MUCOSAL VACCINE FOR

TUMOR THERAPY
Aliasgar Shahiwala, PhD

8th International Symposium, Controlled Release Society-India Chapter

Mucosal system
Covers aerodigestive and urogenital as well as the
eye conjunctiva and the inner ear and the ducts of all
endocrine glands are endowed with powerful
mechanical and chemical cleansing mechanisms
that degrade and repel most foreign matter.
Contributes almost 80% of all immunocytes,
accumulated in, or in transit between, various
mucosa-associated lymphoid organ system.
Comprised of anatomical defined lymphoid
microcompartments such as the Peyer patches, the
mesenteric lymph nodes, the appendix and solitary
follicles in intestine, and the tonsils and adenoids at
the entrance of the aerodigestive tract, which serve
as the principal mucosal inductive sites where
immune responses are initiated.

Vaccines
Live attenuated pathogens
may induce cell mediated immunity, can cause disease in immuno-suppressed
individuals
Some pathogens are difficult or impossible to grow in culture (e.g. HCV)
Whole inactivated organisms
Inactivated toxins
generally proven ineffective at inducing cell mediated immunity which is
necessary for most difficult pathogens, which often establish chronic
infections (e.g., HIV, HCV, TB and Malaria).
Many traditional inactivated vaccines (e.g. Bordetella pertussis) also contains
components that can cause undesirable side effects and safety issues.
New Approaches
Recombinant protein subunits
synthetic peptides
protein polysaccharide conjugates
plasmid DNA
These new approaches may offer important safety advantages, a general problem is
that the vaccines alone are poorly immunogenic. Therefore, there is an urgent need for
the development of potent and safe adjuvants and delivery systems that can be used
with new generation vaccines, including DNA vaccines.

Adjuvants
Advantages
9
Can increase the immunogenicity of weak antigens,
9
Enhance the speed and duration of the immune response,
9
Stimulate cell mediated immunity (CMI),
9
Promote the induction of mucosal immunity,
9
Enhance immune responses in immunologically immature, or senescent individuals,
9
Decrease the dose of antigen in the vaccine and reduce costs,
9
Help to overcome antigen competition in combination vaccines.
Mechanisms of action

For most of the adjuvants still remains only partially understood

Enhances delivery of the antigen to the lymph node

Increase cellular infiltration into the injection site, so that more cells are present to take
up antigen (the principal mode of action of a range of particulate adjuvants, or antigen
delivery systems e.g. microparticles, emulsions, liposomes, etc., may be to promote
uptake of the antigen by APC at the injection site.

In addition, some of these delivery systems may also be capable of moving away from
the injection site in lymph and may deliver antigen directly to the lymph node.

Particulate adjuvants (e.g. emulsions, microparticles, iscoms, liposomes, virosomes and

virus-like particles) have comparable dimensions to the pathogens which the immune
system evolved to combat. Therefore, these particulates are normally taken up efficiently
by phagocytic cells of the innate immune system and function mainly to deliver
associated antigen into these key cells.

Approved Nanocarrier based vaccine

EMULSIONS
Squalene o/w emulsion based Influenza
vaccines approved in Italy in 1997, and for
several additional countries through mutual
recognition in 2000.
LIPOSOMES
Liposomal vaccines based on viral membrane
proteins (virosomes) without additional
immunostimulators, have been extensively
evaluated in the clinic and are approved as
products in Europe for hepatitis A and influenza.

MUCOSAL VACCINATION FOR

TUMOR THERAPY

MULTIPLE EMULSIONS
W/O/W multiple emulsions are small (nanometer sized)
aqueous droplets entrapped within a larger oil droplets,
which are again stabilized and dispersed in a continuous
aqueous phase.
Taste masking
Enzyme immobilization
Sorbent for treatment of drug overdose
Enhances enteral or dermal absorption
Vaccine adjuvants
Can encapsulate several active agents in a single
formulation and sequestering the different agents in
selective compartments for enhanced stability

Composition of an optimized W/O/W emulsion formulation

W/O Emulsion
Squalene Oil

1 mL

Span 80

20%

Saline

0.8 mL
W/O/W Emulsion

W/O Emulsion

2 mL

Tween 80

0.25%

Saline

2 mL

Photograph of Microemulsion (OVA-Emul) Formulation at 40X. Bar in the photograph is equal to 10 m.

