U.S. patent number 10,286,050 [Application Number 15/102,996] was granted by the patent office on 2019-05-14 for multi-epitope tarp peptide vaccine and uses thereof.
This patent grant is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Services. The grantee listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Services, The United States of America, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Jay A. Berzofsky, Brenda D. Roberson, Masaki Terabe, Lauren V. Wood.
United States Patent |
10,286,050 |
Wood , et al. |
May 14, 2019 |
Multi-epitope TARP peptide vaccine and uses thereof
Abstract
Immunogenic T cell receptor .gamma. alternate reading frame
protein (TARP) peptide compositions that include multiple epitopes
of the TARP protein are described. The disclosed compositions can
be used for the treatment of TARP-expressing cancers, such as
prostate cancer, breast cancer and mesothelioma. In some
embodiments, the TARP peptide compositions disclosed herein include
sets of overlapping TARP peptides that each have a length of about
15 to about 25 amino acids, and comprise about 5 to about 15 amino
acids that are identical to at least another overlapping peptide in
the set. In particular examples, the combination of the overlapping
TARP peptides in the set encompasses the complete amino acid
sequence of human TARP. The multi-epitope peptide compositions
described herein include both CD4 and CD8 epitopes, a feature that
is important for eliciting CD4.sup.+ T cell and CD8.sup.+ T cell,
as well as humoral, immune responses.
Inventors: |
Wood; Lauren V. (Bethesda,
MD), Berzofsky; Jay A. (Bethesda, MD), Roberson; Brenda
D. (Frederick, MD), Terabe; Masaki (Potomac, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Services |
Washington |
DC |
US |
|
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Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Services (Washington, DC)
|
Family
ID: |
52432905 |
Appl.
No.: |
15/102,996 |
Filed: |
December 12, 2014 |
PCT
Filed: |
December 12, 2014 |
PCT No.: |
PCT/US2014/070144 |
371(c)(1),(2),(4) Date: |
June 09, 2016 |
PCT
Pub. No.: |
WO2015/089469 |
PCT
Pub. Date: |
June 18, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160310585 A1 |
Oct 27, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61915948 |
Dec 13, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
37/04 (20180101); A61K 9/19 (20130101); A61P
35/00 (20180101); A61K 39/0011 (20130101); A61K
2039/575 (20130101); A61K 2039/70 (20130101); A61K
2039/572 (20130101); A61K 2039/5154 (20130101) |
Current International
Class: |
A61K
39/00 (20060101); A61K 9/19 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2009-541373 |
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Nov 2009 |
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JP |
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WO 01/04309 |
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Jan 2001 |
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WO |
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WO-0116163 |
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Mar 2001 |
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WO |
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WO 2005/000889 |
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Jan 2005 |
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WO |
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Other References
Kobayashi et al. (Clip Cancer Res. May 15, 2005;11(10):3869-78).
(Year: 2005). cited by examiner .
Ferrante, Front Immunol. Oct. 1, 2013;4:308, pp. 1-6 (Year: 2013).
cited by examiner .
Lin et al., BMC Bioinformatics 2008, 9(Suppl 12):S22, pp. 1-10
(Year: 2008). cited by examiner .
Essand et al., "High expression of a specific T-cell receptor
.gamma. transcript in epithelial cells of the prostate," Proc Natl
Acad Sci USA 96:9287-9292, 1999. cited by applicant .
Kenter et al., "Phase I Immunotherapeutic Trial with Long Peptides
Spanning the E6 and E7 Sequences of High-Risk Human Papillomavirus
16 in End-Stage Cervical Cancer Patients Shows Low Toxicity and
Robust Immunogenicity," Clin. Cancer Res., vol. 14:169-177, 2008.
cited by applicant .
Mirshahidi et al., "Overlapping Synthetic Peptides Encoding TPD52
as Breast Cancer Vaccine in Mice: Prolonged Survival," Vaccine,
vol. 27:1825-1833, 2009. cited by applicant .
Oh et al., "Human CTLs to Wild-Type and Enhanced Epitopes of a
Novel Prostate and Breast Tumor-Associated Protein, TARP, Lyse
Human Breast Cancer Cells," Cancer Res., vol. 64:2610-2618, 2004.
cited by applicant .
Welters et al., "Induction of Tumor-Specific CD4+ and CD8+ T-Cell
Immunity in Cervical Cancer Patients by a Human Papillomavirus Type
16 E6 and E7 Long Peptides Vaccine," Clin. Cancer Res., vol.
14:178-187, 2008. cited by applicant .
Wolfgang et al., "T-Cell Receptor .gamma. Chain Alternate Reading
Frame Protein (TARP) Expression in Prostate Cancer Cells Leads to
an Increased Growth Rate and Induction of Caveolins and
Amphiregulin," Cancer Res 61(22):8122-8126, 2001. cited by
applicant .
Wolfgang et al., "TARP: A nuclear protein expressed in prostate and
breast cancer cells derived from an alternate reading frame of the
T cell Receptor .gamma. chain locus," Proc Natl Acad Sci USA
97(17):9437-9442, 2000. cited by applicant .
Wood, "Cancer Vaccines: Current Challenges & Evolving
Concepts," Health Disparities Conference, University of Puerto
Rico, oral presentation Apr. 13, 2013 (93 pages). cited by
applicant .
Wood, "Autologous TARP Peptide Vaccination is Associated with
Slowing in PSA Velocity and a Decrease in Tumor Growth Rates in
Patients with Stage D0 Prostate Cancer," AACR 2013 Annual Meeting,
Abstract 4571, oral presentation Apr. 9, 2013 (18 pages). cited by
applicant .
Wood et al., "Therapeutic Vaccination with Epitope-Enhanced and
Wild Type TARP Peptides in Stage Do Prostate Cancer," AACR 2011
Annual Meeting, Abstract 5520, poster presentation Apr. 6, 2011 (1
page). cited by applicant .
Yamada et al., "Next-Generation Peptide Vaccines for Advanced
Cancer," Cancer Sci., vol. 104:15-21, 2013. cited by
applicant.
|
Primary Examiner: Skelding; Zachary S
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage of Internation
Application No. PCT/US2014/070144, filed Dec. 12, 2014, published
in English under PCT Article 21(2), which claims the benefit of
U.S. Provisional Application No. 61/915,948, filed Dec. 13, 2013,
which is herein incorporated by reference in its entirety.
Claims
The invention claimed is:
1. A composition comprising five non-identical overlapping T cell
receptor .gamma. alternate reading frame protein (TARP) peptides,
wherein the amino acid sequences of the five overlapping TARP
peptides consist of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, and SEQ ID NO: 9.
2. The composition of claim 1, further comprising a TARP peptide
consisting of SEQ ID NO: 3 and a TARP peptide consisting of SEQ ID
NO: 4.
3. The composition of claim 1, further comprising a
pharmaceutically acceptable carrier.
4. The composition of claim 1, further comprising an adjuvant.
5. The composition of claim 1, comprising antigen presenting cells
(APCs) loaded with the TARP peptides.
6. The composition of claim 5, wherein the APCs are dendritic
cells.
7. The composition of claim 1 in unit dose form.
8. The composition of claim 7, comprising a lyophilized powder of
the TARP peptides.
9. A method of treating a subject having prostate cancer or breast
cancer comprising selecting a subject having prostate cancer or
breast cancer that expresses TARP and administering to the subject
a therapeutically effective amount of the composition of claim 1,
thereby treating the subject.
10. The method of claim 9, wherein the composition comprises APCs
loaded with the TARP peptides.
11. The method of claim 10, wherein the APCs are dendritic
cells.
12. The method of claim 10, wherein the APCs are autologous.
13. The method of claim 10, wherein the therapeutically effective
amount of the composition comprises about 1.times.10.sup.6 to about
30.times.10.sup.6 viable APCs.
14. The method of claim 9, comprising administering the composition
intradermally, intravenously, intramuscularly or subcutaneously.
Description
FIELD
This disclosure concerns T cell receptor .gamma. alternate reading
frame protein (TARP) peptides and their use for stimulating an
immune response against TARP-expressing cells, such as
TARP-expressing tumor cells.
BACKGROUND
Historically five categories of tumor antigens have been utilized
in immunotherapy: mutated antigens (e.g., p53 or RAS),
over-expressed self-antigens (e.g., HER2/neu or MUC-1),
differentiation antigens (e.g., gp100, tyrosinase), cancer testis
antigens (e.g., MAGE, BAGE or CAGE families, NY-ESO-1) and viral
antigens (e.g., HPV16 E6 or E7, EBV) (Cheever et al., Clin Canc Res
15:5323-5337, 2009). The advantages of therapeutic cancer vaccines
utilizing proteins and peptides include the simplicity of
production and the relative absence of major safety and regulatory
issues.
All cells that express major histocompatibility complex (MHC) class
I molecules can present short peptides (9-11 mers) from
tumor-associated antigens (TAA) or viruses whose chronic persistent
infection is associated with the development of malignancy (e.g.
human papilloma virus, hepatitis B virus, and hepatitis C virus).
However, co-stimulatory signals essential for T cell stimulation
and the induction of lasting potent and effective immune responses
are often absent due to the lack of induction of specific T-cell
help, resulting in suboptimal and short-lived CD8.sup.+ T-cell
responses caused by a lack of proper T-helper cell-mediated
signaling through dendritic cells (DCs) (Zom et al., Adv Immunol
114: 177-201, 2012). In addition, vaccination with restricted MHC
class I binding peptides can be associated with induction of
peptide-specific tolerance rather than tumor-controlling immunity
(Toes et al., J Immunol 156:3911-3918, 1996; Toes et al., Proc Natl
Acad Sci USA 93:7855-7860, 1996). Furthermore, the use of a limited
number of peptides within any given vaccine platform may allow the
development of immune escape. Recent developments in therapeutic
cancer vaccine research have included the use of TAA synthetic long
peptides (SLPs) (Quakkelaar and Melief, Adv Immunol 114:77-106,
2012), as well as the use of overlapping and/or multi-epitope
peptide vaccines (Walter et al., Nat Med 18:1254-1261, 2012). SLPs
are synthetic peptides of 20-50 amino acids that because of their
length require internalization and processing by DCs. Examples of
multi-epitope peptide cancer vaccine platforms under clinical
investigation include those using folate receptor alpha
(NCT01606241), HER2/neu ((NCT01632332, NCT00266110, NCT00088985)
and melanoma (NCTI00580060, NCT 00071981, NCT00471471, NCT00705640,
NCTI00085137) peptides.
TARP (T-cell receptor .gamma. alternate reading frame protein) is a
58 amino acid protein identified using the expressed sequence
database (Maeda et al., J Biol Chem. 279:24561-24568, 2004). The
mRNA is initiated in the J.gamma. 1 exon of the TCR .gamma. and the
protein expressed is initiated in an alternative reading frame
distinct from that of the TCR .gamma. coding sequence. Prior
studies have shown that TARP is highly expressed in primary as well
as metastatic prostate cancer; is expressed in prostate cancers
with a range of Gleason patterns: and is expressed in both hormone
sensitive and castrate resistant prostate cancer.
SUMMARY
Provided herein are compositions comprising immunogenic TARP
peptides, and their use for eliciting an immune response in a
subject, such as for the treatment of a TARP-expressing cancer.
In some embodiments, the composition includes at least two
non-identical overlapping TARP peptides, wherein the amino acid
sequences of the at least two overlapping TARP peptides consist of
15 to 25 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2,
and wherein each of the at least two overlapping TARP peptides
comprises 5 to 15 consecutive amino acids that are identical to
another of the overlapping TARP peptides. In some examples, the
composition includes three, four, five, six or seven overlapping
TARP peptides. In one non-limiting embodiment, the composition
comprises five overlapping TARP peptides, wherein the amino acid
sequences of the five overlapping TARP peptides consist of 18 to 20
consecutive amino acids of SEQ ID NO: 1, and wherein each of the
five overlapping TARP peptides comprises 10 consecutive amino acids
that are identical to at least one other of the overlapping TARP
peptides, and wherein the combination of the five overlapping TARP
peptides comprises all 58 amino acids of SEQ ID NO: 1. In some
examples, the composition further comprises the TARP peptides of
SEQ ID NO: 3 and SEQ ID NO: 4.
In some embodiments, the compositions comprise antigen presenting
cells (APCs), such as dendritic cells, loaded with the TARP
peptides. In some embodiments, the compositions comprise a
pharmaceutically acceptable carrier and/or an adjuvant.
Also provided is a method of eliciting an immune response in a
subject, by administering to the subject a therapeutically
effective amount of a TARP peptide composition disclosed
herein.
Further provided is a method of treating a subject having a
TARP-expressing cancer, such as prostate cancer, breast cancer or
mesothelioma, comprising selecting a subject having a cancer that
expresses TARP, such as a prostate cancer, breast cancer or
mesothelioma that expresses TARP, and administering to the subject
a therapeutically effective amount of a TARP peptide composition
disclosed herein. In some embodiments, the subject is administered
APCs loaded with TARP peptides.
The foregoing and other objects, features, and advantages of the
invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustrating the study design of a
therapeutic multi-epitope TARP cancer vaccine phase II clinical
trial.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, and three letter code for amino acids, as defined
in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence
is shown, but the complementary strand is understood as included by
any reference to the displayed strand. The Sequence Listing is
submitted as an ASCII text file, created on Jun. 7, 2016, 4.71 KB,
which is incorporated by reference herein. In the accompanying
sequence listing:
SEQ ID NO: 1 is the amino acid sequence of a human TARP protein
comprising a glycine at position 40.
SEQ ID NO: 2 is the amino acid sequence of human TARP, deposited
under GENBANK.TM. Accession No. AAG29337.
SEQ ID NO: 3 is the amino acid sequence of the TARP 27-35
peptide.
SEQ ID NO: 4 is the amino acid sequence of the TARP 29-37-9V
peptide.
SEQ ID NO: 5 is the amino acid sequence of the TARP 1-20
peptide.
SEQ ID NO: 6 is the amino acid sequence of the TARP 11-30
peptide.
SEQ ID NO: 7 is the amino acid sequence of the TARP 21-40
peptide.
SEQ ID NO: 8 is the amino acid sequence of the TARP 31-50
peptide.
SEQ ID NO: 9 is the amino acid sequence of the TARP 41-58
peptide.
