal. In one embodiment, the patient, subject, or individual is
a mammal. In one embodiment, the patient, subject or individual is
a human. In some embodiments the patient, subject or individual is
an animal, such as, but not limited to, domesticated animals, such
as equine, bovine, murine, ovine, canine, and feline.
[0057] The terms "protein transduction domain (PTD)" or
"transporter peptide sequence" (also known as cell permeable
proteins (CPP) or membrane translocating sequences (MTS)) are used
interchangeably herein to refer to small peptides that are able to
ferry much larger molecules into cells independent of classical
endocytosis. In some embodiments, a nuclear localization signal can
be found within the protein transduction domain, which mediates
further translocation of the molecules into the cell nucleus.
[0058] The terms "treating" or "treatment" as used herein covers
the treatment of a disease in a subject, such as a human, and
includes: (i) inhibiting a disease, i.e., arresting its
development; (ii) relieving a disease, i.e., causing regression of
the disease; (iii) slowing progression of the disease; and/or (iv)
inhibiting, relieving, or slowing progression of one or more
symptoms of the disease. With respect to a melanoma, "treating" or
"treatment" also encompasses regression of a tumor, slowing tumor
growth, inhibiting metastasis of a melanoma tumor, inhibiting
relapse or recurrent melanoma and/or maintaining remission.
[0059] It is also to be appreciated that the various modes of
treatment or prevention of medical diseases and conditions as
described are intended to mean "substantial," which includes total
but also less than total treatment or prevention, and wherein some
biologically or medically relevant result is achieved. The
treatment can be a continuous prolonged treatment for a chronic
disease or a single, or few time administrations for the treatment
of an acute condition.
[0060] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of a disease state.
II. Overview
[0061] The present disclosure relates, in part, to the treatment of
melanoma in a subject by administering a composition comprising one
or more immune cells having anti-tumor activity (e.g., immune cells
that modulate a response against a tumor, such as
tumor-infiltrating lymphocytes (TILs)), wherein the one or more
immune cells are contacted with a PTD-MYC fusion polypeptide in
vitro prior to administration to the subject. In some embodiments,
the immune cells are obtained from a donor subject that has a
melanoma tumor. In some embodiments, the cells are autologous to
the subject receiving treatment. In some embodiments, the melanoma
is a superficial spreading melanoma, a nodular melanoma, a lentigo
maligna melanoma, or an acral melanoma.
[0062] The present disclosure is based, at least in part, on the
discovery, that treating lymphocytes isolated from a donor subject
having a melanoma tumor with a MYC fusion polypeptide containing a
MYC polypeptide and a protein transduction domain (PTD), such as
the HIV TAT protein transduction domain, and administering the
treated lymphocytes to a subject bearing a melanoma tumor
significantly increases the survival of the tumor-bearing subject.
The examples provided herein demonstrate that immune cells
extracted from the lymph nodes of a melanoma-bearing mouse had
significantly increased therapeutic efficacy when the cells were
treated with a TAT-MYC fusion protein in vitro prior to
administration to a second melanoma-bearing mice. These data
support that adoptive cell transfer using anti-tumor immune cells
treated with a PTD-MYC fusion polypeptide can be employed in the
treatment of tumors, such as melanoma tumors.
[0063] In some embodiments, the method for the treatment of
melanoma in a subject comprises administering immune cells that
have been contacted in vitro with a PTD-MYC fusion polypeptide. In
some embodiments, the immune cells for use in the present methods
are primed in vivo with melanoma tumor antigen. In some
embodiments, the immune cells are from a donor having melanoma. In
some embodiments, the immune cells are from a donor having a solid
tumor, such as a melanoma tumor. In some embodiments, the immune
cells are contacted in vivo with a melanoma tumor antigen. In some
embodiments, the immune cells are from a donor that has been
exposed to a one or more melanoma tumor antigens. In some
embodiments, the immune cells are from a donor that has been
exposed to an anti-tumor vaccine. In some embodiments, the immune
cells are B cells, T cells, NK cells, or any combination thereof.
In some embodiments, the immune cells are tumor infiltrating
lymphocytes (TIL). In some embodiments, the immune cells are
chimeric antigen receptor (CAR)-T cells.
[0064] In some embodiments, the method for the treatment of
melanoma in a subject comprises administering one or more modified
immune cells to the subject in need thereof, wherein the one or
more modified immune cells comprise a MYC fusion peptide comprising
(i) a protein transduction domain; (ii) a MYC polypeptide sequence
and are reactive to a melanoma tumor-specific antigen.
[0065] In some embodiments, the method for the treatment of
melanoma in a subject comprises the steps of:
[0066] a) contacting immune cells in vitro with a MYC fusion
polypeptide, wherein the immune cells are from a donor that has
been exposed to one or more melanoma tumor antigens and the MYC
fusion peptide comprising (i) a protein transduction domain; (ii) a
MYC polypeptide sequence; and
[0067] b) administering the contacted immune cells to the melanoma
tumor-bearing subject, whereby the melanoma is treated.
[0068] In some embodiments, contacting the immune cells in vitro
with a PTD-MYC fusion polypeptide is performed by culturing the
immune cells in the presence of the MYC fusion polypeptide. In some
embodiments, the immune cells are cultured in the presence of one
or more cytokines and/or growth factors (e.g., interleukin-2
(IL-2), IL-4, IL-7, IL-9, and IL-15). In some embodiments, the
immune cells are not expanded prior to administration. In some
embodiments, the immune cells are expanded prior to administration.
In some embodiments, the donor and subject for treatment are the
same.
[0069] In some embodiments, the immune cells are tumor-infiltrating
lymphocytes. In some embodiments, the tumor-infiltrating
lymphocytes are autologous tumor-infiltrating lymphocytes.
Accordingly, in some embodiments, the method for the treatment of
melanoma in a subject comprises administering lymphocytes that have
been contacted in vitro with a PTD-MYC fusion polypeptide, wherein
the immune cells are from lymphocytes are autologous
tumor-infiltrating lymphocytes from the subject.
[0070] In some embodiments, the method for the treatment of
melanoma in a subject comprises the steps of:
[0071] a) contacting lymphocytes in vitro with a PTD-MYC fusion
polypeptide, wherein the lymphocytes are autologous
tumor-infiltrating lymphocytes from the subject, and
[0072] b) administering the contacted autologous tumor-infiltrating
lymphocytes to the subject, whereby the melanoma is treated.
Methods of Obtaining and Preparing Immune Cells for Transfer
[0073] Immune cells for use in the methods provided herein can be
obtained using any suitable method known in the art. In some
embodiments, the immune cells are primary immune cells. In some
embodiments, the immune cells are lymphocytes, such as T and B
cells. In some embodiments, the immune cells are natural killer
(NK) cells. In some embodiments, the immune cells are a mixture of
lymphocytes and NK cells. In some embodiments, the immune cells are
peripheral blood mononuclear cells (PBMC). In some embodiments, the
immune cells are T cells that have infiltrated a tumor (e.g., tumor
infiltrating lymphocytes). In some embodiments, the T cells are
removed during surgery of a melanoma tumor or a metastatic tumor in
a subject. For example, in some embodiments, the T cells are
isolated after removal of tumor tissue by biopsy. In some
embodiments, the immune cells are modified following isolation from
a donor. In some embodiments, the immune cells are chimeric antigen
receptor (CAR)-T cells.
[0074] In some embodiments, the T cells are isolated from sample
containing a population of cells, such as a blood, lymph or tissue
biopsy sample. T cells can be isolated from a population of cells
by any means known in the art. In one embodiment, the method
comprises obtaining a bulk population of T cells from a tumor
sample by any suitable method known in the art. For example, a bulk
population of T cells can be obtained from a tumor sample by
dissociating the tumor sample into a cell suspension from which
specific cell populations can be selected. Suitable methods of
obtaining a bulk population of T cells can include, but are not
limited to, any one or more of mechanically dissociating (e.g.,
mincing) the tumor, enzymatically dissociating (e.g., digesting)
the tumor, and aspiration (e.g., as with a needle).
[0075] The bulk population of T cells obtained from a tumor sample
can comprise any suitable type of T cell. Preferably, the bulk
population of T cells obtained from a tumor sample comprises tumor
infiltrating lymphocytes (TILs).
[0076] The tumor sample can be obtained from any mammal. Unless
stated otherwise, as used herein, the term "mammal" refers to any
mammal including, but not limited to, mammals of the order
Logomorpha, such as rabbits; the order Carnivora, including Felines
(cats) and Canines (dogs); the order Artiodactyla, including
Bovines (cows) and Swines (pigs); or of the order Perssodactyla,
including Equines (horses). The mammals can be non-human primates,
e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of
the order Anthropoids (humans and apes). In some embodiments, the
mammal can be a mammal of the order Rodentia, such as mice and
hamsters. Preferably, the mammal is a non-human primate or a human.
An exemplary mammal is a human. In some embodiments, the subject to
receive the immune cells is also the donor of the tumor sample
(i.e., autologous ACT)
[0077] T cells can be obtained from a number of sources, including
peripheral blood mononuclear cells, bone marrow, lymph node tissue,
spleen tissue, and tumors. In certain embodiments, T cells can be
obtained from a unit of blood collected from a subject using any
number of techniques known to the skilled artisan, such as Ficoll
separation. In one embodiment, cells from the circulating blood of
an individual are obtained by apheresis or leukopheresis. The
apheresis product typically contains lymphocytes, including T
cells, monocytes, granulocytes, B cells, other nucleated white
blood cells, red blood cells, and platelets. In one embodiment, the
cells collected by apheresis can be washed to remove the plasma
fraction and to place the cells in an appropriate buffer or media
for subsequent processing steps. In one embodiment of the
invention, the cells are washed with phosphate buffered saline
(PBS). In an alternative embodiment, the wash solution lacks
calcium and can lack magnesium or can lack many if not all divalent
cations. Initial activation steps in the absence of calcium lead to
magnified activation. As those of ordinary skill in the art would
readily appreciate, a washing step can be accomplished by methods
known to those in the art, such as by using a semi-automated
"flow-through" centrifuge (for example, the Cobe 2991 cell
processor) according to the manufacturer's instructions. After
washing, the cells can be resuspended in a variety of biocompatible
buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively,
the undesirable components of the apheresis sample can be removed
and the cells directly resuspended in culture media.
