U.S. patent application number 11/391632 was filed with the patent office on 2006-10-05 for mage-a3/hpv 16 peptide vaccines for head and neck cancer.
This patent application is currently assigned to UNIVERSITY OF MARYLAND, BALTIMORE. Invention is credited to Esteban Celis, Scott E. Strome.
Application Number | 20060222656 11/391632 |
Document ID | / |
Family ID | 37070769 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060222656 |
Kind Code |
A1 |
Strome; Scott E. ; et
al. |
October 5, 2006 |
MAGE-A3/HPV 16 peptide vaccines for head and neck cancer
Abstract
The present invention relates to Trojan antigens, and
immunogenic compositions comprising the Trojan antigens. The
present invention also relates to methods of generating an immune
response in a subject using the Trojan antigens or immunogenic
compositions. The present invention further relates to methods of
treating squamous cell carcinoma of the head and neck (SCCHN) using
the Trojan antigens and immunogenic compositions of the present
invention.
Inventors: |
Strome; Scott E.;
(Reisterstown, MD) ; Celis; Esteban; (Tampa,
FL) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
UNIVERSITY OF MARYLAND,
BALTIMORE
|
Family ID: |
37070769 |
Appl. No.: |
11/391632 |
Filed: |
March 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60667060 |
Apr 1, 2005 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
514/19.8; 514/21.3; 530/324 |
Current CPC
Class: |
C12N 2740/16322
20130101; A61K 2039/6075 20130101; A61K 2039/645 20130101; C07K
2319/01 20130101; A61P 35/00 20180101; A61P 37/04 20180101; A61K
39/0011 20130101; A61K 39/12 20130101; A61P 31/00 20180101; C07K
2319/50 20130101; C07K 14/4748 20130101; C12N 2710/20022 20130101;
C12N 2710/20034 20130101; C07K 14/005 20130101; A61K 2039/5154
20130101 |
Class at
Publication: |
424/185.1 ;
514/012; 530/324 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/005 20060101 C07K014/005 |
Claims
1. A isolated polypeptide comprising amino acids 1-35 of SEQ ID
NO:15.
2. A isolated polypeptide comprising amino acids 1-47 of SEQ ID
NO:17.
3. A isolated polypeptide comprising amino acids 1-21 of SEQ ID
NO:19.
4. A isolated polypeptide comprising amino acids 1-43 of SEQ ID
NO:22.
5. The isolated polypeptide of claim 3, wherein X cysteine or
aminobutyric acid.
6. The isolated polypeptide of claim 4, wherein X.sub.6, X.sub.30,
X.sub.31, and X.sub.33 are each independently cysteine or
aminobutyric acid.
7. An immunogenic composition comprising a polypeptide of claim 2
or 4, and a pharmaceutically acceptable carrier, diluent or
adjuvant.
8. An immunogenic composition comprising the polypeptide of claims
2 and 4, and a pharmaceutically acceptable carrier, diluent or
adjuvant.
9. A method of generating an immune response in a subject
comprising administering a polypeptide comprising amino acids 1-47
of SEQ ID NO:17 to a subject in an amount sufficient to induce an
immune response in said subject.
10. A method of generating an immune response in a subject
comprising administering a polypeptide comprising amino acids 1-43
of SEQ ID NO:22, wherein X.sub.6, X.sub.30, X.sub.31 and X.sub.33
are each independently cysteine or aminobutyric acid, to a subject
in an amount sufficient to induce an immune response in said
subject.
11. A method of generating an immune response in a subject
comprising administering (a) a polypeptide comprising amino acids
1-47 of SEQ ID NO:17 and (b) a polypeptide comprising amino acids
1-43 of SEQ ID NO:22, wherein X.sub.6, X.sub.30, X.sub.31 and
X.sub.33 are each independently cysteine or aminobutyric acid, to a
subject in an amount sufficient to induce an immune response in
said subject.
12. The method of claim 9 or 10, wherein said polypeptide is
administered in conjunction with a pharmaceutically acceptable
carrier, diluent or adjuvant.
13. The method of claim 11, wherein said polypeptides are
administered in conjunction with a pharmaceutically acceptable
carrier, diluent or adjuvant.
14. The method of claim 9 or 10, wherein said polypeptide is
administered in an amount of between about 100 ug and about 1.5
mg.
15. The method of claim 11, wherein said polypeptides are
administered in a combined amount of between about 100 ug and about
1.5 mg.
16. The method of claim 9 or 10, wherein said polypeptide is
administered in an amount of about 1 mg.
17. The method of claim 11, wherein said polypeptides are
administered in a combined amount of about 1 mg.
18. The method of claim 9 or 10, wherein said polypeptide is
co-administered with montanide, in an amount of between about 0.5
and 1.5 mL, and GM-CSF, in an amount of between about 50 and 150
ug/m.sup.2.
19. The method of claim 11, wherein said polypeptides are
co-administered with montanide, in an amount of between about 0.5
and 1.5 mL, and GM-CSF, in an amount of between about 50 and 150
ug/m.sup.2.
20. A method of treating squamous cell carcinoma of the head and
neck (SCCHN) comprising administering to a subject in need of such
treatment a therapeutically-effective amount of a polypeptide
comprising amino acids 1-47 of SEQ ID NO:17.
21. A method of treating SCCHN comprising administering to a
subject in need of such treatment a therapeutically-effective
amount of a polypeptide comprising amino acids 1-43 of SEQ ID
NO:22, wherein X.sub.6, X.sub.30, X.sub.31 and X.sub.33 are each
independently cysteine or aminobutyric acid.
22. A method of treating SCCHN comprising administering to a
subject in need of such treatment a therapeutically-effective
amount of (a) a polypeptide comprising amino acids 1-47 of SEQ ID
NO:17 and (b) a polypeptide comprising amino acids 1-43 of SEQ ID
NO:22, wherein X.sub.6, X.sub.30, X.sub.31 and X.sub.33 are each
independently cysteine or aminobutyric acid.
23. The method of claim 20 or 21, wherein said polypeptide is
co-administered with a pharmaceutically acceptable carrier, diluent
or adjuvant.
24. The method of claim 22, wherein said polypeptides are
co-administered with a pharmaceutically acceptable carrier, diluent
or adjuvant.
25. The method of claim 20 or 21, wherein said polypeptide is
administered in an amount between about 100 ug and about 1.5
mg.
26. The method of claim 22, wherein said polypeptides are
administered in a combined amount of between about 100 ug and about
1.5 mg.
27. The method of claim 20 or 21, wherein said polypeptide is
administered in an amount of about 1 mg.
28. The method of claim 22, wherein said polypeptides are
administered in a combined amount of about 1 mg.
29. The method of claim 20 or 21, wherein said polypeptide is
co-administered with montanide, in an amount of between about 0.5
and 1.5 mL, and GM-CSF, in an amount of between about 50 and 150
ug/m.sup.2.
30. The method of claim 22, wherein said polypeptides are
co-administered with montanide, in an amount of between about 0.5
and 1.5 mL, and GM-CSF, in an amount of between about 50 and 150
ug/m.sup.2.
31. An expression vector comprising an isolated polynucleotide
molecule encoding amino acids 1-35 of SEQ ID NO:15.
32. An expression vector comprising an isolated polynucleotide
molecule encoding amino acids 1-47 of SEQ ID NO:17.
33. An expression vector comprising an isolated polynucleotide
molecule encoding amino acids 1-21 of SEQ ID NO:19.
34. An expression vector comprising an isolated polynucleotide
molecule encoding amino acids 1-43 of SEQ ID NO:22.
35. A host cell comprising an expression vector of claim 31.
36. A host cell comprising an expression vector of claim 32.
37. A host cell comprising an expression vector of claim 33.
38. A host cell comprising an expression vector of claim 34.
39. A method of preparing a polypeptide comprising amino acids 1-35
of SEQ ID NO:15, comprising culturing the host cell of claim 35
under conditions promoting expression of said polypeptide and
recovering said polypeptide from the cell culture.
40. A method of preparing a polypeptide comprising amino acids 1-47
of SEQ ID NO:17, comprising culturing the host cell of claim 36
under conditions promoting expression of said polypeptide and
recovering said polypeptide from the cell culture.
41. A method of preparing a polypeptide comprising amino acids 1-21
of SEQ ID NO:19, comprising culturing the host cell of claim 37
under conditions promoting expression of said polypeptide and
recovering said polypeptide from the cell culture.
42. A method of preparing a polypeptide comprising amino acids 1-43
of SEQ ID NO:22, comprising culturing the host cell of claim 38
under conditions promoting expression of said polypeptide and
recovering said polypeptide from the cell culture.
Description
[0001] The present application claims benefit of U.S. provisional
application No. 60/667,060, filed Apr. 1, 2005, incorporated herein
by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Squamous cell carcinoma of the head and neck (SCCHN) effects
43,000 individuals in the United States annually with an estimated
5-year overall survival of 50% (R. M. Byers, Dr. Martin: How are we
doing in 2000? Archives of Otolaryngology-Head and Neck Surgery
127:759-765 (2001)). For some patients who develop local or distant
metastases following primary therapy, surgical salvage is a viable
therapeutic option. The remainder of individuals is forced to
choose between palliative chemotherapy and supportive care. In
order to improve both survival and quality of life for patients
with unresectable disease, new therapeutic alternatives are
mandated.
[0003] One treatment option is the use of T cell-specific
immunotherapy to stimulate a patient's anti-tumor immune response.
Several T cell based strategies have demonstrated clinical efficacy
for the treatment of unresectable tumors of diverse histologic
types (Kugler et al. Regression of human metastatic renal cell
carcinoma after vaccination with tumor cell=dendritic cell hybrids.
Nature Medicine 6(3):332-6 (2000); Nestle et al. Vaccination of
melanoma patients with peptide- or tumor lysate-pulsed dendritic
cells. Nature Medicine 4(3):328-32 (1998); Rosenberg et al.
Immunologic and therapeutic evaluation of a synthetic peptide
vaccine for the treatment of patients with metastatic melanoma.
Nature Medicine 4(3):321-327 (1998)). However, the majority of
trials have failed to demonstrate any therapeutic benefit (Chang et
al. A phase I trial of tumor lysate-pulsed dendritic cells in the
treatment of advanced cancer. Clinical Cancer Research
8(4):1021-1032 (2002)). The causes of these failures are
multi-factorial, but are likely related to well characterized
immunologic defects within the target population, including
aberrant antigen processing and presentation, which restrict T cell
function. Specifically, tumor bearing patients have demonstrable
defects in antigen presentation, both at the tumor cell and
professional antigen presenting cell (APC) levels, e.g., down
regulation of TAP and HLA molecules, which, in some cases, can be
overcome by the administration of interferon (Seliger et al.
Antigen-processing machinery breakdown and tumor growth. Immunology
Today 21(9):455-64 (2000); Marincola et al. Escape of human solid
tumors from T-cell recognition: Molecular mechanisms and functional
significance, Advanced Immunology 74:181-273 (2000)). Additionally,
in cancer patients T cells are found to be tolerized or improperly
activated which might be caused by down regulation of the zeta
chain of the T cell receptor (TCR) and P56LCK signaling molecule
(Mizoguchi et al. Alterations in signal transduction molecules in T
lymphocytes from tumor-bearing mice. Science 258(5089):1795-8
(1992)).
[0004] Classic experiments by Gross in the 1940s created the
foundation for the field of tumor immunology, demonstrating that
C3H mice capable of rejecting a syngeneic murine sarcoma developed
protective immunity to subsequent injections of the same tumor, but
not to a spontaneously arising murine mastocytoma (L. Gross,
Intradermal immunization of C3H mice against a sarcoma that
originated in an animal of the same line. Cancer Res. 326-333
(1943)). This immune response is directed against tumor specific
target antigens and is cellular in nature as lymphocytes, but not
serum, from previously immunized animals are effective in lysing
tumor (Prehn et al. Immunity to methylcolanthrene-induced sarcomas.
Journal of the National Cancer Institute 6:769-778 (1957).
[0005] It is now clear that tumor antigens are presented in the
context of specific class I and class II HLA molecules. Class I
presentation, in the presence of appropriate costimulation, is
thought to stimulate a cytolytic CD8+ T cell response, while
antigen presentation in the context of class II molecules is
postulated to stimulate a CD4+ helper T cell response (Townsend et
al., Antigen recognition by class I-restricted T lymphocytes.
Annual Review of Immunology 7:601-624 (1989)). Tumors can evade the
immune response by manipulating these antigen presentation
pathways. Specifically, direct tumor presentation of antigen in the
absence of costimulation results in T cell anergy (D. M. Pardoll,
Cancer vaccines. Nature Medicine 4(5 supp):525-531 (1998)).
Additionally, down regulation of either HLA molecules on the tumor
surface or tumor antigen expression limits the efficacy of
antigen-specific cytotoxic T cells (Seliger et al.
Antigen-processing machinery breakdown and tumor growth. Immunology
Today 21(9):455-64 (2000)). Finally, tumors can upregulate
non-classical HLA molecules, e.g., HLA G, which are thought to
suppress T cell anti-tumor immunity (Rouas-Freiss et al. HLA-G
promotes immune tolerance. Journal of Biological Regulators &
Homeostatic Agents 14(2):93-8 (2000)). These molecular methods of
protection provide clear evidence that CTLs induce selective
pressure on tumors and can potentially be harnessed with
therapeutic intent.
