Beau S Rough

Beau Kildow & Sean Penn
Immunology Paper-Cancer Vaccines
March 25, 2008
Cancer Vaccines
The development of vaccines is undoubtedly the most important contribution of immunology to the improvement of human health (3, 41). Edward Jenner’s discovery of the smallpox vaccine in 1798 marked the beginning of immunology (3). In addition, Louis Pasteur successfully created the first therapeutic vaccine by curing individuals infected with rabies (36). Currently, there are twenty-six infectious diseases that are preventable through vaccination; however, there are still many other diseases that are not protected by vaccines (3). One disease that has escaped protection by vaccines is cancer. Cancer is a disease that can be described by cells that grow, divide, invade, and spread uncontrollably over the body. Cells become cancerous when a mutation occurs specifically in two of its genes: proto-oncogenes and tumor suppressor genes. Proto-oncogenes control cell division and differentiation** and turn into oncogenes when mutated, which allows the cell to divide quickly and wildly.** Tumor suppressor genes slow down cell division, repair DNA mistakes, and activate apoptosis. When these genes are mutated, they are deactivated, allowing the cell to divide uncontrollably without undergoing apoptosis (42). Mutations of these genes (which genes the tumor suppressor or the proto-oncogenes or both) can occur hereditarily, through environmental factors such as exposure to radiation and toxins, or by viral infection. Today, cancer is the second leading cause of death and over 40% of men and women in the US will develop some type of cancer during his or her lifetime (42). (**I like the opening paragraph and introduction of your topic. Maybe consider dividing this paragraph in half by starting a new paragraph when introducing cancer.)* Well explain introduction

Currently, there are four main types of treatment for cancer: Surgery, radiotherapy, chemotherapy, and immunotherapy. Nevertheless (Consider using however), none of these types of treatments have been totally effective in eliminating cancer. Treatment via surgery is only effective in removing visible tumors, but is ineffective in eliminating cancer that has metastasized to other regions of the body. In addition, chemotherapy and radiotherapy are effective in reducing cancer cells; however, both methods are harmful to normal neighboring cells (42). The future for a possible cancer vaccine lies in the treatment through immunotherapy. The basis behind immunotherapy is to stimulate an immune response to attack cancer cells. In order to stimulate an immune response against cancer, the cancer cells must contain an antigen that is either overexpressed or foreign to the body (27). Since cancer cells are not foreign, they are highly tolerated by the immune system (3,27,36). Many antigens expressed on tumor cells are also expressed in normal cells, which is one challenge in developing a cancer vaccine. However, there have been over 70 antigens that have been discovered that are specific to tumor cells (27). The main challenge for developing an effective cancer vaccine is to present the tumor specific antigens to the immune system in order to induce an immune response that will attack cancer cells only.