[0078] In another embodiment, T cells are isolated from peripheral
blood lymphocytes by lysing the red blood cells and depleting the
monocytes, for example, by centrifugation through a PERCOLL.TM.
gradient. A specific subpopulation of T cells, such as CD28+, CD4+,
CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by
positive or negative selection techniques. For example, in one
embodiment, T cells are isolated by incubation with
anti-CD3/anti-CD28 (i.e., 3.times.28)-conjugated beads, such as
DYNABEADS.RTM. M-450 CD3/CD28 T, or XCYTE DYNABEADS.TM. for a time
period sufficient for positive selection of the desired T cells. In
one embodiment, the time period is about 30 minutes. In a further
embodiment, the time period ranges from 30 minutes to 36 hours or
longer and all integer values there between. In a further
embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours.
In yet another embodiment, the time period is 10 to 24 hours. In
one embodiment, the incubation time period is 24 hours. For
isolation of T cells from patients with leukemia, use of longer
incubation times, such as 24 hours, can increase cell yield. Longer
incubation times can be used to isolate T cells in any situation
where there are few T cells as compared to other cell types, such
in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue
or from immunocompromised individuals. Further, use of longer
incubation times can increase the efficiency of capture of CD8+ T
cells.
[0079] Enrichment of a T cell population by negative selection can
be accomplished with a combination of antibodies directed to
surface markers unique to the negatively selected cells. In one
embodiment, the method is cell sorting and/or selection via
negative magnetic immunoadherence or flow cytometry that uses a
cocktail of monoclonal antibodies directed to cell surface markers
present on the cells negatively selected. For example, to enrich
for CD4+ cells by negative selection, a monoclonal antibody
cocktail typically includes antibodies to CD14, CD20, CD11b, CD16,
HLA-DR, and CD8.
[0080] Further, monocyte populations (i.e., CD14+ cells) can be
depleted from blood preparations by a variety of methodologies,
including anti-CD14 coated beads or columns, or utilization of the
phagocytotic activity of these cells to facilitate removal.
Accordingly, in one embodiment, the invention uses paramagnetic
particles of a size sufficient to be engulfed by phagocytotic
monocytes. In certain embodiments, the paramagnetic particles are
commercially available beads, for example, those produced by Life
Technologies under the trade name Dynabeads.TM.. In one embodiment,
other non-specific cells are removed by coating the paramagnetic
particles with "irrelevant" proteins (e.g., serum proteins or
antibodies). Irrelevant proteins and antibodies include those
proteins and antibodies or fragments thereof that do not
specifically target the T cells to be isolated. In certain
embodiments the irrelevant beads include beads coated with sheep
anti-mouse antibodies, goat anti-mouse antibodies, and human serum
albumin.
[0081] In brief, such depletion of monocytes is performed by
preincubating T cells isolated from whole blood, apheresed
peripheral blood, or tumors with one or more varieties of
irrelevant or non-antibody coupled paramagnetic particles at any
amount that allows for removal of monocytes (approximately a 20:1
bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37
degrees C., followed by magnetic removal of cells which have
attached to or engulfed the paramagnetic particles. Such separation
can be performed using standard methods available in the art. For
example, any magnetic separation methodology can be used including
a variety of which are commercially available, (e.g., DYNAL.RTM.
Magnetic Particle Concentrator (DYNAL MPC.RTM.)). Assurance of
requisite depletion can be monitored by a variety of methodologies
known to those of ordinary skill in the art, including flow
cytometric analysis of CD14 positive cells, before and after
depletion.
[0082] For isolation of a desired population of cells by positive
or negative selection, the concentration of cells and surface
(e.g., particles such as beads) can be varied. In certain
embodiments, it can be desirable to significantly decrease the
volume in which beads and cells are mixed together (i.e., increase
the concentration of cells), to ensure maximum contact of cells and
beads. For example, in one embodiment, a concentration of 2 billion
cells/ml is used. In one embodiment, a concentration of 1 billion
cells/ml is used. In a further embodiment, greater than 100 million
cells/ml is used. In a further embodiment, a concentration of cells
of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
In yet another embodiment, a concentration of cells from 75, 80,
85, 90, 95, or 100 million cells/ml is used. In further
embodiments, concentrations of 125 or 150 million cells/ml can be
used. Using high concentrations can result in increased cell yield,
cell activation, and cell expansion. Further, use of high cell
concentrations allows more efficient capture of cells that can
weakly express target antigens of interest, such as CD28- negative
T cells, or from samples where there are many tumor cells present
(i.e., leukemic blood, tumor tissue, etc). Such populations of
cells can have therapeutic value and would be desirable to obtain.
For example, using high concentration of cells allows more
efficient selection of CD8+ T cells that normally have weaker CD28
expression.
[0083] In a related embodiment, it can be desirable to use lower
concentrations of cells. By significantly diluting the mixture of T
cells and surface (e.g., particles such as beads), interactions
between the particles and cells is minimized. This selects for
cells that express high amounts of desired antigens to be bound to
the particles. For example, CD4+ T cells express higher levels of
CD28 and are more efficiently captured than CD8+ T cells in dilute
concentrations. In one embodiment, the concentration of cells used
is 5.times.10.sup.6/ml. In other embodiments, the concentration
used can be from about 1.times.10.sup.5/ml to 1.times.10.sup.6/ml,
and any integer value in between.
[0084] T cells can also be frozen. The freeze and subsequent thaw
step can provide a more uniform product by removing granulocytes
and to some extent monocytes in the cell population. After a
washing step to remove plasma and platelets, the cells can be
suspended in a freezing solution. While many freezing solutions and
parameters are known in the art and will be useful in this context,
one method involves using PBS containing 20% DMSO and 8% human
serum albumin, or other suitable cell freezing media, the cells
then are frozen to -80.degree. C. at a rate of 1.degree. per minute
and stored in the vapor phase of a liquid nitrogen storage tank.
Other methods of controlled freezing can be used as well as
uncontrolled freezing immediately at -20.degree. C. or in liquid
nitrogen.
[0085] T cells for use in the present invention can also be
antigen-specific T cells. For example, tumor-specific T cells can
be used. In certain embodiments, antigen-specific T cells can be
isolated from a patient of interest, such as a patient afflicted
with a melanoma, such as patient with a melanoma tumor. In some
embodiments, the patient has melanoma.
[0086] In one embodiment neoepitopes are determined for a subject
and T cells specific to these antigens are isolated.
Antigen-specific cells for use in expansion can also be generated
in vitro using any number of methods known in the art, for example,
as described in U.S. Patent Publication No. US 20040224402
entitled, Generation And Isolation of Antigen-Specific T Cells, or
in U.S. Pat. No. 6,040,177. Antigen-specific cells for use in the
present invention can also be generated using any number of methods
known in the art, for example, as described in Current Protocols in
Immunology, or Current Protocols in Cell Biology, both published by
John Wiley & Sons, Inc., Boston, Mass.
[0087] In a related embodiment, it can be desirable to sort or
otherwise positively select (e.g. via magnetic selection) the
antigen specific cells prior to or following one or two rounds of
expansion. Sorting or positively selecting antigen-specific cells
can be carried out using peptide-WIC tetramers (Altman, et al.,
Science. 1996 Oct. 4; 274(5284):94-6). In another embodiment the
adaptable tetramer technology approach is used (Andersen et al.,
2012 Nat Protoc. 7:891-902). Tetramers are limited by the need to
utilize predicted binding peptides based on prior hypotheses, and
the restriction to specific HLAs. Peptide-WIC tetramers can be
generated using techniques known in the art and can be made with
any WIC molecule of interest and any antigen of interest as
described herein. Specific epitopes to be used in this context can
be identified using numerous assays known in the art. For example,
the ability of a polypeptide to bind to WIC class I can be
evaluated indirectly by monitoring the ability to promote
incorporation of .sup.125I labeled .beta.2-microglobulin (.beta.2m)
into WIC class I/.beta.2m/peptide heterotrimeric complexes (see
Parker et al., J. Immunol. 152:163, 1994).
[0088] In some embodiments, the T cells are recombinantly modified
to express a modified or chimeric receptor (e.g., chimeric antigen
receptor (CAR) modified T cells).
[0089] In one embodiment, cells are directly labeled with an
epitope-specific reagent for isolation by flow cytometry followed
by characterization of phenotype and TCRs. In one embodiment, T
cells are isolated by contacting the T cell specific antibodies.
Sorting of antigen-specific T cells, or generally any cells of the
present invention, can be carried out using any of a variety of
commercially available cell sorters, including, but not limited to,
MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAria.TM.,
FACSArray.TM., FACSVantage.TM., BD.TM. LSR II, and FACSCalibur.TM.
(BD Biosciences, San Jose, Calif.).
[0090] In one embodiment, the method comprises selecting cells that
also express CD3. The method can comprise specifically selecting
the cells in any suitable manner. Preferably, the selecting is
carried out using flow cytometry. The flow cytometry can be carried
out using any suitable method known in the art. The flow cytometry
can employ any suitable antibodies and stains. Preferably, the
antibody is chosen such that it specifically recognizes and binds
to the particular biomarker being selected. For example, the
specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 can be
carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3,
anti-4-1BB, or anti-PD-1 antibodies, respectively. The antibody or
antibodies can be conjugated to a bead (e.g., a magnetic bead) or
to a fluorochrome. Preferably, the flow cytometry is
fluorescence-activated cell sorting (FACS). TCRs expressed on T
cells can be selected based on reactivity to autologous tumors.