[0006] Several strategies have been employed to prime the
anti-tumor T cell response. A major advance in the field of
immunotherapy is the clinical application of a class of
professional antigen presenting cells (APC), termed dendritic cells
(DC), into clinical trials. Dendritic cells are thought to
stimulate the T cell anti-tumor response by the presentation of
tumor associated antigens in the context of class I and II MHC,
co-stimulatory molecules, and appropriate chemokines/cytokines
(Gilboa et al. Immunotherapy of cancer with dendritic-cell-based
vaccines. Cancer Immunology, Immunotherapy 46(2):82-7 (1998)).
Presentation of putative tumor antigens by DC is postulated to
effectively overcome tumor induced tolerance (Dallal et al. The
dendritic cell and human cancer vaccines, Current Opinion in
Immunology 12(5):583-8 (2000)). This suggests that vaccine
approaches using DC primed with tumor specific antigens will be an
effective means to stimulate tumor specific immunity.
[0007] Recent evidence suggests that DC primed with
tumor-associated antigens in the form of peptides, tumor lysates,
or tumor RNA are capable of mediating a potent anti-tumor immune
response (Nair et al. Regression of tumors in mice vaccinated with
professional antigen-presenting cells pulsed with tumor extracts.
International Journal of Cancer 70(6):706-15 (1997)). While ex vivo
maturation of DC is one method of priming DC, such maturation is
expensive and can lack reproducibility. Specifically, because large
numbers of DC are required for vaccine preparation, 1-2
leukophoreses are often required.
[0008] One means to potentially eliminate problems associated with
ex vivo maturation is through the use of DC stimulation in vivo.
Monocytes cultured in appropriate concentrations of IL-4 and GM-CSF
transform into immature DC (Banchereau et al. Dendritic cells and
the control of immunity. Nature 392(6673):245-52 (1998); R. M.
Steinman, The dendritic cell system and its role in immunogenicity.
Annual Review of Immunology 9:271-96 (1991)). GM-CSF is the
critical component for stimulating DC maturation, phagocytosis,
migration, and HLA class II expression (J. O. Armitage. Emerging
applications of recombinant human granulocyte-macrophage
colony-stimulating factor. Blood 92(12):4491-508 (1998)). The
majority of clinical trials have cultured DC precursors in
recombinant GM-CSF, with subsequent administration back to the host
as part of a tumor vaccine. Recent data from Disis et al.
demonstrates that the presentation of tumor peptides in a rat model
is enhanced by subcutaneous or intradermal administration of GM-CSF
(Disis et al. Granulocyte-macrophage colony-stimulating factor: an
effective adjuvant for protein and peptide-based vaccines. Blood
88(1):202-10 (1996)). The concept of in vivo administration of
GM-CSF in combination with an autologous vaccine has been
clinically evaluated in patients with advanced melanoma.
Specifically, using an autologous melanoma vaccine in combination
with GM-CSF and BCG, Leong et al. demonstrated a 10% complete
response rate with an equal number of partial responders in a
cohort of 20 patients (Leong et al. Recombinant human granulocyte
macrophage-colony stimulating factor (rhGM-CSF) and autologous
melanoma vaccine mediate tumor regression in patients with
metastatic melanoma. Journal of Immunotherapy 22(2): 166-74
(1999)). Using a modification of this approach, Soiffer et al.
demonstrated that a vaccine composed of irradiated melanoma cells
engineered to secrete GM-CSF, stimulated T cell mediated tumor
destruction in 11/16 patients (Soiffer et al. Vaccination with
irradiated autologous melanoma cells engineered to secrete human
granulocyte-macrophage colony-stimulating factor generates potent
antitumor immunity in patients with metastatic melanoma. PNAS
95(22):13141-13146 (1998)). Finally, Bendandi et al. demonstrated
molecular remission of residual lymphoma in 8/11 patients after
treatment with an idiotype protein vaccine in combination with
either 100 or 500 ug/M2 of GM-CSF (Bendandi et al. Complete
molecular remissions induced by patient-specific vaccination plus
granulocyte-monocyte colony-stimulating factor against lymphoma.
Nature Medicine 5(10):1171-7 (1999)). Importantly, in this trial
the vaccine was given once per month for four cycles with a booster
given 2 months after the final cycle. GM-CSF was given at the time
of vaccination and daily, for three additional doses. These data
suggest that in vivo administration of GM-CSF may supplant the need
for in vitro culture of DC precursors.
[0009] Additional problems with whole tumor based approaches
include the potential for tumor contamination, small cell number,
and limited ability to monitor the immune response. An alternative
approach is the use of tumor associated synthetic antigens for
immunologic priming. Peptide-based strategies for DC priming enable
prior characterization of the immunologic stimulant, facilitating
subsequent analysis of the anti-tumor immune response. Because
specific peptides are ubiquitous in tumors of the same histologic
type, identical peptide vaccines may be employed in allogeneic
hosts bearing the same tumor histology. Additionally, the use of
single peptides for immunization limits the potential induction of
undesired autoimmunity (Nestle et al. Vaccination of melanoma
patients with peptide- or tumor lysate-pulsed dendritic cells.
Nature Medicine 4(3):328-32 (1998); Tsai et al. In vitro
immunization and expansion of antigen-specific cytotoxic T
lymphocytes for adoptive immunotherapy using peptide-pulsed
dendritic cells. Critical Reviews in Immunology 18(1-2):65-75
(1998); Tsai et al. Identification of subdominant CTL epitopes of
the GP100 melanoma-associated tumor antigen by primary in vitro
immunization with peptide-pulsed dendritic cells. Journal of
Immunology 158(4):1796-1802 (1997)). Finally, recent developments
in the use of soluble MHC Class I peptide tetramers/dimers and
Elispot technology have enabled rapid characterization of epitope
specific CTL response (Altman et al. Phenotypic analysis of
antigen-specific T lymphocytes. Science 274(5284):94-96 (1996); V.
Cerundolo. Use of major histocompatibility complex class I
tetramers to monitor tumor-specific cytotoxic T lymphocyte response
in melanoma patients. Cancer Chemotherapy & Pharmacology
46(Suppl):S83-5 (2000)). The primary limitations to peptide-based
vaccine strategies are haplotype restriction; potential for
degradation; the lack of identifiable putative tumor antigens
recognized to induce a CTL response; the potential failure of the
efferent arm of the immune response if smaller numbers of peptides
are employed; and uncertainty regarding which peptides, used alone
or in combination, are the most immunogenic (Nair et al. Regression
of tumors in mice vaccinated with professional antigen-presenting
cells pulsed with tumor extracts. International Journal of Cancer
70(6):706-15 (1997); Amoscato et al. Rapid extracellular
degradation of synthetic class I peptides by human dendritic cells.
Journal of Immunology 161(8):4023-32 (1998)).
[0010] The optimal antigenic target is derived from a protein which
is essential for cell survival, is expressed on all tumor cells, is
tumor specific, is a surface protein, and is not expressed in the
thymus nor during fetal development. No protein identified to date
satisfies all of these criteria with regard to SCCHN tumors.
However, several proteins have been identified with peptide
epitopes capable of stimulating antigen specific CTL against SCCHN
including SART1, SART 3, CASP8, and SCCA 1 (Hamada et al. Molecular
cloning of human squamous cell carcinoma antigen 1 gene and
characterization of its promoter, Biochimica et Biophysica Acta
1518(1-2):124-31 (2001); Nakao et al. Identification of a gene
coding for a new squamous cell carcinoma antigen recognized by the
CTL. Journal of Immunology 164(5):2565-74 (2000); Shichijo et al. A
gene encoding antigenic peptides of human squamous cell carcinoma
recognized by cytotoxic T lymphocytes. J. of Exp. Med.
187(3):277-88 (1998); Yang et al. Identification of a gene coding
for a protein possessing shared tumor epitopes capable of inducing
HLA-A24-restricted cytotoxic T lymphocytes in cancer patients.
Cancer Research 59(16):4056-63 (1999)). The major limitations to
clinical application of these peptide epitopes are both their
limited prevalence in SCCHN and putative epitopes restricted by HLA
types with low population specific frequencies. For example, while
SART-1 is expressed in the majority of SCCHN, the defined peptide
epitopes are HLA26 restricted, limiting potential therapeutic
application (Shichijo et al. A gene encoding antigenic peptides of
human squamous cell carcinoma recognized by cytotoxic T
lymphocytes. J. of Exp. Med. 187(3):277-88 (1998)). In order to
increase therapeutic application, proteins should optimally be
expressed in the majority of SCCHN, with defined epitopes
representative of common HLA haplotypes.
[0011] One attractive candidate antigenic target for use in
treating SCCHN is the MAGE-A3 differentiation antigen (SEQ ID NO:
27) initially identified in the human MZ2E melanoma (van der
Bruggen et al. A gene encoding an antigen recognized by cytolytic T
lymphocytes on a human melanoma. Science 254(5038): 1643-7 (1991)).
This protein is a member of the cancer testis family and is
expressed on tumors of diverse histologic types. MAGE-A3 has high
potential utility for the immunotherapy of SCCHN based on its tumor
specificity, high percent expression, and the existence of
previously defined epitopes. A recent report demonstrated the
presence of MAGE-A3 in 44.4% of freshly isolated SCCHN by PCR and
in 27% of specimens by immunohistochemistry (Kienstra et al.
Identification of NY-ESO-1, MAGE-1, and MAGE-3 in head and neck
squamous cell carcinoma. Head and Neck 25(6):457-463 (2003)).
Additionally, HLA-A2 epitopes have been identified and clinically
evaluated for the treatment of gastrointestinal malignancies and
melanomas. Specifically, Sadanaga et al. utilized DC pulsed with
the FLWGPRALV peptide (SEQ ID NO:1), restricted to the HLA-A2
epitope (van der Bruggen et al. A peptide encoded by human gene
MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that
recognize tumor cells expressing MAGE-3. European Journal of
Immunology 24(12):3038-43 (1994)), and demonstrated the induction
of CTL in 2/5 patients, with 2/6 patients enjoying a mixed clinical
response (Sadanaga et al. Dendritic cell vaccination with MAGE
peptide is a novel therapeutic approach for gastrointestinal
carcinomas. Clinical Cancer Research 7(8):2277-2284 (2001)). In a
similar study in patients with metastatic melanoma, patients were
vaccinated with PBMC pulsed with either MAGE-A3 or MelanA peptides
in combination with IL-12. In eight patients who demonstrated an
increased immune response, there was one complete response, one
partial response, one minor response, and two mixed responses.
Interestingly, in the mixed responders, tumor specimens that did
not respond to treatment did not express the antigen used for
vaccination (Gajewski et al. Immunization of HLA-A2+ melanoma
patients with MAGE-3 or MelanA peptide-pulsed autologous peripheral
blood mononuclear cells plus recombinant human interleukin 12.
Clinical Cancer Research 7(3 Suppl):895s-901s (2001)).
[0012] Recently, a new peptide epitope of MAGE-A3, KVAELVHFL (SEQ
ID NO:2), has been defined (Kawashima et al. The multi-epitope
approach for immunotherapy for cancer: identification of several
CTL epitopes from various tumor-associated antigens expressed on
solid epithelial tumors. Human Immunology 59(1):1-14 (1998)).
Dendritic cells pulsed with this peptide stimulate naive CTL to
lyse MAGE-A3 positive tumors with an HLA-A2.1 phenotype. Although
this peptide has not been clinically evaluated, studies suggest
that it is capable of stimulating a higher percentage of tumor
reactive CTL than FLWGPRALV.
[0013] In addition to class I epitopes, immunogenic HLA-DR
restricted class II epitopes have also been defined for MAGE-A3.
Specifically, Chaux et al. have identified an HLA-DR13
MAGE-A3.sub.114-127 epitope AELVHFLLLKYRAR (SEQ ID NO:3),
recognized by epitope specific HTL clones (Chaux et al.
Identification of MAGE-3 epitopes presented by HLA-DR molecules to
CD4(+) T lymphocytes. Journal of Experimental Medicine
189(5):767-778 1999)). Similarly, Manici et al. used a
bioinformatics based approach to characterize 3 MAGE-A3 epitopes
for HLA-DR11, MAGE-A3.sub.281-295, MAGE-A3.sub.141-155, and
MAGE-A3.sub.146-160 T cells stimulated with MAGE-A3.sub.281-295,
TSYVKVLHHMVKISG (SEQ ID NO:4), were capable of lysing
HLA-DR11/MAGE-A3 positive melanomas (Manici et al. Melanoma cells
present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association
with histocompatibility leukocyte antigen DR11. Journal of
Experimental Medicine 189(5):871-876 (1999)). Additional recent
evaluations by the present inventors demonstrated
MAGE-A3.sub.146-160 (VIFSKASSSLQL; SEQ ID NO:5) can stimulate
T-helper cells restricted by both HLA-DR4 and HLA-DR7.
MAGE-A3.sub.146-160 is naturally processed, as peptide stimulated T
cells react with DC primed with whole tumor preparations (Kobayashi
et al. Tumor-reactive T helper lymphocytes recognize a promiscuous
MAGE-A3 epitope presented by various major histocompatibility
complex class II alleles. Cancer Research 61(12):4773-8 (2001)).
Based on these two studies, it is clear that MAGE-A3.sub.146-160 is
naturally processed peptide epitope and that it is promiscuous for
multiple HLA-DR epitopes, making it an ideal candidate for
therapeutic application.
