Download Mucosal Alpha-Galactosylceramide Delivery

Efficacy of combination mucosal
vaccination and immunotherapy strategies
for the treatment of HPV-associated cancers
Jagan Sastry, PhD
Professor, Department of Immunology
Professor, Department of Veterinary Sciences
The University of Texas MD Anderson Cancer Center
Associate Development Core Director
UT-Baylor Center for AIDS Research
Most pathogens are transmitted via
mucosal routes: Genital, Oral, Nasal
Viruses:
•
•
•
HIV, HPV, Genital Herpes (sexual transmission)
Rota Virus, Hepatitis-A virus (oral)
Influenza Virus, Respiratory syncytial virus (pulmonary)
Bacteria:
•
•
•
•
•
Mycobacterium tuberculosis (pulmonary)
Salmonella (oral)
Helicobacter pylori (oral/GI)
E. coli (oral/GI)
Neisseria gonorrhea (sexual tranmission)
Fungi/Yeast:
•
•
•
•
•
Aspergillus fumigatus: Aspergillosis (pulmonary)
Candida albicans: Candidiasis (oral thrush and vaginitis)
Histoplasma capsulatum: Histoplasmosis (pulmonary)
Coccidioides immitis: Coccidioidomycosis (pulmonary)
Cryptococcus neoformans: Cryptococcosis (pulmonary, GI)
Mucosal tissues are targets for
primary and/or metastatic tumors
Because of the circulatory pattern and the selective affinity
of the endothelium for cancer cells, the lung is the second
most commonly targeted organ for metastases after liver
• Pulmonary metastases are frequent in melanoma,
breast, colorectal, head and neck, prostrate and renal
cancers
Important concern:
• In general, most pre-clinical cancer vaccine studies rely
on extrapolating the observations of protection in mouse
models against subcutaneous tumors to mucosal tumors
Common Mucosal Immune System
• Vaccination at the easily assessable oral/nasal mucosal surfaces
induces immunity at the local as well as distant difficult to reach
genital mucosal tissues
Holmgren J., Czerkinsky C., Nature Medicine 2005.
• Because of the potential to induce more wide-spread immune
responses in addition to the ease of application, the oral and nasal
routes are more popularly explored for mucosal delivery of antigens
Mucosal Immunity
• Mucosal immune cells:
– protect the host from potentially harmful pathogens
– Most mucosal immune cells are educated at specific
inductive sites in the local mucosal-associated
lymphoid tissues (MALT) and subsequently move into
and protect mucosal barriers
However:
– prevent development of immune responses to
commensal microbiota and harmless food and
environmental antigens: tolerogenic
Hence, stimulation of mucosal immunity
necessitates inclusion of ADJUVANTS
Adjuvants
• Adjuvants enhance immune responses to coadministered antigens
– Adjuvants typically function by activating innate
immune cells such as Dendritic Cells (DC)
– Currently, only the alum adjuvant has been FDA
approved for use in vaccines in the US
– Bacterial toxins (and their mutant versions) are
potent mucosal adjuvants; but the toxicity (despite
mutations) is a concern for human use approvals
– There is a need for the development of more
adjuvants, particularly those that can modulate innate
immunity and also administered by mucosal routes
Alpha-Galactosylceramide
Kim S et al., Expert Rev Vaccines 2008.
• The synthetic glycolipid alpha-glactosylceramide
(α-GalCer) is a potent activator of natural killer T
(NKT) cells.
• NKT cells are a major innate immune mediator cell
type effective in inducing maturation of dendritic
cells (DC) for efficient presentation of coadministered antigens
Alpha-Galactosylceramide
•The a-GalCer adjuvant functions
as a ligand to activate NKT cells
when presented by the CD1d
molecule, particularly on dendritic
cells.
•Presentation of a-GalCer by DC
leads to rapid IFN-g production
and proliferation by the NKT cells.