Additionally, T cells that are reactive to tumors can be selected
for based on markers using the methods described in patent
publication Nos. WO2014133567 and WO2014133568, herein incorporated
by reference in their entirety. Additionally, activated T cells can
be selected for based on surface expression of CD107a.
[0091] In one embodiment, the method further comprises expanding
the numbers of T cells in the enriched cell population. Such
methods are described in U.S. Pat. No. 8,637,307 and is herein
incorporated by reference in its entirety. The T cells can be
expanded before or after treatment of the cells with the PTD-MYC
polypeptide. The numbers of T cells can be increased at least about
3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least
about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold),
more preferably at least about 100-fold, more preferably at least
about 1,000 fold, or most preferably at least about 100,000-fold.
The numbers of T cells can be expanded using any suitable method
known in the art. Exemplary methods of expanding the numbers of
cells are described in patent publication No. WO 2003057171, U.S.
Pat. No. 8,034,334, and U.S. Patent Application Publication No.
2012/0244133, each of which is incorporated herein by
reference.
[0092] In one embodiment, ex vivo T cell expansion can be performed
by isolation of T cells and subsequent stimulation or activation
followed by further expansion. In one embodiment of the invention,
the T cells can be stimulated or activated by a single agent. In
another embodiment, T cells are stimulated or activated with two
agents, one that induces a primary signal and a second that is a
co-stimulatory signal. Ligands useful for stimulating a single
signal or stimulating a primary signal and an accessory molecule
that stimulates a second signal can be used in soluble form.
Ligands can be attached to the surface of a cell, to an Engineered
Multivalent Signaling Platform (EMSP), or immobilized on a surface.
In a one embodiment both primary and secondary agents are
co-immobilized on a surface, for example a bead or a cell. In one
embodiment, the molecule providing the primary activation signal
can be a CD3 ligand, and the co-stimulatory molecule can be a CD28
ligand or 4-1BB ligand. In some embodiments, the cells are expanded
by stimulation with one or more antigens, such as a melanoma tumor
antigen or antigens derived from the patient's tumor.
[0093] In some embodiments, the isolated immune cells are
immediately treated with the PTD-MYC fusion polypeptide following
isolation. In other embodiments, the isolated immune cells are
stored in a suitable buffer and frozen prior to treatment with the
PTD-MYC fusion polypeptide. In some embodiments, the isolated
immune cells are immediately treated with the PTD-MYC fusion
polypeptide following isolation and the treated cells are stored in
a suitable buffer and frozen until needed for administration to the
patient.
[0094] In certain embodiments, the isolated immune cells (e.g., a
mixed population immune cells or isolated types, such as tumor
infiltrating lymphocytes) are contacted with a composition
containing a PTD-MYC fusion polypeptide for a period of time
sufficient to be taken up by the cells. In some embodiments, the
immune cells are contacted with a composition containing a PTD-MYC
fusion polypeptide for less than about 24 hours, less than about 23
hours, less than about 22 hours, less than about 21 hours, less
than about 20 hours, less than about 19 hours, less than about 18
hours, less than about 17 hours, less than about 16 hours, less
than about 15 hours, less than about 14 hours, less than about 13
hours, less than about 12 hours, less than about 11 hours, less
than about 10 hours, less than about 9 hours, less than about 8
hours, less than about 7 hours, less than about 6 hours, less than
about 5 hours, less than about 4 hours, less than about 3 hours,
less than about 2 hours, or less than about 1 hour.
[0095] In certain embodiments, the immune cells are contacted with
a composition containing a PTD-MYC fusion polypeptide for less than
about 55 minutes, less than about 50 minutes, less than about 45
minutes, less than about 40 minutes, less than about 35 minutes,
less than about 30 minutes, less than about 29 minutes, less than
about 28 minutes, less than about 27 minutes, less than about 26
minutes, less than about 25 minutes, less than about 24 minutes,
less than about 23 minutes, less than about 22 minutes, less than
about 21 minutes, less than about 20 minutes, less than about 19
minutes, less than about 18 minutes, less than about 17 minutes,
less than about 16 minutes, less than about 15 minutes, less than
about 14 minutes, less than about 13 minutes, less than about 12
minutes, less than about 11 minutes, or less than about 10 minutes.
In certain embodiments, the immune cells are contacted with a
composition containing a PTD-MYC fusion polypeptide for about 1
hour.
[0096] In certain embodiments, the immune cells are contacted with
a composition containing a PTD-MYC fusion polypeptide for 24 hours
or longer. In certain embodiments, the immune cells are contacted
with a composition containing a PTD-MYC fusion polypeptide for less
than about 12 days, less than about 11 days, less than about 10
days, less than about 9 days, less than about 8 days, less than
about 7 days, less than about 6 days, less than about 5 days, less
than about 4 days, less than about 2 days, or less than about 1
day.
[0097] In certain embodiments that may be combined with any of the
preceding embodiments, the cells are contacted with a MYC-fusion
polypeptide at a concentration of 0.5 .mu.m/ml to 500 .mu.g/ml. 0.5
.mu.g/ml, at least 0.6 .mu.m/ml, at least 0.7 .mu.m/ml, at least
0.8 .mu.m/ml, at least 0.9 .mu.m/ml, at least 1 .mu.g/ml, at least
2 .mu.g/ml, at least 3 .mu.g/ml, at least 4 .mu.m/ml, at least 5
.mu.g/ml, at least 6 .mu.g/ml, at least 7 .mu.g/ml, at least 8
.mu.g/ml, at least 9 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 30
.mu.g/ml, at least 35 .mu.g/ml, at least 40 .mu.g/ml, at least 45
.mu.g/ml, at least 50 .mu.g/ml, at least 55 .mu.g/ml, at least 60
.mu.g/ml, at least 65 .mu.g/ml, at least 70 .mu.g/ml, at least 75
.mu.g/ml, at least 80 .mu.g/ml, at least 85 .mu.g/ml, at least 90
.mu.g/ml, at least 95 .mu.g/ml, or at least 100 .mu.g/ml.
MYC Fusion Proteins
[0098] In some embodiments, the PTD-MYC fusion polypeptide
comprises a protein transduction domain (PTD), a MYC polypeptide
that promotes one or more of cell survival or proliferation, and
optionally a protein tag domain, e.g., one or more amino acid
sequences that facilitate purification of the fusion protein. In
some embodiments, a cell contacted with MYC polypeptide exhibits
increased survival time (e.g., as compared to an identical or
similar cell of the same type that was not contacted with MYC),
and/or increased proliferation (e.g., as compared to an identical
or similar cell of the same type that was not contacted with
MYC).
[0099] In some embodiments, the fusion protein comprises (a) a
protein transduction domain; and (b) a MYC polypeptide sequence. In
some embodiments, the fusion peptide is a peptide of Formula
(I):
protein transduction domain-MYC polypeptide sequence.
[0100] In some embodiments, a fusion peptide disclosed herein
comprises (a) a protein transduction domain; (b) a MYC polypeptide
sequence; and (c) one or more molecules that link the protein
transduction domain and the MYC polypeptide sequence. In some
embodiments, the fusion peptide is a peptide of Formula (II):
protein transduction domain-X-MYC polypeptide sequence,
wherein --X-- is molecule that links the protein transduction
domain and the MYC polypeptide sequence. In some embodiments, --X--
is at least one amino acid.
[0101] In some embodiments, a fusion peptide disclosed herein
comprises (a) a protein transduction domain; (b) a MYC polypeptide
sequence; (c) at least two protein tags; and (d) optionally
linker(s). In some embodiments, the fusion peptide is a peptide of
Formula (III-VI):
protein transduction domain-X-MYC polypeptide sequence-X-protein
tag 1-X-protein tag 2 (Formula (III)), or
protein transduction domain-MYC polypeptide sequence-X-protein tag
1-X-protein tag 2 (Formula (IV)), or
protein transduction domain-MYC polypeptide sequence-protein tag
1-X-protein tag 2 (Formula (V)), or
protein transduction domain-MYC polypeptide sequence-protein tag
1-protein tag 2 (Formula (VI)),
wherein --X-- is a linker. In some embodiments, --X-- is one or
more amino acids.
[0102] In some embodiments, a fusion peptide disclosed herein
comprises (a) a protein transduction domain; (b) a MYC polypeptide
sequence; (c) a 6-histidine tag; (d) a V5 epitope tag: and (e)
optionally linker(s). In some embodiments, the fusion peptide is a
peptide of Formula (VII-XIV):
protein transduction domain-X-MYC polypeptide
sequence-X-6-histidine tag-X-V5 epitope tag (Formula (VII)), or
protein transduction domain-MYC polypeptide sequence-X-6-histidine
tag-X-V5 epitope tag (Formula (VIII)), or
protein transduction domain-MYC polypeptide sequence-6-histidine
tag-X-V5 epitope tag (Formula (IX)), or
protein transduction domain-MYC polypeptide sequence-6-histidine
tag-V5 epitope tag (Formula (X)),
protein transduction domain-X-MYC polypeptide sequence-X-V5 epitope
tag-X-6-histidine tag (Formula (XI)), or
protein transduction domain-MYC polypeptide sequence-X-V5 epitope
tag-X-6-histidine tag (Formula (XII)), or
protein transduction domain-MYC polypeptide sequence-V5 epitope
tag-X-6-histidine tag (Formula (XIII)), or
protein transduction domain-MYC polypeptide sequence-V5 epitope
tag-6-histidine tag (Formula (XIV)),
wherein --X-- is a linker. In some embodiments, --X-- is one or
more amino acids.