[0014] A second attractive candidate for peptide-based
immunotherapy is the human papilloma virus (HPV) 16 E7 nuclear
protein (SEQ ID NO:28). HPV 16 E7 protein is a tumor rejection
antigen, which is postulated to play an integral role in the
development of carcinoma of the uterine cervix. Recent studies by
the present inventors, as well as others, have identified HPV 16 as
an independent risk factor for oropharyngeal SCC (Strome et al.
Squamous Cell Carcinoma of the Tonsils: A Molecular Analysis of HPV
Associations, Clinical Cancer Research 18:1093-1100 (2002);
Gillison et al. Evidence for a casual association between human
papillomavirus and a subset of head and neck cancers. Journal of
the National Cancer Institute 92:709-720 (2000); Gillison et al.
Human papillomavirus in head and neck squamous cell carcinoma: are
some head and neck cancers a sexually transmitted disease? Current
Opinion in Oncology 11:191-199 (1999)). While a cause effect
relationship remains to be established, several in vitro studies
suggest that continued expression of the nuclear E6 and E7 proteins
is likely required for malignant transformation of infected cells
(Crook et al. Continued expression of HPV-16 E7 protein is required
for maintenance of the transformed phenotype of cells
co-transformed by HPV-16 plus EJ-ras. EMBO Journal 8(2):513-9
(1989); Munger et al. The E6 and E7 genes of the human
papillomavirus type 16 together are necessary and sufficient for
transformation of primary human keratinocytes. Journal of Virology
63(10):4417-21 (1989)). Because of its association with
malignancies of diverse histologic types, development of HPV-based
peptide immunotherapy platforms can potentially impact the
treatment of multiple disease entities.
[0015] Several groups have now identified HLA-A2 and HLA-DR
restricted antigenic epitopes for the HPV 16 E7 nuclear protein
(Feltkamp et al. Vaccination with cytotoxic T lymphocyte
epitope-containing peptide protects against a tumor induced by
human papillomavirus type 16-transformed cells. European Journal of
Immunology 23(9):2242-2249 (1993); Feltkamp et al. Cytotoxic T
lymphocytes raised against a subdominant epitope offered as a
synthetic peptide eradicate human papillomavirus type 16-induced
tumors. European Journal of Immunology 25(9):2638-42 (1995); Chen
et al. Human papillomavirus type 16 nucleoprotein E7 is a tumor
rejection antigen. PNAS USA 88(1):110-4 (1991); Nijman et al.
Characterization of cytotoxic T lymphocyte epitopes of a
self-protein, p53, and a non-self-protein, influenza matrix:
relationship between major histocompatibility complex peptide
binding affinity and immune responsiveness to peptides. Journal of
Immunotherapy 14(2):121-6 (1993)). Two of these epitopes, E7 12-20
(MLDLQPETT; SEQ ID NO:6) and E7 86-93 (TLGIVCPI; SEQ ID NO:7), have
been evaluated in phase I trials for cervical carcinoma. Treatment
of 18 women who were HLA-A2 positive with high-grade cervical
intraepithelial neoplasia with this regimen resulted in 3 complete
responses and 6 partial responses (Nijman et al. Characterization
of cytotoxic T lymphocyte epitopes of a self-protein, p53, and a
non-self-protein, influenza matrix: relationship between major
histocompatibility complex peptide binding affinity and immune
responsiveness to peptides. Journal of Immunotherapy 14(2):121-6
(1993); Muderspach et al. A phase I trial of a human papillomavirus
(HPV) peptide vaccine for women with high-grade cervical and vulvar
intraepithelial neoplasia who are HPV 16 positive. Clin Cancer Res
6(9):3406-16 (2000)). Additionally, Van der Burg et al. have
recently defined an HPV 16 E7 helper epitope
(PAGQAEPDRAHYNIVTFCCKCD; SEQ ID NO:8) which effectively stimulates
CD4 responses in patients with HPV 16 positive cervical lesions
(van der Burg et al. Natural T-helper immunity against human
papillomavirus type 16 (HPV16) E7-derived peptide epitopes in
patients with HPV16-positive cervical lesions: identification of 3
human leukocyte antigen class II-restricted epitopes. International
Journal of Cancer 91(5):612-8 (2001)).
[0016] Potential pitfalls in the development of peptide-based
immunotherapy for SCCHN including: 1) peptide-induced tolerance, 2)
synthetic peptide degradation, 3) limited antigenic repertoire, and
4) inadequate tools for evaluating treatment response. It is now
clear that depending on the dose and timing of drug delivery, the
same peptide can induce either antigen specific CTL or T cell
deletion (Zinkernagel et al. Antigen localisation regulates immune
responses in a dose- and time-dependent fashion: a geographical
view of immune reactivity. Immunological Reviews 156:199-209
(1997); Mullbacher et al. In vivo administration of major
histocompatibility complex class I-specific peptides from influenza
virus induces specific cytotoxic T cell hyporesponsiveness.
European Journal of Immunology 23(10):2526-31 (1993); Gallimore et
al. Hierarchies of antigen-specific cytotoxic T-cell responses.
Immunological Reviews 164:29-36 (1998); Aichele et al. T cell
priming versus T cell tolerance induced by synthetic peptides.
Journal of Experimental Medicine 182(1):261-6, 1995)). The primary
factors in determining the type of peptide-induced T cell response
appear to be largely pharmacokinetic in nature (Weijzen et al.
Pharmacokinetic differences between a T cell-tolerizing and a T
cell-activating peptide. J. Immunol 166(12):7151-7 (2001)).
Specifically, peptides which achieve high initial tissue
concentrations followed by rapid elimination tend to induce T cell
deletion. Importantly, recent studies by the present inventors have
clearly demonstrated that systemic peptide administration,
recognized to induce T cell deletion, is preceded by T cell
proliferation. In contrast, peptides that achieve gradual tissue
uptake and maintain their presence preferentially induce a CTL
response. In order to limit the potential for rapid systemic
antigen exposure in this study, an approved human adjuvant
(Montanide ISA 51), which is similar to incomplete Freunds adjuvant
(IFA), may be used to regulate the temporal aspects of peptide
release (Aichele et al. T cell priming versus T cell tolerance
induced by synthetic peptides. Journal of Experimental Medicine
182(1):261-6, 1995)).
[0017] The second potential problem with synthetic peptide-based
immunotherapy is the potential for proteolysis. A recent report by
Amoscato et al. has clearly demonstrated that DC induces peptide
degradation through both endo- and ectoproteolysis. Ectocellular DC
mediated proteolysis is primarily mediated through CD13, a molecule
which appears to play a physiologic role in DC migration and
extracellular antigen processing (Amoscato et al. Rapid
extracellular degradation of synthetic class I peptides by human
dendritic cells. Journal of Immunology 161(8):4023-32 (1998)).
Several strategies have been designed to overcome the physiologic
degradation of synthetic peptides including N and C terminal
modifications and dose increases (Amoscato et al. Rapid
extracellular degradation of synthetic class I peptides by human
dendritic cells. Journal of Immunology 161(8):4023-32 (1998)).
While modification of the amino terminus has been demonstrated to
reduce degradation and enhance presentation of a class II epitope,
there are concerns that peptide modification can alter HLA binding
(Dong et al. Modification of the amino terminus of a class II
epitope confers resistance to degradation by CD13 on dendritic
cells and enhances presentation to T cells. Journal of Immunology
164(1):129-35 (2000)). Additionally, as previously mentioned, while
increased doses may enhance peptide availability for presentation,
in specific settings, alteration of peptide pharmacokinetics can
induce T cell deletion.
[0018] An alternative means to overcome the problem of proteolysis
is through the construction of long "Trojan antigens." The present
inventors have recently demonstrated that large synthetic peptides,
up to 50 amino acids in length, which contain multiple epitopes
linked to a translocating region of HIV TAT (RKKRRQRRR; SEQ ID
NO:9) can be internalized and processed. Additionally, these
peptides appear to be highly resistant to proteolysis and do not
require proteosomal processing and transport by TAP, since they
penetrate directly to the ER and Golgi where they form peptide/MHC
complexes (Lu et al. TAP-independent presentation of CTL epitopes
by Trojan antigens. Journal of Immunology 166(12):7063-71 (2001)).
The present inventors have also established that multiple T cell
epitopes can be joined together using furin-cleavable linkers
(RVKR; SEQ ID NO:10), which allow the release of the individual
epitopes in the Golgi, where the furin endopeptidase resides.
[0019] The third potential limiting factor for peptide-based
immunotherapy is related to a defined antigenic repertoire that is
HLA restricted. This factor, inherent to all peptide-based
approaches, restricts patient access. Additionally, because
individual peptides only have the potential to induce epitope
specific CTL, the vast majority of potential tumor antigens are not
targeted. In this setting, tumor down regulation of individual
antigens or HLA epitopes promotes immune evasion. Recent evidence,
however, suggests that this problem of epitope restriction may not
be as physiologically important as was previously postulated.
Specifically, it has now been clearly demonstrated that a T cell
response induced against one epitope can stimulate CTL response to
other target epitopes through a mechanism termed epitope-spreading
(Vanderlugt et al. Epitope spreading in immune-mediated diseases:
implications for immunotherapy. Nature Reviews. Immunology
2(2):85-95 (2002)). Using an experimental autoimmune encephalitis
model, Vanderlugt et al. have demonstrated that disease progression
is associated with the development of epitope specific helper T
cells which are distinct from those initiating the disease.
Transfer of secondary CD4 cells to naive mice induces the disease
phenotype and the disease is abrogated by blocking the secondary T
cell response even though the primary T cell response remains
intact (Prehn et al. Immunity to methylcolanthrene-induced
sarcomas. Journal of the National Cancer Institute 6:769-778
(1957); McRae et al. Functional evidence for epitope spreading in
the relapsing pathology of experimental autoimmune
encephalomyelitis. Journal of Experimental Medicine 182(1):75-85
(1995)). These data suggest that peptide-based approaches to cancer
immunotherapy may indirectly stimulate multiple tumor reactive CTL
against minor antigens in the presence of residual tumor.
[0020] The fourth and final limitation to peptide-based
immunotherapy, is the limited number of diagnostic tools available
to evaluate clinical response. Positron emission tomography (PET)
in combination with systemic administration of a glucose analogue,
FDG, is a relatively new imaging modality that measures metabolic
activity of individual tissues. Tissues with large energy
requirements, e.g. tumors, incorporate higher levels of FDG than
surrounding normal tissue, allowing whole body tumor screening.
Because other pathogenic processes, such as infection, can have
high metabolic requirements, PET is not tumor specific. However, in
patients with known tumors, PET is highly accurate for both staging
head and neck cancer and identifying recurrent disease (Rege et al.
Use of positron emission tomography with fluorodeoxyglucose in
patients with extracranial head and neck cancers, Cancer
73(12):3047-3058 (1994); McGuirt et al. A comparative diagnostic
study of head and neck nodal metastases using positron emission
tomography. Laryngoscope 105((4 Pt 1)): 373-375 (1995);
Laubenbacher et al. Comparison of fluorine-18-fluorodeoxyglucose
PET, MRI and endoscopy for staging head and neck squamous-cell
carcinomas. Journal of Nuclear Medicine 36(10):1747-1757 (1995);
Lapela et al. Head and neck cancer: detection of recurrence with
PET and 2-[F-18]fluoro-2-deoxy-D-glucose. Radiology 197:205-211
(1995); Bailet et al. Positron emission tomography: a new, precise
imaging modality for detection of primary head and neck tumors and
assessment of cervical adenopathy. Laryngoscope 102:281-288
(1992)). In fact, in one recent study, PET had a sensitivity of 88%
compared to 25% for MRI/CT for identifying recurrent head and neck
malignancy (Anzai et al. Recurrence of head and neck cancer after
surgery or irradiation: prospective comparison of
2-deoxy-2-[F-18]fluoro-D-glucose PET and MR imaging diagnoses.
Radiology 200(1):135-141 (1996)). In patients being evaluated for
response to chemotherapy, PET can accurately identify residual
disease--even in some cases where initial biopsies are negative
(Lowe et al. Prediction of Chemotherapy Response in Patients with
Advanced Head and Neck Cancer Using [18F] Fluoro-deoxyglucose
Positron Emission Tomography (FDG-PET). Head and Neck 19:666-674
(1997)). Recent innovations in PET technology that combine PET and
CT machines into one, largely overcome the lack of anatomic detail
traditionally hampering PET scan interpretation.
[0021] In view of the need for additional therapeutic options for
use in the treatment of SCCHN, especially in patients with
unresectable disease, the present invention provides novel Trojan
antigen-based compositions and method for their use in the
treatment of SCCHN. More specifically, MAGE-A3 and HPV 16-based
Trojan antigen compositions, each composed of 1-2 HLA-A2.1
restricted CTL epitopes, HLA-DR helper epitopes joined together
with furin-cleavable linkers and HIV TAT translocating region.
SUMMARY OF THE INVENTION
[0022] The present invention relates to Trojan antigens, and
immunogenic compositions comprising the Trojan antigens. The
present invention also relates to methods of generating an immune
response in a subject using the Trojan antigens or immunogenic
compositions. The present invention further relates to methods of
treating squamous cell carcinoma of the head and neck (SCCHN) using
the Trojan antigens or immunogenic compositions of the present
invention.
