Technical 5 01 11 Final

Triad Immunologies, LLC, all rights reserved 1

Triad Immunologies, LLC, Presents:Hyperthermia + Cytokine Cascade Dendritic Cell Vaccine for Solid Tumors

Presented by:Bruce W. Lyday

Vice President, Product DevelopmentThis presentation is for information purposes only, and this experimental project has notbeen tested to prove its safety or efficacy to treat any disease in approved clinical trials


To Induce a Durable, Complete Response in Metastatic Cancer:

Reduce tumor burden 50% before vaccine8

Active immunotherapy to kill remainder19

Overcome the major immune evasions8,25

Use CD4+, CD8+ CTL, plus LAK/CIK cells8,23

Break T-cell tolerance to self-peptides7

Use prior advantages of low perfusion/high16 mutation rates against tumor clones


Strategic Objective- A Durable, Complete Response Triad plans to achieve this goal using:

1. Dengue-1 Strain #45AZ5- Dengue mortality 1/61,000 primary infections by Manson’s Tropical Diseases (W.B. Saunders Publishers, 1996) Ref. (A)

2. Autologous or Allogeneic peptide-pulsed Dendritic Cells, safety record in >1,000 patients

“Strategies to improve Dendritic-Cell-based Immunotherapy against cancer” Y. Song, et. al., Yonsei Med. Jun. 2004 Suppl. #48-52. Ref. (B)


Triad- 7 Mechanisms of Action in a Single Cell-Based Therapy Hyperthermia-Direct9,17

TNF-alpha Direct17

TNF-alpha on vessels3,20

TNF-beta direct5,6

Specific CTL from DC23

Nonspecific LAK2,11

Activated Macrophage24

Hyperthermia killing tumor cells

CTL killing tumor cell

Net im

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ercial use

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Dengue Virus Type-1 Strain #45AZ5

Group B Arbovirus

+ RNA, 4 serotypes Targets-monocytes,

immature B cells Transmitted by Aedes

Aegypti mosquitoes 5 x 107 cases/year9

Strain #45AZ5 tested in volunteers under FDA IND27

Aedes aegypti Dengue Virus

Dengue Range ’30 N to ’30 S


Dengue Strain #45AZ5 Production and Delivery Seed Virus grown in

fetal Rhesus lung

Final product filtered, bottled, shipped as any other vaccine

0.5 ml injected s.c. in arm before DC infusions

Bio-reactor makes 50,000 doses/batch

Final Product in 2 mlBottle similar to flu vaccine


Dengue as Hyperthermia & Cytokine-Inducing Agent Hyperthermia

Fever of 39.5-41 C over typical 5-day range9

Fever induces selective perfusion reduction of tumor micro-vessels18

In synergy with TNF-a, can kill to 50% of tumors prior to vaccine phase 19, 20

Th1 Cytokines

Pro-inflammatory to Th1 cytokine shift 2,12

Cytokines kill tumor cells directly6, by shutting off blood20, and raise CTL/LAK status to maximum.8,22


Cytokine Levels in Dengue CYTOKINE Control Post-Dengue Increase Data type/Ref.#

IL-1 9.4 pg/ml 745 pg/ml 79X In vitro (4)

IL-2 2.1 U/ml 60.3 U/ml 29X In vivo (2)

IL-12 Undetected 270 pg/ml 270X In vivo (10)

IL-15 5.2 pg/ml 12-31 pg/ml 2-3X In vivo (5)

IFN-alpha <6 U/ml 1600 U/ml 267X In vitro (2)

IFN-gamma <5 pg/ml 275 pg/ml 55X In vivo (2)

TNF-alpha 3 pg/ml 210 pg/ml 70X In vivo (3)

TNF-beta Undetected To 605 pg/ml

600X In vivo (5)

GM-CSF 0.2 pg/ml 10.5 pg/ml 53X In vitro (1)

Cytokines Induced by Dengue and Their Effects on ImmunityIL-1b Fever, selective toxicity to tumor vessels9,3,20

IL-2 CTL proliferation, Raise NK cells to LAK state2,22,23

IL-12 Shift DC to Th1-profile, raise CTL, LAK cytotoxicity2,15

IL-15 Raise cytotoxicity of CTL/LAK cells, protect from AICD2,5

TNF-alpha

Raise cytotoxicity of CTL/LAK, increase gap junctions for CTL diapedesis, kill tumors by TRAIL-ligand, destroy tumor microvasculature in synergy with hyperthermia2,3,19