SEQ ID NO: 10 is the nucleotide sequence of human TARP, deposited
under GENBANK.TM. Accession No. AF51103.
DETAILED DESCRIPTION
I. Abbreviations
APC antigen presenting cell
cGMP current good manufacturing practices
CTL cytotoxic T lymphocyte
DC dendritic cell
DLT dose limiting toxicity
DMSO dimethylsulfoxide
EE epitope enhanced
GM-CSF granulocyte macrophage colony stimulating factor
HLA human leukocyte antigen
ICS intracellular cytokine staining
IFN interferon
IL interleukin
KLH keyhole limpet hemocyanin
LPS lipopolysaccharide
ME multi-epitope
MHC major histocompatibility complex
PBL peripheral blood lymphocyte
PBMC peripheral blood mononuclear cell
PSA prostate specific antigen
PSADT PSA doubling time
PTFE polytetrafluoroethylene
rh recombinant human
SLP synthetic long peptide
TAA tumor-associated antigen
TARP T cell receptor .gamma. alternate reading frame protein
TFA trifluoroacetic acid
TIL tumor infiltrating lymphocyte
WT wild type
II. Terms and Methods
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.). The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
In order to facilitate review of the various embodiments of the
disclosure, the following explanations of specific terms are
provided:
Adjuvant: A vehicle used to enhance antigen presentation and
stimulate an immune response, such as a suspension of minerals
(alum, aluminum hydroxide, or phosphate) on which antigen is
adsorbed; or water-in-oil emulsion in which antigen solution is
emulsified in mineral oil (Freund's incomplete adjuvant), sometimes
with the inclusion of killed mycobacteria (Freund's complete
adjuvant) to further enhance immunogenicity (inhibits degradation
of antigen and/or causes influx of macrophages). Imnmunostimulatory
oligonucleotides (such as those including a CpG motif) can also be
used as adjuvants (for example see U.S. Pat. Nos. 6,194,388;
6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705;
and 6,429,199). In one embodiment, the adjuvant is MONTANIDE.RTM.
ISA 51 VG plus GM-CSF. In some embodiments, the adjuvant is a
non-naturally occurring adjuvant.
Administration: The introduction of a composition into a subject by
a chosen route. For example, if the chosen route is intravenous,
the composition is administered by introducing the composition into
a vein of the subject. Exemplary routes of administration include,
but are not limited to, injection (such as subcutaneous,
intramuscular, intradermal, intraperitoneal, and intravenous),
oral, intraductal, sublingual, rectal, transdermal, intranasal,
vaginal and inhalation routes. In particular embodiments disclosed
herein, the route of administration is intradermal.
Antibody: An immunoglobulin molecule produced by B lymphoid cells
with a specific amino acid sequence. Antibodies are evoked in
humans or other animals by a specific antigen (immunogen).
Antibodies are characterized by reacting specifically with the
antigen in some demonstrable way, antibody and antigen each being
defined in terms of the other. "Eliciting an antibody response"
refers to the ability of an antigen or other molecule to induce the
production of antibodies.
Antigen: A compound, composition, or substance that can stimulate
the production of antibodies and/or a CD4+ or CD8+ T cell response
in an animal, including compositions that are injected or absorbed
into an animal. An antigen reacts with the products of specific
humoral or cellular immunity, including those induced by
heterologous immunogens. The term "antigen" includes all related
antigenic epitopes. "Epitope" or "antigenic determinant" refers to
a site on an antigen to which B and/or T cells respond. In some
embodiments, T cells respond to the epitope, when the epitope is
presented in conjunction with an MHC molecule.
An antigen can be a tissue-specific antigen, or a disease-specific
antigen. These terms are not exclusive, as a tissue-specific
antigen can also be a disease specific antigen. A tissue-specific
antigen is expressed in a limited number of tissues, such as a
single tissue. A disease-specific antigen is expressed
coincidentally with a disease process. Specific non-limiting
examples of a disease-specific antigen are an antigen whose
expression correlates with, or is predictive of, tumor formation,
such as prostate cancer or breast cancer. TARP is one example of a
disease-specific antigen that is overexpressed in prostate cancer,
breast cancer and other types of cancer. A disease-specific antigen
can be an antigen recognized by T cells or B cells.
Antigen presenting cells (APCs): A type of cell that displays
antigens complexed with major histocompatibility complex (MHC)
proteins on their surface. Professional APCs are very efficient at
internalizing antigen, processing it and then displaying small
pieces of the antigen (peptides) bound to a MHC molecule on the
cell membrane surface. The three main types of professional APCs
include DCs, macrophages and certain B cells. DCs have the broadest
range of antigen presentation and are the most important APC in
processing antigens for presentation to T-cells, which recognize
antigen-MHC complexes using their T cell receptors.
Autologous: Derived from the same individual. In the context of the
present disclosure, "autologous" APCs used for treatment of a
subject are APCs originally obtained from the subject.
Breast cancer: A neoplastic condition of breast tissue that can be
benign or malignant. The most common type of breast cancer is
ductal carcinoma. Ductal carcinoma in situ is a non-invasive
neoplastic condition of the ducts. Lobular carcinoma is not an
invasive disease but is an indicator that a carcinoma may develop.
Infiltrating (malignant) carcinoma of the breast can be divided
into stages (I, IIA, IIB, IIIA, IIIB, and IV).
Cancer: A malignant neoplasm that has undergone characteristic
anaplasia with loss of differentiation, increased rate of growth,
invasion of surrounding tissue, and is capable of metastasis. For
example, prostate cancer is a malignant neoplasm that arises in or
from prostate tissue, and breast cancer is a malignant neoplasm
that arises in or from breast tissue (such as a ductal carcinoma).
Residual cancer is cancer that remains in a subject after any form
of treatment given to the subject to reduce or eradicate the
cancer. Metastatic cancer is a cancer at one or more sites in the
body other than the site of origin of the original (primary) cancer
from which the metastatic cancer is derived.
CD4: Cluster of differentiation factor 4, a T cell surface protein
that mediates interaction with the MHC Class II molecule. CD4 also
serves as the primary receptor site for HIV on T cells during HIV
infection. Cells that express CD4 are often helper T cells.
CD8: Cluster of differentiation factor 8, a T cell surface protein
that mediates interaction with the MHC Class I molecule. Cells that
express CD8 are often cytotoxic T cells.
Chemotherapeutic agents: Any cytotoxic chemical agent with
therapeutic usefulness in the treatment of diseases characterized
by abnormal cell growth. Such diseases include tumors, neoplasms,
and cancer as well as diseases characterized by hyperplastic growth
such as psoriasis. In one embodiment, a chemotherapeutic agent is
an agent of use in treating prostate cancer, breast cancer or
another tumor. In one embodiment, a chemotherapeutic agent is a
radioactive compound. One of skill in the art can readily identify
a chemotherapeutic agent of use (e.g., see Slapak and Kufe,
Principles of Cancer Therapy, Chapter 86 in Harrison's Principles
of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.
17 in Abeloff, Clinical Oncology 2.sup.nd ed., .COPYRGT. 2000
Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds):
Oncology Pocket Guide to Chemotherapy. 2nd ed. St. Louis.
Mosby-Year Book, 1995; Fischer. D. S., Knobf, M. F., Durivage. H.
J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis,
Mosby-Year Book, 1993). Combination therapy is the administration
of more than one agent, such as cytotoxic, radioactive or
immunotherapeutic compounds, to treat cancer. One example is the
administration of a TARP peptide vaccine used in combination with a
radioactive or cytotoxic chemical compound.
Conservative variants: "Conservative" amino acid substitutions are
those substitutions that do not substantially affect or decrease an
activity or antigenicity of a protein, such as TARP. For example, a
TARP polypeptide can include at most about 1, at most about 2, at
most about 5, and most about 10, or at most about 15 conservative
substitutions and specifically bind an antibody that binds the
original TARP polypeptide. Specific, non-limiting examples of a
conservative substitution include the following examples:
TABLE-US-00001 Original Residue Conservative Substitutions Ala Ser
Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln
Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met;
Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu
The term conservative variant also includes the use of a
substituted amino acid in place of an unsubstituted parent amino
acid. Non-conservative substitutions are those that reduce an
activity or antigenicity.
Degenerate variant: A polynucleotide encoding an epitope of TARP
that includes a sequence that is degenerate as a result of the
genetic code. There are 20 natural amino acids, most of which are
specified by more than one codon. Therefore, all degenerate
nucleotide sequences are included in this disclosure as long as the
amino acid sequence of the TARP peptide encoded by the nucleotide
sequence is unchanged.
Dendritic cells (DCs): The principle professional antigen
presenting cells (APCs) involved in primary immune responses. They
are potent activators of T helper cell responses because as part of
their composition, they express co-stimulatory molecules on their
cell surface. Their major function is to obtain antigen in tissues,
migrate to lymphoid organs and present the antigen in order to
activate T cells. Immature dendritic cells originate in the bone
marrow and reside in the periphery as immature cells. Dendritic
cell sub-types include plasmacytoid dendritic cells and myeloid
dendritic cells.
Consecutive: Following one after another in a series without
interruption.
Contacting: Placement in direct physical association; includes both
in solid and liquid form.
Epitope: An antigenic determinant. These are particular chemical
groups or peptide sequences on a molecule that are antigenic, i.e.
that elicit a specific immune response. An antibody specifically
binds a particular antigenic epitope on a polypeptide.
Fusion protein: A protein generated by expression of a nucleic acid
sequence engineered from nucleic acid sequences encoding at least a
portion of two different (heterologous) proteins. To create a
fusion protein, the nucleic acid sequences must be in the same
reading frame and contain no internal stop codons.
Heterologous: Originating from separate genetic sources or species.
For example, a polypeptide that is heterologous to TARP originates
from a nucleic acid that does not encode TARP. In some embodiments,
the heterologous amino acid sequence includes a protein tag, such
as .beta.-galactosidase, maltose binding protein, albumin, or an
immunoglobulin amino acid sequence.
Immune response: A response of a cell of the immune system, such as
a B cell, T cell, monocyte, macrophage, dendritic cell or natural
killer cell to a stimulus. In one embodiment, the response is
specific for a particular antigen (an "antigen-specific response"),
also known as an adaptive immune response. In some embodiments, the
adaptive immune response is a T cell response, such as a CD4+
response and/or a CD8+ response. In some embodiments, the adaptive
immune response is a B cell response, and results in the production
of specific antibodies.
Immunogenic peptide: A peptide which comprises an allele-specific
motif or other sequence, such as an N-terminal repeat, such that
the peptide will bind an MHC molecule and induce a CTL response, or
a B cell response (e.g. antibody production) against the antigen
from which the immunogenic peptide is derived.
In one embodiment, immunogenic peptides are identified using
sequence motifs or other methods, such as neural net or polynomial
determinations, known in the art. Typically, algorithms are used to
determine the "binding threshold" of peptides to select those with
scores that give them a high probability of MHC binding at a
certain affinity that will be immunogenic. The algorithms are based
either on the effects on MHC binding of a particular amino acid at
a particular position, the effects on antibody binding of a
particular amino acid at a particular position, or the effects on
binding of a particular substitution in a motif-containing peptide.
Within the context of an immunogenic peptide, a "conserved residue"
is one which appears in a significantly higher frequency than would
be expected by random distribution at a particular position in a
peptide. In one embodiment, a conserved residue is one where the
MHC structure may provide a contact point with the immunogenic
peptide.
Immunogenic composition: In the context of the present disclosure,
a composition comprising a TARP polypeptide that induces a
measurable CTL response against cells expressing TARP polypeptide,
and/or induces a measurable B cell response (e.g. production of
antibodies) against a TARP polypeptide. It further refers to
isolated nucleic acid molecules encoding a TARP polypeptide that
can be used to express the TARP polypeptide (and thus be used to
elicit an immune response against this polypeptide). For in vitro
use, the immunogenic composition may consist of the isolated
protein or peptide. For in vivo use, the immunogenic composition
will typically comprise the protein or peptide in pharmaceutically
acceptable carriers, and/or other agents. Any particular peptide,
TARP polypeptide, or nucleic acid encoding the polypeptide, can be
readily tested for its ability to induce a CTL or B cell response
by art-recognized assays. Immunogenic compositions can include
adjuvants, which are well known to one of skill in the art.
Inhibiting or treating a disease: Inhibiting the full development
of a disease or condition, for example, in a subject who is at risk
for a disease such as a tumor (for example, a prostate or breast
tumor). "Treatment" refers to a therapeutic intervention that
ameliorates a sign or symptom of a disease or pathological
condition after it has begun to develop. As used herein, the term
"ameliorating," with reference to a disease or pathological
condition, refers to any observable beneficial effect of the
treatment. The beneficial effect can be evidenced, for example, by
a delayed onset of clinical symptoms of the disease in a
susceptible subject, a reduction in severity of some or all
clinical symptoms of the disease, a slower progression of the
disease, a reduction in the number of metastases, an improvement in
the overall health or well-being of the subject, or by other
parameters well known in the art that are specific to the
particular disease. A "prophylactic" treatment is a treatment
administered to a subject who does not exhibit signs of a disease
or exhibits only early signs for the purpose of decreasing the risk
of developing pathology.
Isolated: An "isolated" biological component (such as a nucleic
acid or protein or organelle) has been substantially separated or
purified away from other biological components in the cell of the
organism in which the component naturally occurs, such as other
chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
Label: A detectable compound or composition that is conjugated
directly or indirectly to another molecule, such as an antibody or
a protein, to facilitate detection of that molecule. Specific,
non-limiting examples of labels include fluorescent tags, enzymatic
linkages, and radioactive isotopes.
Linker: One or more nucleotides or amino acids that serve as a
spacer between two molecules, such as between two nucleic acid
molecules or two peptides (such as in a fusion protein).
Major histocompatibility complex (MHC): Generic designation meant
to encompass the histocompatibility antigen systems described in
different species, including the human leukocyte antigens ("HLA").
The term "motif" or "epitope" refers to the pattern of residues in
a peptide of defined length, usually about 8 to about 11 amino
acids, which is recognized by a particular MHC allele. The peptide
motifs or epitopes are typically different for each MHC allele and
differ in the pattern of the highly conserved residues and negative
binding residues.
Mesothelioma: A type of neoplasm derived from the lining cells of
the pleura and peritoneum which grows as a thick sheet covering the
viscera, and is composed of spindle cells or fibrous tissue which
may enclose gland-like spaces lined by cuboidal cells.