[0023] More specifically, the present invention related to Trojan
antigens, which include an isolated polypeptide comprising amino
acids 1-35 of SEQ ID NO:15, an isolated polypeptide comprising
amino acids 1-47 of SEQ ID NO:17, an isolated polypeptide
comprising amino acids 1-21 of SEQ ID NO:19, and an isolated
polypeptide comprising amino acids 1-43 of SEQ ID NO:22.
[0024] In the Trojan antigen of SEQ ID NO:19, X may be cysteine or
aminobutyric acid. In the Trojan antigen of SEQ ID NO:22, each X
may independently be cysteine or aminobutyric acid.
[0025] The immunogenic compositions of the present invention
include immunogenic compositions comprising one or more of the
following Trojan antigens: an isolated polypeptide comprising amino
acids 1-35 of SEQ ID NO:15, an isolated polypeptide comprising
amino acids 1-47 of SEQ ID NO:17, an isolated polypeptide
comprising amino acids 1-21 of SEQ ID NO:19, wherein X may be
cysteine or aminobutyric acid, and an isolated polypeptide
comprising amino acids 1-43 of SEQ ID NO:22, wherein X.sub.6,
X.sub.30, X.sub.31 and X.sub.33 are each independently cysteine or
aminobutyric acid. The immunogenic compositions of the present
invention further comprise a pharmaceutically acceptable carrier,
diluent or adjuvant.
[0026] The methods of generating an immune response in a subject of
the present invention comprising administering one or more of the
following Trojan antigens to a subject in an amount sufficient to
induce an immune response in said subject: a polypeptide comprising
amino acids 1-35 of SEQ ID NO:15, a polypeptide comprising amino
acids 1-47 of SEQ ID NO:17, a polypeptide comprising amino acids
1-21 of SEQ ID NO:19, wherein X may be cysteine or aminobutyric
acid, and a polypeptide comprising amino acids 1-43 of SEQ ID
NO:22, wherein X.sub.6, X.sub.30, X.sub.31 and X.sub.33 are each
independently cysteine or aminobutyric acid. The one or more Trojan
antigens may be co-administered with a pharmaceutically acceptable
carrier, diluent or adjuvant.
[0027] In a preferred embodiment, the present invention includes a
method of generating an immune response in a subject comprising
administering the following Trojan antigens to a subject in an
amount sufficient to induce an immune response in said subject: (a)
a polypeptide comprising amino acids 1-47 of SEQ ID NO:17 and (b) a
polypeptide comprising amino acids 1-43 of SEQ ID NO:22, wherein
X.sub.6, X.sub.30, X.sub.31 and X.sub.33 are each independently
cysteine or aminobutyric acid. The Trojan antigens may be
co-administered with a pharmaceutically acceptable carrier, diluent
or adjuvant.
[0028] In preferred embodiments, the one or more Trojan antigens
are administered in a combined amount of between about 100 ug and
about 1.5 mg, more preferably in an amount of about 1 mg.
[0029] In other preferred embodiments, the one or more Trojan
antigens are co-administered with montanide, in an amount of
between about 0.5 and 1.5 mL, and GM-CSF, in an amount of between
about 50 and 150 ug/m.sup.2.
[0030] The methods treating squamous cell carcinoma of the head and
neck (SCCHN) of the present invention comprising administering to a
subject in need of such treatment a therapeutically-effective
amount of one of the following Trojan antigens: a polypeptide
comprising amino acids 1-35 of SEQ ID NO:15, a polypeptide
comprising amino acids 1-47 of SEQ ID NO:17, a polypeptide
comprising amino acids 1-21 of SEQ ID NO:19, wherein X may be
cysteine or aminobutyric acid, and a polypeptide comprising amino
acids 1-43 of SEQ ID NO:22, wherein X.sub.6, X.sub.30, X.sub.3, and
X.sub.33 are each independently cysteine or aminobutyric acid. The
one or more Trojan antigens may be co-administered with a
pharmaceutically acceptable carrier, diluent or adjuvant.
[0031] In a preferred embodiment, the method of treating SCCHN
comprises administering to a subject in need of such treatment a
therapeutically-effective amount of the following Trojan antigens:
(a) a polypeptide comprising amino acids 1-47 of SEQ ID NO:17 and
(b) a polypeptide comprising amino acids 1-43 of SEQ ID NO:22,
wherein X.sub.6, X.sub.30, X.sub.31 and X.sub.33 are each
independently cysteine or aminobutyric acid. The Trojan antigens
may be co-administered with a pharmaceutically acceptable carrier,
diluent or adjuvant.
[0032] In preferred embodiments, the one or more Trojan antigens
are administered in a combined amount of between about 100 ug and
about 1.5 mg, more preferably in an amount of about 1 mg.
[0033] In other preferred embodiments, the one or more Trojan
antigens are co-administered with montanide, in an amount of
between about 0.5 and 1.5 mL, and GM-CSF, in an amount of between
about 50 and 150 ug/m.sup.2.
[0034] The present invention also comprises the following
polynucleotide molecules: a polynucleotide molecule encoding amino
acids 1-35 of SEQ ID NO:15, a polynucleotide molecule encoding
amino acids 1-47 of SEQ ID NO:17, a polynucleotide molecule
encoding amino acids 1-21 of SEQ ID NO:19, and a polynucleotide
molecule amino acids 1-43 of SEQ ID NO:22.
[0035] The present invention further comprises expression vectors
comprising one of the following polynucleotide molecules: a
polynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15,
a polynucleotide molecule encoding amino acids 1-47 of SEQ ID
NO:17, a polynucleotide molecule encoding amino acids 1-21 of SEQ
ID NO:19, and a polynucleotide molecule amino acids 1-43 of SEQ ID
NO:22.
[0036] The present invention additionally includes a host cell
comprising an expression vector, wherein the expression vector
comprises one of the following polynucleotide molecules: a
polynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15,
a polynucleotide molecule encoding amino acids 1-47 of SEQ ID
NO:17, a polynucleotide molecule encoding amino acids 1-21 of SEQ
ID NO:19, and a polynucleotide molecule amino acids 1-43 of SEQ ID
NO:22.
[0037] The present invention further includes methods of preparing
a polypeptide, comprising culturing a host cell comprising an
expression vector, which in turn comprises a polynucleotide
molecule, under conditions promoting expression of the polypeptide
encoded by the polynucleotide molecule and recovering the
polypeptide from the cell culture. In preferred embodiments, the
polynucleotides include: a polynucleotide molecule encoding amino
acids 1-35 of SEQ ID NO:15, a polynucleotide molecule encoding
amino acids 1-47 of SEQ ID NO:17, a polynucleotide molecule
encoding amino acids 1-21 of SEQ ID NO:19, and a polynucleotide
molecule amino acids 1-43 of SEQ ID NO:22.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a graphical representation of the ability of a
MAGE3-specific cytotoxic T lymphocyte (CTL) clone to recognize
different tumor types. An MAGE3[9.sub.112]-specific CTL clone was
tested for cytotoxicity using the following targets: .largecircle.,
0.221A2.1 pulsed with MAGE3[9.sub.112], .largecircle.. 0.221A2.1
without peptide; .DELTA., 624mel (melanoma, A2.sup.+, MAGE3.sup.+);
.quadrature., KATO-III (gastric Ca, A2.sup.+, MAGE3.sup.+);
.diamond., SW403 (colon Ca, A2.sup.+, MAGE3.sup.+); .quadrature.,
WiDr (colon Ca, A2.sup.-, MAGE3.sup.+); .DELTA., 888mel (melanoma,
A2.sup.-, MAGE3.sup.-).
[0039] FIG. 2 is the results of experiments that demonstrated
HLA-DR4-restricted HTL clone 8G9 recognizes naturally processed
MAGE A3 antigen. A: Proliferative T-cell response induced by
MAGE-A3.sub.146-160 (+Peptide), recombinant MAGE-A3 protein
(rMAGE-A3) or recombinant gp100 (rgp100). B: Tissue culture
supernatants from experiment described in panel A, were collected
after 48 hr and the concentration of GM-CSF was measured by ELISA.
C: T-cell clone 8G9 recognizes UV-irradiated melanoma cells that
express MAGE-A3 (HT-144 (+), SKmel-28 (+), 697mel (+)) via antigen
cross-presentation by autologous DC. DC incubated with MAGE-A3
negative melanoma cell line (888mel (-)) did not stimulate the
T-cell clone. Values shown are the means of triplicate
determinations; bars, SD.
[0040] FIG. 3 is the result of an experiment where dendritic cells
where pulsed with a Trojan antigen. C57/BL6 DC were pulsed with
either SIINFEKL (SEQ ID NO:11) or TrojAg (RKKRRQRRRRAAASIINFEKL;
SEQ ID NO:12) for 2 hours, washed three times, and then kept in
37.degree. C. After 48 hours, the ability of peptide loaded DC to
induce IFN-gamma release from OT-1 T cells was determined by ELISA.
The concentration of IFN-gamma was determined at various dilutions
of the supernatant that was collected after 24 hours of incubation
of the DC with the OT-1 T cells.
[0041] FIG. 4 shows the results of an Elispot analysis of IFN y
production by HPV 16 Trojan antigen stimulated T cells.
[0042] FIG. 5 shows the results of an immunohistochemical analysis
of HLA-A2 expression in fresh SCCHN. Panel A: HLA-A2 negative
patient with no staining of the tumor or surrounding parenchyma.
Panel B: HLA-A2 positive patient with positive staining of both the
tumor and surrounding parenchyma, arrows indicate tumor location.
Panel C: HLA-A2 positive patient with positive staining of the
parenchyma, but loss of HLA-A2 reactivity in the tumor. Specimens
were stained with H&E (hematoxylin and eosin), mouse Ig control
or the SB03-111 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention relates to Trojan antigens, and
immunogenic compositions comprising the Trojan antigens. The
present invention also relates to methods of generating an immune
response in a subject using the Trojan antigens or immunogenic
compositions. The present invention further relates to methods of
treating squamous cell carcinoma of the head and neck (SCCHN) using
the Trojan antigens or immunogenic compositions of the present
invention.
Trojan Antigens
[0044] One embodiment of the present invention pertains to Trojan
antigens. Trojan antigens are polypeptides comprising one or more
antigenic epitopes joined together by cleavable linkers, that may
be used as peptide vaccines for administration to a subject.
Therapeutic peptide vaccines may be used to induce a subject's
innate anti-tumor response by using antigenic epitopes derived from
polypeptides expressed by the tumor cells from said subject. Trojan
antigens are processed by antigen presenting cells (APC) which then
display the antigenic epitopes of the Trojan antigens on their
surface. Cytotoxic T lymphocytes (CTL) are activated by the APC
displaying the antigenic epitopes in the context of MHC class I
molecules, which then recognize and destroy tumor cells displaying
the antigenic epitope in the context of a larger polypeptide.
[0045] In addition to the activation of CTL, a CD4+ helper T cell
response is also activated by the APC displaying the antigenic
epitopes in the context of MHC class II molecules.
[0046] In addition to the antigenic epitopes derived from
tumor-expressed proteins, the Trojan antigens of the present
invention may also include a transporter peptide. Transporter
peptides are regions of polypeptides known to be translocated into
cells without first requiring proteosomal processing and transport
by TAP (transporter associated with antigen processing).
Polypeptides comprising transporter peptides are internalized
directly to the endoplasmic reticulum (ER) and Golgi. Inclusion of
a transporter peptide in the Trojan antigens allows the Trojan
antigens to be directly taken up by APC where the antigenic
epitopes can form peptide/MHC complexes. Such Trojan antigens are
more resistant to degradation than smaller constructs and allow
simultaneous stimulation of multiple T cell populations, reducing
the chance of tumor escape through the selection of antigen loss
variants.
[0047] The antigenic epitopes are portions of a polypeptide, or the
entire polypeptide in the case of small proteins, expressed by a
tumor cell. Preferably, the polypeptide is essential for cell
survival, is expressed by all cells of the tumor, is tumor
specific, is a surface protein, and is a protein not expressed in
the thymus nor during fetal development. After uptake and
processing of Trojan antigens by antigen presenting cells, the
antigenic epitopes are displayed on the cell surface in the context
of MHC class I and class II molecules, which in turn induce CD8+
and CD4+ T cells, respectively. The Trojan antigens of the present
invention may comprise antigenic epitopes that include CD8+ T cells
alone, CD4+ T cells alone, or both CD8+ and CD4+ T cells.
[0048] While the composition of the antigenic epitopes is governed
by the factors noted above, peptides homologous to antigenic
epitopes may also be used in Trojan antigens to increased the
spectrum of immune response generated by the Trojan antigens.
Peptide homologues having at least about 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity to a selected antigenic
epitope are included in the present invention. Each reference to an
antigenic epitope herein is also meant to be a reference to a
peptide homologue that may be used in place of the antigenic
epitope. A single Trojan antigen may comprise both a specific
antigenic epitope and a peptide homologue of the specific antigenic
epitope.
[0049] The antigenic epitopes of the present invention may be
comprised of naturally occurring amino acids or amino acid
analogues. Such analogues include those amino acids that produce
cleavage-resistant peptides.