TNF-b Direct killing of tumor cells by death receptors5,8

IFN-a Upregulate Class I MHC on tumor cells, cytostatic agent2,8

IFN-g Up-regulate class I MHC, increase cytoxicity, direct killing of tumor cells, activate macrophages to kill tumor cells2,24

GMCSF Increase DC maturation/CTL stimulation1,15

10

Shifting Cytokines to Th1-Type Systemically

Within Tumor

In node at time of T-cell cloning, critical for hi-avidity, perforin+

CD62LLo CTLLymph Node Cross-section

11

Allogeneic DC Pulsed with HLA-Matched Tumor Peptides DC grown in culture

Stem Cell technology transferable to DC

Economies of scale Cell lines certified path-free

1 DC can prime >103 CTL specific for tumors12

108 DC = 1010-11 CTL for E:T ratio of 50:1

CTL primed by DC net image for non-commercial use

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Allogeneic Dendritic Cells-Towards a More Standardized Therapeutic Business Model

Patient is HLA-typed

Tumor peptides matched

HLA-matched DC pulsed

DC shipped, tested, infused, 50% frozen for 2nd treatment

3 Standard Cell lines for majority of A types

Eliminates patient-specific product logistics

0.14, 16%

0.24, 27%0.52, 57%

1

2

3

A2

A3

A1

Relative % of HLA-A types Caucasians


Hyperthermia Effects Tumor micro-vasculature is inadequate16

High growth/low perfusion= hi [lactate]17

Low pH alters DNA fidelity/ hi mutation17

Up-regulation of Survivin, p100 pump8

At 39.5 C, hypothalamus conserves [O2]18

Tumor vessels shut down=necrosis19

Survivors on hi-flow vessels near CTL19

Effect doubled by hi [TNF-alpha]20

Triad Immunologies, LLC, all rights reserved 14Dengue Fever Range

Tumor Blood Flow decrease

MAJORITY OF CANCER SPONATANEOUS REGRESSIONS OCCUR FOLLOWING A FEVER IN EXCESS OF 39.5 C21

Ref. #17

15

Hyperthermia Killing of Tumor

Before hyperthermia After Hyperthermia

Ref. #17


Hyperthermic Effect on Tumor With low levels of

hyperthermia in the range of dengue fever, surviving cells will be 5-10 cell layers from the vessel lumen9,18

DC-Vaccine creates CTL to eliminate survivors19

Ref. #17

17

Conclusions on Hyperthermia Selective for tumor micro-vasculature18

Kills most dangerous (hi mutation) clones resistant to drugs first17,18

Ideal de-bulking agent before vaccine19

Immune effectors-optimum function11,13

Stage is set for DC infusions, create 109 high avidity/Perforin+/CD62LLo CTL15

18

Documented Tumor Immune Evasion Methods in Literature8

1. Down-regulation of Class I MHC 2. Point mutations in tumor peptides 3. Tumor vessels inhibit killer cells 4. Expression of Fas-ligand to kill CTL 5. Expression of HLA-G to defeat NK 6. Macrophage inhibition by TGF-b 7. Construction of stromal barriers 8. Protease Inhibitor PI-9 defeats CTL25


Evasion #1-Low [MHC Class I] Tumor cells down-

regulate Class I MHC expression to avoid recognition by CTL8

High levels of IFN-a & IFN-g by dengue restore MHC to normal, allowing CTL lysis2,11,22

Net image for non-commercial use

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Evasion #2-Point Mutations in Tumor-Associated Antigens Tumor cells often mutate

a.a. in tumor peptides [gp100], allowing escape from gp-100 CTL8

High levels of IL-1, IL-2, IL-7, and 12 induced by dengue create CIK cells capable of killing any mutant cell2,4,5,15

3-D Image of MHC-peptide Net Image for non-commercial use


TNF-a induced by dengue restores P&E Selectins3

Evasion Method #3- Tumor Vessels Lack P&E-Selectins to Allow CTL Extravasation8

Net imageFor non-Commercial use


Evasion #4 “Counterattack” Fas+

CTL Killed by FasL on Tumor Cells

Fas (CD95) is death receptor on CTL8

High [IL-6] during dengue induce FLIP1,6, protects Fas+ CTL

High levels of IL-15 induced by dengue5 protect CTL from AICD through bcl-x6

FLIP protects CTLNet Image for non-commercial use


Evasion #5-Expression of HLA-G to defeat NK Cells HLA-G is fetal ag-blocks

maternal NK8

Inhibits NK from killing low [MHC] cell

High levels of IL-2, IL-12, & 15 induced by dengue activate NK to LAK state2,5

Activated LAK Cell lysing HLA-G+ Target Cell

Net image for non-Commercial use

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Evasion #6-Stromal Barriers Tumor cells erect

stromal barriers to CTL invasion using connective tissue8

High levels of IFN-g induced by dengue2 activate Macrophage to destroy these through their CD44 receptor24 Connective Barriers