[0103] As noted above, in some embodiments, the MYC fusion protein
comprises one or more linker sequences. The linker sequences can be
employed to link the protein transduction domain, MYC polypeptide
sequence, V5 epitope tag and/or 6-histidine tag of the fusion
protein. In some embodiments, the linker comprises one or more
amino acids. In some embodiments, the amino acid sequence of the
linker comprises KGELNSKLE. In some embodiments, the linker
comprises the amino acid sequence of RTG.
[0104] Protein Transduction Domain (PTD)
[0105] In some embodiments, the MYC fusion protein includes a
protein transduction domain. Peptide transport provides an
alternative for delivery of small molecules, proteins, or nucleic
acids across the cell membrane to an intracellular compartment of a
cell. One non-limiting example and well-characterized protein
transduction domain (PTD) is a TAT-derived peptide. Frankel et al.,
(see, e.g., U.S. Pat. Nos. 5,804,604, 5,747,641, 5,674,980,
5,670,617, and 5,652,122) demonstrated transport of a cargo protein
(.beta.-galactosidase or horseradish peroxidase) into a cell by
conjugating a peptide containing amino acids 48-57 of TAT to the
cargo protein. In some embodiments, TAT comprises an amino acid
sequence of MRKKRRQRRR (SEQ ID NO: 7).
[0106] Another non-limiting example of a PTD is penetratin.
Penetratin can transport hydrophilic macromolecules across the cell
membrane (Derossi et al., Trends Cell Biol., 8:84-87 (1998)
incorporated herein by reference in its entirety). Penetratin is a
16 amino acid peptide that corresponds to amino acids 43-58 of the
homeodomain of Antennapedia, a Drosophila transcription factor
which is internalized by cells in culture.
[0107] Yet another non-limiting example of a PTD is VP22. VP22, a
tegument protein from Herpes simplex virus type 1 (HSV-1), has the
ability to transport proteins and nucleic acids across a cell
membrane (Elliot et al., Cell 88:223-233, 1997, incorporated herein
by reference in its entirety). Residues 267-300 of VP22 are
necessary but cannot be sufficient for transport. Because the
region responsible for transport function has not been identified,
the entire VP22 protein is commonly used to transport cargo
proteins and nucleic acids across the cell membrane (Schwarze et
al., Trends Pharmacol Sci, 21:45-48, 2000).
[0108] In some embodiments, the PTD-MYC fusion polypeptide includes
a protein transduction domain. By way of example, but not by way of
limitation, in some embodiments, the protein transduction domain
comprises the protein transduction domain of one or more of TAT,
penetratin, VP22, vpr, EPTD, R9, R15, VP16, and Antennapedia. In
some embodiments, the protein transduction domain comprises the
protein transduction domain of one or more of TAT, penetratin,
VP22, vpr, and EPTD. In some embodiments, the protein transduction
domain comprises the protein transduction domain of at least one of
TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP16, and Antennapedia.
In some embodiments, the protein transduction domain comprises a
synthetic protein transduction domain (e.g., polyarginine or
PTD-5). In particular embodiments, the protein transduction domain
comprises a TAT protein transduction domain. In some embodiments,
the protein transduction domain is covalently linked to the MYC
polypeptide. In some embodiments, the protein transduction domain
is linked to the MYC polypeptide via a peptide bond. In some
embodiments, the protein transduction domain is linked to the MYC
polypeptide via a linker sequence. In some embodiments, the linker
comprises a short amino acid sequence. By way of example, but not
by way of limitation, in some embodiments, the linker sequences is
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.
[0109] The MYC fusion protein of the present technology can be
arranged in any desired order. For example, in some embodiments,
the MYC fusion protein can be arranged in order of a) the protein
transduction domain linked in frame to the MYC polypeptide, b) the
MYC polypeptide linked in frame to the V5 domain, and c) the V5
domain linked in frame to the 6-histidine epitope tag. In some
embodiments, the MYC fusion protein has an order of components of
a) the MYC polypeptide linked in frame to the protein transduction
domain, b) the protein transduction domain linked in frame to the
V5 domain, and c) the V5 domain linked in frame to the 6-histidine
epitope tag. In some embodiments, additional amino acid sequences
can be included between each of the sequences. In some embodiments,
additional amino acids can be included at the start and/or end of
the polypeptide sequences.
[0110] In some embodiments, the protein transduction domain is a
TAT protein transduction domain. In some embodiments, the protein
transduction domain is TAT.sub.[48-57]. In some embodiments, the
protein transduction domain is TAT.sub.[57-48].
[0111] Protein Tag Domains
[0112] In some embodiments, the MYC fusion protein comprises a
protein tag domain that comprises one or more amino acid sequences
that facilitate purification of the fusion protein. In some
embodiments, the protein tag domain comprises one or more of a
polyhistidine tag, and an epitope tag. By way of example, but not
by way of limitation, exemplary tags include one or more of a V5, a
histidine-tag (e.g., a 6-histidine tag), HA (hemagglutinin) tags,
FLAG tag, CBP (calmodulin binding peptide), CYD (covalent yet
dissociable NorpD peptide), Strepll, or HPC (heavy chain of protein
C). In some embodiments, the protein tag domain comprise about 10
to 20 amino acids in length. In some embodiments, the protein tag
domain comprises 2 to 40 amino acids in length, for example 6-20
amino acids in length. In some embodiments, two of the above listed
tags (for example, V5 and the HIS-tag) are used together to form
the protein tag domain.
[0113] In some embodiments, the histidine tag is a 6-histidine tag.
In some embodiments, the histidine tag comprises the sequence
HHHHHH (SEQ ID NO:8). In some embodiments, the fusion peptide
disclosed herein comprises a V5 epitope tag. In some embodiments,
the V5 tag comprises the amino acid sequence of: GKPIPNPLLGLDST
(SEQ ID NO:9). In some embodiments, the V5 tag comprises the amino
acid sequence of IPNPLLGLD (SEQ ID NO:10).
[0114] The protein tags can be added to the fusion protein
disclosed herein by any suitable method. By way of example, but not
by way of limitation, in some embodiments, a TAT-MYC polypeptide
sequence is cloned into an expression vector encoding one or more
protein tags, e.g., a polyHis-tag and/or a V5 tag. In some
embodiments, a polyhistidine tag and/or a V5 tag is added by PCR
(i.e., the PCR primers comprise a polyhistidine sequence and/or V5
sequence).
[0115] Construction of PTD-MYC Fusion Polypeptides
[0116] PTD-MYC fusion polypeptides (e.g., TAT-MYC fusion
polypeptide) disclosed herein can be constructed by methods well
known in the art. By way of example, but not by way of limitation,
a nucleotide sequence encoding a TAT-MYC fusion polypeptide can be
generated by PCR. In some embodiments, a forward primer for a human
MYC sequence comprises an in frame N-terminal 9-amino-acid sequence
of the TAT protein transduction domain (e.g., RKKRRQRRR). In some
embodiments, a reverse primer for a human MYC sequence is designed
to remove the stop codon. In some embodiments, the PCR product is
cloned into any suitable expression vector. In some embodiments,
the expression vector comprises a polyhistidine tag and a V5
tag.
[0117] In some embodiments, a fusion peptide disclosed herein
comprises (a) TAT, and (b) c-MYC. In some embodiments, a fusion
peptide disclosed herein comprises (a) TAT.sub.[48-57], and (b)
c-MYC. In some embodiments, a fusion peptide disclosed herein
comprises (a) TAT.sub.[57-48], and (b) c-MYC.
[0118] In some embodiments, a fusion peptide disclosed herein
comprises (a) TAT, (b) c-MYC, (c) linker(s), (d) V5 tag, and (e)
6-histidine tag. In some embodiments, a fusion peptide disclosed
herein comprises (a) TAT.sub.[48-57], (b) c-MYC, (c) linker(s), (d)
V5 tag, and (e) 6-histidine tag. In some embodiments, a fusion
peptide disclosed herein comprises (a) TAT.sub.[57-48], (b) c-MYC,
(c) linker(s), (d) V5 tag, and (e) 6-histidine tag.
[0119] In some embodiments, the PTD-MYC fusion polypeptide
comprises SEQ ID NO: 1; in some embodiments, the PTD-MYC fusion
polypeptide is SEQ ID NO: 1.
TABLE-US-00005 (SEQ ID NO: 1)
MRKKRRQRRRPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSEL
QPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGG
GGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGF
SAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASE
CIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPE
PLVLREETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAG
GHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLR
QISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPE
LENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLE
QLRKGELNSKLEGKPIPNPLLGLDSTRTGEITEHTEHH.
[0120] The fusion protein can be modified during or after synthesis
to include one or more functional groups. By way of example but not
by way of limitation, the protein can be modified to include one or
more of an acetyl, phosphate, acetate, amide, alkyl, and/or methyl
group. This list is not intended to be exhaustive, and is exemplary
only. In some embodiments, the protein includes at least one acetyl
group.
[0121] A PTD-MYC fusion polypeptide can be generated by any
suitable method known the art, e.g. by recombinant protein
expression in a cell, such as a bacterial cell, an insect cell, or
mammalian cell. In some embodiments, a PTD-MYC fusion polypeptide
is recombinantly produced by microbial fermentation. In some
embodiments microbial fermentation is performed in a fermentation
volume of from about 1 to about 10,000 liters, for example, a
fermentation volume of about 10 to about 1000 liters. The
fermentation can utilize any suitable microbial host cell and
culture medium. In exemplary embodiments, E. coli is utilized as
the microbial host cell. In alternative embodiments, other
microorganisms can be used, e.g., S. cerevisiae, P. pastoris,
Lactobacilli, Bacilli and Aspergilli. In an exemplary embodiment
the microbial host cell is BL-21 Star.TM. E. coli strain
(Invitrogen). In an exemplary embodiment the microbial host cell is
BLR DE3 E. coli. strain.
