LAMP-vax DNA Vaccine Technology

LAMP Technology Prospectus Immunomic Therapeutics, Inc. info@immunomix.com www.immunomix.com 240-731-5232 www.immunomix.com

LAMP was discovered in 1984 Lysosomal Associated Membrane Protein or “LAMP� was first identified in the laboratory of Dr. J. Thomas August as part of his research program delving into an understanding of compartmental trafficking within cells.

Shown in the figure is an electromicrograph of a cell staining for the LAMP protein.

2 www.immunomix.com

Lysosome-associated membrane protein (LAMP-1)

Source: August Laboratory

www.immunomix.com

Chen et al, J Cell Bio ,101:85, 1985

3

Signal Sequence Identified Targets the Lysosomal compartment Systematic analysis of the transmembrane sequence of the LAMP protein reveal a consensus sequence that directs LAMP and other proteins into the lysosomal compartment.

Shown in the figure is the basic configuration of the LAMP protein and highlights the 4 amino acid residues that are essential for intracellular localization

4 www.immunomix.com

LAMP-1

Following protein synthesis, the amino acid sequence code found on the “tail of the LAMP molecule is used to direct the LAMP molecule to the lysosome in a clathrin-mediated process

Clathrin Adapted from “M. Robinson and J. S. Bonifacino Cur. Op. in Cell Bio. 2001 444-453

5 www.immunomix.com

LAMP Chimeric Proteins Localize to the MHC-II Compartment LAMP has been shown in a number of studies to mediate the localization of foreign proteins to the MHC-II compartment. As shown in the next figure, a LAMP-gag plasmid construct was transfected into cells and following protein synthesis, the cells were stained for the Gag protein and for MHC-II. In the first panel, MHC-II staining in shown in green; in the center panel, anti-Gag staining is shown in red. When the two stains are super-imposed upon each other, the Gag protein is shown to be found in the same cellular compartments at the MHC-II molecule indicated by yellow staining.

6 www.immunomix.com

Co-Localization of LAMP Chimera with MHC-II

Anti-MHCII

Anti-Gag

Merged

7 www.immunomix.com

LAMP Vaccines Activates the Immune System – An Overview When a DNA vaccine is introduced to the system, antigen presenting cells will take up the DNA and will produce the encoded protein sequence inside the cell. In the absence of LAMP, this protein is present in the cytoplasm of the cells and is presented to the immune system through the MHC-I pathway on the left. This results in primarily a cell – mediated response through CD8+ cells. (left side of diagram) However, when LAMP is included in the vaccine design, the synthesized protein is directed into the lysosome and the MHC-II pathway. As a result, the immune system is primarily activated through the MHC-II / CD4+ helper T-cell pathway (right side of diagram) resulting in a more complete immune response including antibody production, cytokine release and immunological memory. Interestingly, there is no decrease observed in CD8+ cell mediated activation. Thus, LAMP vaccines active the entire immune system.

8 www.immunomix.com

No LAMP

+ LAMP

9 www.immunomix.com

LAMP Chimeric Proteins Stabilize Expression of Protein The LAMP gene element does more than just provide for enhanced immune system presentation – it also stabilizes the gene product.

Shown in the next figure are results that show that by incorporating LAMP into the plasmid design, levels of gag produced in transfected cells is much greater than that observed with plasmids that lack LAMP.

10 www.immunomix.com

LAMP lumen

ss Gag TM/Cyto.

B

72bp

108bp

pITR

pcDNA3.1 ø

1503bp

pcDNA3.1

A

1

2

3

4

5

6

7

8

kDa

1 GagN 2 SS/Gag/lamp

220

3 GagDINS

1113bp

4 SS/GagDINS/lamp 5 LAMP/GagN

97.4 66

pITR

6 GagN 7 SS/GagDINS /lamp 8 LAMP/GagN

45 From Marques, et al 2002

11 www.immunomix.com

LAMP Vaccines Activate the Immune System – Cytokines & Interferon One of the key elements of a successful DNA vaccine is the ability to activate cytokine production in the immune system. Shown in the next figure are the results from cells stimulated with gag following immunization with DNA +/- LAMP. As can be clearly seen, interferon-gamma, IL-2 and IL-4 can all be detected following stimulation of the cells.