Surrounding tumor cellsNet image for non-commercial use

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Evasion #7-Inhibition of Macrophage, DC by TGF-beta Tumor cells secrete

TGF-b; tumor Macs secrete IL-1012,14 blunts CTL8

Pro-inflammatory and Th1 cytokines induced by dengue reverse1,2

IFN-gamma activitiesNet Image for non-commercial use

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Evasion #8-Expression of PI-9 PI-9- serine protease

inhib. protects tumors from Perforin+ CTL25

Dengue cytokines induce high levels of CD8 and ICAM-1 on CTL, allowing lysis of PI-9+ tumors5,25,26

Stable target-CTL Interactions through CD8 Co-receptor/ICAM-1 Net image for non-commercial use


Breaking T-Cell Tolerance from Clonal Selection/Deletion Most tumor antigens(PSA, gp100, etc.), are

“self”, protected from CTL7,8

Dengue Cytokine Cascade raises [ICAM-1] and [CD8] allowing lysis by low-affinity, specific-TCR CTL25,26 Only low-affinity

CTL survive

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CTL Quantity and Quality Estimated E:T during

dengue- 50:1 CTL, 20:1 LAK/CIK23 varies with n

Most vaccines create CD4+ 25+Fox3P+ TReg12

CTL need CD62LLo Perforin+ “License to kill”22

SEM of 20:1 E:T Ratio CTL:Tumor Target Cell

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Clinical Safety & ManagementOf Dengue Fever in Humans9

Proper Fluid Intake reduces adverse events in first infection to <1%

Bed rest, support care for 5 day fever Hepatic, hemorrhage toxicities primary Both are manageable in ward setting

Most toxic effects due to TNF-a, agonist drugs Enbrel, Infliximab can bind TNF

“with proper patient selection & fluid management, I estimate the chance of a serious adverse event is <1%”. Col. (Ret.), Dr. David Vaughn, M.D., Ph.D., Dengue Researcher, personal communication.

Dengue in Cancer Patients 23 cancer patients had dengue fever in Taiwan

epidemic of 2002-200328.

1/23 had SAE (Acute Renal Failure), 4.2%

Authors found that cancer was not a risk factor for severe dengue (O.R. 0.8); raised risk for ARF (1.8).


“Impact of Renal Failure on the outcome of dengue viral infection”. M. Kuo, et. al., Clinical J. of American Society of Nephrology, 3, pp.1350-56, 2008.


Triad’s Principal Investigators Dr. Boris Minev,

M.D., of UC San Diego Cancer Center

Formerly with Dr. Steven Rosenberg’s melanoma unit at N.C.I.-Bethesda MD

Chief Science Officer-Triad

Dr. Gregory Daniels, M.D., Ph.D. of UCSD

formerly with Mayo Clinic P.I. at U.C.S.D. and V.A. Medical Center

Experienced in cancer immunotherapy trials under FDA-approved IND


Triad Management Team C.E.O.- Dr. John Bonfiglio, Ph.D., former C.E.O. of Argos Therapeutics Currently a consultant to Triad, will be CEO upon funding

Chief Science Officer- Dr. Boris Minev, M.D.

Regulatory Dr. Bruce Mackler, J.D., Ph.D

Business Advisors-:9. William Gerhart, CEO, Elevation Pharmaceuticals10. Mark Wilson, Licensing and Marketing Professional11. David Geigel. Ph.D., Drug Development


Intellectual Property U.S. Patent #6,524,587, “Hyperthermia and

Immunotherapy for Leukemias, Lymphomas, and Solid Tumors”, issued 2/25/2003, Bruce Lyday, Inventor, assigned to Triad Immunologies, LLC

Method of Use Patent covering use of any strain of dengue virus to treat any form of cancer.

Pending Bio-License for Strain 45AZ5 with Army MEDCOM will extend I.P. beyond patent expiration.

