CANCER AND IMMUNITY — THE NEXT FRONTIER IN

CANCER AND IMMUNITY - THE NEXT FRONTIER IN CANCER TREATMENT Zheng Cui, MD/Ph. D Section of Tumor Biology Department of Pathology Wake Forest University School of Medicine Winston-Salem, NC, USA Telephone: 336 -716 -6185 Email: zhengcui@wfubmc. edu 1

Acknowledgement This presentation is made possible by: Gailyn Waldron and Threshold, Inc. Barbara Shook and the Barbara Ingalls Shook Foundation 2

Current cancer therapies: Chemo Surgeries Kill growing cells systemically Remove cancer at early stage Immunotherapies Repair a weak/bad immune system Radiation Kill live cells locally Targeted therapies: Herceptin, Gleevec 3

Cancer Stats: The US Population: ~6, 000, 000 300, 000 1/20 Yearly cancer death: ~6, 000 600, 000 1/10 Cancer mortality rate: (age-adjusted) ~100/100 K ~200/100 K The outlook of cancer treatment is not so good! Mortality rate (per 100 k population) Worldwide >1600 cancer deaths a day in the US! 600 400 200 1950 US/W 2 -fold CDC data Heart disease Cancer Death Rate 2004 4

May 8 th, 2006 5

May 8 th, 2006 The second most blogged news story in May, 2006 6

The most blogged news story in May: Britney Spears! 7

Since then, she continues to make news…. 8

Lung cancer rate 0. 08% 8% Smoking General population Smokers Why don’t most smokers have lung cancer in the face of intense carcinogen exposure? 9

Cause of cancer: cell damage and aging Host protection Cell Damage DNA repair Clearance of cancer cells Cell Damage Host protection Younger and healthier Older 10

Pathways to cancer treatment Pathway #1: why do we get cancer Modeling cancer in animals Understanding Cancer mechanisms Finding Molecular targets Testing Targets Developing human therapy Pathway #2: why don’t we get cancer Finding Cancer. Resistant Humans Developing human therapy 11

Modeling Cancers in mice Aggressiveness Transplantable mouse cancer cell lines Too aggressive, too lethal, too rapid S 180, EL 4, L 5178, L 1210, J 774, LL 2, Meth. A etc. Cancer type Carcinogen-induced cancers Takes months if not year Genetically engineered tumors Takes months if not year and often not malignant Transplantable xenograft-human tumor cell lines Often not showing malignant properties Spontaneous tumors at old age: 81% of laboratory mice have cancers at their natural death but often not aggressive Survival time 12

Serendipitous discovery of a cancer-resistant mouse Body Weight in Grams No exception 40 WT WT WT 30 20 CR 10 WT 0 1 9 17 25 33 41 Days After S 180 Injections 49 57 CR=cancer-resistant WT=cancer-sensitive Completely unexpected CR 13

Survival after cancer challenge is based on inheritance: it is all in the DNA M F SR 24 37% 9 15 WT 40 18 22 14

From a single mouse to a great legacy! Finally, they are available free to research community! 15

Cancer cells are specifically killed by white cells that formed “rosettes” 16

Cancer cell killing is dependent on cell-cell contact. 17

Natural killer cells form rosettes and aggregates with tumor cells 18

The surface of cancer cell is damaged before dying 19

An overview of two arms of immune system Presence of pathogens Innate immunity: Natural Killer cells (NK) Macrophages (MΦ) Neutrophils (PMN) Complements (Within hours) Adaptive Immunity: B cells (antibodies) T cells (CTL) Dendritic cells (DC) Macrophages (Weeks) 20

