Efficacy of combination mucosal vaccination and immunotherapy strategies for the treatment of
HPV-associated cancers
Jagan Sastry, PhD
Professor, Department of ImmunologyProfessor, Department of Veterinary Sciences
The University of Texas MD Anderson Cancer CenterAssociate Development Core Director UT-Baylor Center for AIDS Research
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)
Most pathogens are transmitted via mucosal routes: Genital, Oral, Nasal
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
Holmgren J., Czerkinsky C., Nature Medicine 2005.
• Vaccination at the easily assessable oral/nasal mucosal surfaces induces immunity at the local as well as distant difficult to reach genital mucosal tissues
• 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
Common Mucosal Immune System
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 enhance immune responses to co-administered 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
Adjuvants
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 co-administered antigens
• 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
NKT-Cell
IL-4, IFNγ, IL-2
αGalCer
DC
T-Cell
+ Antigen
T-CellT-CellT-CellT-Cell
Fujii, SI et. al. Activation of Natural Killer T Cells by a-Galactosylceramide 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.
Alpha-Galactosylceramide
Intranasal Immunization using α-GalCer Adjuvant
Courtney AN, et al. Vaccine 2009.
Antibody responses
T cell responses
IFNγ ELISPOT Assay
1
10
100
1000
10000
Spleen MLN
IFN
-γS
FU
/ 1x
10
6C
ells
Tissue
Day 0 Day 5
Immunize Immunize/Sacrifice
Day 10
Immunize/Sacrifice
Day 15
Sacrifice
Multiple immunizations by the intranasal mucosal route using aGalCer adjuvant Induces Progressively Increasing Antigen Specific Immune Responses
1
2
3
4
5
6
7
Ova Ova + aGC
Va
gin
al I
gA
(R
eci
pro
ca
l lo
g5
Tit
er)
1D 2D 3D*
**
*
*
2
3
4
5
6
Ova Ova + aGC
IgG
Se
rum
Ig
G
(R
eci
pro
ca
l lo
g10
Tit
er)
1D 2D 3D
High Risk
HPV16, 18,
(Cervical Cancer)
Low Risk
HPV6, 11,
(Warts)
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
The 150+ different types of HPV are broadly classified as
Mucosal vaccination againstHuman papillomavirus (HPV)-associated cancers
Human papillomavirus (HPV)
· 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 HPV genome encodes for six different early proteins (E1, E2, E4, E5, E6, and E7) and two late proteins (L1 and L2).
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
Schiffman Lancet (2007) 370:890-907
L1, L2
E6 and E7
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 post-treatment for HPV-associated CIN.
• To test this hypothesis we conducted a cross-sectional study in HPV patients
Cross-sectional Study population
Group 1. (HPV-/CIN-)
Control women: negative for both HPV and cervical intraepithelial neoplasia (CIN-): n=6
Group 2. (HPV+/CIN+)
Women HPV+ and with newly diagnosed CIN lesions (CIN+): n = 33
Group 3. (Recur-)
Disease-free after excisional/ablative treatment for HPV-CIN (at least six months post-treatment): n = 22
Group 4. (Recur+)
Exhibiting recurrence or persistence of disease after excisional/ablative treatment for HPV-CIN (at least six months post-treatment): n = 10