Mesotheliomas often originate in the tissue lining the lung, heart
or abdomen. In some cases, mesotheliomas are caused by exposure to
asbestos.
Operably linked: A first nucleic acid sequence is operably linked
with a second nucleic acid sequence when the first nucleic acid
sequence is placed in a functional relationship with the second
nucleic acid sequence. For instance, a promoter is operably linked
to a coding sequence if the promoter affects the transcription or
expression of the coding sequence. Generally, operably linked DNA
sequences are contiguous and, where necessary to join two
protein-coding regions, in the same reading frame.
Overlapping peptide: A peptide that at least partially overlaps in
amino acid sequence with another peptide. In the context of the
present disclosure. "overlapping TARP peptides" are peptides
comprising a portion of the amino acid sequence of human TARP (SEQ
ID NO: 1), wherein each overlapping TARP peptide contains about 5
to about 15 consecutive amino acids that are identical to at least
one other overlapping TARP peptide in any given set of overlapping
peptides. In some embodiments, the TARP peptides are 15 to 25 amino
acids in length and have an amino acid sequence consisting of 15 to
25 consecutive amino acids of SEQ ID NO: 1. In particular examples,
the set of overlapping TARP peptides contains all 58 amino acid
residues of SEQ ID NO: 1.
Peptide or polypeptide: Any chain of amino acids regardless of
length or post-translational modification (such as glycosylation or
phosphorylation). In some embodiments, a polypeptide is between 5
and 100 amino acids in length, including 5 to 58, 5 to 50, 5 to 30,
8 to 20, 8 to 10, or 18 to 20 amino acids in length. In particular
examples, a TARP polypeptide is 8, 9, 10, 18 or 20 amino acids in
length.
A "TARP polypeptide" or "TARP peptide" is a series of contiguous
amino acid residues from a TARP protein. In one example, with
respect to immunogenic compositions comprising a TARP peptide, the
term further refers to variations of these peptides in which there
are conservative substitutions of amino acids, so long as the
variations do not alter by more than about 20% (such as no more
than about 1%, about 5%, or about 10%) the ability of the peptide
to produce a B cell response, or, when bound to a MHC class I
molecule, to activate cytotoxic T lymphocytes against cells
expressing wild-type TARP protein. Induction of CTLs using
synthetic peptides and CTL cytotoxicity assays are taught in. e.g.,
U.S. Pat. No. 5,662,907.
A "residue" refers to an amino acid or amino acid mimetic
incorporated in a polypeptide by an amide bond or amide bond
mimetic.
Peptide or polypeptide modifications: TARP peptides include
synthetic embodiments of the peptides described herein. In
addition, analogs (non-peptide organic molecules), derivatives
(chemically functionalized peptide molecules obtained starting with
the disclosed peptide sequences) and variants (homologs or
paralogs) of these proteins can be utilized in the methods
described herein. Each polypeptide is comprised of a sequence of
amino acids, which may be either L- and/or D-amino acids, naturally
occurring and otherwise.
Peptides may be modified by a variety of chemical techniques to
produce derivatives having essentially the same activity as the
unmodified peptides, and optionally having other desirable
properties. For example, carboxylic acid groups of the protein,
whether carboxyl-terminal or side chain, may be provided in the
form of a salt of a pharmaceutically-acceptable cation or
esterified to form a C.sub.1-C.sub.16 ester, or converted to an
amide of formula NR.sub.1R.sub.2 wherein R.sub.1 and R.sub.2 are
each independently H or C.sub.1-C.sub.16 alkyl, or combined to form
a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups
of the peptide, whether amino-terminal or side chain, may be in the
form of a pharmaceutically-acceptable acid addition salt, such as
the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric
and other organic salts, or may be modified to C.sub.1-C.sub.16
alkyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the peptide side chains may be converted to
C.sub.1-C.sub.16 alkoxy or to a C.sub.1-C.sub.16 ester using
well-recognized techniques. Phenyl and phenolic rings of the
peptide side chains may be substituted with one or more halogen
atoms, such as fluorine, chlorine, bromine or iodine, or with
C.sub.1-C.sub.16 alkyl, C.sub.1-C.sub.16 alkoxy, carboxylic acids
and esters thereof, or amides of such carboxylic acids. Methylene
groups of the peptide side chains can be extended to homologous
C.sub.2-C.sub.4 alkylenes. Thiols can be protected with any one of
a number of well-recognized protecting groups, such as acetamide
groups. Those skilled in the art will also recognize methods for
introducing cyclic structures into the TARP peptides to select and
provide conformational constraints to the structure that result in
enhanced stability.
Peptidomimetic and organomimetic embodiments are envisioned,
whereby the three-dimensional arrangement of the chemical
constituents of such peptido- and organomimetics mimic the
three-dimensional arrangement of the peptide backbone and component
amino acid side chains, resulting in such peptido- and
organomimetics of a TARP polypeptide having measurable or enhanced
ability to generate an immune response. For computer modeling
applications, a pharmacophore is an idealized, three-dimensional
definition of the structural requirements for biological activity.
Peptido- and organomimetics can be designed to fit each
pharmacophore with current computer modeling software (using
computer assisted drug design or CADD). See Walters,
"Computer-Assisted Modeling of Drugs", in Klegerman & Groves,
eds., 1993, Pharmaceutical Biotechnology, Interpharm Press, Buffalo
Grove, Ill., pp. 165-174 and Principles of Pharmacology Munson
(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.
Also included are mimetics prepared using such techniques.
Polypeptide modifications also include amino acid substitutions,
such as those that alter binding affinity of the polypeptide to MHC
molecules. Exemplary amino acid substitutions for altering MHC
binding affinity have been described in the art (see, for example,
Berzofsky et al., Nat. Rev. Immunol. 1(3):209-219, 2001).
Pharmaceutically acceptable carrier: The pharmaceutically
acceptable carriers of use are conventional. Remington's
Pharmaceutical Sciences, by E. W. Martin. Mack Publishing Co.,
Easton, Pa., 15th Edition, 1975, describes compositions and
formulations suitable for pharmaceutical delivery of proteins, such
as those disclosed herein.
In general, the nature of the carrier will depend on the particular
mode of administration being employed. For instance, parenteral
formulations usually comprise injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate. In some
embodiments, the pharmaceutical carrier is sterile, particularly
when in an injectable form. In some embodiments, the pharmaceutical
carrier is non-naturally occurring.
Polynucleotide: The term polynucleotide or nucleic acid sequence
refers to a polymeric form of nucleotide at least 10 bases in
length. A recombinant polynucleotide includes a polynucleotide that
is not immediately contiguous with both of the coding sequences
with which it is immediately contiguous (one on the 5' end and one
on the 3' end) in the naturally occurring genome of the organism
from which it is derived. The term therefore includes, for example,
a recombinant DNA which is incorporated into a vector: into an
autonomously replicating plasmid or virus; or into the genomic DNA
of a prokaryote or eukaryote, or which exists as a separate
molecule (e.g., a cDNA) independent of other sequences. The
nucleotides can be ribonucleotides, deoxyribonucleotides, or
modified forms of either nucleotide. The term includes single- and
double-stranded forms of DNA.
Promoter: A promoter is an array of nucleic acid control sequences
that directs transcription of a nucleic acid. A promoter includes
necessary nucleic acid sequences near the start site of
transcription, such as in the case of a polymerase II type promoter
(a TATA element). A promoter also optionally includes distal
enhancer or repressor elements which can be located as much as
several thousand base pairs from the start site of transcription.
Both constitutive and inducible promoters are included (see e.g.,
Bitter et al., Methods in Enzymology 153:516-544, 1987).
Specific, non-limiting examples of promoters include promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the retrovirus long
terminal repeat; the adenovirus late promoter, the vaccinia virus
7.5K promoter) can be used. Promoters produced by recombinant DNA
or synthetic techniques can also be used. A polynucleotide can be
inserted into an expression vector or a viral vector that contains
a promoter sequence which facilitates the efficient transcription
of the inserted genetic sequence of the host. The expression vector
typically contains an origin of replication, a promoter, as well as
specific nucleic acid sequences that allow phenotypic selection of
the transformed cells.
Prostate Cancer: A malignant tumor, generally of glandular origin,
of the prostate. Prostate cancers include adenocarcinomas and small
cell carcinomas. Many prostate cancers express prostate specific
antigen (PSA), prostate stem cell antigen (PSCA), PSMA (prostate
specific membrane antigen), prostatic acid phosphatase (PAP) as
well as other tumor antigens.
Purified: The term purified does not require absolute purity;
rather, it is intended as a relative term. Thus, for example, a
purified peptide preparation is one in which the peptide or protein
is more enriched than the peptide or protein is in its natural
environment within a cell. In one embodiment, a preparation is
purified such that the protein or peptide represents at least 50%
of the total peptide or protein content of the preparation. A
substantially purified protein is at least 60%, 70%, 80%, 90%, 95%
or 98% pure. Thus, in one specific, non-limiting example, a
purified protein is 90% free of other proteins or cellular
components. The TARP polypeptides disclosed herein can be purified
by any of the means known in the art (see, e.g., Guide to Protein
Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press,
San Diego, 1990; and Scopes, Protein Purification: Principles and
Practice, Springer Verlag, New York, 1982).
Recombinant: A recombinant nucleic acid or protein is one that has
a sequence that is not naturally occurring or has a sequence that
is made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination is often
accomplished by chemical synthesis or by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques.
Sequence identity: The similarity between amino acid sequences is
expressed in terms of the similarity between the sequences,
otherwise referred to as sequence identity. Sequence identity is
frequently measured in terms of percentage identity (or similarity
or homology); the higher the percentage, the more similar the two
sequences are. Homologs or variants of a TARP polypeptide will
possess a relatively high degree of sequence identity when aligned
using standard methods.
Methods of alignment of sequences for comparison are well known in
the art. Various programs and alignment algorithms are described
in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and
Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237,
1988: Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic
Acids Research 16:10881, 1988: and Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85:2444, 1988. In addition, Altschul et al.,
Nature Genet. 6:119, 1994, presents a detailed consideration of
sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et
al., J. Mol. Biol. 215:403, 1990) is available from several
sources, including the National Center for Biotechnology
Information (NCBI. Bethesda, Md.) and on the internet, for use in
connection with the sequence analysis programs blastp, blastn,
blastx, tblastn and tblastx. A description of how to determine
sequence identity using this program is available on the NCBI
website on the internet.
Homologs and variants of a TARP polypeptide are typically
characterized by possession of at least about 75%, for example at
least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity
counted over the full length alignment with the amino acid sequence
of TARP or a TARP paralog using the NCBI Blast 2.0, gapped blastp
set to default parameters. For comparisons of amino acid sequences
of greater than about 30 amino acids, the Blast 2 sequences
function is employed using the default BLOSUM62 matrix set to
default parameters, (gap existence cost of 11, and a per residue
gap cost of 1). When aligning short peptides (fewer than around 30
amino acids), the alignment should be performed using the Blast 2
sequences function, employing the PAM30 matrix set to default
parameters (open gap 9, extension gap 1 penalties). Proteins with
even greater similarity to the reference sequences will show
increasing percentage identities when assessed by this method, such
as at least 80%, at least 85%, at least 90%, at least 95%, at least
98%, or at least 99% sequence identity. When less than the entire
sequence is being compared for sequence identity, homologs and
variants will typically possess at least 80% sequence identity over
short windows of 10-20 amino acids, and may possess sequence
identities of at least 85% or at least 90% or 95% depending on
their similarity to the reference sequence. Methods for determining
sequence identity over such short windows are available at the NCBI
website on the internet. One of skill in the art will appreciate
that these sequence identity ranges are provided for guidance only;
it is entirely possible that strongly significant homologs could be
obtained that fall outside of the ranges provided.
Subject: Living multi-cellular vertebrate organisms, a category
that includes both human and veterinary subjects, including human
and non-human mammals.
Synthetic: Produced by artificial means in a laboratory, for
example a synthetic nucleic acid or peptide can be chemically
synthesized in a laboratory.
T cell: A white blood cell critical to the immune response. T cells
include, but are not limited to, CD4.sup.+ T cells and CD8.sup.+ T
cells. A CD4.sup.+ T lymphocyte is an immune cell that carries a
marker on its surface known as "cluster of differentiation 4"
(CD4). These cells, also known as helper T cells, help orchestrate
the immune response, including antibody responses as well as killer
T cell responses. In another embodiment, a CD4.sup.+ T cell is a
regulatory T cell that also expresses CD25 and Foxp3 ("CD4+CD25+
regulatory T cells or Tregs"). CD8.sup.+ T cells carry the "cluster
of differentiation 8" (CD8) marker. In one embodiment. CD8.sup.+ T
cells are cytotoxic T lymphocytes.
T cell receptor .gamma. alternate reading frame protein (TARP): A
polypeptide that is translated from a form of the T cell receptor
.gamma. gene. TARP is known to be expressed or overexpressed in
several different types of cancer, including prostate cancer,
breast cancer and mesothelioma. TARP is disclosed in PCT
Publication No. WO 01/04309, which is incorporated herein by
reference. Human TARP amino acid and nucleic acid sequences are set
forth herein as SEQ ID NO: 1 and SEQ ID NO: 2 (protein), and SEQ ID
NO: 10 (nucleic acid). TARP is also known as CD3G, TCRG, TCRGC1,
TCRGC2, T-cell receptor gamma-chain constant region and TCR gamma
alternate reading frame protein.
Therapeutically effective amount: A quantity of a specified agent
sufficient to achieve a desired effect in a subject, cell or
culture being treated with that agent. In the context of the
present disclosure, a therapeutically effective amount of a TARP
peptide is an amount of TARP peptide that causes induction of an
immune response, as measured by clinical response (for example
increase in a population of immune cells, production of antibody
that specifically binds the peptide, or measurable reduction of
tumor burden). In one embodiment, a therapeutically effective
amount of a TARP peptide is an amount used to generate an immune
response, or to treat cancer (such as breast cancer, mesothelioma
or prostate cancer) in a subject.
Transduced: A transduced cell is a cell into which has been
introduced a nucleic acid molecule by molecular biology techniques.
As used herein, the term transduction encompasses all techniques by
which a nucleic acid molecule might be introduced into such a cell,
including transfection with viral vectors, transformation with
plasmid vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration.