White cells are tracking down cancer cells Cell aggregation 21

Cancer cells are ruptured by white cells Mixed pop-rosettes 22

Cancer cells are ruptured by white cells Mixed pop 23

A much larger cancer cell killed by a small white cell (neutrophil) Pmn-apop 24

SR/CR, n=14 WT, n=8 2500 Tumor Volume (mm 3) Tumor Volume relative to Controls (%) Cure established cancers in WT mice by systemic WBC therapy 2000 1500 AT 1000 500 0 1 3 5 7 9 11 13 15 Days after adoptive transfer of leukocytes 200 160 AT 140 120 100 80 60 40 20 0 17 WT-AT SR/CR-AT 180 0 2 4 100 Survival (%) 80 6 8 10 12 14 16 18 20 Days After Cancer Establishment Still alive after 18 months AT (day 4) WT-AT, n=5 SR-AT, n=7 60 40 20 0 0 4 8 12 16 20 24 28 32 36 40 44 Days Post-Tumor Injection SR AT 1 -day 8 -day 12 -day 18 -day 23 -day 25

Cure of mouse prostate cancer by white cell infusion White cell infusion Cancer cell challenges n=7 100 Survival % Dr. Yong Chen Cancer Biology WFUSM SR/CR AT 80 60 No AT 40 20 n=10 0 0 1 2 3 4 5 6 7 8 Age (Months) 9 10 11 12 26

It is a permanent, complete cure of cancer! WT Normal PTEN-KO + SR Cancer Scars 27

The innate immunity of cancer-resistant mice can cure cancer Genetically defined No need for further manipulation Involves the innate immunity that has been mostly ignored Highly effective: survival of million times more lethal doses No side effect Kill different cancers Transferable to treat established cancers in ordinary mice 28

Cancer-resistance and humans Why do we have natural protection against cancers: Humans and other large, long-living animals must have exceptional natural resistance and immunity against malignancy in order to survive beyond reproductive ages Mouse Human Difference Body size 25 g 75, 000 g 3000 x Lifespan 2. 5 (<1) y 75 y 30 x Pre-reproduction 8 w 800 w 100 x % with ca at death 81% 25% 3. 2 x Cancer Surveillance ± +++++ Why don’t normal mice have meaningful resistance or immunity against malignancy? 29

Mice in the wild have a tough life and don’t live long enough to get cancer. Therefore, there was no natural selection for resistance against cancers. 30

If humans do have cancer-resistance, can we measure it? Can we inject cancer cells into humans to find out? Probably not!? 31

Cancer Cells White blood cells Incubation % killed 70 30 40 / 70 = 57. 3% Killed 32

Manual test of cancer cell killing activity: How well the white cells can kill cancer cells in test tubes Control: cancer cells without white cells (100% survival) Cancer cells with white cells (Ratio at 50: 1) (some survival) 33

Automated real-time recording of cancer cells killed by WBC Hela + non-killing control cells: 1: 20 Hela control 1 Hela + WBC: 1: 20 2 3 21 42 63 84 105 Time in h Plating target cells Time point of manual assays 1 =0% kill Addition of effector cells 2 =30% kill 34 3 =50% kill

Identifying cancer-resistant mice without a cancer cell challenge Litter mates 1 2 3 4 5 6 7 Killing in Test tubes Resistant/Survival after challenge + + + 35

Test-tube cancer resistance results using cancer-resistant and non-resistant mice 100. 0 87. 5 In vitro cancer kill activity (target cells killed %) 75. 0 62. 5 50. 0 37. 5 25. 0 12. 5 0 WT Mice SR mice 36

Mouse cancer-resistance by age 100. 0 S 180 cells 87. 5 In vitro cancer kill activity (S 180 cells killed %) 75. 0 62. 5 50. 0 37. 5 25. 0 12. 5 0 WT Mice Young SR mice Old SR mice cancer cells 37

Human cancer-resistance by age 100. 0 87. 5 Hela cells In vitro cancer kill activity (target cells killed %) 75. 0 62. 5 50. 0 37. 5 25. 0 12. 5 0 WT Mice SR mice Humans <50 y Humans >50 y, Ca 38

Automated recording of cancer cell kill White cells that don’t kill cancer cells White cells that kill cancer cells 39