12 www.immunomix.com

2 Gag

SS/GagDINS/lamp

4

LAMP/GagN 0

D

mRNA IL-2/HPRT 0

2

4

6

8

10

12

14

16

5 4 3 2 1

E

n=6 n=8 n=11

0 -1

LAMP/GagN

n=2 4

LAMP GagN SS/GagDINS/lamp

LAMP/GagN

mRNA IL-4/HPRT

100 80

medium Gag

SS/GagDINS/lamp

GagN

7 6

LAMP/GagN

medium Gag

6

Gag N

4.5

LAMP

4

IFN- (ng/ml)

LAMP

C

B 3.5

Medium

1

Gag +Anti-CD8

.5

Gag + Anti-CD4

0

IFN- (ng/ml)

IFN- (ng/ml) 1.5 2 2.5 3

A

60 40 20

0

LAMP

Gag N

LAMP/GagN Marques, et al 2006

13

LAMP-gag Construct activates CD8+ cytotoxic T Cells One aspect of utilizing LAMP vectors that is particularly important is their ability to maintain activation of the CD8+ cell mediated immunity pathway. Shown in the next figure, following gag immunization + or – LAMP, cells were tested for cytotoxic activity.

Clearly, the inclusion of the LAMP gene in the construct does not adversely affect the percent cells killed – in fact, the response is heightened.

14 www.immunomix.com

C 60

0

1

2

3

4

5

% IFN-  + /CD8 2 2.5 3 3.5 4 4.5 5 7 0 .5 1 1.5

50

B

%Tetramer/CD8+ 6

%Killing

A

-

LAMP

Control

Control

tetramer

IFN- 

30 20 10

GagN

0 -10

SS/GagDINS/lamp

0

LAMP/GagN

20 40 60 80 100 120 Effector:Target

LAMP GagN SS/GagDINS/lamp LAMP/GagN

D 0 LAMP

40

1

%Tetramer/CD8+ 2 3 4 5

6

E 7 0

10

F %Killing (E:T 100:1) 12 0 5 10 15 20 25 30 35 40 45

n=17

n=14

n=4

n=9 n=25

n=22

LAMP/GagN

n=13 n=9

n=9

GagN

SS/GagDINS/lamp

2

% IFN-  /CD8+ 4 6 8

n=4

n=23 n=4 Marques, et al 2002

15 www.immunomix.com

DNA Prime – Protein Boost Induces an Enhanced Response DNA vaccines have shown promise as an effective method to “primer” the immune system for subsequent immunization with a weakly reactive protein. LAMP vaccines, since they use the CD4+ helper T-cell pathway, are ideal candidates for this approach.

Shown in the next figure is the immune response following an initial LAMP-gag immunization and a subsequent boost with gag protein.

16 www.immunomix.com

IFN- production (ng/ml)

A 0

.5

1

1.5

2

2.5

3

3.5

LAMP

4

4.5

medium Gag

GagN

mRNA IL-2/HPRT

B

LAMP/GagN

0

2

4

6

8

10 12

14

LAMP GagN LAMP/GagN LAMP/GagN+GagN

LAMP/GagN+GagN CTL 0 20 40 60 80 100

E

0

LAMP

1

% Tet+/CD8+ 2 3 4 5

80

D 6

7

0

1

% IFN- +/CD8+ 2 3 4 5

6

% killing

C

60 40

GagN

20

LAMP/GagN

0

LAMP/GagN + GagN

E:T ratio LAMP GagN LAMP/GagN LAMP/GagN + GagN

17 www.immunomix.com

Fig. 5; Marques, et al

Luminal Domain Of LAMP Is Key To Protein Expression Much of the early work on LAMP containing vaccines emphasized the signal sequence region of LAMP, however, it is clear that the intra-vesicle luminal domain is key to protein stability and co-localization with the MHC-II compartment. The next few slides show the value of including the luminal domain of LAMP in the design of future DNA vaccines, even those that already have shown effectiveness with only the targeting domain included in the construct.

The next figure shows a series of DNA vectors that have increasing segments of the luminal domain of LAMP included in the construct for a gag vaccine.

18 www.immunomix.com

Vectors to Evaluate Luminal Domain of LAMP in Gag Vaccine

19 www.immunomix.com

The Addition Of Luminal Domain Enhances Gene Product Levels In Cells Shown in the next figure are blots detecting the presence of the Gag protein following transfection with the various LAMP-gag constructs from the previous slide. As the size of the luminal domain included in the vector increases, so does the amount of protein produced in the cells.

