Aliasgar Shahiwala

7/23/2019 Aliasgar Shahiwala

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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 allendocrine glands are endowed with powerful

mechanical and chemical cleansing mechanismsthat 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 solitaryfollicles in intestine, and the tonsils and adenoids atthe entrance of the aerodigestive tract, which serveas the principal mucosal inductive sites where

immune responses are initiated.







VaccinesLive attenuated pathogens•may induce cell mediated immunity, can cause disease in immuno-suppressedindividuals

•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 isnecessary for most difficult pathogens, which often establish chronicinfections (e.g., HIV, HCV, TB and Malaria).

•Many traditional inactivated vaccines (e.g. Bordetella pertussis) also containscomponents 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 isthat 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 usedwith new generation vaccines, including DNA vaccines.



Adjuvants

Advantages Can increase the immunogenicity of weak antigens,

Enhance the speed and duration of the immune response,

Stimulate cell mediated immunity (CMI),

Promote the induction of mucosal immunity,

Enhance immune responses in immunologically immature, or senescent individuals, Decrease the dose of antigen in the vaccine and reduce costs,

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 takeup antigen (the principal mode of action of a range of particulate adjuvants, or antigendelivery 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 fromthe injection site in lymph and may deliver antigen directly to the lymph node.

• Particulate adjuvants (e.g. emulsions, microparticles, iscoms, liposomes, virosomes andvirus-like particles) have comparable dimensions to the pathogens which the immunesystem evolved to combat. Therefore, these particulates are normally taken up efficiently

by phagocytic cells of the innate immune system and function mainly to deliverassociated antigen into these key cells.







Approved Nanocarrier based vaccine

• EMULSIONSSqualene o/w emulsion based Influenza

vaccines approved in Italy in 1997, and for

several additional countries through mutualrecognition 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 continuousaqueous 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 singleformulation and sequestering the different agents in

selective compartments for enhanced stability



Composition of an optimized W/O/W emulsion formulation

2 mLSaline

0.25%Tween® 80

2 mLW/O Emulsion

W/O/W Emulsion

0.8 mLSaline20%Span

®

80

1 mLSqualene Oil

W/O Emulsion



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

Internal aqueous phase

Oil globules

External aqueous

phase

Scale bar = 10 µm



schematic representation of the in vivo treatment protocols

Immunization Protocol

NasalOral

Group 1 Group 2

1st Immunization

(day 0)

2nd Immunization

(day 14)

1st Immunization

(day 0)

Evaluation of

Immune response(day 14)

Evaluation of Immune response

(day 21)

Group 1 Group 2

1st Immunization

(day 0)

2nd Immunization

(day 14)

1st Immunization

(day 0)

Evaluation of

Immune response(day 14)

Evaluation of Immune response

(day 21)



0

5

10

15

20

25

30

35

OVA Saline OVA-Emul OVA-Emul-Chi

OVA Formulati on (100 mcg dose)

O V A - S p e c i f i c A n t i b o d y

I g G ( m c g / m L )

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.

0

20

40

60

80

100


OVA Formulation (100 mcg dose)

O V A - S p e c i f i c A n t i b o d y

I g G ( m c g / m L )


a

b



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.

a

b

0

500

1000

1500

2000

2500

3000

3500

4000



O V A - S

p e c i f i c A n t i b o d y I g A ( m c g )

.


0

0.4

0.8

1.2

1.6



O V A - S p e c i f i c A n t i b o d y I g A ( m

c g )




• 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 numeroustherapeutic 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 disciplinein the management of melanoma . Based on evidence that the immune

system plays a natural role in melanoma regression, there is well-

founded 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 abili ty to overcome self-tolerance and to

specifically activate tumor-specific killer cells (i.e. cytotoxic T cells).

Melanoma



• 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 humanmelanoma cells and can also be recognized by immune cells

including melanoma-derived tumor-infiltrating lymphocytes.

• Adoptive transfer of autologous, GP100-reactive, tumor-

infiltrating lymphocytes into melanoma patients, along with IL-2treatment, 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.