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Triad’s Collaboration Partners

University of California, San Diego, Moore’s Cancer Center

Walter Reed Army Institute of Research

Veterans Affairs Medical Center- San Diego, CA

Principal Investigators Dr. Minev and Dr. Daniels, mouse models

GMP-Dengue Virus Production, CRADA

Clinical Trial Site

Partner Collaborative Extent

35

Triad Phase I Costs- n=24-$1.5-1.67MTriad Phase I Trial Budget $1,677,508

$1,677,508 Visit

1/Screen Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8 Visit 9 Visit 10 Visit 11 Visit 12

Visit 13 Study

Complete/ Off Study

Study Procedures $ $ $ $ $ $ $ $ $ $ $ $ $

Consent, Complete physical exam with ECOG, evaluations of measurable disease,medical history, cancer history TX, vital signs-BP, HR, RR, temp, height & weight $83.25 $135.00

MRI scan with contrast $1,076.10

CT scan with contrast-chest, abdomen, & pelvis $1,062.00 $1,062.00 $1,062.00 $1,062.00

Interim physical exam with ECOG, adverse event assessment, concomitant medications, eligibility criteria (screening) $83.25 $83.25 $83.25 $83.25 $83.25 $83.25 $83.25 $83.25 $83.25 $83.25

Viral screen-HBSAG, HCAB, HIV $92.00

Baseline clinical labs $185.00 $114.00

RF, ANA $49.00 $49.00

BHCG $17.00

Dengue Antibody (IGG, IGM) $48.00

Leukapheresis $850.00

Standard clinical labs $45.00 $22.00 $22.00 $22.00 $22.00 $22.00 $22.00

HLA A2 $185.00

Total Per Patient Visit Cost $2,842.35 $850.00 $83.25 $105.25 $83.25 $105.25 $105.25 $1,167.25 $83.25 $105.25 $154.25 $1,145.25 $1,311.00

Total Per Pateint Cost of All Visits $8,138.35Estimated Costs for 24 Patients 195312

$108Total Costs Part "A" $195,312 $195,312

Costs "B" PATIENT CARE COSTS "D"CRC (Aor D) - "D" = inpt. $720/day, outpt. $25 + $32/nurse , labs as above $43,500.00

Shipping - Dengue IGG, IGM labs on ice overnight $250 Melanoma n=12

DTH - Supplies antigen (candida 1ml = 100 dose vial $89 x 2, trichophyton 2 ml = 20 dose vial $39.50 x 1) $160.00

Pharmacy services - DTH prep x 2, dengue injection x 1, orders $750.00 Bed Rate at CTRI $1.097.25

CRO Support Clinical Phase II $62,750.00 #days/patient 12

CRO Support Pre-Clinical $34,250.00 # patient days 144

Courier transport to lab $150.00 CTRI costs $157,968

Total Costs "B" $141,810

Prostate n=12

Total Costs "C"

One-Time Study Costs $157,968

Start-up Cost (non-refundable) $16,500

Study Coordination/Data management $32,000 Total Costs "D" $315,936

Liability insurance $128,000

Dengue-1 Strain 45AZ5 (500 Vials) $381,500 Total A+B+C+D= $1,461,508Dendritics (Tech. + 24 Patients@$7,000/patient $420,000

HLA-A2 Peptides (3 Melanoma +3 Prostate) Estimated Peptide Costs (from stock) $81,000

Total Study Costs Part "C" 978,000.00$ Estimated Peptide Costs (new synthesis) $216,000

Grand total for 24 Stage IV patients=$1,542,508

,

IGG, IGM - 3 employees at $36ea x 3 tests $108.00 (Occupational Health)

Business Consideration Points Advanced Prostate and Melanoma currently have no

effective/approved treatments; markets for these indications >$6.4 B worldwide

Triad’s Adjuvant can be used to complement any existing active immunotherapy platform: DC, oncolytic viruses, etc., allowing sub-licensing for multiple, separate indications after Phase I.

CRADA with U.S. Army allows FDA to x-reference IND on strain 45AZ5, speeding up regulatory review.



Contact Information

Bruce W. Lyday, V.P., Product Development

phone (714) 585-7485 email: [email protected]

38

Reference List A Kurnae, I., et. al., Dengue virus infection of human skin fibroblasts in vitro production of

IFN-Beta, IL-6, and GM-CSF. Archives of Virology 124: pp.21-30, 1992. Kurane, I., Innis, B. L., Nimmannitya, S., Nisalak, A., Meager, A., Janus, J. and Ennis, F.