[0122] In some embodiments the host cells are modified to provide
tRNAs for rare codons, which are employed to overcome host
microbial cell codon bias to improve translation of the expressed
proteins. In exemplary embodiments, the host cells (e.g., E. coli)
transformed with a plasmid, such as pRARE (CamR), which express
tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons. Additional, suitable
plasmids or constructs for providing tRNAs for particular codons
are known in the art and can be employed in the methods
provided.
[0123] Integrative or self-replicative vectors can be used for the
purpose of introducing the PTD-MYC fusion polypeptide expression
cassette into a host cell of choice. In an expression cassette, the
coding sequence for the PTD-MYC fusion polypeptide is operably
linked to promoter, such as an inducible promoter. Inducible
promoters are promoters that initiate increased levels of
transcription from DNA under their control in response to some
change in culture conditions, e.g., the presence or absence of a
nutrient or a change in temperature. In some embodiments, the
nucleic acid encoding the PTD-MYC fusion polypeptide is codon
optimized for bacterial expression.
[0124] Exemplary promoters that are recognized by a variety of
potential host cells are well known. These promoters can be
operably linked to PTD-MYC fusion polypeptide-encoding DNA by
removing the promoter from the source DNA, if present, by
restriction enzyme digestion and inserting the isolated promoter
sequence into the vector. Promoters suitable for use with microbial
hosts include, but are not limited to, the .beta.-lactamase and
lactose promoter systems (Chang et al, (1978) Nature, 275:617-624;
Goeddel et al., (1979) Nature, 281: 544), alkaline phosphatase, a
tryptophan (trp) promoter system (Goeddel (1980) Nucleic Acids Res.
8: 4057; EP 36,776), and hybrid promoters such as the tac promoter
(deBoer et al, (1983) Proc. Natl. Acad. Sci. USA 80: 21-25). Any
promoter for suitable for expression by the selected host cell can
be used. Nucleotide sequences for suitable are published, thereby
enabling a skilled worker operably to ligate them to DNA encoding
PTD-MYC fusion polypeptide (see, e.g., Siebenlist et al., (1980)
Cell 20: 269) using linkers or adaptors to supply any required
restriction sites. In exemplary embodiments, promoters for use in
bacterial systems can contain a Shine-Dalgarno (S.D.) sequence
operably linked to the coding sequence. In some embodiments, the
inducible promoter is the lacZ promoter, which is induced with
Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG), as is well-known
in the art. Promoters and expression cassettes can also be
synthesized de novo using well known techniques for synthesizing
DNA sequences of interest. In an exemplary embodiment, the
expression vector for expression of the PTD-MYC fusion polypeptides
herein is pET101/D-Topo (Invitrogen).
[0125] For expression of the PTD-MYC fusion polypeptides, the
microbial host containing the expression vector encoding the
PTD-MYC fusion polypeptide is typically grown to high density in a
fermentation reactor. In some embodiments, the reactor has
controlled feeds for glucose. In some embodiments, a fermenter
inoculum is first cultured in medium supplemented with antibiotics
(e.g., overnight culture). The fermenter inoculum is then used to
inoculate the fermenter culture for expression of the protein. At
an OD600 of at least about 15, usually at least about 20, at least
25, at least about 30 or higher, of the fermenter culture,
expression of the recombinant protein is induced. In exemplary
embodiments, where the inducible promoter is the lacZ promoter,
IPTG is added to the fermentation medium to induce expression of
the PTD-MYC fusion polypeptide. Generally, the IPTG is added to the
fermenter culture at an OD600 which represents logarithmic growth
phase.
[0126] In certain embodiments of the methods provided, induced
protein expression is maintained for around about 2 to around about
5 hours post induction, and can be from around about 2 to around
about 3 hours post-induction. Longer periods of induction may be
undesirable due to degradation of the recombinant protein. The
temperature of the reaction mixture during induction is preferably
from about 28.degree. C. to about 37.degree. C., usually from about
30.degree. C. to about 37.degree. C. In particular embodiments,
induction is at about 37.degree. C.
[0127] The PTD-MYC fusion polypeptide is typically expressed as
cytosolic inclusion bodies in microbial cells. To harvest inclusion
bodies, a cell pellet is collected by centrifugation of the
fermentation culture following induction, frozen at -70.degree. C.
or below, thawed and resuspended in disruption buffer. The cells
are lysed by conventional methods, e.g., sonication,
homogenization, etc. The lysate is then resuspended in
solubilization buffer, usually in the presence of urea at a
concentration effective to solubilize proteins, e.g., from around
about 5M, 6M, 7M, 8M, 9M or greater. Resuspension may require
mechanically breaking apart the pellet and stirring to achieve
homogeneity. In some embodiments, the cell pellet is directly
resuspended in urea buffer and mixed until homogenous. In some
embodiments, the resuspension/solubilization buffer is 8M Urea, 50
mM Phosphate pH 7.5 and the suspension is passed through a
homogenizer.
[0128] In some embodiments, the homogenized suspension is
sulfonylated. For example, in some embodiments, the homogenized
suspension is adjusted to include 200 mM Sodium Sulfite and 10 mM
Sodium Tetrathionate. The solution is then mixed at room
temperature until homogeneous. The mixed lysate is then mixed for
an additional period of time to complete the sulfonylation (e.g.,
at 2-8.degree. C. for .gtoreq.12 hours). The sulfonylated lysate
was then centrifuged for an hour. The supernatant containing the
sulfonylated PTD-MYC fusion polypeptides is then collected by
centrifugation and the cell pellet discarded. The supernatant is
then passed through a filter, e.g., 0.22 .mu.m membrane filter to
clarify the lysate.
[0129] The solubilized protein is then purified. Purification
methods may include affinity chromatography, reverse phase
chromatography, gel exclusion chromatography, and the like. In some
embodiments, affinity chromatography is used. For example, the
protein is provided with an epitope tag or histidine 6 tag for
convenient purification. In the present methods, exemplary PTD-MYC
fusion polypeptide comprise histidine 6 tag for purification using
Ni affinity chromatography using Ni-resin.
[0130] In exemplary embodiments, the Ni-resin column is
equilibrated in a buffer containing urea. In some embodiments, the
equilibration buffer is 6M Urea, 50 mM Phosphate, 500 mM NaCl, and
10% Glycerol solution. The sulfonylated and clarified supernatant
comprising the PTD-MYC fusion polypeptide is then loaded onto the
Ni-resin column. The column is then washed with a wash buffer,
e.g., 6M Urea, 50 mM Phosphate, 10% Glycerol, 500 mM NaCl, pH 7.5.
The column was then washed with sequential wash buffers with
decreasing salt concentration. For example, exemplary subsequent
washed can include 6M Urea, 50 mM Phosphate, 10% Glycerol, and 2M
NaCl, pH 7.5, followed another wash of 6M Urea, 50 mM Phosphate,
10% Glycerol, 50 mM NaCl, and 30 mM Imidazole, pH 7.5.
[0131] Following sequential application of the wash buffers the
PTD-MYC fusion polypeptide is eluted from the column by addition of
elution buffer, e.g., 6M Urea, 50 mM Phosphate, 10% Glycerol, and
50 mM NaCl, pH 7.5 with a gradient from 100 to 300 mM Imidazole,
and collecting fractions. The protein containing fractions to be
pooled are then filtered through a 0.22 .mu.m membrane. Assessment
of protein yield can be measured using any suitable method, e.g.,
spectrophotometry at UV wavelength 280.
[0132] In some embodiments, one or more additional purification
methods can be employed to further purify the isolated PTD-MYC
fusion polypeptides. In exemplary embodiments, the pooled fractions
from the Ni-Sepharose chromatography step are further purified by
anion exchange chromatography using a Q-Sepharose resin. In some
embodiments, the pool is prepared for loading onto the Q-Sepharose
column by diluting the samples to the conductivity of the Q
sepharose buffer (17.52+/-1 mS/cm) with the second wash buffer
(e.g., 6M Urea, 50 mM Phosphate, 10% Glycerol, 2M NaCl, pH 7.5)
from the Ni Sepharose chromatography step. The diluted pool is then
loaded onto the Q-Sepharose column, followed by two chase steps
using a chase buffer (e.g., 6M Urea, 50 mM Phosphate, 300 mM NaCl,
and 10% Glycerol), with further sequential applications of the
chase buffer until the UV trace reaches baseline, indicating that
the protein has eluted from the column.
Methods of Treatment
[0133] The PTD-MYC fusion polypeptide-modified immune cells are
administered for the treatment of a melanoma in a patient. In some
embodiments, the patient has a metastatic melanoma. In some
embodiments, the patient has received one or more agents for the
treatment of the melanoma prior to administration of the PTD-MYC
fusion polypeptide-modified immune cells. In some embodiments, the
melanoma is a relapsed or refractory melanoma. In some embodiments,
the melanoma is a metastatic melanoma. In some embodiments, the
melanoma is a superficial spreading melanoma, a nodular melanoma, a
lentigo maligna melanoma, or an acral melanoma. In some
embodiments, the melanoma is resistant to one or more agents for
the treatment of the melanoma.
[0134] In some embodiments, administration of the PTD-MYC fusion
polypeptide-modified immune cells inhibits growth of a melanoma
tumor or reduces the volume of a melanoma tumor. In some
embodiments, administration of the PTD-MYC fusion
polypeptide-modified immune cells to a subject having a melanoma
alleviates one or more symptoms of the melanoma. In some
embodiments, administration of the PTD-MYC fusion
polypeptide-modified immune cells to a subject having melanoma
increases the overall survival of the subject. In some embodiments,
administration of the PTD-MYC fusion polypeptide-modified immune
cells to a subject having melanoma increases the regression of the
melanoma.