Unit Dose: A drug or pharmaceutical composition in a single or
metered dose form, such as a table, capsule, powder or solution to
be administered as a single dose, or multiple preselected doses. In
the context of the present disclosure, a TARP peptide composition
in unit dose form contains a preselected therapeutic amount of
peptide appropriate for a single dose, or one of multiple
preselected metered doses, such as the amount necessary to elicit
an immune response against TARP-expressing tumor cells. In some
examples, the unit dose is a liquid contained in a sterile vial, or
a powder in a sterile vial capable of being reconstituted for
administration by introduction of a liquid into the vial. In other
examples, the unit dosage form is provided in a syringe suitable
for administration, for example injection into a subject.
Vector: A vector is a nucleic acid molecule allowing insertion of
foreign nucleic acid without disrupting the ability of the vector
to replicate and/or integrate in a host cell. A vector can include
nucleic acid sequences that permit it to replicate in a host cell,
such as an origin of replication. A vector can also include one or
more selectable marker genes and other genetic elements. An
expression vector is a vector that contains the necessary
regulatory sequences to allow transcription and translation of
inserted gene or genes. In some embodiments, the vector is a
plasmid vector. In other embodiments, the vector is a viral
vector.
Viable (cell): Alive and capable of growth and/or biological
functions (such as antigen presentation, cytokine production
etc.).
Unless otherwise explained, 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. The
singular terms "a," "an," and "the" include plural referents unless
context clearly indicates otherwise. "Comprising A or B" means
including A, or B, or A and B. It is further to be understood that
all base sizes or amino acid sizes, and all molecular weight or
molecular mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
III. Multi-Epitope TARP Peptide Compositions
T cell receptor .gamma. alternate reading frame protein (TARP) is a
polypeptide that is translated from a form of the T cell receptor
.gamma. gene. TARP is known to be overexpressed in several types of
cancer, including prostate cancer, breast cancer and mesothelioma.
In particular, TARP is highly expressed in primary and metastatic
prostate cancer, and in both hormone sensitive and castrate
resistant prostate cancer. In addition, TARP expression is
associated with unfavorable and more aggressive tumor behavior.
Thus, TARP is an ideal tumor antigen for use in a cancer
vaccine.
In one embodiment, the TARP protein has a sequence set forth
as:
TABLE-US-00002 (SEQ ID NO: 1) MQMFPPSPLFFFLQLLKQSSRRLEHTFVFL
RNFSLMLLRGIGKKRRATRFWDPRRGTP.
In another embodiment, the TARP protein has a sequence set forth
as:
TABLE-US-00003 (SEQ ID NO: 2) MQMFPPSPLFFFLQLLKQSSRRLEHTFVFL
RNFSLMLLRYIGKKRRATRFWDPRRGTP.
In other embodiments, TARP has an amino acid sequence at least 90%
identical to SEQ ID NO: 1 or SEQ ID NO: 2, for example at least
about 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99% identical to
SEQ ID NO: 1 or SEQ ID NO: 2. Additional TARP variants have been
described (see PCT Publication No. WO 01/04309, which is
incorporated herein by reference).
In some embodiments, TARP is encoded by a nucleic acid having a
sequence set forth as SEQ ID NO: 10.
In some embodiments, the TARP peptide compositions provided herein
comprise at least one, such as two or more, overlapping TARP
peptides selected from:
TABLE-US-00004 TARP 1-20: (SEQ ID NO: 5) MQMFPPSPLFFFLQLLKQSS TARP
11-30: (SEQ ID NO: 6) FFLQLLKQSSRRLEHTFVFL TARP 21-40: (SEQ ID NO:
7) RRLEHTFVFLRNFSLMLLRG TARP 31-50: (SEQ ID NO: 8)
RNFSLMLLRGIGKKRRATRF TARP 41-58: (SEQ ID NO: 9)
IGKKRRATRFWDPRRGTP
The overlapping TARP peptides listed above are 18 or 20 amino acids
in length and are HLA non-restricted.
In some embodiments, the overlapping TARP peptides are administered
in combination with TARP 27-35 (FVFLRNFSL; SEQ ID NO: 3) and/or the
epitope enhanced TARP 29-37-9V (FLRNFSLMV; SEQ ID NO: 4), both of
which are 9 mer peptides that bind HLA-A*0201.
A prospective, randomized pilot study of a first generation TARP
peptide vaccine that utilized TARP 27-35 (SEQ ID NO: 3) and TARP
29-37-9V (SEQ ID NO: 4) peptides was previously conducted in
HLA-A*0201 positive men with stage D0 prostate cancer (PSA
biochemical recurrence without evidence of visceral or bony
metastatic disease). TARP vaccination was found to be immunogenic,
safe and well tolerated, with adverse events limited to injection
site reactions. TARP vaccination was also associated with a
decreased slope log PSA compared to pre-vaccination baseline in 72%
of subjects reaching 24 weeks and in 74% of subjects reaching 48
weeks. The PSA slope or velocity is a validated measure of tumor
growth in stage D0 prostate cancer in which there is no macroscopic
tumor to measure. TARP vaccination also resulted in a 50% decrease
in calculated tumor growth rate constant. TARP-specific interferon
(IFN)-.gamma. ELISPOT responses were detected in the majority of
subjects but did not correlate with decreases in slope log
(PSA).
The multi-epitope TARP peptide vaccine approach disclosed herein
has several distinct advantages over previously reported TARP
peptide vaccines. When all five overlapping TARP peptides are used,
the peptides cover the entire TARP protein, resulting in potential
for induction of a multivalent anti-TARP immune response, not
limited to a single HLA type. In addition, the longer overlapping
TARP peptides (18-20 amino acids in length) include TARP-specific
MHC class II CD4+ T cell helper epitopes that allow for the
generation of improved CD8.sup.+ T cell responses with enhanced
functional avidity and longevity, as well as the induction of
humoral anti-TARP antibody responses.
Provided herein are compositions that comprise at least two
non-identical overlapping TARP peptides. In some embodiments, the
amino acid sequences of the at least two overlapping TARP peptides
consist of 15 to 25 consecutive amino acids of SEQ TD NO: 1 or SEQ
ID NO: 2, and each of the at least two overlapping TARP peptides
comprises 5 to 15 consecutive amino acids that are identical to at
least another of the overlapping TARP peptides (for example, each
of the at least two overlapping TARP peptides comprise 5 to 15
consecutive amino acids that are identical to 5 to 15 consecutive
amino acids of at least another of the overlapping TARP peptides).
In some examples, the at least two overlapping TARP peptides
consist of 16 to 22, or 18 to 20 consecutive amino acids of SEQ ID
NO: 1 or SEQ ID NO: 2. In particular examples, the at least two
overlapping TARP peptides consist of 15, 16, 17, 18, 19, 20, 21,
22, 23, 24 or 25 consecutive amino acids of SEQ ID NO: 1 or SEQ ID
NO: 2. In some examples, the overlapping TARP peptides comprise 8
to 12, or 9 to 11 consecutive amino acids that are identical to at
least another of the overlapping TARP peptides. In particular
examples, the at least two overlapping TARP peptides consist of 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
consecutive amino acids that are identical to at least one other of
the overlapping TARP peptides.
In one non-limiting example, the at least two overlapping TARP
peptides consist of 18 or 20 consecutive amino acids of SEQ ID NO:
1 or SEQ ID NO: 2, and each of the at least two overlapping TARP
peptides comprises 10 consecutive amino acids that are identical to
at least another of the overlapping TARP peptides (such as one or
two of the other overlapping TARP peptides).
In some embodiments, the composition comprises three, four, five,
six or seven overlapping TARP peptides. In particular examples, the
composition comprises five overlapping TARP peptides.
In some embodiments, the combination of the three, four, five, six
or seven overlapping TARP peptides comprises all 58 amino acids of
SEQ ID NO: 1 or SEQ ID NO: 2.
In one non-limiting example, the composition comprises five
overlapping TARP peptides, wherein the amino acid sequences of the
five overlapping TARP peptides consist of 18 to 20 (such as 18 or
20) consecutive amino acids of SEQ ID NO: 1, and wherein each of
the five overlapping TARP peptides comprises 10 consecutive amino
acids that are identical to at least one other of the overlapping
TARP peptides, and wherein the combination of the five overlapping
TARP peptides comprises all 58 amino acids of SEQ ID NO: 1.
In some examples, the TARP peptide composition comprises at least
two overlapping TARP peptides, wherein each peptide consists of a
different amino acid sequence selected from SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
In some embodiments, the TARP peptide compositions further include
a TARP peptide consisting of SEQ ID NO: 3 and/or a TARP peptide
consisting of SEQ ID NO: 4. In some examples, the composition
comprises the TARP peptides of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:
9.
Also provided herein are compositions comprising polynucleotides
encoding the TARP peptides disclosed herein. Such compositions can
include polynucleotides encoding any combination of the TARP
peptides disclosed herein. In some embodiments, the composition
comprises a polynucleotide(s) encoding at least two overlapping
TARP peptides having an amino acid sequence that consists of 15 to
25 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, wherein
each of the at least two overlapping TARP peptides comprises 5 to
15 consecutive amino acids that are identical to at least one other
of the overlapping TARP peptides. In some examples, the composition
comprises a polynucleotide(s) encoding at least two overlapping
TARP peptides selected from SEQ ID NO: 5, SEQ ID NO: 6. SEQ ID NO:
7, SEQ ID NO: 8 and SEQ ID NO: 9. In some examples, the
compositions further include a polynucleotide(s) encoding the
peptide of SEQ ID NO: 3 and/or the peptide of SEQ ID NO: 4. In
particular examples, the polynucleotide comprises at least a
portion of the nucleotide sequence of SEQ ID NO: 10. In some
examples, the polynucleotides comprise vectors, such as plasmid
vectors or viral vectors. Exemplary viral vectors include
adenovirus vectors, adeno-associated virus vectors, retrovirus
vectors and lentivirus vectors.
Polynucleotides include DNA, cDNA and RNA sequences which encode
the peptide of interest. The polynucleotides encoding an
immunogenic TARP peptide include recombinant DNA which is
incorporated into a vector into an autonomously replicating plasmid
or virus or into the genomic DNA of a prokaryote or eukaryote, or
which exists as a separate molecule (e.g., a cDNA) independent of
other sequences. The nucleotides can be ribonucleotides,
deoxyribonucleotides, or modified forms of either nucleotide. The
term includes single-stranded and double-stranded forms of DNA.
A polynucleotide sequence encoding an immunogenic TARP peptide can
be operatively linked to expression control sequences. An
expression control sequence operatively linked to a coding sequence
is ligated such that expression of the coding sequence is achieved
under conditions compatible with the expression control sequences.
The expression control sequences include, but are not limited to,
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signal for introns, maintenance of the correct reading
frame of that gene to permit proper translation of mRNA, and stop
codons.
The polynucleotide sequences encoding an immunogenic TARP peptide
can be inserted into an expression vector including, but not
limited to, a plasmid, virus or other vehicle that can be
manipulated to allow insertion or incorporation of sequences and
can be expressed in a host cell. Methods of expressing DNA
sequences having eukaryotic or viral sequences in prokaryotes are
well known in the art. Biologically functional viral and plasmid
DNA vectors capable of expression and replication in a host are
known in the art.
In some embodiments, the compositions further include a
pharmaceutically acceptable carrier. In some embodiments, the
compositions further include an adjuvant. In some examples,
particularly when the composition to be administered comprises
isolated peptides, the adjuvant is MONTANIDE.RTM. ISA 51 VG, and
may further include GM-CSF. In other examples, the adjuvant
comprises poly-ICLC.
In some embodiments, the compositions comprise antigen presenting
cells (APCs) loaded with the TARP peptides. In some examples, the
APCs are professional APCs. In particular examples, the APCs are
dendritic cells. APCs loaded with TARP peptide can be generated,
for example, by pulsing or co-incubating APCs with the TARP
peptides. Alternatively, APCs can be transduced with a vector
encoding the TARP peptide. The TARP peptide will then be expressed
and processed by the APC for presentation on the APC surface.
In some embodiments, the compositions are in unit dose form. In
some examples, the composition in unit dose form comprises a
lyophilized powder of the TARP peptides.
Immunogenic TARP peptides can be chemically synthesized by standard
methods. If desired, polypeptides can also be chemically
synthesized by emerging technologies. One such process is described
in W. Lu et al., Federation of European Biochemical Societies
Letters. 429:31-35, 1998. Polypeptides can also be produced using
molecular genetic techniques, such as by inserting a nucleic acid
encoding TARP or an epitope thereof into an expression vector,
introducing the expression vector into a host cell, and isolating
the polypeptide.
IV. Administration and Use of TARP Peptide Compositions
The immunogenic TARP peptides disclosed herein can be administered
to a subject in order to generate an immune response. Thus,
provided herein is a method of eliciting an immune response in a
subject by administering to the subject a therapeutically effective
amount of a TARP peptide composition disclosed herein. In some
instances, the immune response comprises a CD4+ T cell response, a
CD8+ T cell response, or both. The immune response can also include
an anti-TARP antibody response. In some embodiments, the
composition is administered intradermally, intramuscularly or
subcutaneously.
The TARP peptide compositions disclosed herein can be administered
to a subject to treat a cancer that expresses TARP, such as
prostate cancer, mesothelioma, breast cancer, or any other tumor
that expresses TARP. Thus, in some embodiments, the subject has
prostate cancer, breast cancer or mesothelioma, or any other cancer
that is identified to express or over-express TARP. In one example,
the subject with breast cancer has triple-negative breast cancer.
In one example, the subject with prostate cancer has
hormone-sensitive prostate cancer. In another example, the subject
with prostate cancer has castration-resistant prostate cancer. In
some examples, the cancer is metastatic. In some examples, the
immune response inhibits the growth of the TARP-expressing cancer.
In some cases, the subject has undergone or will undergo other
cancer-specific treatments, including surgery, chemotherapy or
radiation therapy.
Also provided are methods of treating a subject with cancer, by
selecting a subject with a cancer that expresses TARP, and
administering to the subject a therapeutically effective amount of
a TARP peptide composition disclosed herein. In some embodiments,
the TARP-expressing cancer is prostate cancer, breast cancer or
mesothelioma. In some embodiments, the composition is administered
intradermally, intravenously, intramuscularly or
subcutaneously.