[0050] The size of the antigenic epitopes used in the Trojan
antigens is not limited, though preferably the antigenic epitopes
are of a size that readily forms a complex with MHC class I and
class II molecules in antigen presenting cells. Preferably, the
antigenic epitopes for class I molecules are peptides of between
about 8 amino acids and about 10 amino acids in length. More
preferably, the antigenic epitopes for class I molecules are
peptides of about 9 amino acids in length. Preferably, the
antigenic epitopes for class II molecules are peptides of between
about 10 amino acids and about 20 amino acids in length. More
preferably, the antigenic epitopes for class II molecules are
peptides of about 15 amino acids in length.
[0051] The use of cleavable linkers to join antigenic epitopes
(when two or more antigenic epitopes are used in a Trojan antigen)
or one or more antigenic epitopes and the transporter peptide,
allows the release of the individual components of the Trojan
antigen upon internalization of the antigen into a cell.
[0052] Preferably, the cleavable linkers are furin-sensitive
linkers which allow the components of the Trojan antigen to be
separated from each other in the Golgi, where the furin
endopeptidase resides. Furin is a very specific protease that
recognizes a motif consisting of RX(R/K)R, where R and K are the
positive-charged amino acids lysine and arginine, respectively, and
X is any amino acid residue. A preferred furin linker used herein
is RVKR (SEQ ID NO:10). Through the action of furin in the Golgi,
the Trojan antigen is first cleaved separating the antigenic
epitopes from the transporter peptide. The antigenic epitopes are
then trimmed via amino- and carboxy-peptidases that are present in
the ER and Golgi, until the appropriately-sized peptide is formed
and binds to MHC molecules in these compartments, protecting it
from further degradation. The skilled artisan will understand that
any furin-cleavable linker may be used, where the first, third and
fourth residues are positively charged amino acids such as lysine
and arginine. Other suitable linkers will be readily apparent to
the skilled artisan.
[0053] The portion of the Trojan antigen that allows direct
internalization of the antigens into a cell is a transporter
peptide. The transporter peptide is any peptide that allows
transport of the Trojan antigen directly into a cell without first
requiring proteolytic processing of the antigen. An example of a
transporter peptide is the penetrin peptide of HIV TAT (Frankel et
al. Cellular uptake of the tat protein from human immunodeficiency
virus. Cell 55:1189-1193 (1988)). Several other cell penetrating
sequences have been described from proteins, including the VP22
protein of herpes simplex virus (Elliott et al. Intercellular
trafficking and protein delivery by a herpesvirus structural
protein. Cell 88:223-233 (1997); Phelan et al. Intercellular
delivery of functional p53 by the herpesvirus protein VP22. Nat.
Biotechnol. 16:440-443 (1998)), the fibroblast growth factor (Lin
et al. Inhibition of nuclear translocation of transcription factor
NF-kappa B by a synthetic peptide containing a cell
membrane-permeable motif and nuclear localization sequence. J.
Biol. Chem. 270:14255-14258 (1995); Rojas et al. Genetic
engineering of proteins with cell membrane permeability. Nat.
Biotechnol. 16:370-375 (1998)) and the Drosophila antennapedia
homeodomain protein (Schutze-Redelmeier et al. Introduction of
exogenous antigens into the MHC class I processing and presentation
pathway by Drosophila antennapedia homeodomain primes cytotoxic T
cells in vivo. J. Immunol. 157:650 (1996)).
[0054] Preferably, the transporter protein is the penetrin peptide
of HIV TAT: RKKRRQRRR (SEQ ID NO:9). The transporter peptide may be
coupled to either the amino- or the carboxy-terminal end of the
antigenic epitopes.
[0055] In one embodiment of the invention, the Trojan antigen is
based on one or more antigenic epitopes from the MAGE-A3
differentiation antigen linked to a transporter peptide. MAGE-A3 is
expressed by cells of SCCHN tumors. When more than one antigenic
epitope from MAGE-A3 is used, the epitopes are linked by a
cleavable linker, preferably a furin-sensitive linker. In this
embodiment, a cleavable linker is also used to link the selected
MAGE-A3 antigenic epitopes to the transporter peptide. In a
preferred embodiment, the MAGE-A3.sub.112-120 antigenic epitope
KVAELVHFL (SEQ ID NO:2) is linked to the MAGE-A3.sub.271-279
antigenic epitope FLWGPRALV (SEQ ID NO:1) using the furin-sensitive
linker RVKR (SEQ ID NO:10), to produce the linked peptide
KVAELVHFL-RVKR-FLWGPRALV (SEQ ID NO:13). Upon action by furin, the
linked peptide is cleaved into KVAELVHFLRVKR (SEQ ID NO:14) and
FLWGPRALV (SEQ ID NO:1) in the Golgi. KVAELVHFLRVKR (SEQ ID NO:14)
is then trimmed by exopeptidases into the MHC-binding peptide
KVAELVHFL (SEQ ID NO:2).
[0056] In this preferred embodiment, the linked peptide
KVAELVHFLRVKRFLWGPRALV (SEQ ID NO:13) is joined to the penetrin
transporter peptide from HIV TAT: RKKRRQRRR (SEQ ID NO:9). In this
embodiment, the Trojan antigen is
KVAELVHFLRVKRFLWGPRALVRVKRRKKRRQRRR (SEQ ID NO:15). Trojan antigens
comprising the MAGE-A3.sub.112-120 antigenic epitope linked by a
furin-sensitive linker to HIV TAT penetrin (SEQ ID NO:9), or the
MAGE-A3.sub.271-279 antigenic epitope linked by a furin-sensitive
linker to HIV TAT penetrin (SEQ ID NO:9), are also include in this
invention.
[0057] In a further preferred embodiment of the invention, the
Trojan antigen is comprised of antigenic epitopes from MAGE-A3 that
induce both CD8+ and CD4+ T cells responses, i.e., the antigenic
epitopes induce the formation of both class I and class II MHC
complexes by APC. In this preferred embodiment, the MAGE class I
antigenic epitopes MAGE-A3.sub.112-120 (KVAELVHFL; SEQ ID NO:2) and
MAGE-A3.sub.271-279 (FLWGPRALV; SEQ ID NO:1) are linked with the
class II MAGE-A3.sub.149-160 antigenic epitope (VIFSKASSSLQL (SEQ
ID NO:5) using the furin-sensitive linker RVKR (SEQ ID NO:10), to
produce the linked peptide KVAELVHFLRVKRFLWGPRALVRVKRVIFSKASSSLQL
(SEQ ID NO:16).
[0058] In this preferred embodiment, the linked peptide (SEQ ID
NO:16) is joined to the penetrin transporter peptide from HIV TAT:
RKKRRQRRR (SEQ ID NO:9). In this embodiment, the Trojan antigen is
KVAELVHFLRVKRFLWGPRALVRVKRVIFSKASSSLQL-RKKRRQRRR (SEQ ID
NO:17).
[0059] Trojan antigens comprising the MAGE-A3.sub.149-160 antigenic
epitope linked by a furin-sensitive linker to HIV TAT penetrin (SEQ
ID NO:9) are also include in this invention, as are Trojan antigens
comprising the MAGE-A3.sub.112-120 (SEQ ID NO:2) and
MAGE-A3.sub.149-160 (SEQ ID NO:5) antigenic epitopes linked to each
other and to HIV TAT penetrin (SEQ ID NO:9) by a furin-sensitive
linkers. Trojan antigens comprising the MAGE-A3.sub.271-279 (SEQ ID
NO:1) and MAGE-A3.sub.149-160 (SEQ ID NO:5) antigenic epitopes
linked to each other and to HIV TAT penetrin (SEQ ID NO:9) by a
furin-sensitive linkers are also included in the present
invention.
[0060] In a related embodiment of the invention, the Trojan antigen
is based on one or more antigenic epitopes from the human papilloma
virus (HPV) 16 E7 nuclear protein. HPV 16 E7 protein is a tumor
rejection antigen, which is postulated to play an integral role in
the development of carcinoma of the uterine cervix. When more than
one antigenic epitope from HPV 16 E7 is used, the epitopes are
linked by a cleavable linker, preferably a furin-sensitive linker.
A cleavable linker is further used to link one or more HPV 16 E7
antigenic epitopes to a transporter peptide. In a preferred
embodiment, the HPV 16 E7.sub.86-93 antigenic epitope TLGIVXPI (SEQ
ID NO:18), where X is cysteine or aminobutyric acid, preferably
aminobutyric acid, is linked to the penetrin peptide sequence from
HIV TAT: RKKRRQRRR (SEQ ID NO:9). In this embodiment, the Trojan
antigen is TLGIVXPIRVKR-RKKRRQRRR (SEQ ID NO:19), where X is
cysteine or aminobutyric acid, preferably aminobutyric acid.
[0061] In a preferred embodiment of an HPV 16 E7-based Trojan
antigen, the HPV 16 E7.sub.86-93 antigenic epitope TLGIVXPI (SEQ ID
NO:18) is linked via a furin-sensitive linker to the HPV 16
E7.sub.41-62 antigenic epitope PAGQAEPDRAHYNIVTFXXKXD (SEQ ID
NO:20), to form the linked peptide
TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXD (SEQ ID NO:21), where each X is
independently cysteine or aminobutyric acid, preferably each X is
aminobutyric acid. This linked peptide (SEQ ID NO:21) is joined to
the HIV TAT transporter peptide RKKRRQRRR (SEQ ID NO:9) to create
the Trojan antigen TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXDRKKRRQRRR (SEQ
ID NO:22), where again each X is independently cysteine or
aminobutyric acid, preferably each X is aminobutyric acid.
[0062] In addition to the HLA-A2 antigenic epitopes from the
MAGE-A3 and HPV 16 E7 polypeptides, antigenic epitopes from these
two polypeptides that associate with alternative HLA alleles may be
used in the present invention. Similarly, antigenic epitopes from
other tumor expressed polypeptides, such as telomerase, may be used
in the present invention, both those that associate with HLA-A2,
and those that associates with other HLA alleles.
[0063] The each of the antigenic epitopes, polypeptides, peptides,
linkers and Trojan antigens of the present invention may be
prepared based on methods well known to those of skill in the art.
For example, these amino acid sequences can be produced by using
recombinant DNA techniques easily identified and well known by
those of skill in the art. For example, DNA molecules encoding the
Trojan antigens are prepared using generally available methods such
as PCR mutagenesis, site-directed mutagenesis, and/or restriction
digestion and ligation. The hybrid DNA is then inserted into
expression vectors and introduced into suitable host cells.
Preferred expression vectors include plasmids and cosmids. An
expression vector containing one or more polynucleotides encoding
one or more of the Trojan antigens of this invention can be used to
transfect or transform a suitable host cell (prokaryotic or
eukaryotic) to produce the protein or to produce an immune
response, or for some other purpose.
[0064] A recombinant virus can also be used as the expression
vector. Exemplary viruses include the adenoviruses,
adeno-associated viruses, herpes viruses, vaccinia, CMV, BLUESCRIPT
(Stratagene, San Diego, Calif.), baculovirus, or an RNA virus such
as a retrovirus or an alphavirus. Preferably, the retroviral vector
is a derivative of a murine or avian retrovirus. The alphavirus
vector is preferably derived from Sindbis or Semliki Forest Virus.
All of these expression vectors can transfer or incorporate a gene
for a selectable marker so that transduced cells can be identified
and generated.
[0065] The viral vector can be made target specific by inserting
one or more sequences of interest into the viral vector, along with
another polynucleotide encoding a Trojan antigen. For example,
retroviral vectors can be made target specific by inserting a
polynucleotide encoding a sugar, a glycolipid, or a protein. Those
of skill in the art will know of, or can readily ascertain without
undue experimentation, specific polynucleotide sequences which can
be inserted into the retroviral genome to allow target specific
delivery of the retroviral vector containing the polynucleotides of
interest.
[0066] It will be appreciated that the same techniques that are
utilized to incorporate the nucleotide sequences encoding a Trojan
antigen, and optionally other immunostimulatory polynucleotides,
into viral gene expression vectors can be used to incorporate the
sequences into live and attenuated live viruses for use as
immunogenic compositions.
[0067] Construction of suitable expression vectors containing
desired coding, non-coding, and control sequences employ standard
ligation techniques. Isolated plasmids or DNA fragments are
cleaved, tailored, and re-ligated in the form desired to construct
the required plasmids. To confirm correct sequences in the plasmids
constructed, the ligation mixtures can be used, for example, to
transform a host cell and successful transformants selected by
antibiotic resistance where appropriate. Plasmids from the
transformants are prepared, analyzed by restriction and/or
sequenced by, for example, by the method disclosed in Messing, et
al. (Nucleic Acids Res., 9:309 (1981)), Maxam, et al. (Methods in
Enzymology 65:499 (1980)), or other suitable methods which will be
known to those skilled in the art. Size separation of cleaved
fragments can be performed using conventional gel electrophoresis
as described, for example, by Maniatis, et al. (Molecular Cloning,
pp. 133-134 (1982)).
[0068] Host cells can be transformed with the expression vectors
described herein and cultured in conventional nutrient media
modified as is appropriate for inducing promoters, selecting
transformants, or amplifying genes. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0069] Steps involved in the purification of one or more of the
polypeptides, such as the Trojan antigens, of this invention
include (1) solubilization of the desired protein, (2) the
development of one or more isolation and concentration procedures,
(3) stabilization of the protein following purification, and (4)
development of a suitable assay to determine the presence of the
desired protein. Various aspects of protein isolation and
purification are discussed in detail in Cooper, T. G., "The Tools
of Biochemistry," John Wiley & Sons, New York, 1977. As the
techniques of protein isolation and purification are notoriously
well known in the art, this disclosure will refrain from discussing
them in detail. Nevertheless, elements of the cited reference are
summarized and discussed below.