External aqueous
phase

Internal aqueous phase

Oil globules

Scale bar = 10 m

schematic representation of the in vivo treatment protocols

Immunization Protocol

Nasal

Group 1

1st Immunization
(day 0)

Evaluation of
Immune response
(day 14)

Oral

Group 2

1st Immunization
(day 0)

2nd Immunization
(day 14)

Evaluation of Immune response

(day 21)

Group 1

1st Immunization
(day 0)

Evaluation of
Immune response
(day 14)

Group 2

1st Immunization
(day 0)

2nd Immunization
(day 14)

Evaluation of Immune response

(day 21)

a
35
OVA-Specific Antibody
IgG (mcg/mL)

30
25
20
15
10
5
0
OVA Saline

OVA-Emul

OVA-Emul-Chi

OVA Formulation (100 mcg dose)

1st Immunization

2nd Immunization

80
IgG (mcg/mL)

OVA-Specific Antibody

100

60
40
20
0
OVA Saline

OVA-Emul

OVA-Emul-Chi

OVA Formulation (100 mcg dose)

1st Immunization

2nd Immunization

OVA Specific IgG Response following Nasal and Oral Administration of Different Formulations. a and b showing OVA Specific IgG
Response following nasal and oral administration of different formulations respectively.

OVA-Specific Antibody IgA (mcg)

1.6

1.2

0.8

0.4

0
OVA Saline

OVA-Emul

OVA-Emul-Chi

OVA Formulation (100 mcg dose)

1st Immunization

2nd Immunization

OVA-SpecificAntibodyIgA(mcg)

4000
3500
3000
2500
2000
1500
1000
500
0
OVA Saline

OVA-Emul

OVA-Emul-Chi

OVA Formulation (100 mcg dose)

1st Immunization

2nd Immunization

OVA Specific IgA (mucosal immune response) following Nasal and Oral Administration of Different Formulations. a and b showing
OVA Specific IgA Response following nasal and oral administration of different formulations respectively.

Melanoma

Melanoma is a malignant tumor of melanocytes that often starts

from harmless-looking moles. Once the tumor has started to
metastasize, the prognosis is generally poor. The metastases may
spread to any organ. Despite intensive research and numerous
therapeutic approaches, no satisfactory treatment is available today.
The first treatment of choice is, whenever possible, surgical removal
of the tumor.

Active vaccination strategies represent a relatively new discipline

in the management of melanoma . Based on evidence that the immune
system plays a natural role in melanoma regression, there is wellfounded hope that the power of the immune system could be
enhanced by the use of melanoma vaccines. The key to an effective
tumor vaccine lies in the ability to overcome self-tolerance and to
specifically activate tumor-specific killer cells (i.e. cytotoxic T cells).

Gp100
Recent advances in the molecular identification of melanoma
specific antigens have given a significant boost to the study of
novel cancer vaccines.
Glycoprotein GP100 is an antigen expressed in most human
melanoma cells and can also be recognized by immune cells
including melanoma-derived tumor-infiltrating lymphocytes.
Adoptive transfer of autologous, GP100-reactive, tumorinfiltrating lymphocytes into melanoma patients, along with IL-2
treatment, has been associated with tumor regression in some
patients.
The antigen has been shown to be highly immunogenic and an
important target for active-specific immunotherapy in humans.