In exemplary applications, the disclosed compositions are
administered to a patient suffering from a disease, such as
prostate cancer, mesothelioma, breast cancer, or any other cancer
that expresses TARP, in an amount sufficient to raise an immune
response to TARP-expressing cells. Administration induces a
sufficient immune response to slow the proliferation of such cancer
cells or to inhibit their growth, or to reduce a sign or a symptom
of the tumor. Amounts effective for this use will depend upon the
severity of the disease, the general state of the patient's health,
and the robustness of the patient's immune system. A
therapeutically effective amount of the composition is that which
provides either subjective relief of a symptom(s) or an objectively
identifiable improvement as noted by the clinician or other
qualified observer, including alterations in laboratory parameters
such as kinetics of PSA value change.
In some embodiments, the subject is HLA-A2 positive. In other
embodiments, the subject is HLA-A2 negative.
In some embodiments, the compositions comprise antigen presenting
cells (APCs), such as dendritic cells, loaded with the TARP
peptides. In some examples, the APCs are autologous cells. In other
examples, the APCs are allogeneic. In these methods, APCs can be
pulsed or co-incubated with immunogenic TARP peptides in vitro.
Alternative, the APCs can be transduced with a vector encoding the
immunogenic TARP peptides, which leads to processing and display of
the peptide in complex with MCH. Regardless of the method used to
generate APCs loaded with TARP peptides, a therapeutically
effective amount of the APCs can then be administered to a subject.
In some examples, the therapeutically effective amount of the
composition comprises about 1.times.10.sup.6 to about
30.times.10.sup.6 viable APCs, such as about 5.times.10.sup.6 to
about 25.times.10.sup.6 viable APCs, or about 10.times.10.sup.6 to
about 20.times.10.sup.6 viable APCs. In non-limiting examples, the
therapeutically effective amount of the composition comprises about
1.times.10.sup.6, 2.times.10.sup.6, 3.times.10.sup.6,
4.times.10.sup.6, 5.times.10.sup.6, 6.times.10.sup.6,
7.times.10.sup.6, 8.times.10.sup.6, 9.times.10.sup.6,
10.times.10.sup.6, 15.times.10.sup.6, 20.times.10.sup.6,
25.times.10.sup.6, or 30.times.10.sup.6 viable APCs. In some cases,
when multiple peptides are to be administered to a subject using
APCs, individual pools of cells are each pulsed with one peptide
and subsequently pooled together for administration. The pooled
APCs can be administered in a single injection, or in multiple
injections, such as in two injections. In most cases, the
peptide-loaded APCs are administered intradermally, but can be
administered using any suitable route for generating an immune
response, such as subcutaneously, intravenously or
intramuscularly.
As discussed above, the immunogenic TARP peptide(s) can be
delivered to the dendritic cells or to dendritic cell precursors
via any method known in the art, including, but not limited to,
pulsing dendritic cells directly with antigen, or utilizing a broad
variety of antigen delivery vehicles, such as, for example,
liposomes, or other vectors known to deliver antigen to cells. In
one specific, non-limiting example an antigenic formulation
includes about 0.1 .mu.g to about 1,000 .mu.g, or about 1 to about
100 .mu.g of a selected immunogenic TARP peptide. The immunogenic
TARP peptide can also be administered with agents that promote
dendritic cell maturation. Specific, non-limiting examples of
agents of use are interleukin-4 (IL-4), interferon-gamma
(IFN-.gamma.), endotoxin/lipopolysaccharide (LPS), keyhole limpet
hemocyanin (KLH), granulocyte/macrophage colony stimulating factor
(GM-CSF), or flt-3 ligand (flt-3L). The preparation can also
contain buffers, excipients, and preservatives, amongst other
ingredients.
In one embodiment, mature antigen presenting cells are generated to
present the immunogenic TARP peptide(s). These dendritic cells are
then administered alone to a subject with a tumor that expresses
TARP, such as a prostate cancer, mesothelioma or breast cancer. In
another embodiment, the mature dendritic cells are administered in
conjunction with a chemotherapeutic agent or other immune-based
therapies targeting negative regulation such as anti-CLA-4
(cytotoxic T-lymphocyte antigen 4), anti-PD-1 (programmed cell
death protein 1) or anti-PD-L1 (programmed cell death 1 ligand
1).
Alternatively, the APCs are used to sensitize CD8+ cells, such as
tumor infiltrating lymphocytes (TILs) from prostate, mesothelioma
or breast tumors (or another type of cancer) or peripheral blood
lymphocytes (PBLs). The TILs or PBLs can be from the same subject
(autologous) that is to be treated. Alternatively, the TILs or PBLs
can be heterologous. However, they should at least be MHC class-I
restricted to the HLA types the subject possesses. An effective
amount of the sensitized cells are then administered to the
subject.
Peripheral blood mononuclear cells (PBMCs) can be used as the
responder cell source of CTL precursors. The appropriate
antigen-presenting cells are incubated with peptide, after which
the peptide-loaded antigen-presenting cells are then incubated with
the responder cell population under optimized culture conditions.
Positive CTL activation can be determined by assaying the culture
for the presence of CTLs that kill radio-labeled target cells, both
specific peptide-pulsed targets as well as target cells expressing
endogenously processed forms of the antigen from which the peptide
sequence was derived, such as TARP (e.g. SEQ ID NO: 1 or SEQ ID NO:
2).
The cells can be administered to a subject to inhibit the growth of
cells of TARP expressing tumors. In these applications, a
therapeutically effective amount of activated antigen presenting
cells, or activated lymphocytes, are administered to a subject
suffering from a disease, in an amount sufficient to raise an
immune response to TARP-expressing cells. The resulting immune
response is sufficient to slow the proliferation of such cancer
cells or to inhibit their growth, or to reduce a sign or a symptom
of the tumor.
In other embodiments, compositions comprising isolated TARP
peptides are administered to the subject. An immunogenic TARP
peptide can be administered by any means known to one of skill in
the art (see Banga. A., "Parenteral Controlled Delivery of
Therapeutic Peptides and Proteins." in Therapeutic Peptides and
Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995)
such as by intradermal, intramuscular, subcutaneous, or intravenous
injection. In one embodiment, administration is by intradermal or
intramuscular injection. To extend the time during which the
peptide(s) is available to stimulate a response, the peptide(s) can
be provided as an implant, an oily injection, or as a particulate
system. The particulate system can be a microparticle, a
microcapsule, a microsphere, a nanocapsule, or similar particle. A
particulate carrier based on a synthetic polymer has been shown to
act as an adjuvant to enhance the immune response, in addition to
providing a controlled release. Aluminum salts can also be used as
adjuvants to produce an immune response.
In some embodiments, an immunogenic TARP peptide composition is
administered in a manner to direct the immune response to a
cellular response (that is, a CTL response). A number of means for
inducing cellular responses, both in vitro and in vivo, are known.
Lipids have been identified as agents capable of assisting in
priming CTLs in vivo against various antigens. For example, as
described in U.S. Pat. No. 5,662,907, palmitic acid residues can be
attached to the alpha and epsilon amino groups of a lysine residue
and then linked (e.g., via one or more linking residues, such as
glycine, glycine-glycine, serine, serine-serine, or the like) to an
immunogenic peptide. The lipidated peptide can then be injected
directly in a micellar form, incorporated in a liposome, or
emulsified in an adjuvant. As another example, E. coli
lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine
can be used to prime tumor specific CTL when covalently attached to
an appropriate peptide (see, Deres et al., Nature 342:561, 1989).
Further, as the induction of neutralizing antibodies can also be
primed with the same molecule conjugated to a peptide that displays
an appropriate epitope, two compositions can be combined to elicit
both humoral and cell-mediated responses where that is deemed
desirable.
In another embodiment, to induce a CTL response to an immunogenic
TARP peptide composition, a MHC class II-restricted T-helper
epitope is added to the immunogenic TARP polypeptide to induce
T-helper cells to secrete cytokines in the microenvironment to
activate CTL precursor cells. The overlapping TARP peptides
disclosed herein include MHC class II epitopes for inducing
T-helper cells. The technique further involves adding short lipid
molecules to retain the construct at the site of the injection for
several days to localize the antigen at the site of the injection
and enhance its proximity to dendritic cells or other
"professional" antigen presenting cells over a period of time (see
Chesnut et al., "Design and Testing of Peptide-Based Cytotoxic
T-Cell-Mediated Immunotherapeutics to Treat Infectious Diseases and
Cancer," in Powell et al., eds., Vaccine Design, the Subunit and
Adjuvant Approach, Plenum Press, New York. 1995).
Pharmaceutical compositions including immunogenic TARP peptides are
provided herein. In one embodiment, the immunogenic TARP peptides
are mixed with an adjuvant containing a stabilizing detergent, a
micelle-forming agent, and/or an oil. In one embodiment, the
adjuvant is MONTANIDE.RTM. ISA 51 VG, and may further include
granulocyte macrophage colony stimulating factor (GM-CSF). Suitable
stabilizing detergents, micelle-forming agents, and oils are
detailed in U.S. Pat. No. 5,585,103; U.S. Pat. No. 5,709,860; U.S.
Pat. No. 5,270,202: and U.S. Pat. No. 5,695,770. A stabilizing
detergent is any detergent that allows the components of the
emulsion to remain as a stable emulsion. Such detergents include
polysorbate, 80 (TWEEN)
(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl;
manufactured by ICI Americas, Wilmington, Del.), TWEEN 40.TM.,
TWEEN 20.TM., TWEEN 60.TM., ZWITTERGENT.TM. 3-12, TEEPOL HB7.TM.,
and SPAN 85.TM.. These detergents are usually provided in an amount
of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle
forming agent is an agent which is able to stabilize the emulsion
formed with the other components such that a micelle-like structure
is formed. Such agents generally cause some irritation at the site
of injection in order to recruit macrophages to enhance the
cellular response. Examples of such agents include polymer
surfactants described by BASF Wyandotte publications, e.g.,
Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al.,
J. Immunol 129:1244, 1981, PLURONIC.TM. L62LF, L101, and L64,
PEG1000, and TETRONIC.TM. 1501, 150R1, 701, 901, 1301, and 130R1.
The chemical structures of such agents are well known in the art.
In one embodiment, the agent is chosen to have a
hydrophile-lipophile balance (HLB) of between 0 and 2, as defined
by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be
provided in an effective amount, for example between 0.5 and 10%,
or in an amount between 1.25 and 5%.
The oil included in the composition is chosen to promote the
retention of the antigen in oil-in-water emulsion (i.e., to provide
a vehicle for the desired antigen) and may have a melting
temperature of less than 65.degree. C. such that an emulsion is
formed either at room temperature (about 20.degree. C. to
25.degree. C.), or once the temperature of the emulsion is brought
down to room temperature. Examples of such oils include squalene,
squalane, EICOSANE.TM., tetratetracontane, glycerol, and peanut oil
or other vegetable oils. In one specific, non-limiting example, the
oil is provided in an amount between 1 and 10%, or between 2.5 and
5%. The oil should be both biodegradable and biocompatible so that
the body can break down the oil over time, and so that no adverse
effects, such as granulomas, are evident upon use of the oil.
An adjuvant can be included in the composition. In one embodiment,
the adjuvant is MONTANIDE.RTM. ISA 51 VG plus GM-CSF. In other
embodiments, the adjuvant is a mixture of stabilizing detergents,
micelle-forming agent, and oil available under the name PROVAX.RTM.
(Biogen Idec, San Diego, Calif.). An adjuvant can also be an
immunostimulatory nucleic acid, such as a nucleic acid including a
CpG motif.
In another embodiment, the TARP composition includes one or more
nucleic acids encoding one or more immunogenic TARP peptides. A
therapeutically effective amount of the nucleic acid(s) encoding
the TARP peptide(s) can be administered to a subject in order to
generate an immune response. In one specific, non-limiting example,
a therapeutically effective amount of the nucleic acid(s) is
administered to a subject to treat prostate cancer, mesothelioma or
breast cancer, or any other tumor that expresses TARP. One approach
to administration of nucleic acids to a subject is direct
immunization with plasmid DNA, such as with a mammalian expression
plasmid. The nucleotide sequence encoding an immunogenic TARP
peptide can be placed under the control of a promoter to increase
expression of the molecule.
Immunization by nucleic acid constructs is well known in the art
and taught, for example, in U.S. Pat. No. 5,643,578 (which
describes methods of immunizing vertebrates by introducing DNA
encoding a desired antigen to elicit a cell-mediated or a humoral
response), and U.S. Pat. Nos. 5,593,972 and 5,817,637 (which
describe operably linking a nucleic acid sequence encoding an
antigen to regulatory sequences enabling expression). U.S. Pat. No.
5,880,103 describes several methods of delivery of nucleic acids
encoding immunogenic peptides or other antigens to an organism. The
methods include liposomal delivery of the nucleic acids (or of the
synthetic peptides themselves), and immune-stimulating constructs,
or ISCOMS.TM., negatively charged cage-like structures of 30-40 nm
in size formed spontaneously on mixing cholesterol and QUIL A.TM.
(saponin). Protective immunity has been generated in a variety of
experimental models of infection, including toxoplasmosis and
Epstein-Barr virus-induced tumors, using ISCOMS.TM. as the delivery
vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383,
1991). Doses of antigen as low as 1 .mu.g encapsulated in
ISCOMS.TM. have been found to produce MHC class 1 mediated CTL
responses (Takahashi et al., Nature 344:873, 1990).
In another approach to using nucleic acids for immunization,
immunogenic TARP peptides can be expressed by attenuated viral
hosts or vectors or bacterial vectors. Recombinant vaccinia virus,
adeno-associated virus, herpes virus, retrovirus, or other viral
vectors can be used to express the peptide, thereby eliciting a CTL
response. For example, vaccinia vectors and methods useful in
immunization protocols are described in U.S. Pat. No. 4,722,848.
BCG (Bacillus Calmette Guerin) provides another vector for
expression of the peptides (see Stover, Nature 351:456-460,
1991).
In one embodiment, one or more nucleic acids encoding one or more
immunogenic TARP peptides are introduced directly into cells. For
example, the nucleic acid(s) can be loaded onto gold microspheres
by standard methods and introduced into the skin by a device such
as Bio-Rad's HELIOS.TM. Gene Gun. The nucleic acids can be "naked,"
consisting of plasmids under control of a strong promoter.