A.: Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. Journal of Clinical Investigation 88: 1473-80, 1991.

Hober, D., et. al., High levels of sTNFR p75 and TNF alpha in dengue-infected patients. Microbiol. Immunol. 40: pp.569-73, 1996.

Chang, D., and Shaio, M., Production of IL-1 by human monocytes exposed to dengue virus. J. Infectious Disease 170: pp.811-817, 1994.

“NK Cells, displaying early activation, cytotoxicity, and adhesion molecules, are associated with mild dengue disease”. E. Azeredo, et. al. Clin. And Exp. Immunology 143 2003, pp. 345-456.

Kovalovich, K., et. al. IL-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2, and Bcl-xl. J. Biol. Chem. 276: pp.26605-13, 2001.

den Boer, A., et. al., Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL Immunity Vs. Tolerance.J. Immunol. 167: pp.2252-2258, 2001.

Ellem, K., et. al. The labyrinthine ways of cancer immunotherapy-T cell, tumor cell encounter: “How do I lose thee? Let me count the ways”. Advances in Cancer Research 1998, Academic Press.

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Reference List B

1. George, R., and Lum, L., Clinical spectrum of dengue infection. In Dengue and Dengue Hemorrhagic Fever, ed. by D.. Gubler, CAB International, 1997.

2. Pasca, A., et. al., Role of Interleukin-12 in patients with dengue hemorrhagic fever. FEMS Immunol. Med. Microbiol. 28: pp.151-155, 2000.

3. X. Bai, et. al. Local co-stimulation reinvigorates tumor-specific CTL for experimental therapy in mice with large tumor burdens. J. Immunol. 167: pp. 3936-43, 2001.

4. Chakraborty, N., et. al., Emergence of regulatory CD4+ T cell responses to repetitive stimulation with antigen-presenting cells in vitro: implications in designing tumor vaccines. J. Immunol. 162, pp.5576-83, 1999.

5. Razvi, E., et. al. In vivo state of antiviral CTL precursors. J. Immunol. 154: pp. 620-632, 1995.

6 K. Steinbrink, et. al., IL-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8+ T cells resulting in a failure to lyse tumor cells. Blood 93: pp.1634-42, 1999.

7 Wagner, S., et. al. Immune response against primary human malignant melanoma: a distinct cytokine mRNA profile associated with spontaneous regression. Lab. Invest. 78: pp.541-550, 1998.

8 Durand, R. Intermittent blood flow in solid tumors, an under-appreciated source of ‘drug resistance”. Cancer and Metastasis Reviews 20: pp. 57-61, 2001.

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Reference List C1. Vaupel, P., and Kallinowski, F. Physiological effects of hyperthermia. In Recent Results

in Cancer Research 104: Springer-Verlag, Heidelberg, 1987. 2. Dudar, T., and Jain, R. Differential response of normal and tumor microcirculation to

hyperthermia. Cancer Research 44: pp.605-612, 1984. 3. Ganss, R., et. al., Combination of T-cell therapy and trigger of inflammation induces

remodeling of the vasculature and tumor eradication. Cancer Research 62: pp.1462-1470, 2002.

4. Watanabe, N., et. al. Toxic effect of TNF-alpha on tumor microvasculature in mice. Cancer Research 48: pp.2179-83, 1988.

5. Nikan, B. A mechanism of the spontaneous remission and regression of cancer. Cancer Biother. Radiopharm. 13: pp.209-10, 1998.

6. Overwijk, W., et. al., Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive T cells. J. Exp. Med. 198: pp. 569-80, 2003.

7. Butz, E., and Bevan, M. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8: pp. 167-75, 1998.

8. Chen, Y., and Wang, S., Activation of terminally differentiated human monoctyes by dengue virus. J. Virology 76: pp.9877-9887, 2002.

9. Medema, J., et. al., Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9 constitutes an escape mechanism for tumors. PNAS 98: 11515-20, 2001.

10. “ICAM-1 has a critical role in the regulation of Metastatic Melanoma tumor susceptibility to CTL lysis by interfering with PI3K/AKT pathway”. A, Hamai,et. al., Cancer Res. 68, Dec. 2008, pp. 9854-9864

27. “Lack of attenuation of a candidate Dengue-1 vaccine (45AZ5) in human volunteers”. K. Eckels, et.al., Am. J. Trop. Med. Hyg.. Mar. 1987, pp. 435-42.

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