[0135] The administration of the PTD-MYC fusion
polypeptide-modified immune cells (e.g. PTD-MYC fusion polypeptide
treated tumor infiltrating lymphocytes) according to the methods
provided herein can be carried out in any suitable manner for
administering cells to a subject, including but not limited to
injection, transfusion, implantation or transplantation. In some
embodiments, the PTD-MYC fusion polypeptide-modified immune cells
are administered to a patient subcutaneously, intradermally,
intratumorally, intranodally, intramedullary, intramuscularly,
intrathecally, by intravenous or intralymphatic injection, or
intraperitoneally. In some embodiments, the PTD-MYC fusion
polypeptide-immune cells are administered into a cavity formed by
the resection of tumor tissue (i.e. intracavity delivery) or
directly into a tumor prior to resection (i.e. intratumoral
delivery). In one embodiment, the MYC-fusion polypeptide-immune
cells are administered by intravenous injection.
[0136] In addition to the PTD-MYC fusion polypeptide-modified
immune cells, compositions for administration can comprise any
other agents such as pharmaceutically acceptable carriers, buffers,
excipients, adjuvants, additives, antiseptics, filling, stabilizing
and/or thickening agents, and/or any components normally found in
corresponding products. Selection of suitable ingredients and
appropriate manufacturing methods for formulating the compositions
for particular routes of administration generally known in the
art.
[0137] The adoptive cell therapeutic composition comprising PTD-MYC
fusion polypeptide-modified immune cells can be in any form, such
as solid, semisolid or liquid form, suitable for administration. A
formulation can be selected from a group consisting of, but not
limited to, solutions, emulsions, suspensions, tablets, pellets and
capsules. The compositions are not limited to a certain
formulation, instead the composition can be formulated into any
known pharmaceutically acceptable formulation. The pharmaceutical
compositions may be produced by any conventional processes known in
the art.
[0138] In some embodiments, the administration of the MYC-fusion
polypeptide-modified immune cells comprises administering of
10.sup.4-10.sup.10 of the cells per kg body weight, including
10.sup.5 to 10.sup.6 cells/kg body weight, including all integer
values of cell numbers within those ranges. In some embodiments,
the cells are administered with or without a course of
lymphodepletion, for example with cyclophosphamide.
[0139] The MYC-fusion polypeptide-modified immune cells can be
administrated in one or more doses. In one embodiment, the
therapeutically effective amount of PTD-MYC fusion
polypeptide-modified immune cells are administrated as a single
dose. In some embodiments, administering a single dose of the
PTD-MYC fusion polypeptide-modified immune cells has a therapeutic
effect. In another embodiment, the effective amount of MYC-fusion
polypeptide-modified immune cells are administrated as more than
one dose over a period time. Timing of administration is within the
judgment of managing physician and depends on various factors,
including, but not limited to the age, gender, or clinical
condition of the patient and characteristics of the melanoma,
including type, degree or location of melanoma. While individual
needs vary, determination of optimal ranges of effective amounts of
a MYC-fusion polypeptide-modified immune cell for treatment of a
particular disease or conditions are within the skill of one in the
art.
[0140] PTD-MYC fusion polypeptide-modified immune cells can be
administered for example from 1 to 10 times in the first 2 weeks, 3
weeks, 4 weeks, monthly or during the treatment period. In some
embodiments, PTD-MYC fusion polypeptide-modified immune cells are
administered 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some
embodiments, PTD-MYC fusion polypeptide-modified immune cells are
administered weekly, every 2 weeks, every 3 weeks or monthly.
[0141] A therapeutically effective amount means an amount which
provides a therapeutic or prophylactic benefit. The dosage
administrated will be dependent upon the age, health and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment and the nature of the effect desired.
[0142] In some embodiments, a patient receiving PTD-MYC modified
immune cells are first pretreated with one or more cytokines and/or
other immunomodulatory agents. In some embodiments, a patient
receiving PTD-MYC modified immune cells is lymphodepleted prior to
administration of the PTD-MYC modified immune cells. The purpose of
lymphodepletion is to make room for the infused lymphocytes, in
particular by eliminating regulatory T cells and other non-specific
T cells which compete for homeostatic cytokines.
[0143] In some embodiments, the PTD-MYC modified immune cells are
administered with an additional therapeutic agent. In some
embodiments, additional therapeutic agent is administered prior to,
simultaneously with, intermittently with, or following treatment
with the PTD-MYC modified immune cells. In some embodiments, the
additional therapeutic agent is an immunomodulator, such as an
interleukin (e.g. IL-2, IL-7, IL-12), a cytokine, a chemokine, or
and immunomodulatory drug. In some embodiments, the cytokine is
selected from among cytokine is selected from a group consisting of
interferon alpha, interferon beta, interferon gamma, complement
C5a, IL-2, TNF.alpha., CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11,
CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16,
CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1,
CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3,
CCL3L1, CCL4, CCL4L1, CCL5 (=RANTES), CCL6, CCL7, CCL8, CCL9,
CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR,
CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16,
CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9,
CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2. In some
embodiments, the additional therapeutic agent is an anticancer
agent, such as chemotherapy or radiation therapy.
[0144] In some embodiments, the modified immune cells administered
for the treatment of melanoma are T cells with genetically modified
antigen receptors, including chimeric antigen receptor (CAR)-T
cells. Various strategies can, for example, be employed to
genetically modify T cells by altering the specificity of the T
cell receptor (TCR), for example, by introducing new TCR .alpha.
and .beta. chains with selected peptide specificity (see, e.g.,
U.S. Pat. No. 8,697,854; PCT Patent Publications: WO2003020763,
WO2004033685, WO2004044004, WO2005114215, WO2006000830,
WO2008038002, WO2008039818, WO2004074322, WO2005113595,
WO2006125962, WO2013166321, WO2013039889, WO2014018863,
WO2014083173; U.S. Pat. No. 8,088,379). Chimeric antigen receptors
(CARs) can be used in order to generate immunoresponsive cells,
such as T cells, specific for selected targets, such as malignant
cells, with a wide variety of receptor chimera constructs having
been described (see, e.g. U.S. Pat. Nos. 5,843,728; 5,851,828;
5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162;
8,211,422; and, PCT Publication WO9215322). Methods for the
preparation of CART cells are known in the art and can be used in
combination with the methods provided herein to generate modified
CAR T cells comprising a MYC fusion polypeptide (e.g. PTD) as
described herein.
[0145] In general, CARs are comprised of an extracellular domain, a
transmembrane domain, and an intracellular domain, wherein the
extracellular domain comprises an antigen-binding domain that is
specific for a predetermined target. While the antigen-binding
domain of a CAR is often an antibody or antibody fragment (e.g., a
single chain variable fragment, scFv), the binding domain is not
particularly limited so long as it results in specific recognition
of a target. For example, in some embodiments, the antigen-binding
domain may comprise a receptor, such that the CAR is capable of
binding to the ligand of the receptor. Alternatively, the
antigen-binding domain may comprise a ligand, such that the CAR is
capable of binding the endogenous receptor of that ligand.
[0146] In some embodiments, the T cells expressing a desired CAR
are selected through co-culture with .gamma.-irradiated activating
and propagating cells (AaPC), which co-express the melanoma antigen
and co-stimulatory molecules. In some embodiments, the engineered
CAR T- cells are expanded, for example by co-culture on AaPC in
presence of soluble factors, such as IL-2 and IL-21. This expansion
can for example be carried out so as to provide memory CAR+ T
cells. In this way, CAR T cells can be provided that have specific
cytotoxic activity against antigen-bearing tumors (optionally in
conjunction with production of desired chemokines such as
interferon-.gamma.).
[0147] In some embodiments, the CAR T-cells are contacted with a
PTD-MYC fusion polypeptide provided herein in vitro to generation a
modified CAR T cells for the treatment of a melanoma. The modified
CAR T cells can be administered according any suitable method,
including the methods for administration of the PTD-MYC fusion
polypeptide-modified immune cells as described above.
Kits
[0148] Pharmaceutical compositions comprising MYC-fusion
polypeptides and/or MYC-fusion polypeptide-modified immune cells
provided herein can be assembled into kits or pharmaceutical
systems for use in treating a melanoma. Kits according to this
embodiment can comprise a carrier means, such as a box, carton,
tube, having in close confinement therein one or more containers,
such as vials, tubes, ampoules, bottles, syringes, or bags. The
kits can also comprise associated instructions for using the
MYC-fusion polypeptides and/or MYC-fusion polypeptide-modified
immune cells.
[0149] In some embodiments, the kit comprises an effective amount
of an adoptive cell therapy, such as MYC-fusion
polypeptide-modified immune cells. In some embodiments, the kit
comprises one for more reagents for the detection of the
administered MYC-fusion polypeptides and/or MYC-fusion
polypeptide-modified immune cells. In some embodiments, the kit
comprises cells for treatment with a MYC-fusion polypeptide
provided herein, for example, hematopoietic stem cells, donor
leukocytes, T cells, or NK cells. In some embodiments, the kit
further comprises an effective amount of a therapeutic agent to be
administered in combination with MYC-fusion polypeptides and/or
MYC-fusion polypeptide-modified immune cells provided herein. In
some embodiments, therapeutic agent is an anti-cancer agent.
[0150] Kits provided herein also can include a device for
administering MYC-fusion polypeptides and/or MYC-fusion
polypeptide-modified immune cells provided herein to a subject. Any
of a variety of devices known in the art for administering
polypeptides and cells to a subject can be included in the kits
provided herein. Exemplary devices include a hypodermic needle, an
intravenous needle, a catheter, a needle-less injection, but are
not limited to, a hypodermic needle, an intravenous needle, a
catheter, a needle-less injection device, an inhaler and a liquid
dispenser such as an eyedropper. Typically the device for
administering the MYC-fusion polypeptides and/or MYC-fusion
polypeptide-modified immune cells of the kit will be compatible
with the desired method of administration of the composition. For
example, a composition to be delivered intravenously can be
included in a kit with a hypodermic needle and a syringe.
