Fight for life.
Introduction to Cancer Immunity One out of four people born in the USA will be diagnosed with cancer at some point. A long-standing question has been: what about the other three? Why do they not get cancer? Are they merely lucky, or do they harbor some sort of immunity to cancer? Several lines of evidence suggest that the latter may be the case: Susceptibility to cancer and freedom from cancer in old-age appear to run in families 1, suggesting a genetic basis for cancer resistance. Cancer incidence increases exponentially throughout the life-span, but levels off in extreme old-age, suggesting the existence of a cancer-resistant sub-population2. Finally, a group of experiments that would find difficulty to get approved today suggest that healthy people have intrinsic resistance to transplanted cancers, and such resistance is lost in cancer patients 3. The immune system appeared to be a natural place to search for a mechanism underlying this apparent natural human cancer resistance. Broadly, the immune system can be divided in two strands: The adaptive arm, which “learns” to discriminate between self-and non-self, using antibodies and T-cells, and the innate arm, which “automatically” recognizes a fixed set of hardwired targets, such as invading bacteria, or possibly cancers. Adaptive Immunity to Cancer The use of adaptive immunity to target cancer is a burgeoning field. Several anti-cancer antibodies are currently on the market 4, and cancer vaccines are not far behind 5. These therapies are called “targeted”, because the immune system can be trained to target more or less only the cancer, and leave healthy cells alone. Indeed, they have far fewer side-effects than first-generation chemotherapy and radiation therapy, which kills dividing cells, whether or not they are cancerous 6
Innate Immunity to Cancer
Thus, it seems to us, that using the more “stubborn” cells of innate immunity could potentially circumvent these problems. Certain innate immune cells can still recognize targets selectively, but are harder to embezzle with corrupt signaling molecules. They have multifunctional “pattern recognition” receptors, which are hard to fool by reducing the expression of single proteins. For the killing mechanism, they use free radicals, which no biological system can resist.
There is indeed limited evidence suggesting that innate immunotherapies may be far more effective than any other known types of therapies. The figures below compare survival rates for different rates of intervention.
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A mixture of chemotherapy drugs slightly extends survival in a mouse model of spontaneous leukemia, (while causing horrible side-effects).12
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In humans, typical chemotherapy drugs slightly extend survival, do not cure the disease and have devastating side-effects (Pemetrexed, also known as Alimta, is shown here as one example)13
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Provenge, a human cancer vaccine, is more slightly effective than chemotherapy and avoids most of its side-effects. This is certainly a great accomplishment. However, most patients still die only months after when they would have died without Provenge. Even this futuristic biological treatment does not cure the disease. 14
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Avastin,
one of the world’s best anti-cancer antibodies is more effective than
chemotherapy, with few side-effects. However, like the vaccine, it
does not cure the disease, and delays the inevitable by no more than a
few months.15
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In stark contrast, recent research with innate immune cell transplantation has completely cured lethal cancers in mice. 16
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In humans, Bacillus Calmette-Guerin (BCG), an activator of innate immunity, causes dramatically extended patient survival with many patients achieving a complete, sustainable cure.
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Only
when using innate immunotherapy, the survival curves seem to stabilize and
never reach zero, suggesting a permanent, sustainable cure in many patients
(BCG) or all research animals (cell transplantation). Results like this
suggest to us that innate immunotherapy is the only approach that has shown the
promise to permanently cure advanced cancer. Ironically, it is also one
of the most neglected areas in contemporary research. We felt that the
case was sufficiently compelling to make this Livly’s first internal research
project: To develop biotechnology therapeutics based on innate immunity
to cancer.
Research Results In our cell culture facility, we have done some preliminary experimentation to determine whether human innate immune cells would be able to destroy cancer cells in a Petri dish.
Innate immune cells appear to associate with cancer cells in conspicuous ways
We purified granulocytes from Eri’s blood and exposed them to cervical cancer cells in a culture dish. The granulocytes are small and round. Cervical cancer cells are large and firmly attached to the culture dish. When the cancer cells die, they are known to detach from the dish material, balloon and then burst. One interpretation of these images could be that Eri’s granulocytes can seek out (A), attack (B) and kill (C) the cervical cancer cells. However, these events were not frequent, and we have yet to develop a method for their precise quantification. Cancer killing can be confirmed by time-lapse video microscopy To gain a clearer image of whether the cancer killing is actually caused by the immune cells, we pointed a time-lapse camera into our incubated tissue culture microscope, and recorded the cellular activity in petri-dishes containing our purified immune cells and cancers. We found that indeed sometimes a group of immune cells approached a cancer cell, surrounded it and killed it. However, these events were rare. At most a few percent of the cancers seemed to be killed. Several of these cancer killings can be seen in our video gallery. Implementation of a quantitative high throughput cancer killing assay The microscopic analyses were limited to showing us qualitative events at low throughput. We could not precisely tell how many cancers were killed, and can only record one single culture per day, because we have only one microscope. We needed a method to measure the killing quantitatively that could allow us to test many different conditions in parallel, so that we can determine under what conditions the killing activity is best. For this purpose, we “paint” the cancer cells with a green fluorescent dye, Calcein-AM before the immune cell treatment. 