The immune system contains many different cells, each with a specific role in attacking foreign particles. The main goal is to stimulate T cells that are effective in lysing a cancer cell. One type of T cell that is directly involved in lysing cancer cells are CD8+ T cells. CD8+ T cells are responsible for recognizing foreign antigens expressed on MHC class I molecules on the cell surface of a particular cell. MHC class I molecules are transmembrane glycoproteins that acquire internal antigens from a cell and present them on the surface of the cell for recognition by the immune system. Once the CD8+ T cell-MHC class I interaction occurs, antigen-specific cytotoxic T lymphocytes (CTL) clonally expand and lyse any cell with the particular antigen-MHC class I complex (21). In addition, CD4+ T cells are involved in antibody production, macrophage stimulation, and CD8+ T cell proliferation (3). CD4+ T cells are responsible for recognizing antigens presented on MHC class II molecules. MHC class II molecules, like MHC class I, are transmembrane proteins on the surface of Dendritic cells (DCs). Dendritic cells are antigen presenting cells (APCs) that function by engulfing foreign and infected cells, breaking down them down, and presenting the cells’ antigens on its surface via MHC class I and class II molecules. Dendritic cells are so important in an immune response because they have such a large surface area full of MHC class I and class II and costimulatory molecules (B7-1/CD80, B7-2/CD86, and CD40) that increase presentation of antigens and stimulation of T cells. In addition, Dendritic cells are able to move to secondary lymphoid tissue effectively in order to present antigens to T cells (41). After a CD4+ T cell interacts with an antigen presented on an MHC class II molecule in presence of a costimulatory molecule, it clonally expands and interacts with a particular B-cell. The B-cell then differentiates into a plasma cell which then clones itself and secretes antibodies against the particular antigen that was presented on the DC. The antibodies then interact with all the cells that contain the particular antigen which then are killed through phagocytosis or by the complement cascade. Moreover, You can use Also or Besides the particular antigen that is expressed on the MHC class I molecule of the Dendritic cell is allowed to interact with CD8+ T cells, allowing them to proliferate and lyse any cell containing that particular antigen. Dendritic cells also recruit natural killer cells (NK) through the production of cytokines. Cytokines are proteins that are released by immune cells once a foreign particle is recognized by its receptors. Dendritic cells release interleukin-12 (IL-12), which recruit NK cells. NK cells recognize allogenic MHC molecules or cells that express low levels of MHC molecules. Cancer cells express little if any MHC molecules, ( Why do cancer cells express little MHC molecules? Is that a fact?) making NK cells important in cancer immunotherapy. Also, T helper cells (CD4+) secrete IL-2 and interferon gamma (IFN-γ) once activated. IL-2 is important in the survival of activated T cells and promotion of CTL activity (3, 21). If IL-2 is not present, T-cells begin to die and clonal expansion becomes inefficient. After an immune attack eliminates most cells containing a particular antigen, the lymphocytes involved with the attack die. However, there are very few lymphocytes that enter a quiescent state. These lymphocytes are termed memory cells and become re-activated if a pathogen/cancer cell containing the particular antigen would re-appear. Eliciting memory cells is another challenge in designing an effective cancer vaccine. In order for a cancer vaccine to work effectively, it must stimulate an immune response that will effectively kill cancer cells, and retain memory cells. However, stimulation of an immune response against cancer cells is difficult due to the fact that cancer cells are not recognized as foreign and are poor immunogens (30). The main goal of a cancer vaccine is to effectively present tumor specific antigens in order to stimulate a powerful immune response capable of destroying cancer cells. This paragraph is FULL of definitions and terminology, you may need to break it down a bit for the reader, because I know that I was overwhelmed. ** I agree there is a lot of different terms in here that can be divided in paragraphs. The reader can get confused with so many terms**

Recruitment and/or manipulation of dendritic cells(I do not believe that dendritic needs to be capitalized) is the main focus of many types of cancer vaccines. One type of cancer vaccine involves the modification of dendritic cells in vivo. Kim et al. created a vaccine by injecting naked DNA coding for the tumor specific antigen, HPV-16 E7 via gene gun into Langerhans cells (a type of DC). HPV-16 E7 antigen is targeted because HPV is present in many cases of cervical cancer and a vaccination against E7 will help reduce HPV infections. In addition, the DNA also contains small interfering RNA that inhibits the expression of Bax and Bac proteins, which are DC proapoptotic proteins. As a result, it increased the life of Dendritic cells along with an increased anti-tumor immune response which was seen in murine models (14). On the same note, Dendritic Is dendritic going to have capitalize D or not, because earlier on the paper you wrote it with lower case d ;) ** cells can also be injected with cytokines in order to stimulate an immune response against tumor cells. Nishitani et al. created a cancer vaccine by injecting DC’s with DNA encoding for IL-12 with a gene gun (20). As previously mentioned IL-12 activates NK cells, inhibits angiogenesis, and primes Cytotoxic T lymphocytes. In concert with IL-12, mice were inoculated with irradiated murine renal cancer cells. Irradiated tumor cells easily release its antigens, allowing DC’s to present them in greater number. Results revealed that nearly 80% of mice vaccinated showed tumor rejection. Furthermore, the vaccinated mice showed tumor resistance when challenged again with tumor cells. This observation revealed that memory cells were maintained, which is essential for an effective cancer vaccine (20). More recently, naked DNA fusion gene vaccines have been used to attack cancer. The goal of this particular vaccine is to load DNA with genes encoding for cytokines, Fc receptors, complement, and antibodies against DC receptors in order to increase uptake for presentation of antigens that are also encoded in the DNA (21, 33). More importantly, naked DNA fusion gene vaccines are widely used against B cell malignancies. Similar to that of DC uptake, this mechanism involves B-cell uptake of the DNA which encodes for a specific antigen on the B-cell (36). The B-cell then presents the antigen via MHC class II molecules which are recognized by CD4+ T cells. This interaction allows for antibody production, thus producing immunity against B-cell malignancies. Significant antibody production against Ig (the main B-cell associated antigen)-FrC (tetanus toxin) complex along with existence of memory B-cells was observed in an experiment conducted by Savelyeva et al. (29)
(You are flipping back and forth between DC and dendritic cells, once you have established dendritic cell (DC) you can simply use DC or DC's throughout the rest of the paper, it's much easier on the reader if you keep it consistent)**