Typically, the DNA is injected into muscle, although it can also be
injected directly into other sites, including tissues in proximity
to metastases. Dosages for injection are usually around 0.5
.mu.g/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to
about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).
The compositions (e.g., TARP peptides. APCs loaded with TARP
peptides, or nucleic acids or vectors encoding TARP peptides) can
be administered for therapeutic treatments. In therapeutic
applications, a therapeutically effective amount of the composition
is administered to a subject suffering from a disease, such as
prostate cancer, mesothelioma or breast cancer, or any other cancer
that expresses TARP. Single or multiple administrations of the
compositions are administered depending on the dosage and frequency
as required and tolerated by the subject. In one embodiment, the
composition is administered in multiple doses, such as two, three,
four, five, six, seven or eight doses. Generally, the dose is
sufficient to treat or ameliorate symptoms or signs of disease
without producing unacceptable toxicity to the subject. Systemic or
local administration can be utilized. For each dose, the
composition can be administered using a single injection (a single
site of injection) or can be administered using two or more
injections (two or more sites of injection). In particular
examples, the TARP peptide compositions are administered using two
injections (such as one in each arm). In other cases, particularly
if isolated TARP peptides are being administered (i.e. administered
in the absence of APCs), one site per peptide may be required.
In some embodiments, any of the immunotherapies discussed above is
augmented by administering a cytokine, such as IL-2, IL-3. IL-6,
IL-10, IL-12, IL-15, GM-CSF, or interferons, or a combination of
two or more cytokines, such as 2, 3, 4, 5, 6, 7 or more
cytokines.
Administration of the immunogenic TARP peptide compositions
disclosed herein can also be accompanied by administration of other
anti-cancer agents or therapeutic treatments (such as surgical
resection of a tumor). Any suitable anti-cancer agent can be
administered in combination with the compositions disclosed herein.
Exemplary anti-cancer agents include, but are not limited to,
cytotoxic chemotherapeutic agents, such as, for example, mitotic
inhibitors, alkylating agents, anti-metabolites, intercalating
antibiotics, growth factor inhibitors, cell cycle inhibitors,
enzymes, topoisomerase inhibitors, anti-survival agents, biological
response modifiers, anti-hormones (e.g. anti-androgens) and
anti-angiogenesis agents. Other anti-cancer treatments include
radiation therapy, antibodies that specifically target cancer
cells, or antibodies to other immune modulating proteins such as
CTLA-4, PD-1, PD-L1 or TGF-.beta. (transforming growth
factor-beta).
Non-limiting examples of alkylating agents include nitrogen
mustards (such as mechlorethamine, cyclophosphamide, melphalan,
uracil mustard or chlorambucil), alkyl sulfonates (such as
busulfan), nitrosoureas (such as carmustine, lomustine, semustine,
streptozocin, or dacarbazine).
Non-limiting examples of antimetabolites include folic acid analogs
(such as methotrexate), pyrimidine analogs (such as 5-FU or
cytarabine), and purine analogs, such as mercaptopurine or
thioguanine.
Non-limiting examples of natural products include vinca alkaloids
(such as vinblastine, vincristine, or vindesine),
epipodophyllotoxins (such as etoposide or teniposide), antibiotics
(such as dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicamycin, or mitomycin C), and enzymes (such as
L-asparaginase).
Non-limiting examples of miscellaneous agents include platinum
coordination complexes (such as cis-diamine-dichloroplatinum II
also known as cisplatin), substituted ureas (such as hydroxyurea),
methyl hydrazine derivatives (such as procarbazine), and
adrenocrotical suppressants (such as mitotane and
aminoglutethimide).
Non-limiting examples of hormones and antagonists include
adrenocorticosteroids (such as prednisone), progestins (such as
hydroxyprogesterone caproate, medroxyprogesterone acetate, and
magestrol acetate), estrogens (such as diethylstilbestrol and
ethinyl estradiol), antiestrogens (such as tamoxifen), and
androgens (such as testerone proprionate and fluoxymesterone).
Examples of the most commonly used chemotherapy drugs include
Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCN LU, Carboplatinum,
Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine,
Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,
Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes,
such as docetaxel), Velban, Vincristine, VP-16, while some more
newer drugs include Gemcitabine (Gemzar), Herceptin.RTM.,
Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan
STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine),
Zevelin and calcitriol.
Non-limiting examples of immunomodulators that can be used include
AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon
(Genentech), GM-CSF (granulocyte macrophage colony stimulating
factor; Genetics Institute). IL-2 (Cetus or Hoffman-LaRoche), human
immune globulin (Cutter Biological). IMREG (from Imreg of New
Orleans, La.), SK&F 106528. TNF (tumor necrosis factor;
Genentech) and anti-CTLA-4 (ipilimumab, Bristol-Myers Squibb).
Another common treatment for some types of cancer is surgical
treatment, for example surgical resection of the cancer or a
portion of it. Another example of a treatment is radiotherapy, for
example administration of radioactive material or energy (such as
external beam therapy) to the tumor site to help eradicate the
tumor or shrink it prior to surgical resection.
In particular embodiments, a subject having prostate cancer is
administered a TARP peptide composition disclosed herein in
combination with radiation therapy, brachytherapy, or cryotherapy.
In other specific embodiments, a subject having prostate cancer,
such as metastatic castration-resistant prostate cancer, is
administered a TARP peptide composition disclosed herein in
combination with chemotherapy.
In other embodiments, a subject with a TARP-expressing cancer, such
as prostate cancer, is administered a TARP peptide composition
disclosed herein in combination with an agent that targets negative
regulation of the immune system, such as anti-CTLA4, anti-PD-1,
anti-PD-L1 or anti-TGF.beta..
V. Wild-Type and Epitope-Enhanced TARP Peptides as Cancer
Vaccines
Two HLA-A2 epitopes that produce cytolytic T cell responses were
previously identified (Oh et al., Cancer Res. 64:2610-2618, 2004).
These sequences map to amino acids 27-35 and 29-37 of human TARP
(SEQ ID NO: 2). TARP 27-35 was found to bind with an affinity that
was 10 times greater than that of TARP 29-37. Both peptides were
shown to be immunogenic by immunizing A2K.sup.b transgenic mice
(expressing human HLA-A*0201) with dendritic cells pulsed with the
peptides or with DNA encoding the peptides. Dendritic cell
immunization produced a higher level of immunity than DNA
immunization, and as expected due to its higher binding affinity,
TARP 27-35 produced a higher level of CD8+ T cell response than
TARP29-37.
A. Epitope Enhancement
Modification of the amino acid sequence of epitopes, commonly
referred to as epitope enhancement, can improve the efficacy of
vaccines through several means: (1) increasing affinity of peptide
for MHC molecules; (2) increasing T cell receptor (TCR) triggering;
or (3) inhibiting proteolysis of the peptide by serum peptidases.
Epitope-enhanced subdominant peptides can bypass self-tolerance
because subdominant epitopes do not generally induce tolerance but
can be made more immunogenic by epitope enhancement.
Epitope enhancement of TARP peptides was previously performed to
increase the level of immunity that could be generated with these
peptides. As described in U.S. Pat. Nos. 7,541,035 and 8,043,623
(incorporated herein by reference), amino acid substitutions in the
TARP 27-35 peptide did not increase binding affinity, but two amino
acid substitutions in TARP 29-37 did produce higher binding
affinity peptides. For TARP 29-37, Arg at position 3 and Leu at
position 9 were substituted with Ala (TARP 29-37-3A) and Val (TARP
29-37-9V), respectively. Substitution at position 3 with Ala in
TARP 29-37 resulted in the greatest increase in the binding
affinity of the peptide. Although TARP29-37-9V showed a lower
binding affinity to HLA-A2 than TARP29-37-3A, substitution of Leu
at position 9 with Val did enhance the binding affinity compared
with the wild-type peptide, TARP 29-37. When the immunogenicity of
these peptides was evaluated in A2K.sup.b transgenic mice, both of
the epitope-enhanced peptides produced a higher percentage of
TARP-specific CD8.sup.+ T cells than the wild type sequence. It was
also shown that T cells generated with the epitope-enhanced TARP
29-37 sequences reacted with targets pulsed with the wild type TARP
29-37 peptide in the mouse.
Studies of these peptides in human cells showed that TARP 29-37,
TARP 29-37-3A, and TARP 29-37-9V were immunogenic in human T cells.
TARP 29-37-9V specific T cells recognized targets pulsed with all
three peptides equally well, whereas TARP 29-37-3A specific T cells
recognized only targets pulsed with TARP 29-37-3A. This suggested
that the TARP 29-37-3A peptide would not be appropriate for
immunization in humans, whereas the TARP 29-37-9V would be more
likely to generate T cells that recognize the wild type sequence.
Human T cells specific for TARP 27-35 recognized targets pulsed
with that sequence. In addition to their ability to kill targets
pulsed with TARP peptides. CD8.sup.+ T cells specific for TARP
peptides were able to kill human tumor targets that were HLA-A2
positive and that expressed TARP sequences, confirming that TARP
was endogenously processed and presented in human tumor cells. The
availability of tetramers that react with CD8.sup.+ T cells
specific for TARP provided a simple means of evaluating the ability
to stimulate immunity to the TARP peptides. In one survey, tetramer
positive cells ranged from 0.66% to 3.9% of the CD8.sup.+ T cells
in prostate and breast cancer patients compared with 0.01-0.6% in
normal controls.
B. Therapeutic Vaccination Utilizing Wild Type (WT) and
Epitope-enhanced (EE) TARP Peptides (NCI 09-C-0139)
NCI 09-C-0139 is a prospective, randomized pilot clinical study
examining TARP vaccination in HLA-A*0201 positive men with Stage D0
prostate cancer (PSA biochemical recurrence without evidence of
visceral or bony metastatic disease). Since the optimal method for
therapeutic immunization with peptide vaccines in patients with
cancer is unclear, patients were randomized to receive vaccination
with TARP peptides in MONTANIDE.RTM. ISA 51 VG adjuvant plus GM-CSF
(Arm A) or as an autologous, TARP peptide-pulsed dendritic cell
(DC) vaccine. The primary objective was to determine the safety and
immunogenicity (as measured by IFN-.gamma. ELISPOT, intracellular
cytokine staining (ICS) and tetramer assays) of TARP vaccination.
The secondary objectives were to determine the effect of TARP
peptide vaccination on PSA doubling time (PSADT) (Arlen et al., J
Urol 179:2181-2186, 2008) and PSA growth rate and regression rate
constants. All study participants had to have a baseline PSADT
(calculated using PSA values within 12 months of study entry)>3
months and .ltoreq.15 months.
TARP vaccine was administered by deep subcutaneous injection (Arm
A) or intradermally (Arm B, 20.times.10.sup.6 viable cells/vaccine)
at Weeks 3, 6, 9, 12, and 15, with an optional sixth dose of
vaccine at Week 36 based on changes in PSADT (.gtoreq.50% increase
over pre-vaccine PSADT) or immune parameters (3-fold increase in
TARP-specific reactivity as measured by IFN-.gamma. ELISPOT at
least two time points) at Week 24. TARP vaccination was found to be
safe and well tolerated, with adverse events limited to injection
site reactions.ltoreq.Grade 2. There were no systemic or immediate
hypersensitivity reactions or laboratory abnormalities associated
with vaccination. TARP vaccination was also shown to be associated
with a slowing in the rise of PSA levels (PSA velocity), measured
as PSA doubling time (PSADT) or slope log (PSA), a surrogate marker
for clinical outcomes and how well patients will do. A highly
statistically significant decrease was observed in the slope log
PSA (i.e. there was significant slowing in how fast the patients'
PSAs were rising compared to their pre-vaccination baseline at both
3-24 and 3-48 weeks). In addition, this effect of decreased slope
log (PSA)/slowing in PSA velocity at Weeks 3-24 didn't wane
significantly over time and wasn't impacted by an additional
vaccine dose at Week 36.
C. Multi-Epitope (ME) TARP Vaccine Design
The second generation ME TARP vaccine is based on the amino acid
sequence of the entire TARP protein (SEQ ID NO: 1). The vaccine
platform includes the original two 9-mer HLA-A*0201 binding TARP
peptide epitopes (WT TARP 27-35 and EE TARP 29-37-9V) utilized in
NCI 09-C-0139 as well as an additional five 20-mer TARP peptides
overlapping by 10 amino acids for a total of 7 peptides that span
the entire TARP sequence.
SLPs are synthetic peptides of 20-50 amino acids that because of
their length require internalization and processing by DCs.
Processing by these professional antigen presenting cells avoids
presentation by non-professional antigen-presenting cells that
could potentially induce tolerance instead of immunity. Overlapping
SLPs contain both CD4 and CD8 epitopes, which results in parallel
stimulation of both CD4+ and CD8+ T cells and a stronger, more
effective immune response. In addition, since overlapping SLPs
contain all potential epitopes irrespective an individual's MHC
type, the use of SLPs is a highly attractive approach to maximize
the therapeutic applicability of any given vaccine in a genetically
diverse human population such as that of the United States. Protein
vaccination is highly suitable for the induction of CD4+ T cell
responses and antibodies, but it generally induces responses
against dominant epitopes and often fails to induce proper and
effective CD8+ T cell immunity, in contrast to long peptides that
induce both. In addition, processing and uptake of SLPs by DCs is
more efficient compared to processing and uptake of intact protein.
For these reasons, SLPs and multi-epitope vaccines are able to
induce a broader repertoire of T cell responses, thereby maximizing
the diversity of epitopes potentially associated with anti-tumor
effector function while minimizing the risk of tumor antigen
escape.
The advantage of the multi-epitope TARP peptide vaccine platform
disclosed herein is that the overlapping epitopes cover the entire
TARP protein, eliminating the need for HLA restriction, thus
allowing any and all patient populations with a TARP-expressing
tumor to be candidates for therapeutic vaccination. In addition,
these longer synthetic peptides include MHC class II CD4+ T cell
helper epitopes that will allow generation of better CD8+ T cell
responses with improved functional avidity and longevity as well as
humoral anti-TARP antibody responses. The peptide sequences
encompassing the whole protein will have all the possible epitopes
that can be presented by any HLA molecule and therefore would be
suitable for vaccinating the entire population of prostate cancer
patients, making adequate accrual feasible. Overlapping long
peptides, such as the 20-mers overlapping by 10 residues, have been
used to represent a whole protein because they contain all the
potential epitopes of the whole protein but are more amenable to
processing for both class I and class II HLA presentation to
CD4.sup.+ and CD8.sup.+ T cells (Jiang et al., Vaccine
24:6356-6365, 2006; Mirshahidi et al., Vaccine 27:1825-1833, 2009:
Dong et al., Vaccine 23:3630-3633, 2005; Zhang et al., J Biol Chem
284:9184-9191, 2009).