Gp100



Immunization Protocol

Treatment Prophylactic

DAY 0

5X105 B16F10 melanoma

cells injected

DAY 22

2nd Immunization

DAY 16

1st Immunization

DAY 0

1st Immunization

DAY 225X105 B16F10

melanoma cells

injected

DAY 16

2nd Immunization



Physical characterization of w/o/w emulsions

4.68 ± 0.517.4 ± 1.219.8 ± 1.7gp100 w/o/w

Emulsion with

Chitosan

1.97 ± 0.5-18.6 ± 1.317.9 ±1.3gp100 w/o/w

Emulsion

2.46 ± 0.4-13.9 ± 1.119.1 ± 1.6Blank w/o/w

Emulsion

Creaming

(%)

Surface

Charge (mV)

Size (µm)Formulation

Mean ± SD (n=3)



0

1000

2000

3000

4000

5000

6000

12 14 16 18 20 22 24 26 28 30Days

T u m o

r v o l u m e ( m m 3

)

Control GP100 solution GP100-Emul-Chi GP100-Emul

1st dose

2nd dose

Change in tumor volume as a function of time: Treatment

Group



Gp100 specific IgG Antibody titer- Treatment Group

0

100

200

300

400

500

600

700

800

7 14 21 28 35 42

Time (days)

G P 1 0 0 s p e c i f i c I g G t i t e

r ( m c g / m l )

GP100 solution GP100-Emul GP100-Emul-Chi



Gp100 specific IgG Antibody titer- Prophylactic Group

0

100

200

300

400

500

600

700

7 14 21 28 35 42Time (days)

G P 1 0 0 s p

e c i f i c I g G t i t e

r ( m c g / m l )

GP100 solution GP100-Emul GP100-Emul-Chi



Mean Tumor Doubling Time & Growth Delay in Treatment

Group

03.12 ± 0.3gp100-Emul-Chi

7 ± 0.55.54 ± 0.5gp100-Emul

02.88 ± 0.3gp100 solution

--2.76 ± 0.2Control

Mean Tumor Growth Delay

(days)

Mean Doubling Time

(days)



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

Treatment group

0

0.2

0.40.6

0.8

1

1.21.4

1.6

1.8

16 18 20 22 24 26 28

Time (days)

T u m o r t o c o n t r o l v o l u m e r a t i o

GP100 solution GP100-Emul-Chi GP100-Emul Control



The weights of the excised tumor mass-Treatment Group

0

0.5

1

1.52

2.5

3

3.5

4

4.5

Control gp100 solution gp100-Emul-Chi gp100-Emul

T u m o r W e i g h t ( g )



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

-25

-20

-15

-10

-5

0

5

0 4 8 12 16 20 24 28

Time (days)

P

e r c e n t C h a n g e i n B o d y w e i g

h t ( % )

Control GP100 Solution GP100 Emul GP100 Emul-Chi



0

1000

2000

3000

4000

5000

6000

0 7 10 22 24 26 28 30 32 34 36

Days

T u m o r

v o l u m e ( m m 3 )

Control GP100 solution GP100-Emul-Chi GP100-Emul

1st

dose2nd

doseMelanomacells

Injected

Change in tumor volume as a function of time:

Prophylactic Group



Mean Tumor Growth Delay in Prophylactic Group

4 ± 0.5Gp100-Emul-Chi

8 ± 0.5Gp100-Emul

0gp100 solution

0Control

Mean Tumor Growth Delay

(days)



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

Prophylactic group

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

22 24 26 28 30 32 34 36

Time (days)

T u m o r t o c o n t r o l v o l u m

e r a t i o




The weights of the excised tumor mass- Prophylactic Group

00.5

1

1.5

22.5

3

3.5

44.5

5


T u m o r W

e i g h t ( g )



Percent change in body weight as a function of time:

Prophylactic Group

-25

-20

-15

-10

-5

0

5

0 5 10 15 20 25 30 35

Time (days)

P e r c e n t C h a n g

e i n B o d y w e i

g h t ( % )

Control GP100 Solution GP100 Emul GP100 Emul-Chi



Control Gp100 Emul

Prophylactic Group



Excised Tumors-Prophylactic Group

Control Gp100 Solution

Gp100 Emul-Chi Gp100 Emul



Before After

(Treatment Group)

HMB Antibody staining in tumor section before and after

treatment: Treatment and Prophylactic group

After

(Prophylactic Group)



Thank you

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Aliasgar Shahiwala