20 www.immunomix.com

LAMP-1 luminal domain targets HIV-Gag to cellular secretory pathway

Gagwt

cell

sup

ssGag/ lamp

cell

sup

tLAMP1/ Gag

cell

sup

tLAMP2/ Gag

cell

sup

tLAMP3/ Gag

cell

sup

LAMP/ Gag

cell

sup

21 www.immunomix.com

Sub-cellular Localization As A Function Of The Size Of The Luminal Domain Of LAMP The presence of the luminal domain also facilitates the trafficking of the LAMP-gag chimeric protein into the MHC-II – containing lysosome.

Shown in the next figure are series of transfected cells showing the localization of the gag protein in the MHC-II compartment as a function of the length of the LAMP luminal domain included in the vector design. As with the previous slide, yellow staining is evidence of gag and MHC-II co-localization.

22 www.immunomix.com

#3 - p43 tLAMP 1/Gag

#2 - p43 Gag/lamp

Anti-MHCII

Anti-Gag

Merged

Anti-Gag

Anti-Gag

Merged

#5 - p43 tLAMP 3/Gag

#4 - p43 tLAMP 2/Gag

Anti-MHCII

Anti-MHCII

Merged Merged

Anti-MHCII

Anti-Gag

Merged 23

In Vivo, The Improved LAMP Chimera Elicits A Strong Response When mice are immunized with the DNA vaccine construct including the increasing segment of the luminal domain, the result is a significant increase in anti-gag antibody titer and increased interferonÎł production.

24 www.immunomix.com

High Protein Expression And Targeting To Secretory Pathway Induced By LAMP Luminal Domain Potentiates The Immune Response To Hiv-gag Serum IgG IgG O.D. 450nm

3

Pre-bleed vector Gag wt ssGag/lamp tLAMP/Gag 1 tLAMP2/Gag tLAMP3/Gag LAMP/Gag

2.5 2 1.5 1 .5 0

100

1000

10000

Dilution IFN-ď § ELISPOT SFC/106 splenocytes

2000 1800

1600 1400 1200 1000 800 600 400 200 0

Gag wt

www.immunomix.com

Gag/lamp

tLg1

tLg2

Experimental Group

tLg3

Lg

25

DC-LAMP provides a second pathway to immune activation

The August Laboratory also discovered that a second form of LAMP, “DC” or “Dendritic Cell” LAMP could be utilized to induce an immune response with a somewhat different response profile. Shown in the next figure are diagrams of LAMP-1 and DC-LAMP as well as vectors designed to evaluate the two forms of the protein.

26 www.immunomix.com

MHC-II Targeting with LAMP and DC-LAMP

A

LAMP-1

B pITR gag wt

DC-LAMP

p55gag

pITR LAMP/gag

Luminal domain

Transmembrane & cytoplasm

LAMP luminal domain p55gag

pITR DC-LAMP/gag Cytoplasmic domain

YQTI-COOH

YQRI-COOH N-glycan

O-glycan

Transmembrane DC-LAMP luminal & cytoplasm domain p55gag

Source: August Laboratory

27 www.immunomix.com

DC-LAMP and LAMP-1 both activate an immune response; DCLAMP activates IgG2A Synthesis Shown in the next figure are the results from mice immunized with either the LAMP-1 or the DC-LAMP constructs. It was observed that the LAMP and DC-LAMP chimeric proteins follow different pathways in the cell and they generate different antibody response profiles. Animals receiving DC-LAMP had lower IL-4 response and elevated IgG2A levels indicating an enhanced Th1 response. This suggests that DC-LAMP may have specific applications in allergy immunotherapy.

28 www.immunomix.com

DC-LAMP/Gag and LAMP Immune responses A

B

Secreted IFN- (ng/ml) 0 2 4 6 0

C % Tetramer+/CD8+ 1 2 3 4 5 6

7 0

D % IFN- +/CD8+ 2 4 6 8 10 12 0 5

% killing 15 25 35

45

DC-LAMP/gag LAMP/gag gagN control

E

F

IL-4 SFC/106 splenocytes 0 10 30 50 70 90

LAMP/gag

control

O.D. (450 nm)

DC-LAMP/gag

IgG1 1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0

IgG2a LAMP/gag DC-LAMP/gag

100 300 900 2700 100 300 900 2700 Sera dilution

29 www.immunomix.com

Acknowledgements Immunomic Therapeutics would like to thank the Laboratory of Dr. J. Thomas August for their advice and contributions to this publication.

30 www.immunomix.com

Additional information about LAMP vaccines and LAMP Technology in general is available for Immunomic Therapeutics.

Immunomic Therapeutics, Inc. info@immunomix.com www.immunomix.com 240-731-5232 www.immunomix.com