• This is followed by activation of
DC that are activated to present
antigens to T cells and their
proliferation and function
•A-GalCer is safe for human use
DC
NKT-Cell
αGalCer
IL-4, IFNγ, IL-2
+ Antigen
T-Cell
T-Cell
T-Cell
T-Cell
T-Cell
Fujii, SI et. al. Activation of Natural Killer T Cells by aGalactosylceramide Rapidly Induces the Full maturation
of Dendritic Cells in vivo and Thereby Acts as an
Adjuvant for Combined CD4 and CD8 T Cell Immunity
to a Coadministered Protein. J. Exp. Med. 2003.
T cell responses
Intranasal Immunization
using α-GalCer Adjuvant
Day 5
Immunize
Immunize/
Sacrifice
IFN-γ SFU/ 1x 106 Cells
Day 0
10000
Day 10
Day 15
Immunize
/Sacrifice
Sacrifice
IFNγ ELISPOT Assay
1000
100
10
1
Spleen
Antibody responses
6
Tissue
Courtney AN, et al.
Vaccine 2009.
7
1D
2D
MLN
3D
1D
2D
3D
*
**
6
Vaginal IgA
(Reciprocal log5 Titer)
Serum IgG
(Reciprocal log10 Titer)
5
4
*
5
*
4
3
3
2
2
1
Ova
Ova + aGC
IgG
Ova
Ova + aGC
Multiple
immunizations by the
intranasal mucosal
route using aGalCer
adjuvant Induces
Progressively
Increasing Antigen
Specific Immune
Responses
Mucosal vaccination against
Human papillomavirus (HPV)-associated cancers
The 150+ different types of HPV are broadly classified as
Low Risk
High Risk
HPV6, 11,
HPV16, 18,
(Warts)
(Cervical Cancer)
The pre-cancerous lesions are described as cervical intraepithelial
neoplasia (CIN) and are classified based on disease severity:
CIN I:
low-grade dysplasia
CIN II:
moderate dysplasia
CIN III:
high-grade dysplasia
CIS:
carcinoma in situ
ICC:
Cervical Cancer
Human papillomavirus (HPV)
• The HPV genome encodes for six different
early proteins (E1, E2, E4, E5, E6, and E7) and
two late proteins (L1 and L2).
Schiffman Lancet (2007) 370:890-907
E6 and E7


L1, L2
The L1 and L2 proteins are important
for virus binding and entry into
epithelial cells.
In infected cells, the E6 and E7 proteins
of high-risk serotypes cause degradation
of cellular tumor suppressor proteins p53
and pRB and oncogenic transformation
The currently approved vaccines are based on the L1 gene and therefore can prevent
initial infection but can not protect against the pre- and cancer lesions where only the
E6 and E7 genes of the virus are expressed
Human papillomavirus (HPV)
The pre-cancerous lesions of the cervix:
Cervical Intraepithelial Neoplasia (CIN)
• Typically, these precancerous lesions regress
spontaneously
• Under conditions of immunodeficiency
(AIDS/Transplatation) — CIN may eventually
progress to invasive cervical cancer (ICC)
HPVs also cause some cancers of the anus, vulva,
vagina, penis, and the oropharynx (throat, soft
palate, the base of the tongue, and the tonsils)
Treatments for HPV-CIN
 Methods commonly used to treat cervical lesions include
cryosurgery (freezing that destroys tissue), LEEP (loop
electrosurgical excision procedure, or the removal of tissue
using a hot wire loop), and conization (surgery to remove a
cone-shaped piece of tissue from the cervix and cervical canal)
 However, a significant number of patients (13-19%) experience
recurrence and it is not clear what the reasons are or what if
any is the relation to HPV-specific immunity.
Hypothesis: Immune memory to HPV, specifically to the E6 and E7
oncoproteins, is necessary for recurrence-free survival posttreatment for HPV-associated CIN.