Rosette formation leading to cancer cell kill Mouse S 180 cells SR mouse WBC Hela cells Human WBC Cancer patient No rosettes 40

Surveillance by human white cells 41

Cancer cell killed by a smart bomb (white cell) 42

Stability of cancer-killing activity in human WBC Recovery in 3 days 100 % of cancer cells killed 80 60 40 20 0 2 4 6 8 10 12 14 16 Time span in weeks Stress 43

Stability of cancer-killing activity in human WBC Recovery in 3 months 100 % of cancer cells killed 80 60 40 20 0 2 4 6 8 10 12 14 16 Time span in weeks Stress 44

Seasonality of CKA activity? 100 % of cancer cells killed 80 60 Higher mortality rate in elderly Influenza season Lower immune responses Severe mood swing (SAD) 40 20 0 Aug Sep Oct Nov Dec Jan Feb Mar Northern Hemisphere: Shorter daylight Lower intensity of sunlight radiation Lower temperature Vitamin D deficiency 45

Chester Southam: Injecting human subjects (Inmates and ca patients) with live cancer cells Healthy humans were resistant to cancer cells. Cancer patients lost cancer resistance while retained antimicrobeial resistance. Ethical and legal problems Elected to the president of AACR in 1968 Science 25 January 1957 125: 158 -160 46

Possible effects of aging and stress on CKA of human WBC 75% 25% 47

Anticancer protection can be lost due to: 1. Genetics 2. Aging 3. Stress 4. Seasonal changes 5. Too many cancer cells (increased carcinogenesis) 6. Too few immune cells (immune suppression) 48

A new cancer treatment concept: To replace a weak/bad immune system with a validated, functional one rather than to repair it 49

Healthy volunteers CKA screening White cells 50

The new cancer treatment concept Donor selection: CKA, Availability, ABO, Virus status Patient selection: reasonable condition Dose escalation Safety control: GVHD Routes of delivery: systemic, local Combination with existing therapies 51

Uniqueness of this treatment: No drug involved Technology- and knowledge-based Minimal side-effect is expected Involves innate immunity (macrophages and neutrophils) but not adaptive immunity (T cells) 52

Adoptive Transfer in different age groups Young=3 -6 months Old=22 -26 months 80 Young to Young (n=16) 60 *n=14 40 Old to Young (n=16) 20 Young to Old (n=15) Old to Old (n=16) 0 0 20 40 0 120 Old cells 20 0 Young cells Old recipients Young recipients 40 Old recipients 60 Young recipients 20 Young recipients 40 100 80 Old recipients 60 Young recipients 80 60 80 Days after S 180 100 Old recipients Survival Percentage 100 Young cells 53

Percent Survival Mismatched Adoptive Transfer in Mice n=12 in each group 100 80 60 100 40 83. 3 58. 3 20 41. 7 Percent Survival 0 100 80 Male recipients 60 Female recipients 40 20 0 Control Sex Mismatch MHC Mismatch Sex+MHC Mismatch 54

Why hasn't this treatment for cancer been tested yet? Differences with conventional funding philosophy No mechanism: rational design vs empirical approach. (Pathways of development) A radical departure from textbook: innate immunity vs adaptive immunity Too good to be true Not from a well-known establishment Worsening funding environment 55

Funding requirements to treat human cancer patients $10 million is needed for: Clinical trials in humans for different cancers Clinical trials in dogs Developing techniques for long-term storage of cancer-killing white cells Refinement of CKA assay: automation 56

Cancer and Immunity: the Next Frontier in Cancer Treatment The Promise No more disfiguring surgery No more toxic chemotherapy No more damaging radiation therapies No more cancer recurrence Permanent cures for cancer using the immune system We can cure cancer using what we have learned from cancer-resistant mice 57

Funding: CRI NCI WFU Charlotte Geyer 58

Many thanks again to: Gailyn Waldron and Threshold, Inc. Barbara Shook and the Barbara Ingalls Shook Foundation 59

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