The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1
A Randomized, Placebo-Controlled Phase II Study of Multi-Epitope
TARP (ME TARP) Peptide Autologous Dendritic Cell Vaccination in Men
with Stage D0 Prostate Cancer
Study Design Eligible patients are prospectively randomized 2:1 to
receive either autologous TARP multi-epitope DC vaccine or an
autologous elutriated monocyte vaccine placebo after safety and
immunogenicity have been established through 12 weeks in an initial
lead-in cohort of 6 patients as outlined in FIG. 1. Enrollment of
this lead-in cohort is staggered every three weeks for the first
three patients to allow a 3-week interval for safety assessment
before the next enrolled patient is scheduled to receive their
first dose of ME TARP vaccine. If there is no adverse safety signal
identified in these first 3 patients, enrollment of the remaining 3
lead-in subjects and subsequent randomization of study subjects
proceeds on or after 9 weeks after the first study subject has
received their first ME TARP vaccine dose and 3 weeks after the
third study subject has received their first ME TARP vaccine dose.
All patients receive a total of 6 doses of vaccine
(20.times.10.sup.6 viable cells/dose) delivered intradermally at
Weeks 3, 6, 9, 12, 15, and 24. All patients undergo restaging at
Weeks 48 and 96 to confirm maintenance of Stage D0 disease. The
study monitoring schedule of clinical assessments, laboratory and
imaging studies is identical for all patients as outlined in FIG.
1.
All patients have a history and physical exam, routine monitoring
labs. PSA and testosterone levels performed at the study week
visits shown in FIG. 1. PSA Doubling Time (PSADT) and slope log
(PSA) are calculated at every study visit using the PSADT Memorial
Sloane Kettering nomogram (available online). Immunologic responses
(IFN-.gamma. ELISPOT, ICS and tetramer assays, anti-TARP
antibodies) to multi-epitope TARP peptide vaccination are examined
at the following timepoints: Weeks 0, 12, 18, 24, 48, 72 and
96.
Vaccine Administration
All patients undergo 15-18 L apheresis to remove peripheral blood
monocytes for dendritic cell preparation as well as peripheral
blood mononuclear cells for flow cytometry and immunologic studies
at their Week 0 visit. Cells used for subsequent dendritic cell
maturation are derived from monocytes frozen during the initial
apheresis. Eligible subjects receive autologous ME TARP dendritic
cell or elutriated monocyte placebo vaccine beginning at Week 3.
For patients receiving active ME TARP DC vaccine, each peptide is
pulsed on dendritic cells separately in order to assure adequate
binding of the peptide and cells are not washed to remove free
peptide after pulsing. Following verification of mature dendritic
cell validation markers and release standards, the separately
peptide-pulsed dendritic cells are recombined for administration.
Autologous ME TARP DC and elutriated monocyte placebo vaccine
preparations are assessed for release standards (nucleated cell
content and concentration, appearance, flow cytometric verification
of DC validation markers, viability.gtoreq.60%, and product
sterility and safety testing) prior to release for vaccine
administration to the patient. For both groups, vaccines are
administered intradermally in two vaccination sites on the forearm
with a maximum volume of 0.5 ml per injection. Vaccination is
alternated between the left and right forearm with each
vaccination. All patients receive a total of 6 doses of vaccine
(20.times.10.sup.6 viable cells/dose) delivered at Weeks 3, 6, 9,
12, 15, and 24 and undergo restaging at Weeks 48 and 96 to confirm
maintenance of Stage D0 disease. Patients are monitored for
immediate adverse event vaccine reactions for 1 hour following
their first TARP peptide vaccine dose. If no adverse reactions are
observed with the first vaccination, patients are monitored for 15
minutes for all subsequent vaccinations. If an adverse reaction is
observed following the first vaccine, the reaction is characterized
and a determination made as to whether it is considered a dose
limiting toxicity (DLT). If the adverse reaction is determined not
to be a DLT, the duration of post-vaccination monitoring for
subsequent vaccinations is determined as clinically indicated
depending on the severity of the initial vaccine reaction. All
patients are given a ME TARP DC Vaccine Report Card and instructed
on how to complete it, following each ME TARP DC or placebo vaccine
dose.
Since this protocol involves multi-epitope TARP vaccination in
humans for the first time, enrollment of randomized subjects does
not begin until safety and immunogenicity through 12 weeks are
established in an initial staggered enrollment lead in of 6
patients. If no adverse events are observed through Week 12
following the first vaccination in these 6 patients, enrollment of
additional patients may proceed as quickly as is logistically
feasible.
Autologous Multi-Epitope (ME) TARP Dendritic Cell Vaccine
Description
The 2.sup.nd generation ME TARP vaccine is based on the amino acid
sequence of the entire TARP protein (SEQ ID NO: 1). The vaccine
platform includes the original two 9-mer HLA-A*0201 binding TARP
peptide epitopes (WT TARP 27-35 and EE TARP 29-37-9V) utilized in
NCI 09-C-0139 as well as an additional five 20-mer TARP peptides
overlapping by 10 amino acids for a total of 7 peptides that span
the entire TARP sequence:
TABLE-US-00005 TARP 27-35: (SEQ ID NO: 3; HLA-A*0201 restricted)
FVFLRNFSL TARP 29-37-9V: (SEQ ID NO: 4; HLA-A*0201 restricted)
FLRNFSLMV TARP 1-20: (SEQ ID NO: 5; HLA non-restricted)
MQMFPPSPLFFFLQLLKQSS; TARP 11-30: (SEQ ID NO: 6; HLA
non-restricted) FFLQLLKQSSRRLEHTFVFL TARP 21-40: (SEQ ID NO: 7; HLA
non-restricted) RRLEHTFVFLRNFSLMLLRG TARP 31-50: (SEQ ID NO: 8; HLA
non-restricted) RNFSLMLLRGIGKKRRATRF TARP 41-58: (SEQ ID NO: 9; HLA
non-restricted) IGKKRRATRFWDPRRGTP
Autologous ME TARP DC vaccine and autologous elutriated monocyte
placebo vaccine are generated utilizing current good manufacturing
practices (cGMP) as outlined in Example 2.
Study Drugs
Interleukin-4 CELLGENIX.TM.
Product Description: Interleukin-4 (IL-4) used in this study is
manufactured and supplied by CellGenix (Freiburg, Germany). It is
used as an ancillary product to mature dendritic cells in vitro and
is not administered directly to patients. IL-4 exerts important
effects on B cells, T cells, macrophages, eosinophils,
hematopoietic progenitor cells, endothelial cells and promotes the
maturation of dendritic cells. The complementary DNA clone (cDNA),
when expressed in E. coli yields a 129 amino acid protein with a
molecular weight of 14,957 daltons. IL-4 is a highly purified
(.gtoreq.95% chromatographically pure), sterile, water-soluble
protein.
Formulation and Preparation: RhIL-4 Sterile Powder for Injection is
supplied in 100 mcg and 200 mcg vials (containing a total of 120
mcg and 240 mcg of IL-4, respectively) as a sterile lyophilized
powder formulated with glycine, human serum albumin, citric acid,
and sodium citrate. Un-reconstituted IL-4 is kept refrigerated at
2-8.degree. C. 1.2 mL of Sterile Water for Injection USP is added
to each vial of rhIL-4 Sterile Powder for Injection. The vial is
gently agitated to completely dissolve the powder and is inspected
visually for discoloration and particulates prior to use.
Stability and Storage: The reconstituted product is refrigerated at
2-8.degree. C. and used within 24 hours.
Administration Procedures: To be used in dendritic cell culture,
not administered directly to patients.
KLH (Keyhole Limpet Hemocyanin)
Product Description: Stellar Biotechnology's KLH is a potent form
of clinical grade KLH that is manufactured by Sigma-Aldrich. It is
purified from the hemocyanin of the giant keyhole limpet, Megathura
crenulata. The denatured subunit of KLH is a glycoprotein with a
molecular weight of 400-450,000 daltons. The native form of KLH is
a dodecamer, which consists of twenty (20) subunits of KLH with a
molecular weight of 6-9000.000 daltons. In the hemocyanin, at least
50% of the KLH exists as a dodecamer and the remainder can be found
as dodecamer aggregates. Stellar Biotechnology's KLH is purified as
native molecules with high molecular weight and designated as
KLH-HMW.
Formulation and Preparation: Stellar Biotechnology's KLH is
provided in soluble form in a buffer solution that is composed of
10 mM sodium phosphate, 135 mM NaCl, 1 mM CaCl.sub.2 and 0.5 mM
MgCl.sub.2. It is provided by the manufacturer in 600 mg containers
at 5 mg/mL. It has re-vialed into single use vials at 2 mg/mL, 250
microliter/vial.
Stability and Storage: KLH-HMW is stable for at least 12 months
when stored at 2 to 8.degree. C.
Administration Procedures: KLH-HMW is used in vitro at a
concentration of 10 mcg/mL for the generation of dendritic cells.
Cells are extensively washed before administration.
TARP 27-35 (Wild Type) Peptide NSC#740703
Product Description: TARP 27-35 is a synthetic HLA-A2-restricted
9-amino acid epitope of the tumor-associated protein TARP.
Amino acid sequence:
Phenylalanine-Valine-Phenylalanine-Leucine-Arginine-Asparagine-Phenylalan-
ine-Serine-Leucine (FVFLRNFSL: SEQ ID NO: 3).
Molecular Weight: 1142.4.
Formulation and Preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 5 mL
siliconized sterile amber molded glass vial containing a sterile
white lyophilized powder. Each vial contains 1.1 mg of TARP 27-35
peptide and Mannitol.
Stability and Storage: The finished injectable dosage forms are
stored in the freezer (-70'C) for long-term storage. Intact vials
are stable for at least 6 months when stored at controlled room
temperature (15.degree. C.-30.degree. C.) or in the refrigerator
(2.degree. C.-8.degree. C.), and for at least 36 months when stored
in the freezer (-10.degree. C. to -25.degree. C. and -70.degree.
C.). The peptide vial contains no preservatives; once the peptide
vial is entered, unused peptide solution is discarded after 3
hours.
Administration Procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with
lipopolysaccharide (LPS) and IFN-.gamma.. A fraction of autologous
dendritic cells are pulsed separately with TARP 27-35 peptide.
After removing peptide-pulsing media, individual fractions of
dendritic cells are combined and concentrated down at
40.times.10.sup.6 cells/ml in infusion media (Plasma-Lyte A
containing 10% autologous heat inactivated plasma). The final
peptide-loaded, volume-reduced mature dendritic cell product is
prepared in sterile syringes for fresh administration
intradermally.
TARP 29-37-9V Peptide (Epitope-Enhanced) NSC #740704
Product Description: TARP 29-37-9V is a synthetic HLA-A2-restricted
9-amino acid epitope of the tumor associated protein TARP, with a
single amino acid substitution (valine at position 37, instead of
leucine) to increase its binding affinity and immunogenicity.
Amino acid sequence:
Phenylalanine-Leucine-Arginine-Asparagine-Phenylalanine-Serine-Leucine-Me-
thionine-Valine (FLRNFSLMV; SEQ ID NO: 4).
Molecular Weight: 1126.4.
Formulation and Preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 5 mL
siliconized sterile amber type 1 glass vial with a Teflon-lined
stopper containing 0.5 mL of a sterile clear solution. Each mL
contains 2.2 mg of TARP 29-37(37V) Peptide and 0.5 mcL of
trifluoroacetate 0.05% v/v.
Stability and Storage: The finished injectable dosage forms are
stored in the freezer (-70.degree. C.) for long-term storage.
Intact vials are stable for at least 6 months when stored at
controlled room temperature (15.degree. C.-30.degree. C.), at least
9 months when stored in the refrigerator (2.degree. C.-8.degree.
C.), and for at least 36 months when stored in the freezer
(-10.degree. C. to -25.degree. C. and -70.degree. C.). The peptide
vial contains no preservatives; once the peptide vial is entered,
unused peptide solution is discarded after 3 hours.
Administration Procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with LPS and
IFN-.gamma.. A fraction of autologous dendritic cells are pulsed
separately with TARP 29-37-9V peptide. After removing
peptide-pulsing media, individual fractions of dendritic cells are
combined and concentrated down at 40.times.10.sup.6 cells/ml in
infusion media (Plasma-Lyte A containing 10% autologous heat
inactivated plasma). The final peptide-loaded, volume-reduced
mature dendritic cell product is prepared in sterile syringes for
fresh administration intradermally.
TARP 1-20 Peptide
Amino Acid Sequence:
H-Met-Gln-Met-Phe-Pro-Pro-Ser-Pro-Leu-Phe-Phe-Phe-Leu-Gln-Leu-Leu-Lys-Gly-
n-Ser-Ser-OH Acetate (MQMFPPSPLFFFLQLLKQSS; SEQ ID NO: 5).
Formulation and preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 2 mL clear
type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl,
polytetrafluoroethylene (PTFE) "Teflon" lined stopper, and a 13 mm
aluminum flip-off seal. Vial contains 1.2 mL of a 1 mg/mL sterile
solution of TARP 1-20 Peptide (MPS-479) in dimethylsulfoxide (DMSO)
with 0.1% trifluoroacetic acid (TFA).
Storage: Peptide is stored at -70.degree. C.
Administration procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with LPS and
IFN-.gamma.. A fraction of autologous dendritic cells is pulsed
separately with TARP 1-20 peptide. After removing peptide-pulsing
media, individual fractions of dendritic cells are combined and
concentrated down at 40.times.10.sup.6 cells/ml in infusion media
(Plasma-Lyte A containing 10% autologous heat inactivated plasma).
The final peptide-loaded, volume-reduced mature dendritic cell
product is prepared in sterile syringes for fresh administration
intradermally.
TARP 11-30 Peptide
Amino Acid Sequence:
H-Phe-Phe-Leu-Gln-Leu-Leu-Lys-Gln-Ser-Ser-Arg-Arg-Leu-Glu-His-Thr-Phe-Val-
-Phe-Leu-OH Acetate (FFLQLLKQSSRRLEHTFVFL; SEQ ID NO: 6).