We can use our new FMAX fluorescence microplate reader, to measure the total amount of green fluorescence in 96 microcultures simultaneously. This method gives us the ability to measure the number of living cancer cells growing in different wells in a 96-well plate, before and after a killing treatment. We tested this using alcohol for killing: A nice correlation is seen between the fluorescence measurement and the amount of alcohol used: Near Future Now we are beginning to test immune cells from different people for cancer killing ability. This experience should allow us to drug or engineer this trait and begin the development of innate immune cell based cancer therapeutics.
Scientific References 1 McDuffie HH, Pahwa P, Karunanayake CP, Spinelli JJ, Dosman JA. Clustering of cancer among families of cases with Hodgkin Lymphoma (HL), Multiple Myeloma (MM), Non-Hodgkin's Lymphoma (NHL), Soft Tissue Sarcoma (STS) and control subjects. BMC Cancer. 2009; 9:70. 2 Caruso C, Lio D, Cavallone L, Franceschi C. Aging, longevity, inflammation, and cancer. Ann N Y Acad Sci. 2004; 1028:1-13. 3 Chester MS, Alice EM, Cornelius PR. Homotransplantation of Human Cell Lines. Science , 1957; 158-160. 4 Gerber DE. Targeted therapies: a new generation of cancer treatments. Am Fam Physician. 2008; 77(3):311-9 5 Finke LH, Wentworth K, Blumenstein B, Rudolph NS, Levitsky H, Hoos A. Lessons from randomized phase III studies with active cancer immunotherapies--outcomes from the 2006 meeting of the Cancer Vaccine Consortium (CVC). Vaccine. 2007; 25 Suppl 2:B97-B109 6 Hait WN, Hambley TW. Targeted cancer therapeutics. Cancer Res. 2009; 69(4): 1263-7; discussion 1267 7 Bubeník J. MHC class I down-regulation: tumour escape from immune surveillance? Int J Oncol. 2004; 25(2):487-91. 8 Stuge TB, Holmes SP, Saharan S, Tuettenberg A, Roederer M, Weber JS, Lee PP. Diversity and recognition efficiency of T cell responses to cancer. PLoS Med. 2004; 1(2):e28 9 Beyer M, Schultze JL. Immunoregulatory T cells: role and potential as a target in malignancy. Curr Oncol Rep. 2008; 10(2):130-6 10 Keilholz U. CTLA-4: negative regulator of the immune response and a target for cancer therapy. J Immunother. 2008; 31(5):431-9 11 Bertrand J, Begaud-Grimaud G, Bessette B, Verdier M, Battu S, Jauberteau MO. Cancer stem cells from human glioma cell line are resistant to Fas-induced apoptosis. Int J Oncol. 2009; 34(3):717-27. 12 Bekesi JG, Roboz JP, Zimmerman E, Holland JF. Treatment of spontaneous leukemia in AKR mice with chemotherapy, immunotherapy, or interferon. Cancer Res. 1976 Feb;36(2 pt 2):631-9. 13 Ciuleanu T, Brodowicz T, Zielinski C, Kim JH, Krzakowski M, Laack E, Wu YL, Bover I, Begbie S, Tzekova V, Cucevic B, Pereira JR, Yang SH, Madhavan J, Sugarman KP, Peterson P, John WJ, Krejcy K, Belani CP. Maintenance pemetrexed plus best supportive care versus placebo plus best supportive care for non-small-cell lung cancer: a randomised, double-blind, phase 3 study. Lancet. 2009 Oct 24;374(9699):1432-40. 14 Higano CS, Schellhammer PF, Small EJ, Burch PA, Nemunaitis J, Yuh L, Provost N, Frohlich MW. Integrated data from 2 randomized, double-blind, placebo-controlled, phase 3 trials of active cellular immunotherapy with sipuleucel-T in advanced prostate cancer. Cancer. 2009 Aug 15;115(16):3670-9. 15 Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004 Jun 3;350(23):2335-42. 16 Hicks AM, Riedlinger G, Willingham MC, Alexander-Miller MA, Von Kap-Herr C, Pettenati MJ, Sanders AM, Weir HM, Du W, Kim J, Simpson AJ, Old LJ, Cui Z. Transferable anticancer innate immunity in spontaneous regression/complete resistance mice. Proc Natl Acad Sci U S A. 2006; 103(20):7753-8. Livly
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