0
20
40
60
80
100
Co
ntr
ol
(H
PV
-/C
IN-)
Un
tre
ate
d
(HP
V+
CIN
+)
Tre
ate
d
(Re
cu
rre
nc
e-)
Tre
ate
d
(Re
cu
rre
nc
e+)
% P
op
ula
tio
n w
ith
po
sit
ive
H
PV
-sp
eci
fic
imm
un
ity
E6 Peptides E7 Peptides
0
20
40
60
80
100
Co
ntr
ol
(H
PV
-/C
IN-)
Un
tre
ate
d
(HP
V+
CIN
+)
Tre
ate
d
(Re
cu
rre
nc
e-)
Tre
ate
d
(Re
cu
rre
nc
e+)
% P
op
ula
tio
n w
ith
po
sit
ive
H
PV
-sp
eci
fic
imm
un
ity
E6 Peptides E7 Peptides
0
20
40
60
80
100
Co
ntr
ol
(H
PV
-/C
IN-)
Un
tre
ate
d
(HP
V+
CIN
+)
Tre
ate
d
(Re
cu
rre
nc
e-)
Tre
ate
d
(Re
cu
rre
nc
e+)
% P
op
ula
tio
n w
ith
po
sit
ive
H
PV
-sp
eci
fic
imm
un
ity
E6 Peptides E7 Peptides
0
20
40
60
80
100
Co
ntr
ol
(H
PV
-/C
IN-)
Un
tre
ate
d
(HP
V+
CIN
+)
Tre
ate
d
(Re
cu
rre
nc
e-)
Tre
ate
d
(Re
cu
rre
nc
e+)
% P
op
ula
tio
n w
ith
po
sit
ive
H
PV
-sp
eci
fic
imm
un
ity
E6 Peptides E7 Peptides
0
20
40
60
80
100
Co
ntr
ol
(H
PV
-/C
IN-)
Un
tre
ate
d
(HP
V+
CIN
+)
Tre
ate
d
(Re
cu
rre
nc
e-)
Tre
ate
d
(Re
cu
rre
nc
e+)
% P
op
ula
tio
n w
ith
po
sit
ive
H
PV
-sp
eci
fic
imm
un
ity
E6 Peptides E7 Peptides
T cell proliferation response in the blood
Dominant proliferative responses
in Recur- subjects
• 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 TH2-
or immunosuppressive-type (e.g. IL-4 and IL-10) of cytokine production
0
20
40
60
80
100
120
Q15L Q19D Q15L Q19D
Recurrence-free With recurrence
% P
os
itiv
e p
op
ula
tio
n
IFN-g IL-12 IL-4 IL-10
0
20
40
60
80
100
Co
ntr
ol
(H
PV
-/C
IN-)
Un
tre
ate
d
(HP
V+
CIN
+)
Tre
ate
d
(Re
cu
rre
nc
e-)
Tre
ate
d
(Re
cu
rre
nc
e+)
% P
op
ula
tio
n w
ith
po
sit
ive
H
PV
-sp
eci
fic
imm
un
ity
E6 Peptides E7 Peptides
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 18 Mo 24 Mo
Diagnosis
CIN II or CIN III
LEEP = Loop Electrosurgical Excision Procedure
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10
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30
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50
60
70
80
Pre-treatment
Post-treatment
Pre-treatment
Post-treatment
Pilot cross-sectional study Prospective study
% R
esp
on
din
g P
ati
en
ts
Fig. 1 E6 Peptide (Q15L)
E7 Peptide (Q19D)
0
10
20
30
40
50
60
70
Gained responses Lost responses
Prospective study
% P
atie
nts
Tes
ted
Fig. 2 E6 Peptide (Q15L)E7 Peptide (Q19D)
Treatment influence on HPV immunityE6 Peptide: Q15L (43-57) QLLRREVYDFAFRDLE7 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
0
10
20
30
40
50
60
70
80
Pre-treatment
Post-treatment
Pre-treatment
Post-treatment
Pilot cross-sectional study Prospective study
% R
esp
on
din
g P
ati
en
ts
Fig. 1 E6 Peptide (Q15L)
E7 Peptide (Q19D)
0
10
20
30
40
50
60
70
Gained responses Lost responses
Prospective study
% P
atie
nts
Tes
ted
Fig. 2 E6 Peptide (Q15L)E7 Peptide (Q19D)
0.1
1
10
CLN VALT CLN VALT
E7 Peptide E6 Peptide
% IF
Ng
+ c
ells
CD8 CD4
0
10
20
30
40
50
60
70
Spleen CLN
% S
pe
cif
ic k
illin
g
HPV tumor
Control
Unrelated tumor
0
50
100
150
200
250
0 10 20 30 40
Tu
mo
r siz
e (m
m^
2)
Days
Prophylactic vaccination study
Unrelated tumorHPV tumor
Intranasal immunization with HPV peptide
Day 0 Day 5 Day 10
Immunize Sacrifice/Tumor ChallengeImmunize
