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

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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

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Tit

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1D 2D 3D*

**

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*

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Ova Ova + aGC

IgG

Se

rum

Ig

G

(R

eci

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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

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(Re

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E6 Peptides E7 Peptides

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(Re

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(Re

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% P

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E6 Peptides E7 Peptides

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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

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Q15L Q19D Q15L Q19D

Recurrence-free With recurrence

% P

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IFN-g IL-12 IL-4 IL-10

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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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Fig. 1 E6 Peptide (Q15L)

E7 Peptide (Q19D)

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Gained responses Lost responses

Prospective study

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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

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% R

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Fig. 1 E6 Peptide (Q15L)

E7 Peptide (Q19D)

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Prospective study

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Fig. 2 E6 Peptide (Q15L)E7 Peptide (Q19D)

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CLN VALT CLN VALT

E7 Peptide E6 Peptide

% IF

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+ c

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CD8 CD4

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HPV tumor

Control

Unrelated tumor

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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

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Days after tumor challenge

αGalCer

αGalCer + HPV peptides

HPV peptides

PBS

d 6

d1

2

Immunizations

* p<0.05

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Days after tumor challenge

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αGalCer + HPV peptides

HPV peptides

PBS

d 6

d1

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Immunizations

* p<0.05

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Days after tumor challenge

αGalCer

αGalCer + HPV peptides

HPV peptides

PBS

d 6

d1

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d2

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d3

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Immunizations

* p<0.05

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D4 D7 D9 D11 D14 D18 D21 D24 D28 D30 D32 D35 D37

% S

urv

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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

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PBSVaccineaPD-1Vaccine + PD-1

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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