EXAMPLES
Example 1. Immune Cells Treated with TAT-MYC to Generate
TAT-MYC-Treated Lymphocytes for Immunotherapy of Melanoma
Tumors
[0151] In this example, the ability of a PTD-MYC fusion polypeptide
comprising the protein transduction domain of HIV-1 transactivation
protein (TAT) and MYC to modulate an immune response against
melanoma cells in vivo was examined. Specifically, the ability of
lymphoid cells, derived from melanoma-bearing mice and treated with
TAT-MYC, to treat mice harboring melanoma tumors was studied. The
object of these studies was to determine whether immune cells
derived from melanoma bearing mice and treated with TAT-MYC to
generate TAT-MYC lymphocytes would be an effective treatment for
melanoma tumors upon transplantation into melanoma bearing
mice.
[0152] Materials and Methods
[0153] C57BL/6J is the most widely used inbred strain and the first
to have its genome sequenced. Although this strain is refractory to
many tumors, it is a permissive background for maximal expression
of most mutations. C57BL/6J mice are resistant to audiogenic
seizures, have a relatively low bone density, and develop
age-related hearing loss. They are also susceptible to diet-induced
obesity, type 2 diabetes, and atherosclerosis. Macrophages from
this strain are resistant to the effects of anthrax lethal
toxin.
[0154] Treatment Groups
[0155] Fifteen C57BL/6 mice (Jackson Laboratory Stock#000664)
weighing approximately 25 g and harboring melanoma tumors were
generated and divided into 3 cohorts of 5 animals, one cohort of
one mouse as a no treatment control, one cohort treated with
Lymphoid cells derived from tumor-bearing mice and treated with
control TAT-fusion protein, and one cohort treated with TAT-MYC
lymphocytes.
[0156] Generation of Tumor-Bearing Donor Mice and Preparation of
Donor Cells
[0157] B16-F10 melanoma cells (ATCC CRL 6475, mouse skin melanoma)
for implantation were cultured in D10 media (DMEM, 10% FBS,
Pen/Strep (10,000 units per/ml) (Gibco Cat#15140); L-glutamine (200
mM) (Gibco Cat#25030); MEM Non-essential Amino Acids (Gibco
Cat#11140)).
[0158] The C57BL/6j mice (Jackson Laboratory #003548) were
implanted with 1.times.10.sup.4 B16-F10 melanoma cells in 250 .mu.L
PBS via tail vein injection. Prior to injection, each test mouse
was placed under a 250W heat lamp for 1-2 minutes and then injected
intravenously with the melanoma cells. At 14 days post-transplant,
lymph nodes from the injected mice were harvested and ground with
the plunger of a 10 mL syringe.
[0159] For the first study, lymph nodes were harvested from 5 mice.
For the second lymph nodes were harvested from 10 mice. The cells
were washed with C10, collected and spun at 260.times.g for 5 min.
After discarding the supernatant, the cells were resuspended in 10
mL sterile TAC, spun at 260.times.g for 5 minutes. After discarding
the supernatant, the cells were resuspended in 2 mL of sterile
filtered PBS with 5% BSA.
[0160] The lymph node cells were treated with TAT-MYC to generate
TAT-MYC lymphocytes or treated with a control TAT-Fusion protein.
The cells were split into 2, 15 mL conical tubes (1 mL each),
treated with 1 mL of 25 ug/ml of a control protein (TAT-CRE for
experiment 1, TAT-GFP for experiment 2) or 1 mL of 25 ug/ml of
TAT-MYC lot C18. After one hour of room temp incubation, each tube
was washed with sterile PBS three times, transferred to 5 mL
sterile tubes and placed on ice.
[0161] The test mice were prepared by injecting
1.times.10.sup.4B16-F10 melanoma cells in 250 uL PBS into the tail
vein for each cohort of 5 C57BL/6j mice. After injection, the mice
were observed once daily. Changes in body weight, food consumption,
activity, and mortality were monitored. At 7 days post-transplant,
TAT-MYC lymphocytes or control lymphoid cells were then
transplanted into melanoma cell injected mice.
[0162] Symptoms were monitored daily. The mice were euthanized when
severe symptoms presented and deaths were recorded. Mice were
either found dead or euthanized if found with severe symptoms such
as heavy breathing, hunched back and immobility. Day of death was
recorded with day of treatment as Day 0.
[0163] The results from Experiments 1 and 2 are shown in FIGS. 1
and 2, respectively. As shown in the figures, treating
melanoma-bearing mice with TAT-MYC lymphocytes (TBX-3400) generated
by contacting mouse lymphoid cells derived from melanoma bearing
mice with TAT-MYC, significantly improved the overall survival of
the mice compared to transplanting lymphoid cells treated with
control TAT-Fusion protein. These results suggest that TAT-MYC
treatment of immune cells are useful in the treatment of melanoma
using adoptive cell transfer.
Example 2. Dose Response Effect of TAT-MYC-Treated Lymphocytes for
Immunotherapy of Melanoma Tumors
[0164] In this example, the therapeutic effects of different
amounts administered TAT-MYC-treated lymphocytes for immunotherapy
of melanoma tumors was examined. This experiment was performed as
described above in Example 1, except that several different doses
of the TAT-MYC-treated lymphocytes were injected and compared. Two
experiments were performed. In the first experiment, Experiment 3,
TAT-MYC lymphocytes were administered to the melanoma-bearing mice
via tail vein injection according to the following dosing groups:
3.0.times.10.sup.6 cells/kg, 6.0.times.10.sup.6 cells/kg,
14.0.times.10.sup.6 cells/kg, and 70.0.times.10.sup.6 cells/kg. For
the control groups, the mice were administered 70.0.times.10.sup.6
TAT-Cre treated or no cells (NT). In the second experiment,
Experiment 4, TAT-MYC lymphocytes were administered to the
melanoma-bearing mice via tail vein injection according to the
following dosing groups: 4.0.times.10.sup.3 cells/kg,
4.0.times.10.sup.4 cells/kg, 4.0.times.10.sup.5 cells/kg,
4.0.times.10.sup.6 cells/kg and 4.0.times.10.sup.7 cells/kg. For
the control groups, the mice were administered 4.0.times.10.sup.6
TAT-Cre treated or no cells (NT). The results from Experiments 3
and 4 are shown in FIGS. 3 and 4, respectively. As shown in the
figures, treating melanoma-bearing mice with increasing amounts of
TAT-MYC lymphocytes (TBX-3400) led to a significantly improved
overall survival rate in both experiments. These experiments
demonstrate the both the reproducibility and efficacy of TAT-MYC
lymphocytes for treating melanoma-bearing subjects.
[0165] While preferred embodiments of the present disclosure have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
disclosure. It should be understood that various alternatives to
the embodiments of the disclosure described herein may be employed
in practicing the disclosure. It is intended that the following
claims define the scope of the disclosure and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
[0166] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0167] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
131476PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro
Leu Asn Val Ser Phe1 5 10 15Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp
Ser Val Gln Pro Tyr Phe 20 25 30Tyr Cys Asp Glu Glu Glu Asn Phe Tyr
Gln Gln Gln Gln Gln Ser Glu 35 40 45Leu Gln Pro Pro Ala Pro Ser Glu
Asp Ile Trp Lys Lys Phe Glu Leu 50 55 60Leu Pro Thr Pro Pro Leu Ser
Pro Ser Arg Arg Ser Gly Leu Cys Ser65 70 75 80Pro Ser Tyr Val Ala
Val Thr Pro Phe Ser Leu Arg Gly Asp Asn Asp 85 90 95Gly Gly Gly Gly
Ser Phe Ser Thr Ala Asp Gln Leu Glu Met Val Thr 100 105 110Glu Leu
Leu Gly Gly Asp Met Val Asn Gln Ser Phe Ile Cys Asp Pro 115 120
125Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile Ile Gln Asp Cys Met Trp
130 135 140Ser Gly Phe Ser Ala Ala Ala Lys Leu Val Ser Glu Lys Leu
Ala Ser145 150 155 160Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser Pro
Asn Pro Ala Arg Gly 165 170 175His Ser Val Cys Ser Thr Ser Ser Leu
Tyr Leu Gln Asp Leu Ser Ala 180 185 190Ala Ala Ser Glu Cys Ile Asp
Pro Ser Val Val Phe Pro Tyr Pro Leu 195 200 205Asn Asp Ser Ser Ser
Pro Lys Ser Cys Ala Ser Gln Asp Ser Ser Ala 210 215 220Phe Ser Pro
Ser Ser Asp Ser Leu Leu Ser Ser Thr Glu Ser Ser Pro225 230 235
240Gln Gly Ser Pro Glu Pro Leu Val Leu His Glu Glu Thr Pro Pro Thr
245 250 255Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu Asp Glu Glu Glu
Ile Asp 260 265 270Val Val Ser Val Glu Lys Arg Gln Ala Pro Gly Lys
Arg Ser Glu Ser 275 280 285Gly Ser Pro Ser Ala Gly Gly His Ser Lys
Pro Pro His Ser Pro Leu 290 295 300Val Leu Lys Arg Cys His Val Ser
Thr His Gln His Asn Tyr Ala Ala305 310 315 320Pro Pro Ser Thr Arg
Lys Asp Tyr Pro Ala Ala Lys Arg Val Lys Leu 325 330 335Asp Ser Val
Arg Val Leu Arg Gln Ile Ser Asn Asn Arg Lys Cys Thr 340 345 350Ser
Pro Arg Ser Ser Asp Thr Glu Glu Asn Val Lys Arg Arg Thr His 355 360
365Asn Val Leu Glu Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Phe Phe
370 375 380Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu Asn Asn Glu Lys
Ala Pro385 390 395 400Lys Val Val Ile Leu Lys Lys Ala Thr Ala Tyr
Ile Leu Ser Val Gln 405 410 415Ala Glu Glu Gln Lys Leu Ile Ser Glu
Glu Asp Leu Leu Arg Lys Arg 420 425 430Arg Glu Gln Leu Lys His Lys
Leu Glu Gln Leu Arg Lys Gly Glu Leu 435 440 445Asn Ser Lys Leu Glu
Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu 450 455 460Asp Ser Thr
Arg Thr Gly His His His His His His465 470 4752439PRTHomo sapiens
2Met Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr1 5
10 15Asp Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe
Tyr 20 25 30Gln Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser
Glu Asp 35 40 45Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu
Ser Pro Ser 50 55 60Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala
Val Thr Pro Phe65 70 75 80Ser Leu Arg Gly Asp Asn Asp Gly Gly Gly
Gly Ser Phe Ser Thr Ala 85 90 95Asp Gln Leu Glu Met Val Thr Glu Leu
Leu Gly Gly Asp Met Val Asn 100 105 110Gln Ser Phe Ile Cys Asp Pro
Asp Asp Glu Thr Phe Ile Lys Asn Ile 115 120 125Ile Ile Gln Asp Cys
Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu 130 135 140Val Ser Glu
Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly145 150 155
160Ser Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu
165 170 175Tyr Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp
Pro Ser 180 185 190Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser
Pro Lys Ser Cys 195 200 205Ala Ser Gln Asp Ser Ser Ala Phe Ser Pro
Ser Ser Asp Ser Leu Leu 210 215 220Ser Ser Thr Glu Ser Ser Pro Gln
Gly Ser Pro Glu Pro Leu Val Leu225 230 235 240His Glu Glu Thr Pro
Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln 245 250 255Glu Asp Glu
Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala 260 265 270Pro
Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser 275 280
285Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp
Tyr Pro305 310 315 320Ala Ala Lys Arg Val Lys Leu Asp Ser Val Arg
Val Leu Arg Gln Ile 325 330 335Ser Asn Asn Arg Lys Cys Thr Ser Pro
Arg Ser Ser Asp Thr Glu Glu 340 345 350Asn Val Lys Arg Arg Thr His
Asn Val Leu Glu Arg Gln Arg Arg Asn 355 360 365Glu Leu Lys Arg Ser
Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu 370 375 380Glu Asn Asn
Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr385 390 395
400Ala Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
405 410 415Glu Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys
Leu Glu 420 425 430Gln Leu Arg Asn Ser Cys Ala 4353450PRTHomo
sapiens 3Met Asp Phe Phe Arg Val Val Glu Asn Gln Gln Pro Pro Ala
Thr Met1 5 10 15Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu
Asp Tyr Asp 20 25 30Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu
Asn Phe Tyr Gln 35 40 45Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala
Pro Ser Glu Asp Ile 50 55 60Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro
Pro Leu Ser Pro Ser Arg65 70 75 80Arg Ser Gly Leu Cys Ser Pro Ser
Tyr Val Ala Val Thr Pro Phe Ser 85 90 95Leu Arg Gly Asp Asn Asp Gly
Gly Gly Gly Ser Phe Ser Thr Ala Asp 100 105 110Gln Leu Glu Met Val
Thr Glu Leu Leu Gly Gly Asp Met Val Asn Gln 115 120 125Ser Phe Ile
Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile 130 135 140Ile
Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val145 150
155 160Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly
Ser 165 170 175Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser
Ser Leu Tyr 180 185 190Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys
Ile Asp Pro Ser Val 195 200 205Val Phe Pro Tyr Pro Leu Asn Asp Ser
Ser Ser Pro Lys Ser Cys Ala 210 215 220Ser Gln Asp Ser Ser Ala Phe
Ser Pro Ser Ser Asp Ser Leu Leu Ser225 230 235 240Ser Thr Glu Ser
Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His 245 250 255Glu Glu
Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu 260 265
270Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro
275 280 285Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His
Ser Lys 290 295 300Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His
Val Ser Thr His305 310 315 320Gln His Asn Tyr Ala Ala Pro Pro Ser
Thr Arg Lys Asp Tyr Pro Ala 325 330 335Ala Lys Arg Val Lys Leu Asp
Ser Val Arg Val Leu Arg Gln Ile Ser 340 345 350Asn Asn Arg Lys Cys
Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn 355 360 365Val Lys Arg
Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu 370 375 380Leu
Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu385 390
395 400Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr
Ala 405 410 415Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile
Ser Glu Glu 420 425 430Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys
His Lys Leu Glu Gln 435 440 445Leu Arg 4504434PRTHomo sapiens 4Pro
Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp1 5 10
15Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr Gln
20 25 30Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp
Ile 35 40 45Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro
Ser Arg 50 55 60Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr
Pro Phe Ser65 70 75 80Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser
Phe Ser Thr Ala Asp 85 90 95Gln Leu Glu Met Val Thr Glu Leu Leu Gly
Gly Asp Met Val Asn Gln 100 105 110Ser Phe Ile Cys Asp Pro Asp Asp
Glu Thr Phe Ile Lys Asn Ile Ile 115 120 125Ile Gln Asp Cys Met Trp
Ser Gly Phe Ser Ala Ala Ala Lys Leu Val 130 135 140Ser Glu Lys Leu
Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser145 150 155 160Pro
Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu Tyr 165 170
175Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser Val
180 185 190Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser
Cys Ala 195 200 205Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp
Ser Leu Leu Ser 210 215 220Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro
Glu Pro Leu Val Leu His225 230 235 240Glu Glu Thr Pro Pro Thr Thr
Ser Ser Asp Ser Glu Glu Glu Gln Glu 245 250 255Asp Glu Glu Glu Ile
Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro 260 265 270Gly Lys Arg
Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser Lys 275 280 285Pro
Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His 290 295
300Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro
Ala305 310 315 320Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu
Arg Gln Ile Ser 325 330 335Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser
Ser Asp Thr Glu Glu Asn 340 345 350Val Lys Arg Arg Thr His Asn Val
Leu Glu Arg Gln Arg Arg Asn Glu 355 360 365Leu Lys Arg Ser Phe Phe
Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu 370 375 380Asn Asn Glu Lys
Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala385 390 395 400Tyr
Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu 405 410
415Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln
420 425 430Leu Arg567PRTHomo sapiens 5Glu Leu Lys Arg Ser Phe Phe
Ala Leu Arg Asp Gln Ile Pro Glu Leu1 5 10 15Glu Asn Asn Glu Lys Ala
Pro Lys Val Val Ile Leu Lys Lys Ala Thr 20 25 30Ala Tyr Ile Leu Ser
Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu 35 40 45Glu Asp Leu Leu
Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu 50 55 60Gln Leu
Arg65614PRTUnknownDescription of Unknown E-box DNA binding domain
sequence 6Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn1
5 10710PRTHuman immunodeficiency virus 1 7Met Arg Lys Lys Arg Arg
Gln Arg Arg Arg1 5 1086PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6xHis tag 8His His His His His His1
5914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp
Ser Thr1 5 10109PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Ile Pro Asn Pro Leu Leu Gly Leu Asp1
511454PRTHomo sapiens 11Met Asp Phe Phe Arg Val Val Glu Asn Gln Gln
Pro Pro Ala Thr Met1 5 10 15Pro Leu Asn Val Ser Phe Thr Asn Arg Asn
Tyr Asp Leu Asp Tyr Asp 20 25 30Ser Val Gln Pro Tyr Phe Tyr Cys Asp
Glu Glu Glu Asn Phe Tyr Gln 35 40 45Gln Gln Gln Gln Ser Glu Leu Gln
Pro Pro Ala Pro Ser Glu Asp Ile 50 55 60Trp Lys Lys Phe Glu Leu Leu
Pro Thr Pro Pro Leu Ser Pro Ser Arg65 70 75 80Arg Ser Gly Leu Cys
Ser Pro Ser Tyr Val Ala Val Thr Pro Phe Ser 85 90 95Leu Arg Gly Asp
Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala Asp 100 105 110Gln Leu
Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn Gln 115 120
125Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile
130 135 140Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys
Leu Val145 150 155 160Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg
Lys Asp Ser Gly Ser 165 170 175Pro Asn Pro Ala Arg Gly His Ser Val
Cys Ser Thr Ser Ser Leu Tyr 180 185 190Leu Gln Asp Leu Ser Ala Ala
Ala Ser Glu Cys Ile Asp Pro Ser Val 195 200 205Val Phe Pro Tyr Pro
Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys Ala 210 215 220Ser Gln Asp
Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu Ser225 230 235
240Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His
245 250 255Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu
Gln Glu 260 265 270Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys
Arg Gln Ala Pro 275 280 285Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser
Ala Gly Gly His Ser Lys 290 295 300Pro Pro His Ser Pro Leu Val Leu
Lys Arg Cys His Val Ser Thr His305 310 315 320Gln His Asn Tyr Ala
Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala 325 330 335Ala Lys Arg
Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile Ser 340 345 350Asn
Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn 355 360
365Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu
370 375 380Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu
Leu Glu385 390 395 400Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu
Lys Lys Ala Thr Ala 405 410 415Tyr Ile Leu Ser Val Gln Ala Glu Glu
Gln Lys Leu Ile Ser Glu Glu 420 425 430Asp Leu Leu Arg Lys Arg Arg
Glu Gln Leu Lys His Lys Leu Glu Gln 435 440 445Leu Arg Asn Ser Cys
Ala 450129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Lys Gly Glu Leu Asn Ser Lys Leu Glu1
5139PRTHuman immunodeficiency virus 1 13Arg Lys Lys Arg Arg Gln Arg
Arg Arg1 5
* * * * *