[0070] Solubilization is required of most proteins that are to be
purified, as most isolation procedures commonly used operate in
aqueous solutions. In some cases, solubilization can be achieved by
merely lysing a host cell within which a desired protein has been
expressed. In other situations, additional steps, such as
extracting the desired protein from a subcellular organelle, may be
required. Osmotic lysis, grinding, the use of blenders, ultrasonic
waves, presses, and other well known techniques of protein
solubilization can be used with the methods disclosed herein.
[0071] There are a variety of techniques available that are well
known in the art for the isolation and concentration of the
proteins of this invention. These techniques include, but are not
limited to, (1) differential solubility, (2) ion exchange
chromatography, (3) absorption chromatography, (4) molecular sieve
techniques, (5) affinity chromatography, (6) electrophoresis, and
(7) electrofocusing. Each of these techniques can also be useful in
the purification of a protein of this invention.
[0072] Stabilizing and maintaining a purified protein product in a
functional state warrants attention to a number of different
conditions such as (1) pH, (2) degree of oxidation, (3) heavy metal
concentration, (4) medium polarity, (5) protease concentration, and
(6) temperature. One of ordinary skill in the art would readily
know which of the available techniques to use to maintain purified
protein in an active form without undue experimentation.
[0073] The Trojan antigens and other polypeptides of the present
invention can further be prepared using an synthetic peptide
synthesizer.
[0074] Also included in the present invention are polynucleotide
molecules encoding the Trojan antigens and other polypeptides,
including: a polynucleotide molecule encoding amino acids 1-35 of
SEQ ID NO:15, a polynucleotide molecule encoding amino acids 1-47
of SEQ ID NO:17, a polynucleotide molecule encoding amino acids
1-21 of SEQ ID NO:19, and a polynucleotide molecule amino acids
1-43 of SEQ ID NO:22.
[0075] The present invention further comprises expression vectors
comprising one of the following polynucleotide molecules: a
polynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15,
a polynucleotide molecule encoding amino acids 1-47 of SEQ ID
NO:17, a polynucleotide molecule encoding amino acids 1-21 of SEQ
ID NO:19, and a polynucleotide molecule amino acids 1-43 of SEQ ID
NO:22.
[0076] The present invention additionally includes a host cell
comprising an expression vector, wherein the expression vector
comprises one of the following polynucleotide molecules: a
polynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15,
a polynucleotide molecule encoding amino acids 1-47 of SEQ ID
NO:17, a polynucleotide molecule encoding amino acids 1-21 of SEQ
ID NO:19, and a polynucleotide molecule amino acids 1-43 of SEQ ID
NO:22.
[0077] The present invention further includes methods of preparing
a polypeptide, comprising culturing a host cell comprising an
expression vector, which in turn comprises a polynucleotide
molecule, under conditions promoting expression of the polypeptide
encoded by the polynucleotide molecule and recovering the
polypeptide from the cell culture. In preferred embodiments, the
polynucleotides include: a polynucleotide molecule encoding amino
acids 1-35 of SEQ ID NO:15, a polynucleotide molecule encoding
amino acids 1-47 of SEQ ID NO:17, a polynucleotide molecule
encoding amino acids 1-21 of SEQ ID NO:19, and a polynucleotide
molecule amino acids 1-43 of SEQ ID NO:22.
Immunogenic Compositions
[0078] Included within the present invention are immunogenic
compositions comprising one or more Trojan antigens and a
pharmaceutically acceptable carrier, diluent or adjuvant.
[0079] The immunogenic compositions of the present invention may
comprise one Trojan antigen, or two or more different Trojan
antigens. Embodiments of immunogenic compositions of the present
invention include those comprising one or more of the specific
Trojan antigens discussed herein.
[0080] In a preferred example of an immunogenic composition, the
composition comprises the MAGE-A3 Trojan antigen
KVAELVHFLRVKRFLWGPRALVRVKRVIFSKASSSLQLRKKRRQRRR (SEQ ID NO:17) and
the HPV 16 E7 Trojan antigen
TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXDRKKRRQRRR (SEQ ID NO:22), where
each X is independently cysteine or aminobutyric acid, preferably
each X is aminobutyric acid, and a pharmaceutically acceptable
carrier, diluent or adjuvant.
[0081] Other preferred embodiments of immunogenic compositions of
the present invention comprises one or more of the following Trojan
antigens: SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID
NO:22, and a pharmaceutically acceptable carrier, diluent or
adjuvant. In SEQ ID NO:19, X may be cysteine or aminobutyric acid,
preferably aminobutyric acid. In SEQ ID NO:22, each X may
independently be cysteine or aminobutyric acid, preferably
aminobutyric acid.
[0082] Preferred examples of pharmaceutically acceptable carriers,
diluents and adjuvants include: (1) Dulbecco's phosphate buffered
saline, pH .about.7.4, containing about 1 mg/ml to 25 mg/ml human
serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v)
dextrose. Other acceptable carriers, diluents and adjuvants
include, but are not limited to saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof, buffers,
antioxidants such as ascorbic acid, low molecular weight (less than
about 10 residues) polypeptides, proteins, amino acids,
carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and
excipients commonly employed in pharmaceutical compositions. The
composition may be formulated as a lyophilizate using appropriate
excipient solutions (e.g. sucrose) as diluents.
[0083] In addition to the immunogenic compositions comprising
polypeptides, such as the Trojan antigen, included in the present
invention are additional forms of immunogenic compositions. In one
embodiment, nucleotide-containing immunogenic compositions are
contemplated. For example, in one embodiment a Trojan
antigen-encoding polynucleotide preparation including DNA or RNA
that encodes an Trojan antigenic may be used for administration to
a subject. Nucleotide-containing immunogenic compositions also
include live viral immunogenic compositions. The viruses for use in
the viral immunogenic compositions may include immunostimulatory
polynucleotides.
Method of Generating an Immune Response
[0084] The present invention also includes methods of generating an
immune response in a subject. The methods of generating an immune
response generally involves administration of a Trojan antigen, or
an immunogenic composition comprising a Trojan antigen, to a
subject.
[0085] A preferred embodiment of the present invention is a method
of generating an immune response in a subject comprising
administering a Trojan antigen comprising amino acids 1-35 of SEQ
ID NO:15 to a subject in an amount sufficient to induce an immune
response in said subject.
[0086] Another preferred embodiment of the present invention is a
method of generating an immune response in a subject comprising
administering a Trojan antigen comprising amino acids 1-47 of SEQ
ID NO:17 to a subject in an amount sufficient to induce an immune
response in said subject.
[0087] A further preferred embodiment of the present invention is a
method of generating an immune response in a subject comprising
administering a Trojan antigen comprising amino acids 1-21 of SEQ
ID NO:19 to a subject in an amount sufficient to induce an immune
response in said subject. In SEQ ID NO:19, X may be cysteine or
aminobutyric acid, preferably aminobutyric acid.
[0088] A equally preferred embodiment of the present invention is a
method of generating an immune response in a subject comprising
administering a Trojan antigen comprising amino acids 1-43 of SEQ
ID NO:22 to a subject in an amount sufficient to induce an immune
response in said subject. In SEQ ID NO:22, each X may independently
be cysteine or aminobutyric acid, preferably aminobutyric acid.
[0089] A further preferred embodiment of the present invention is a
method of generating an immune response in a subject comprising
administering a (a) Trojan antigen comprising amino acids 1-47 of
SEQ ID NO:17 and (b) a Trojan antigen comprising amino acids 1-43
of SEQ ID NO:22 to a subject in an amount sufficient to induce an
immune response in said subject. In SEQ ID NO:22, each X may
independently be cysteine or aminobutyric acid, preferably
aminobutyric acid.
[0090] In each of these embodiments, the Trojan antigens may
co-administered with a pharmaceutically acceptable carrier, diluent
or adjuvant, in the form of an immunogenic composition.
[0091] Preferred examples of pharmaceutically acceptable carriers,
diluents and adjuvants include: (1) Dulbecco's phosphate buffered
saline, pH .about.7.4, containing about 1 mg/ml to 25 mg/ml human
serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v)
dextrose. Other acceptable carriers, diluents and adjuvants
include, but are not limited to saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof, buffers,
antioxidants such as ascorbic acid, low molecular weight (less than
about 10 residues) polypeptides, proteins, amino acids,
carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and
excipients commonly employed in pharmaceutical compositions. The
composition may be formulated as a lyophilizate using appropriate
excipient solutions (e.g. sucrose) as diluents.
[0092] The Trojan antigens may be administered (alone or in the
context of an immunogenic composition) in a dosage containing
between about 5 ug and about 100 mg of peptide, more preferably in
a dosage containing between about 300 ug and about 1 mg of peptide.
More preferred dosages are 300 ug, 350 ug, 400 ug, 450 ug, 500 ug,
550 ug, 600 ug, 650 ug, 700 ug, 750 ug, 800 ug, 850 ug, 900 ug, 950
ug and 1 mg of peptide.
[0093] The methods of generating an immune response as disclosed
herein may also include the administration of additional compounds
to augment the generation of an immune response. For example, in
addition to the Trojan antigen and immunogenic compositions
comprising a Trojan antigen, additional compounds may be
administered before or after the Trojan antigen or immunogenic
composition, or co-administered with the Trojan antigen or
immunogenic composition.
[0094] Such additional compounds include Montanide and GM-CSF.
Montanide (Montanide ISA-51) is a human-approved adjuvant, similar
to incomplete Freunds adjuvant, recognized to regulate the temporal
aspects of peptide release (Aichele et al. T cell priming versus T
cell tolerance induced by synthetic peptides. Journal of
Experimental Medicine 182(1):261-6 (1995)), used in human vaccine
therapy to stimulate the immune system. GM-CSF (granulocyte
macrophage-colony stimulating factor) is a cytokine involved in the
growth and differentiation of myeloid and monocytic lineage cells,
including dendritic cells, monocytes and tissue macrophages and
cells of the granulocyte lineage.
[0095] Additional compounds include adjuvants of the Toll-like
receptor family. These adjuvants include CpG-containing
oligodeoxynucleotides, bacterial DNA, polyinosinic-polycytidylic
acid, synthetic double-stranded RNA, synthetic IMIQUOMOD.TM., and
RNA of viral or bacterial origin, or a viral RNA mimic.
[0096] In a preferred embodiment, both Montanide and GM-CSF are
co-administered to a subject with the immunogenic composition.
Montanide may be administered at a dosage of between about 0.1 mL
and 10 mL, preferably at between 0.25 mL and 2 mL, more preferably
at 1.2 mL. GM-CSF may be administered at a dosage of between about
5 ug/m.sup.2 and 1 mg/m.sup.2, preferably at between 20 ug/m.sup.2
and 500 ug/m.sup.2, more preferably at 100 ug/m.sup.2.
Methods of Treatment
[0097] Also included in the present invention are methods of
treating a subject having SCCHN. The methods of treatment generally
involved administration of a Trojan antigen, or an immunogenic
composition comprising a Trojan antigen, to a subject having
SCCHN.
[0098] A preferred embodiment of the present invention is a method
of treating SCCHN comprising administering to a subject in need of
such treatment a therapeutically-effective amount of a Trojan
antigen comprising amino acids 1-35 of SEQ ID NO:15.
[0099] Another preferred embodiment of the present invention is a
method of treating SCCHN comprising administering to a subject in
need of such treatment a therapeutically-effective amount of a
Trojan antigen comprising amino acids 1-47 of SEQ ID NO:17.
[0100] A further preferred embodiment of the present invention is a
method of treating SCCHN comprising administering to a subject in
need of such treatment a therapeutically-effective amount of a
Trojan antigen comprising amino acids 1-21 of SEQ ID NO:19. In SEQ
ID NO:19, X may be cysteine or aminobutyric acid, preferably
aminobutyric acid.
[0101] A equally preferred embodiment of the present invention is a
method of treating SCCHN comprising administering to a subject in
need of such treatment a therapeutically-effective amount of a
Trojan antigen comprising amino acids 1-43 of SEQ ID NO:22. In SEQ
ID NO:22, each X may independently be cysteine or aminobutyric
acid, preferably aminobutyric acid.
[0102] A further preferred embodiment of the present invention is a
method of treating SCCHN comprising administering to a subject in
need of such treatment a therapeutically-effective amount of (a) a
Trojan antigen comprising amino acids 1-47 of SEQ ID NO:17 and (b)
a Trojan antigen comprising amino acids 1-43 of SEQ ID NO:22. In
SEQ ID NO:22, each X may independently be cysteine or aminobutyric
acid, preferably aminobutyric acid.
[0103] In each of these embodiments, the Trojan antigen may be
co-administered with a pharmaceutically acceptable carrier, diluent
or adjuvant, in the form of an immunogenic composition.
[0104] Preferred examples of pharmaceutically acceptable carriers,
diluents and adjuvants include: (1) Dulbecco's phosphate buffered
saline, pH .about.7.4, containing about 1 mg/ml to 25 mg/ml human
serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v)
dextrose. Other acceptable carriers, diluents and adjuvants
include, but are not limited to saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof, buffers,
antioxidants such as ascorbic acid, low molecular weight (less than
about 10 residues) polypeptides, proteins, amino acids,
carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and
excipients commonly employed in pharmaceutical compositions. The
composition may be formulated as a lyophilizate using appropriate
excipient solutions (e.g. sucrose) as diluents.