Formulation and preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 2 mL clear
type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl,
PTFE "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.
Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 11-30
Peptide (MPS-480) in DMSO with 0.1% TFA.
Storage: Peptide is stored at -70.degree. C.
Administration procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with LPS and
IFN-.gamma.. A fraction of autologous dendritic cells is pulsed
separately with TARP 11-30 peptide. After removing peptide-pulsing
media, individual fractions of dendritic cells are combined and
concentrated down at 40.times.10.sup.6 cells/ml in infusion media
(Plasma-Lyte A containing 10% autologous heat inactivated plasma).
The final peptide-loaded, volume-reduced mature dendritic cell
product is prepared in sterile syringes for fresh administration
intradermally.
TARP 21-40 Peptide
Amino Acid Sequence:
H-Arg-Arg-Leu-Glu-His-Thr-Phe-Val-Phe-Leu-Arg-Asn-Phe-Ser-Leu-Met-Leu-Leu-
-Arg-Gly-OH Acetate (RRLEHTFVFLRNFSLMLLRG; SEQ ID NO: 7).
Formulation and preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 2 mL clear
type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl,
PTFE "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.
Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 21-40
Peptide (MPS-481) in sterile water for injection.
Storage: Peptide is stored at -70.degree. C.
Administration procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with LPS and
IFN-.gamma.. A fraction of autologous dendritic cells is pulsed
separately with TARP 21-40 peptide. After removing peptide-pulsing
media, individual fractions of dendritic cells are combined and
concentrated down at 40.times.10.sup.6 cells/ml in infusion media
(Plasma-Lyte A containing 10% autologous heat inactivated plasma).
The final peptide-loaded, volume-reduced mature dendritic cell
product is prepared in sterile syringes for fresh administration
intradermally.
TARP 31-50 Peptide
Amino Acid Sequence:
H-Arg-Asn-Phe-Ser-Leu-Met-Leu-Leu-Arg-Gly-lle-Gly-Lys-Lys-Arg-Arg-Ala-Thr-
-Arg-Phe-OH Acetate (RNFSLMLLRGIGKKRRATRF: SEQ ID NO: 8).
Formulation and preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 2 mL clear
type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl,
PTFE "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.
Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 31-50
Peptide (MPS-482) in sterile water for injection.
Storage: Peptide is stored at -70.degree. C.
Administration procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with LPS and
IFN-.gamma.. A fraction of autologous dendritic cells is pulsed
separately with TARP 31-50 peptide. After removing peptide-pulsing
media, individual fractions of dendritic cells are combined and
concentrated down at 40.times.10.sup.6 cells/ml in infusion media
(Plasma-Lyte A containing 10% autologous heat inactivated plasma).
The final peptide-loaded, volume-reduced mature dendritic cell
product is prepared in sterile syringes for fresh administration
intradermally.
TARP 41-58 Peptide
TABLE-US-00006 Amino Acid Sequence: (IGKKRRATRFWDPRRGTP; SEQ ID NO:
9) H-Ile-Gly-Lys-Lys-Arg-Arg-Ala-Thr-Arg-
Phe-Trp-Asp-Pro-Arg-Arg-Gly-Thr-Pro- OH Acetate.
Formulation and preparation: The peptide is manufactured by NeoMPS,
Inc. (San Diego, Calif.). The peptide is vialed as a 2 mL clear
type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl,
PTFE "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.
Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 41-58
Peptide (MPS-483) in sterile water for injection.
Storage: Peptide is stored at -70.degree. C.
Administration procedures: Autologous peptide-pulsed dendritic cell
vaccines are prepared under GMP conditions from cryopreserved
patient monocytes. After thaw, the monocytes are placed into a 5
day culture with rhIL-4 and rhGM-CSF to generate immature dendritic
cells, followed by pulse with KLH and maturation with LPS and
IFN-.gamma.. A fraction of autologous dendritic cells is pulsed
separately with TARP 41-58 peptide. After removing peptide-pulsing
media, dendritic cells, individual fractions are combined and
concentrated down at 40.times.10.sup.6 cells/ml in infusion media
(Plasma-Lyte A containing 10% autologous heat inactivated plasma).
The final peptide-loaded, volume-reduced mature dendritic cell
product is prepared in sterile syringes for fresh administration
intradermally.
Detection of Anti-TARP Antibody and Cellular Responses
To determine the immunogenicity of autologous multi-epitope TARP
dendritic cell vaccination, quantitative anti-TARP antibody testing
is performed at Weeks 0, 12, 18, 24, 48, 72 and 96. Immunogenicity
is indicated by a 3-fold increase in anti-TARP antibody
concentration (measured as mcg/ml) or a 4-fold increase in antibody
dilution titers over baseline.
Vaccine-induced anti-TARP and anti-PSA antibody profiles also are
evaluated at Weeks 0, 12, 18, 24, 48, 72 and 96 by peptide
microarray.
At weeks 0, 12, 48, 24 and 48, TARP-specific cellular responses are
evaluated. CFSE proliferation, ICS, ELISPOT (IFN-.gamma., granzyme
B, perforin) and tetramer assays are performed.
Example 2
Dendritic Cell Vaccine Preparation
ME TARP Vaccine Preparation
Autologous Cell Harvest
Blood collection is by standard lymphapheresis; 15 to 18 liters of
whole blood is processed in order to collect peripheral blood
mononuclear cells (MNC) with a target number of at least
2.2.times.10.sup.9 monocytes. Lymphocytes are also cryopreserved.
Apheresis is performed using approved standard operating
procedures. Bilateral peripheral venous access is used for
apheresis whenever possible. Alternatively, a temporary femoral
central venous catheter (CVL) is placed as an outpatient, if
indicated, for collection on the day of apheresis. Prophylactic
intravenous CaCl.sub.2 and MgSO.sub.4 infusions may be administered
during apheresis to treat or prevent citrate toxicity.
Multi Epitope TARP Peptide-Pulsed Dendritic Cells
Autologous dendritic cells prepared from peripheral blood monocytes
are loaded with the following 7 different TARP-derived
peptides:
TABLE-US-00007 TARP 27-35 (SEQ ID NO: 3) TARP 29-37-9V (SEQ ID NO:
4) TARP 1-20: (SEQ ID NO: 5) MQMFPPSPLFFFLQLLKQSS TARP 11-30: (SEQ
ID NO: 6) FFLQLLKQSSRRLEHTFVFL TARP 21-40: (SEQ ID NO: 7)
RRLEHTFVFLRNFSLMLLRG TARP 31-50: (SEQ ID NO: 8)
RNFSLMLLRGIGKKRRATRF TARP 41-58: (SEQ ID NO: 9)
IGKKRRATRFWDPRRGTP
Different fractions of autologous dendritic cells are pulsed
individually with only one of these peptides and the seven
fractions are combined before administration to the patient.
Formulation and Preparation ME TARP DC Vaccine
Autologous peptide-pulsed dendritic cell vaccines are prepared
under cGMP conditions from cryopreserved patient monocytes obtained
during the original Week 0 apheresis. Autologous monocytes for
dendritic cell culture are enriched from peripheral blood MNC
apheresis collections by counter-flow elutriation, aliquoted into
at least 8 vials with .about.333.times.10.sup.6 cells/vial and
cryopreserved for future preparation of the dendritic cell
products. After thaw, the monocytes are placed into a 5 day culture
with rhIL-4 and rhGM-CSF to generate immature dendritic cells,
followed by pulse with KLH and maturation with LPS and IFN-.gamma.,
and pulsed with TARP peptide. After removing peptide-pulsing media,
dendritic cells are concentrated down at 40.times.10.sup.6 cells/ml
in infusion media (Plasma-Lyte A containing 10% autologous heat
inactivated plasma). The final peptide-loaded, volume-reduced
mature dendritic cell product is prepared in sterile syringes for
fresh administration intradermally.
Elutriated Monocyte Placebo Vaccine Preparation
Autologous Cell Harvest
Blood collection is by standard lymphapheresis; 10 to 15 liters of
whole blood is processed in order to collect peripheral blood
mononuclear cells (PBMC). Lymphocytes are also be cryopreserved.
Apheresis is performed using approved standard operating
procedures. Bilateral peripheral venous access is used for
apheresis whenever possible. Alternatively, a temporary femoral
central venous catheter (CVL) is placed as an outpatient, if
indicated, for collection on the day of apheresis. Prophylactic
intravenous CaCl.sub.2 and MgSO.sub.4 infusions may be administered
during apheresis to treat or prevent citrate toxicity.
Formulation and Preparation Elutriated Monocyte Placebo Vaccine
Autologous elutriated monocyte placebo cell vaccines are prepared
under cGMP conditions from cryopreserved patient PBMCs obtained
during the original Week 0 apheresis and aliquoted into at least 8
vials with .about.333.times.10.sup.6 cells/vial and cryopreserved
for future preparation of elutriated monocyte placebo cell
products. Elutriated monocytes are thawed the morning of scheduled
vaccine delivery. After thaw, elutriated monocytes are concentrated
down at 40.times.10.sup.6 cells/ml in infusion media (Plasma-Lyte A
containing 10% autologous heat inactivated plasma). The final,
volume-reduced elutriated monocyte product is prepared in sterile
syringes for fresh administration intradermally.
Stability and Storage
Autologous ME TARP peptide-pulsed dendritic cell vaccines are
harvested from the 5-day culture product and autologous elutriated
monocyte placebo vaccine from the single day thaw product. Both are
packaged for fresh administration on the same day. A fixed
autologous ME TARP peptide-pulsed dendritic cell or elutriated
monocyte placebo vaccine dose of 20.times.10.sup.6 viable cells/in
0.25 ml or 0.5 ml is administered immediately upon receipt in the
clinical setting. Post packaging tests indicated that the product
is stable for at least 2 hours.
In view of the many possible embodiments to which the principles of
the disclosed invention may be applied, it should be recognized
that the illustrated embodiments are only preferred examples of the
invention and should not be taken as limiting the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
SEQUENCE LISTINGS
1
10158PRTArtificial SequenceSynthetic peptide 1Met Gln Met Phe Pro
Pro Ser Pro Leu Phe Phe Phe Leu Gln Leu Leu 1 5 10 15 Lys Gln Ser
Ser Arg Arg Leu Glu His Thr Phe Val Phe Leu Arg Asn 20 25 30 Phe
Ser Leu Met Leu Leu Arg Gly Ile Gly Lys Lys Arg Arg Ala Thr 35 40
45 Arg Phe Trp Asp Pro Arg Arg Gly Thr Pro 50 55 258PRTHomo sapiens
2Met Gln Met Phe Pro Pro Ser Pro Leu Phe Phe Phe Leu Gln Leu Leu 1
5 10 15 Lys Gln Ser Ser Arg Arg Leu Glu His Thr Phe Val Phe Leu Arg
Asn 20 25 30 Phe Ser Leu Met Leu Leu Arg Tyr Ile Gly Lys Lys Arg
Arg Ala Thr 35 40 45 Arg Phe Trp Asp Pro Arg Arg Gly Thr Pro 50 55
39PRTArtificial SequenceSynthetic peptide 3Phe Val Phe Leu Arg Asn
Phe Ser Leu 1 5 49PRTArtificial SequenceSynthetic peptide 4Phe Leu
Arg Asn Phe Ser Leu Met Val 1 5 520PRTArtificial SequenceSynthetic
peptide 5Met Gln Met Phe Pro Pro Ser Pro Leu Phe Phe Phe Leu Gln
Leu Leu 1 5 10 15 Lys Gln Ser Ser 20 620PRTArtificial
SequenceSynthetic peptide 6Phe Phe Leu Gln Leu Leu Lys Gln Ser Ser
Arg Arg Leu Glu His Thr 1 5 10 15 Phe Val Phe Leu 20
720PRTArtificial SequenceSynthetic peptide 7Arg Arg Leu Glu His Thr
Phe Val Phe Leu Arg Asn Phe Ser Leu Met 1 5 10 15 Leu Leu Arg Gly
20 820PRTArtificial SequenceSynthetic peptide 8Arg Asn Phe Ser Leu
Met Leu Leu Arg Gly Ile Gly Lys Lys Arg Arg 1 5 10 15 Ala Thr Arg
Phe 20 918PRTArtificial SequenceSynthetic peptide 9Ile Gly Lys Lys
Arg Arg Ala Thr Arg Phe Trp Asp Pro Arg Arg Gly 1 5 10 15 Thr Pro
101027DNAHomo sapiens 10gggcaagagt tgggcaaaaa aatcaaggta tttggtcccg
gaacaaagct tatcattaca 60gataaacaac ttgatgcaga tgtttccccc aagcccacta
tttttcttcc ttcaattgct 120gaaacaaagc tccagaaggc tggaacatac
ctttgtcttc ttgagaaatt tttccctgat 180gttattaaga tacattggca
agaaaagaag agcaacacga ttctgggatc ccaggagggg 240aacaccatga
agactaacga cacatacatg aaatttagct ggttaacggt gccagaaaag
300tcactggaca aagaacacag atgtatcgtc agacatgaga ataataaaaa
cggagttgat 360caagaaatta tctttcctcc aataaagacg gatgtcatca
caatggatcc caaagacaat 420tgttcaaaag atgcaaatga tacactactg
ctgcagctca caaacacctc tgcatattac 480atgtacctcc tcctgctcct
caagagtgtg gtctattttg ccatcatcac ctgctgtctg 540cttagaagaa
cggctttctg ctgcaatgga gagaaatcat aacagacggt ggcacaagga
600ggccatcttt tcctcatcgg ttattgtccc tagaagcgtc ttctgaggat
ctagttgggc 660tttctttctg ggtttgggcc atttcagttc tcatgtgtgt
actattctat cattattgta 720taacggtttt caaaccagtg ggcacacaga
gaacctcact ctgtaataac aatgaggaat 780agccacggcg atctccagca
ccaatctctc catgttttcc acagctcctc cagccaaccc 840aaatagcgcc
tgctatagtg tagacatcct gcggcttcta gccttgtccc tctcttagtg
900ttctttaatc agataactgc ctggaagcct ttcattttac acgccctgaa
gcagtcttct 960ttgctagttg aattatgtgg tgtgtttttc cgtaataagc
aaaataaatt taaaaaaatg 1020aaaagtt 1027
* * * * *