• To test this hypothesis we conducted a cross-sectional study
in HPV patients
Cross-sectional Study population
-/CIN-)
Group 1. (HPV
100
% Population with positive
HPV-specific immunity
100
80
E6 Peptides
E6 Peptides
E7 Peptides
E7 Peptides
% Population with positive
HPV-specific immunity
Control women: negative for both HPV and cervical
80
60 (CIN-): n=6
intraepithelial neoplasia
60+/CIN+)
Group 2. (HPV
40
+ and with newly diagnosed CIN lesions
Women HPV
40
20
+
(CIN ): n = 33
Treated
(Recurrence+)
Treated
(Recurrence+)
Treated
(Recurrence-)
0
Treated
(Recurrence-)
Untreated
(HPV+CIN+)
Group 3. (Recur-)
Untreated
(HPV+CIN+)
Control
(HPV-/CIN-)
20
Group 4. (Recur+)
Control
(HPV-/CIN-)
0
Disease-free
after excisional/ablative treatment for
HPV-CIN (at least six months post-treatment): n = 22
Exhibiting recurrence or persistence of disease after
excisional/ablative treatment for HPV-CIN (at least six
months
post-treatment):
n = 10 in the blood
T cell
proliferation
response
E6 Peptides
60
40
20
•
Treated
(Recurrence-)
Untreated
(HPV+CIN+)
0
Treated
(Recurrence+)
•
E7 Peptides
80
Control
(HPV-/CIN-)
Dominant proliferative
responses
in Recur- subjects
% Population with positive
HPV-specific immunity
100
E6 peptide:
– Q15L (43-57) QLLRREVYDFAFRDL
E7 peptide:
– Q19D (44-62) QAEPDRAHYNIVTFCCKCD
It has been reported that:
• Production of TH1-type of cytokines (e.g. IL-12 and IFN-g) was
defective in women with extensive HPV infection.
• Progression to CIN was associated with a shift from TH1- to TH2or immunosuppressive-type (e.g. IL-4 and IL-10) of cytokine
production
Outcome from the cross-sectional study
 Peptides Q15L and Q19D, corresponding to the E6 and
E7 oncoproteins of HPV-16, respectively could
potentially be useful as:
 Indicators of protective immunity, (prognostic bio-markers)
 Immunotherapy (therapeutic vaccine)
To validate these results from the cross-sectional study we
performed a prospective study with 250 patients
BL 1 Mo
4 Mo
6 Mo
9 Mo
12 Mo
Diagnosis
CIN II or
CIN III
LEEP = Loop Electrosurgical Excision Procedure
18 Mo
24 Mo
Treatment influence on HPV immunity
E6 Peptide: Q15L (43-57) QLLRREVYDFAFRDL
E7 Peptide: Q19D (44-62) QAEPDRAHYNIVTFCCKCD
•Vaccination with these HPV E6 & E7 peptides to induce/enhance HPV-specific
immunity for protection against HPV lesions is a potential option
•The immunity needs to be specifically at the genital mucosal tissues: i.e.