[0105] The Trojan antigens may be administered (alone or in the
context of an immunogenic composition) in a dosage containing
between about 5 ug and about 100 mg of peptide, more preferably in
a dosage containing between about 300 ug and about 1 mg of peptide.
A preferred dosage is between 300 ug and 1 mg. More preferred
dosages are 300 ug, 350 ug, 400 ug, 450 ug, 500 ug, 550 ug, 600 ug,
650 ug, 700 ug, 750 ug, 800 ug, 850 ug, 900 ug, 950 ug and 1 mg of
peptide. A physician may determine the actual dosage that will be
most suitable for a subject, which may vary with the age, weight
and response of the particular subject. The above dosages are
exemplary of the average case. There can, of course, be individual
instances where higher or lower dosage ranges are merited, and such
are within the scope of this invention.
[0106] The methods of generating an immune response as disclosed
herein may also include the administration of additional compounds
to augment the generation of an immune response. For example, in
addition to the Trojan antigen and immunogenic compositions
comprising a Trojan antigen, additional compounds may be
administered before or after the Trojan antigen or immunogenic
composition, or co-administered with the Trojan antigen or
immunogenic composition.
[0107] Such additional compounds include Montanide and GM-CSF.
Montanide (Montanide ISA-51) is a human-approved adjuvant, similar
to incomplete Freunds adjuvant, recognized to regulate the temporal
aspects of peptide release (Aichele et al. T cell priming versus T
cell tolerance induced by synthetic peptides. Journal of
Experimental Medicine 182(1):261-6 (1995)), used in human vaccine
therapy to stimulate the immune system. GM-CSF (granulocyte
macrophage-colony stimulating factor) is a cytokine involved in the
growth and differentiation of myeloid and monocytic lineage cells,
including dendritic cells, monocytes and tissue macrophages and
cells of the granulocyte lineage. Additional compounds include
adjuvants of the Toll-like receptor family. These adjuvants include
CpG-containing oligodeoxynucleotides, bacterial DNA,
polyinosinic-polycytidylic acid, synthetic double-stranded RNA,
synthetic IMIQUOMOD.TM., and RNA of viral or bacterial origin, or a
viral RNA mimic.
[0108] In a preferred embodiment, both Montanide and GM-CSF are
co-administered to a subject with the immunogenic composition.
Montanide may be administered at a dosage of between about 0.1 mL
and 10 mL, preferably at between 0.25 mL and 2 mL, more preferably
at 1.2 mL. GM-CSF may be administered at a dosage of between about
5 ug/m.sup.2 and 1 mg/m.sup.2, preferably at between 20 ug/m.sup.2
and 500 ug/m.sup.2, more preferably at 100 ug/m.sup.2.
[0109] In each of the methods described herein, the Trojan antigens
and immunogenic compositions comprising the Trojan antigens may be
administered to a subject by any effective, convenient manner
including, for instance, administration by topical, oral, anal,
vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal or intradermal routes among others. Formulations
suitable for parenteral administration include aqueous and
non-aqueous sterile injection solutions presented in unit-dose or
multi-dose containers. It should be also understood that, in
addition to the ingredients mentioned above, formulations of this
invention might include other agents conventional in the art having
regard to the type of formulation in question.
[0110] In each of the methods described herein, the Trojan antigens
and immunogenic compositions comprising the Trojan antigens may be
administered to a subject as a one-time dose, or as a series of two
or more doses over prolonged periods of time. In a preferred
embodiment, the Trojan antigens and immunogenic compositions of the
present invention are administered as a series of four doses, with
one dose administered each month for four months. In this
embodiment, up to four additional doses can be administered over a
further four month period. Other preferred dosing schedules include
administration daily, once a week, twice a week, 3-4 times per
week, weekly, twice a month and three times per month. The number
of doses administered varies depending on the dosing schedule, but
dosing can continue under one of the dosing schedules above for 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.
[0111] Appropriate doses for each can readily be determined by
techniques well known to those of ordinary skill in the art. Such a
determination will be based, in part, on the tolerability and
efficacy of a particular dose using techniques similar to those
used to determine proper vaccine doses. The skilled artisan will
understand that combinations of the dosing schedules indicated
above can also be used.
[0112] Each of the methods described herein may be practiced in
vitro, in vivo, or ex vivo.
[0113] In addition to the methods for generating an immune response
and methods for the treatment of SCCHN described herein, the
following additional methods are included in the present invention.
In one embodiment, a nucleotide sequence encoding a Trojan antigen
is introduced into an exogenous organism using standard molecular
biology techniques well known to those of ordinary skill in the
art, such as through the use of an expression vector described
herein. Exemplary molecular biology techniques are discussed in
Ausubel, et al., "Short Protocols in Molecular Biology." The
resulting recombinant organism can then be used as an immunogen
composition in the methods described herein. In a preferred
embodiment, an attenuated pathogenic organism serves as the
exogenous organism.
[0114] The methods of the present invention may also be practiced
using a nucleotide-containing immunogenic composition. For example,
in one embodiment, an immune response may be generated in a
subject, or a subject may be treated, by administering an Trojan
antigen-encoding polynucleotide preparation including DNA or RNA
that encodes an Trojan antigenic to the host. Preferably, the
polynucleotide preparation is administered to a mucosal inductor
site in the mucosal tissue of the host. Naked DNA may be
administered directly to the mucosa (e.g., in saline drops) or in a
recombinant gene expression vector.
[0115] Nucleotide-containing immunogenic compositions also include
live viral immunogenic compositions. The viruses for use in the
viral immunogenic compositions include immunostimulatory
polynucleotides. Preferably, a Trojan antigen is administered
through its expression by a recombinant gene expression vector.
[0116] U.S. Pat. No. 6,110,898, to Malone, et al., entitled, "DNA
vaccines for eliciting a mucosal immune response" provides detailed
teaching for the generation of such immunogenic compositions. In
particular, Malone teaches obtaining a recombinant alphavirus
vector system as described in Malone, J. G., et al., "Mucosal
immune responses associated with polynucleotide vaccination",
Behring Inst Mitt 98:63-72 (1997 February). DNA encoding a Trojan
antigen (for example) is substituted for the lacZ gene in the
vector.
[0117] Alternatively, one or more polynucleotides encoding a Trojan
antigen can be introduced to an attenuated EAEC, Salmonella spp.,
Shigella spp., Lactobacillus spp., or other attenuated bacteria
which is invasive for mucosal tissue, which then expresses the
particular Trojan antigen encoded by the polynucleotide. The
bacteria is administered to an animal to generate an immune
response to the particular Trojan antigen encoded.
Pre-Screening
[0118] Preferably, the methods of the present invention are
practiced on a subject that has a SCCHN tumor that expresses (a)
HLA-A2 antigens and (b) either MAGE-A3 or HPV 16 E7 proteins, or
(c) HLA-A2 antigens and both MAGE-A3 and HPV 16 E7 proteins.
[0119] A subject may first be typed to determine whether they are
HLA-A2 positive. A subject can be typed, for example, by obtaining
a blood sample, followed by PBL typing using a commercially
available HLA-A detection kit (Dynal) according to manufacturer's
instructions. PBLs may be also typed by PCR or flow cytometric
analysis (using, for example, BB7.2 mouse anti-human HLA-A2 mAb,
available from the ATCC), with known positive and negative controls
(Hoffmann et al. Frequencies of Tetramer+T Cells Specific for the
Wild-Type Sequence p53264-272 Peptide in the Circulation of
Patients with Head and Neck Cancer. Cancer Research
62(12):3521-3529 (2002)).
[0120] Tumors from patients who are HLA-A2 positive may then be
evaluated for the expression of HLA-A2, MAGE-A3, and HPV16 E7.
HLA-A2 expression may be evaluated by immunohistochemistry. For
example, following biopsy tumors can be frozen in OCT blocks,
sectioned onto glass slides, and stained with hematoxylin and eosin
(H&E), anti-HLA-A2 antibody and secondary antibody, or
secondary antibody alone per the DAKO EnVision+Protocol (DAKO
Corporation, Carpinteria, Calif.). Normal lung parenchyma from a
known HLA-A2 positive patient may serve as a positive control.
[0121] HPV 16 E7 and MAGE-A3 detection may be performed using
techniques previously published (Strome et al. Squamous Cell
Carcinoma of the Tonsils: A Molecular Analysis of HPV Associations.
Clinical Cancer Research 18:1093-1100 (2002); Kienstra et al.
Identification of NY-ESO-1, MAGE-1, and MAGE-3 in head and neck
squamous cell carcinoma. Head and Neck 25(6):457-463 (2003)).
Briefly, samples may be amplified using specific primers to:
TABLE-US-00001 E6 region of HPV 16 (325 bp) (SEQ ID NO:23)
5'-CCACAGTTATGCACAGAGCTGCAAACAACTATACAT (HPV16-E6-140-36D) (SEQ ID
NO:24) 5'-TTGTCCAGATGTCTTTGCTTTTCTTCAGGACACAGT (HPV16-E6-465-36U)
MAGE-A3 primer (423 bp) (SEQ ID NO:25) 5'-GAAGCCGGCCCAGGCTCG (SEQ
ID NO:26) 5'-GGAGTCCTCATAGGATTGGCTCC
[0122] The amplification reaction may be performed in 50 .mu.l
containing 10 mM Tris pH 8.3, 50 mM KCl, 2.0-mM MgCl2, 200 .mu.M
each dNTP (100 .mu.M dUTP and 100 .mu.M dTTP), 2.5 units Amplitag
gold (5 U/.mu.l, Perkin Elmer), 0.1% bovine serum albumin (BSA),
19.5 .mu.l RNase free water, 0.5 .mu.m of each primer and 5 .mu.l
of sample DNA. PCR cycling conditions may be 95.degree. C. for 10
min followed by 40 cycles of 95.degree. C. for 1 min, 55.degree. C.
for 1 min, 72.degree. C. for 1 min followed by 10 min at 72.degree.
C. After amplification, 15 .mu.l of each sample may be run on a 2%
agarose gel containing 20 .mu.g ethidium bromide in an 100 ml gel
to visualize products. DNA from the Caski cell line and DNA from a
known MAGE-A3 positive tumor may be used as a positive PCR control
to assess the success of the amplification. PCR reagents lacking
DNA (no sample added) may serve as a negative control in each PCR
amplification.
EXAMPLES
Example 1
Identification of MAGE-A3 Antigenic Epitopes
[0123] Using a predictive algorithm based on the presence of MHC
binding motifs, an HLA-A1-binding peptide from MAGE-A3 that induced
in vitro anti-tumor CTL responses with lymphocytes from normal
individuals was identified (Celis et al. Induction of anti-tumor
cytotoxic T lymphocytes in normal humans using primary cultures and
synthetic peptide epitopes, PNAS USA 91(6):2105-2109 (1994)). In
addition to the HLA-A1-restricted epitope, an HLA-A2 restricted CTL
epitope, which is more frequently found in the general population
than HLA-A1, was identified (Kawashima et al. The multi-epitope
approach for immunotherapy for cancer: identification of several
CTL epitopes from various tumor-associated antigens expressed on
solid epithelial tumors. Human Immunology 59(1):1-14 (1998)).
CTL-induced by peptide MAGE-A3.sub.112-120 (KVAELVHFLL; SEQ ID
NO:2) were quite effective in recognizing tumor cells expressing
MAGE-A3 antigen and HLA-A2 (FIG. 1).
[0124] Boon et al. reported the existence of another
HLA-A2-restricted epitope from MAGE-A3, namely MAGE-A3.sub.271-279
(FLWGPRALV; SEQ ID NO:1). This epitope was also efficient in
inducing CTL responses to tumors expressing MAGE-A3 antigen (van
der Bruggen et al. A peptide encoded by human gene MAGE-3 and
presented by HLA-A2 induces cytolytic T lymphocytes that recognize
tumor cells expressing MAGE-3. European Journal of Immunology
24(12):3038-43 (1994)).
[0125] In summary, the two HLA-A2-restricted CTL epitopes described
above, MAGE-A3.sub.112-120 and MAGE-A3.sub.271-279, have been
proven to be effective in generating cytotoxic responses against
tumors expressing the MAGE-A3 antigen.
Example 2
Identification of a Promiscuous T Helper Epitope from MAGE-A3
[0126] A promiscuous T helper epitope that is presented to T cells
by HLA-DR4 and HLA-DR7, two of the most frequently found MHC class
II alleles, was identified. Peptide MAGE-A3.sub.149-160
(VIFSKASSSLQL; SEQ ID NO:5) was found to stimulate T helper
lymphocytes that recognized recombinant MAGE-A3 protein or cell
lysates from tumors expressing MAGE-A3 antigen (Kobayashi et al.
Tumor-reactive T helper lymphocytes recognize a promiscuous MAGE-A3
epitope presented by various major histocompatibility complex class
II alleles. Cancer Research 61(12):4773-8 (2001)). As shown in FIG.
2, HLA-DR4-restricted HTL clone 8G9 recognizes naturally processed
MAGE-A3 antigen. A: Proliferative T-cell response induced by
MAGE-A3.sub.146-160 (+Peptide), recombinant MAGE-A3 protein
(rMAGE-A3) or recombinant gp100 (rgp100). B: Tissue culture
supernatants from experiment described in panel A, were collected
after 48 hr and the concentration of GM-CSF was measured by ELISA.