Immunization Protocol

Prophylactic

Treatment

5X105

DAY 0
B16F10 melanoma
cells injected

DAY 16
1st Immunization

DAY 22
2nd Immunization

DAY 0
1st Immunization

2nd

DAY 16
Immunization

DAY 22
5X105 B16F10
melanoma cells
injected

Physical characterization of w/o/w emulsions

Formulation

Size (m)

Surface
Charge (mV)

Creaming
(%)

Blank w/o/w
Emulsion

19.1 1.6

-13.9 1.1

2.46 0.4

gp100 w/o/w
Emulsion

17.9 1.3

-18.6 1.3

1.97 0.5

gp100 w/o/w
Emulsion with
Chitosan

19.8 1.7

17.4 1.2

4.68 0.5

Mean SD (n=3)

Change in tumor volume as a function of time: Treatment

Group

Tumor volume (mm3)

6000
5000
4000
2nd dose

3000
2000
1st dose

1000
0
12

Control

14

16

18

GP100 solution

20
22
Days

24

26

GP100-Emul-Chi

28

30

GP100-Emul

GP100 specific IgG titer (mcg/ml)

Gp100 specific IgG Antibody titer- Treatment Group

800
700
600
500
400
300
200
100
0
7

14

21

28

35

Time (days)
GP100 solution

GP100-Emul

GP100-Emul-Chi

42

GP100 specific IgG titer (mcg/ml)

Gp100 specific IgG Antibody titer- Prophylactic Group

700
600
500
400
300
200
100
0
7

14

21

28

35

Time (days)
GP100 solution

GP100-Emul

GP100-Emul-Chi

42

Mean Tumor Doubling Time & Growth Delay in Treatment

Group

Mean Doubling Time

(days)

Mean Tumor Growth Delay

(days)

Control

2.76 0.2

--

gp100 solution

2.88 0.3

gp100-Emul

5.54 0.5

7 0.5

gp100-Emul-Chi

3.12 0.3

Tumor to control volume ratio

Figure 5. Change in tumor to control volume ratio as a function of time:

Treatment group

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
16

18

20

22

24

26

28

Time (days)

GP100 solution

GP100-Emul-Chi

GP100-Emul

Control

The weights of the excised tumor mass-Treatment Group

4.5

Tumor Weight (g)

4
3.5
3
2.5
2
1.5
1
0.5
0
Control

gp100 solution

gp100-Emul-Chi

gp100-Emul

Percent Change in Body weight (%)

Percent change in body weight as a function of time: Treatment Group

5
0
-5
-10
-15
-20
-25
0

12

16

20

24

28

Time (days)
Control

GP100 Solution

GP100 Emul

GP100 Emul-Chi

Change in tumor volume as a function of time:

Prophylactic Group

Tumor volume (mm3)

6000
5000
4000
3000
2000

1st
dose

Melanoma
2nd
dose cells
Injected

1000
0
10

22

24

26

28

30

32

34

36

Days
Control

GP100 solution

GP100-Emul-Chi

GP100-Emul

Mean Tumor Growth Delay in Prophylactic Group

Mean Tumor Growth Delay

(days)
Control

gp100 solution

Gp100-Emul

8 0.5

Gp100-Emul-Chi

4 0.5

Change in tumor to control volume ratio as a function of time:

Prophylactic group

Tumor to control volume ratio

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2

22

24

26

28

30

32

34

36

Time (days)
Control

gp100 solution

gp100-Emul-Chi

gp100-Emul

The weights of the excised tumor mass- Prophylactic Group

5
4.5
Tumor Weight (g)

4
3.5
3
2.5
2
1.5
1
0.5
0
Control

gp100 solution

gp100-Emul-Chi

gp100-Emul

Percent Change in Body weight (%)

Percent change in body weight as a function of time:

Prophylactic Group
5
0
-5
-10
-15
-20
-25
0

10

15

20

25

30

35

Time (days)
Control

GP100 Solution

GP100 Emul

GP100 Emul-Chi

Prophylactic Group

Control

Gp100 Emul

Excised Tumors-Prophylactic Group

Control

Gp100 Emul-Chi

Gp100 Solution

Gp100 Emul

HMB Antibody staining in tumor section before and after

treatment: Treatment and Prophylactic group

Before

After
(Treatment Group)

After
(Prophylactic Group)

Thank you