Mucosal T cell Immunity
Intranasal immunization with HPV peptide
Day 0
Day 5
Day 10
Prophylactic vaccination study
250
Immunize
Sacrifice/Tumor Challenge
Immunize
Tumor size (mm^2)
200
150
Unrelated tumor
HPV tumor
100
% IFNg+ cells
10
CD8
CD4
1
50
0
0
10
20
Days
30
40
0.1
CLN
VALT
CLN
E7 Peptide
% Specific killing
70
E6 Peptide
HPV peptide vaccine
primes mucosal
immunity and Tumor
protection
HPV tumor
60
Control
50
Unrelated
tumor
40
30
20
10
0
Spleen
VALT
CLN
Therapeutic intranasal immunization
with HPV vaccine against HPV tumors
250
350
αGalCer
αGalCer + HPV peptides
300
200
PBS
Antigen
Adjuvant
Vaccine
HPV peptides
120
d32
d24
d6
d12
PBS
250
100
150
200
% Survival
Tumor size (mm2)
Peptides Q15D and Q19D
with aGalCer adjuvant
Immunizations
150
100
80
60
3/6
100
40
50
50
20
0/5
0/4
0/4
* p<0.05
* p<0.05
* p<0.05
0
0
D4 D7 D9 D11 D14 D18 D21 D24 D28 D30 D32 D35 D37
444
7 7 79 9 11 11
9 14 14
18
11 18
21 14
24
18 30 2832
21 3035 24
37
21 2824
32
Days after tumor challenge
Days after tumor challenge
HPV peptide vaccine significantly reduced HPV tumor growth resulting in
survival advantage
While effective in reducing tumor growth, the HPV peptide vaccine was
inefficient in eliminating the tumor
This may be because of the immunosuppressive tumor microenvironment
• Immune suppressive with
• Accumulated regulatory T cells
• Decreased/compromised
Antigen presentation, and
• Exhausted/inhibited Effector T
cell responses
Tumor
microenvironment
Or
Tumor cell
Professor and Chair
Department of Immunology
The UT MD Anderson
Cancer Center
400
350
300
250
200
150
200
150
50
50
0
0
4
6 8 11 13 15 18 20 22 25
Post-tumor-challenge (days)
4
450
PBS
Vaccine
aCTLA-4
Vaccine + aCTLA-4
400
350
300
250
200
150
6 8 11 13 15 18 20 22 25
Post-tumor-challenge (days)
PBS
Vaccine
a4-1BB
Vaccine + a4-1BB
400
Tumor size (mm2)
350
Tumor size (mm2)
d12 (V+Ab)
d9 (Ab)
d6 (V+Ab)
250
100
300
250
200
150
100
100
*
50
**
50
0
0
4
Scheme
300
100
450
Agonistic antibody
to 4-1BB
PBS
Vaccine
aPD-1
Vaccine + PD-1
400
350
Intraperitoneal
injections of
Immune check point
antibodies:
Antagonistic
antibodies to
CTLA-4 and PD-1
450
PBS
Vaccine
Tumor size (mm2)
Intranasal Vaccine:
E6 and E7 peptides
(100ug each in PBS)
450
Tumor size (mm2)
Vaccine +
immunotherapy of
HPV tumors
6 8 11 13 15 18 20 22 25
Post-tumor-challenge (days)
4
6 8 11 13 15 18 20 22 25
Post-tumor-challenge (days)
Collaborators: Michael Curran, PhD, James Allison, PhD; Immunology
Vaccine immunotherapy of
Vaginal HPV tumors
Vaccine immunotherapy of
Vaginal HPV tumors
1.E+08
Avg Radiance
1.E+07
PBS
Vaccine
Vaccine + a4-1BB
Vaccine + aCTLA-4
a4-1BB
aCTLA-4
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
6
8
11
15
Days Post Tumor Challenge
19
Vaccine immunotherapy of
HPV tumors
Combination of 4-1BB and
CTLA-4 antibodies
This combination
augmented HPV E6/E7
vaccine by increasing
CD8 infiltration and
decreasing Tregs in tumors
Acknowledgements
Drs. Michael Curran and James Allison
Immunology
Seth Wardell
Res. Technician
Corinne Bell
MS student
Ameerah
Wishahi
Graduate
Student
Shailabala
Singh
Post-Doctoral Guojun Yang
Fellow
Research
Investigator
Amy Courtney
PhD student
Danielle Fonenot
PhD student
Dr. Hong (Helen) He
Res. Investigator
Dr. Michael Barry, Mayo Clinic, Rochester, MN
Dr. Chun Wang, Univ. Minnesota, Minneapolis, MN
Pramod Nehete, PhD, Assoc. Prof
Bharti Nehete, Research Asst.