C: T-cell clone 8G9 recognizes UV-irradiated melanoma cells that
express MAGE-A3 (HT-144 (+), SKmel-28 (+), 697mel (+)) via antigen
cross-presentation by autologous DC. DC incubated with MAGE-A3
negative melanoma cell line (888mel (-)) did not stimulate the
T-cell clone. Autologous DC were incubated with irradiated melanoma
cells at a 1:1 ratio for 48 hours. The antigen-pulsed DC were then
mixed with HTL (at a 1:20 ratio) and 2 days later culture
supernatants were collected and assayed for the presence of GM-CSF.
Values shown are the means of triplicate determinations; bars,
SD.
Example 3
Identification and Selection of CTL and T Helper Epitopes from HPV
16 E7
[0127] Two HLA-A2-restricted CTL epitopes and one T helper epitope
were selected. The identification and description of these epitopes
have been published (de Jong et al. Frequent detection of human
papillomavirus 16 E2-specific T-helper immunity in healthy
subjects. Cancer Research 62(2):472-479 (2002); Kast et al. Role of
HLA-A motifs in identification of potential CTL epitopes in human
papillomavirus type 16 E6 and E7 proteins. Journal of Immunology
152(8):3904-3912 (1994)). Furthermore, the HLA-A2-restricted CTL
epitopes were shown to induce CTL responses (Jager et al.
Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral
and cellular immune responses. PNAS USA 97(9):4760-4765 (2000); den
Haan et al. Identification of a graft versus host
disease-associated human minor histocompatibility antigen. Science
268(5216):1476-1480 (1995); Bennouna et al. Application of IL-5
ELISPOT assays to quantification of antigen-specific T helper
responses. Journal of Immunological Methods 261(1-2):145-156
(2002)).
Example 4
Control Experiment Using Ovalbumin Antigenic Epitope
[0128] A Trojan antigen comprising the mouse CTL epitope from
ovalbumin (SIINFEKL; SEQ ID NO:11), coupled to the HIV TAT
transporter peptide (RKKRRQRRR; SEQ ID NO:9) via an AAA linked was
preparing, yielding the RKKRRQRRRAAASIINFEKL (SEQ ID NO:12).
[0129] The capacity of the ovalbumin Trojan antigen to be processed
and presented by DC to OT-1 T cells specific for the SIINFEKL (SEQ
ID NO:11) epitope was studied. As shown in FIG. 3, DC that were
pulsed with the ovalbumin Trojan antigen remained stimulatory for T
cells after a 48 hour period of incubation, while the DC that were
incubated with the antigenic epitope alone had lost most of their
stimulatory activity. This experiment demonstrated that pulsing the
DC with Trojan antigen allowed these APC to present the epitope for
a longer period of time, compared with APC pulsed with only the
SIINFEKL (SEQ ID NO:11) antigenic epitope. C57/BL6 DC were pulsed
with either SIINFEKL or TrojAg (RKKRRQRRRRAAASIINFEKL) for 2 hours,
washed three times, and then kept in 37.degree. C. After 48 hours,
the ability of peptide-loaded DC to induce IFN-gamma release from
OT-1 T cells was determined by ELISA. The concentration of
IFN-gamma was determined at various dilutions of the supernatant
that was collected after 24 hours of incubation of the DC with the
OT-1 T cells.
Example 5
HPV 16 E7 Trojan Antigens Stimulate Interferon Gamma Release From
HLA-A2 T cells
[0130] To study the ability of the HPV 16 E7 Trojan antigen to
stimulate functional T cell reactivity, levels of Interferon Gamma
(IFN .gamma.) release from Trojan antigen-stimulated HLA-A2
positive T cells versus IFN .gamma. production from T cells
stimulated with the constituent peptide epitopes, were determined.
Briefly, freshly isolated HLA-A2 positive CD8+ (CTLs) and CD4+
(HTLs) were positively selected by Dynal magnetic bead separation
(Dynal ASA). T cells were stimulated with irradiated autologous
CD8-/CD4-PBLs (flow-through from the magnetic bead separation)
pulsed with TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXDRKKRRQRRR (SEQ ID
NO:22) or the constituent HLA-A2.1 epitope TLGIVXPI (SEQ ID NO:18),
where each X is aminobutyric acid, for 48 hours in 96-well
Immobilon-P membrane multiscreen plate (Millipore) coated with IFN
.gamma.-specific capture antibody (BD Biosciences for IFN .gamma.).
The wells were washed, treated with Biotin-conjugated detection
antibody and 3-amino-9-ethylcarbazole (AEC) for substrate
development.
[0131] Spots were counted by first obtaining digitized images of
the wells (performed by C.T.L. Analyzers, Cleveland, Ohio) and then
analyzing these images with software purchased from C.T.L.
Analyzers. The frequency of spots (250,000 cells per well) obtained
with the Trojan and constituent peptides was compared and found to
be equivalent at 0.02% T cell reactivity for both peptides. The
results of this experiment are shown in FIG. 4.
Example 6
Expression Patterns of HLA-A2 in SCCHN
[0132] To demonstrated the ability to quantitate HLA-A2 expression
on fresh SCCHN, immunohistochemical analysis was performed on five
freshly isolated SCCHN, using the HLA-A2 specific mAb SBO3-111
(kindly provided by Dr. Soldano Ferrone, Roswell Park Cancer
Institute, Buffalo, N.Y.).
[0133] As shown in FIG. 5, stromal tissue demonstrated a membranous
cytoplasmic-staining pattern. Tissue from patients who were
negative for HLA-A2 by PCR, did not display immunoreactivity to
this mAb. In one HLA-A2 positive patient, the parenchyma was
observed to express HLA-A2, but the tumor was negative.
[0134] The samples were prepared using freshly isolated tumors snap
frozen in October 5 micron sections were cut onto charged glass
slides. Specimens were stained with H&E, mouse Ig control or
SB03-111, using previously published techniques (Dong et al.
Tumor-associated B7-H1 promotes T-cell apoptosis. A potential
mechanism of immune evasion. Nature Medicine 8:793-800 (2002)). The
optimal dilution for SB03-111 was 1/2500 (data not shown).
[0135] All documents and publications referenced herein are hereby
expressly incorporated by reference in their entirety. In
particular, Grant Application No. DE015324 is hereby expressly
incorporated by reference in its entirety.
[0136] The invention of this application has been described above
both generically and with regard to specific embodiments. Although
the invention has been set forth in what is believed to be the
preferred embodiments, a wide variety of alternatives known to
those of skill in the art can be selected within the generic
disclosure. The invention is not otherwise limited, except for the
recitation of the claims set forth below.
Sequence CWU 1
1
28 1 9 PRT Homo sapiens 1 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 2
9 PRT Homo sapiens 2 Lys Val Ala Glu Leu Val His Phe Leu 1 5 3 14
PRT Homo sapiens 3 Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg
Ala Arg 1 5 10 4 15 PRT Homo sapiens 4 Thr Ser Tyr Val Lys Val Leu
His His Met Val Lys Ile Ser Gly 1 5 10 15 5 12 PRT Homo sapiens 5
Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu 1 5 10 6 9 PRT
Human papillomavirus 6 Met Leu Asp Leu Gln Pro Glu Thr Thr 1 5 7 8
PRT Human papillomavirus 7 Thr Leu Gly Ile Val Cys Pro Ile 1 5 8 22
PRT Human papillomavirus 8 Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala
His Tyr Asn Ile Val Thr 1 5 10 15 Phe Cys Cys Lys Cys Asp 20 9 9
PRT Human immunodeficiency virus 9 Arg Lys Lys Arg Arg Gln Arg Arg
Arg 1 5 10 4 PRT Artificial Sequence Chemically-synthesized furin
cleavable linker 10 Arg Val Lys Arg 1 11 8 PRT Artificial Sequence
Chemically-synthesized antigenic peptide 11 Ser Ile Ile Asn Phe Glu
Lys Leu 1 5 12 21 PRT Artificial Sequence Chemically-synthesized
antigenic peptide 12 Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Ala
Ala Ala Ser Ile Ile 1 5 10 15 Asn Phe Glu Lys Leu 20 13 22 PRT
Artificial Sequence Chemically-synthesized antigenic peptide 13 Lys
Val Ala Glu Leu Val His Phe Leu Arg Val Lys Arg Phe Leu Trp 1 5 10
15 Gly Pro Arg Ala Leu Val 20 14 13 PRT Artificial Sequence
Chemically-synthesized antigenic peptide 14 Lys Val Ala Glu Leu Val
His Phe Leu Arg Val Lys Arg 1 5 10 15 35 PRT Artificial Sequence
Chemically-synthesized antigenic peptide 15 Lys Val Ala Glu Leu Val
His Phe Leu Arg Val Lys Arg Phe Leu Trp 1 5 10 15 Gly Pro Arg Ala
Leu Val Arg Val Lys Arg Arg Lys Lys Arg Arg Gln 20 25 30 Arg Arg
Arg 35 16 38 PRT Artificial Sequence Chemically-synthesized
antigenic peptide 16 Lys Val Ala Glu Leu Val His Phe Leu Arg Val
Lys Arg Phe Leu Trp 1 5 10 15 Gly Pro Arg Ala Leu Val Arg Val Lys
Arg Val Ile Phe Ser Lys Ala 20 25 30 Ser Ser Ser Leu Gln Leu 35 17
47 PRT Artificial Sequence Chemically-synthesized antigenic peptide
17 Lys Val Ala Glu Leu Val His Phe Leu Arg Val Lys Arg Phe Leu Trp
1 5 10 15 Gly Pro Arg Ala Leu Val Arg Val Lys Arg Val Ile Phe Ser
Lys Ala 20 25 30 Ser Ser Ser Leu Gln Leu Arg Lys Lys Arg Arg Gln
Arg Arg Arg 35 40 45 18 8 PRT Human papillomavirus MISC_FEATURE
(6)..(6) "X" is cysteine or aminobutyric acid 18 Thr Leu Gly Ile
Val Xaa Pro Ile 1 5 19 21 PRT Artificial Sequence
Chemically-synthesized antigenic peptide 19 Thr Leu Gly Ile Val Xaa
Pro Ile Arg Val Lys Arg Arg Lys Lys Arg 1 5 10 15 Arg Gln Arg Arg
Arg 20 20 22 PRT Human papillomavirus MISC_FEATURE (18)..(18) "X"
is cysteine or aminobutyric acid 20 Pro Ala Gly Gln Ala Glu Pro Asp
Arg Ala His Tyr Asn Ile Val Thr 1 5 10 15 Phe Xaa Xaa Lys Xaa Asp
20 21 34 PRT Artificial Sequence Chemically-synthesized antigenic
peptide 21 Thr Leu Gly Ile Val Xaa Pro Ile Arg Val Lys Arg Pro Ala
Gly Gln 1 5 10 15 Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr
Phe Xaa Xaa Lys 20 25 30 Xaa Asp 22 43 PRT Artificial Sequence
Chemically-synthesized antigenic peptide 22 Thr Leu Gly Ile Val Xaa
Pro Ile Arg Val Lys Arg Pro Ala Gly Gln 1 5 10 15 Ala Glu Pro Asp
Arg Ala His Tyr Asn Ile Val Thr Phe Xaa Xaa Lys 20 25 30 Xaa Asp
Arg Lys Lys Arg Arg Gln Arg Arg Arg 35 40 23 36 DNA Human
papillomavirus 23 ccacagttat gcacagagct gcaaacaact atacat 36 24 36
DNA Human papillomavirus 24 ttgtccagat gtctttgctt ttcttcagga cacagt
36 25 18 DNA Homo sapiens 25 gaagccggcc caggctcg 18 26 23 DNA Homo
sapiens 26 ggagtcctca taggattggc tcc 23 27 314 PRT Homo sapiens 27
Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu 1 5
10 15 Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro
Ala 20 25 30 Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu
Val Glu Val 35 40 45 Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro
Asp Pro Pro Gln Ser 50 55 60 Pro Gln Gly Ala Ser Ser Leu Pro Thr
Thr Met Asn Tyr Pro Leu Trp 65 70 75 80 Ser Gln Ser Tyr Glu Asp Ser
Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95 Thr Phe Pro Asp Leu
Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 100 105 110 Val Ala Glu
Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120 125 Pro
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 130 135
140 Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu
145 150 155 160 Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly
His Leu Tyr 165 170 175 Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp
Gly Leu Leu Gly Asp 180 185 190 Asn Gln Ile Met Pro Lys Ala Gly Leu
Leu Ile Ile Val Leu Ala Ile 195 200 205 Ile Ala Arg Glu Gly Asp Cys
Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215 220 Leu Ser Val Leu Glu
Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly 225 230 235 240 Asp Pro
Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu 245 250 255
Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 260
265 270 Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu
His 275 280 285 His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr
Pro Pro Leu 290 295 300 His Glu Trp Val Leu Arg Glu Gly Glu Glu 305
310 28 98 PRT Human papillomavirus 28 Met His Gly Asp Thr Pro Thr
Leu His Glu Tyr Met Leu Asp Leu Gln 1 5 10 15 Pro Glu Thr Thr Asp
Leu Tyr Cys Tyr Glu Gln Leu His Asp Ser Ser 20 25 30 Glu Glu Glu
Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp 35 40 45 Arg
Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr 50 55
60 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu
65 70 75 80 Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys
Ser Gln 85 90 95 Lys Pro
* * * * *