HPV peptide vaccine primes mucosal
immunity and Tumor protection
Therapeutic intranasal immunization with HPV vaccine against HPV tumors
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
Peptides Q15D and Q19D with aGalCer adjuvant
This may be because of the immunosuppressive tumor microenvironment
0
50
100
150
200
250
4 7 9 11 14 18 21 24
Tum
or
siz
e (m
m2 )
Days after tumor challenge
αGalCer
αGalCer + HPV peptides
HPV peptides
PBS
d 6
d1
2
Immunizations
* p<0.05
0
50
100
150
200
250
300
350
4 7 9 11 14 18 21 24 28 30 32
Tum
or
siz
e (m
m2 )
Days after tumor challenge
αGalCer
αGalCer + HPV peptides
HPV peptides
PBS
d 6
d1
2
d2
4
Immunizations
* p<0.05
0
50
100
150
200
250
300
350
4 7 9 11 14 18 21 24 28 30 32 35 37
Tum
or
siz
e (m
m2 )
Days after tumor challenge
αGalCer
αGalCer + HPV peptides
HPV peptides
PBS
d 6
d1
2
d2
4
d3
2
Immunizations
* p<0.05
0
20
40
60
80
100
120
D4 D7 D9 D11 D14 D18 D21 D24 D28 D30 D32 D35 D37
% S
urv
iva
lDays after tumor challenge
PBS Antigen Adjuvant Vaccine
3/6
0/5
0/40/4
• Immune suppressive with
• Accumulated regulatory T cells• Decreased/compromised
Antigen presentation, and • Exhausted/inhibited Effector T
cell responses
Tumor microenvironment
Professor and ChairDepartment of ImmunologyThe UT MD AndersonCancer Center
OrTumor cell
0
50
100
150
200
250
300
350
400
450
4 6 8 11 13 15 18 20 22 25Tu
mo
r s
ize
(mm
2)Post-tumor-challenge (days)
PBS
Vaccine
0
50
100
150
200
250
300
350
400
450
4 6 8 11 13 15 18 20 22 25
Tum
or
siz
e (m
m2)
Post-tumor-challenge (days)
PBSVaccineaCTLA-4Vaccine + aCTLA-4
0
50
100
150
200
250
300
350
400
450
4 6 8 11 13 15 18 20 22 25
Tum
or
siz
e (m
m2)
Post-tumor-challenge (days)
PBSVaccineaPD-1Vaccine + PD-1
0
50
100
150
200
250
300
350
400
450
4 6 8 11 13 15 18 20 22 25Tu
mo
r s
ize
(mm
2)Post-tumor-challenge (days)
PBSVaccinea4-1BBVaccine + a4-1BB
Vaccine + immunotherapy of
HPV tumors
Intranasal Vaccine:E6 and E7 peptides(100ug each in PBS)
Intraperitoneal injections ofImmune check point antibodies:
Antagonistic antibodies toCTLA-4 and PD-1
Agonistic antibody to 4-1BB
d6
(V+
Ab
)
d9
(Ab
)
d12
(V
+A
b)
Scheme
* **
Collaborators: Michael Curran, PhD, James Allison, PhD; Immunology
Vaccine immunotherapy of Vaginal HPV tumors
Vaccine immunotherapy of Vaginal HPV tumors
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
6 8 11 15 19
Avg
Rad
ian
ce
Days Post Tumor Challenge
PBS Vaccine
Vaccine + a4-1BB Vaccine + aCTLA-4
a4-1BB aCTLA-4
Combination of 4-1BB and CTLA-4 antibodies
This combination augmented HPV E6/E7 vaccine by increasing CD8 infiltration and decreasing Tregs in tumors
Vaccine immunotherapy of HPV tumors
Acknowledgements
Pramod Nehete, PhD, Assoc. ProfBharti Nehete, Research Asst.
Dr. Hong (Helen) HeRes. Investigator
Amy CourtneyPhD student
Danielle FonenotPhD student
Corinne BellMS student
Seth WardellRes. Technician
Ameerah Wishahi
Graduate Student
Shailabala Singh
Post-Doctoral Fellow
Guojun YangResearch
Investigator
Dr. Michael Barry, Mayo Clinic, Rochester, MNDr. Chun Wang, Univ. Minnesota, Minneapolis, MN
Drs. Michael Curran and James AllisonImmunology
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