Additionally, viral vectors can be used for effective tumor specific antigen expression by Dendritic cells. DNA encoding for a specific tumor antigen is inserted into a virus, particularly a pox virus or a mutated virus that cannot replicate in a human host. Once the virus is introduced into its host, DC’s recognize its specific motifs leading to engulfment (21). Once the virus is engulfed, it expresses the tumor specific antigen for presentation on the Dendritic cell. It has been shown that pox virus vectors can stimulate a T-cell response. For example, a study conducted by Antonia et al. used an adenovirus as opposed to a pox virus as a vector. The adenovirus coded for the tumor associated antigen p53. P53 is an antigen found in lung cancer cells enabling them to grow and differentiate. In brief, (Consider:57.1% of small cell lung cancer patients having a 5 year survival rate of less than 10 %…) 57.1% of patients with small cell lung cancer, a cancer with a 5 year survival rate of less than 10%, showed p53-specific T cell response (1). Conversely You can use "On the other hand" too, many studies using a viral vector vaccine did not show a stimulation of a T-cell response against tumor cells. This can be explained by the fact that the immune system may pay more attention to the viral antigen epitopes as opposed to the tumor associated antigen epitopes, which is the downfall to this particular type of vaccine.
Similar to viral vectors as an in vivo based DC cancer vaccine, bacterial vectors can also be used to recruit and modify DC’s. One advantage of a bacterial vector is that it can be administered orally (21). Once the bacteria reach the gut, they can immediately be transferred to the Peyer’s Patches via M cells, which then can efficiently migrate to surrounding lymph nodes and be presented to DC’s (41). A study done by Luo et al. showed positive results in a prophylactic cancer vaccine using a bacterial vector. Luo et al. used a mutant strain of S. typhimurium with a DNA plasmid encoding for Fos-related antigen 1 (Fra-1) and IL-18. Fra-1 is an antigen overexpressed in murine breast carcinoma and other epithelial carcinoma cells found in the thyroid, kidney, and esophagus. IL-18 is an immunoregulatory cytokine that up-regulates MHC class I antigen expression, which enhances CTL, NK cell, and macrophage activation in lysing tumor cells. IL-18 also inhibits angiogenesis of tumor cells. Results from this experiment showed the lifespan of 60% of vaccinated mice was (delete was)tripled after a lethal dose of aggressive breast cancer cells, through the activation of T, NK, and Dendritic cells and the inhibition of tumor angiogenesis (18). As well, Zhou used S. typhimurium with an injected DNA plasmid coding for the overexpressed tumor antigen CEA and an NKG2D ligand. NKG2D is a receptor located on NK cells, activated T cells, and macrophages. Once NKG2D is activated by its ligand, it sparks NK cell-mediated immunity and primes CTL’s. Results from this study demonstrate the ability for this vaccine to induce both innate and acquired immune responses against tumors in mice (37)*.

As opposed to cell manipulation, DC’s can be recruited in vivo to initiate an immune response by a direct injection of tumor associated antigens. Many studies use antigens coupled with bacterial proteins to serve as a valuable vaccine for recruiting DC’s. For example, Miconnet et al. designed a cancer vaccine by binding two melanoma specific antigens (Melan-A and TRP-2) with an adjuvant (19). Currently, nearly all studied cancer vaccines involve the use of an adjuvant. An adjuvant is a molecule used to stimulate an immune response not specific towards the molecule itself. Adjuvants are used because tumor associated antigens are poor immunogens, and will not stimulate much of an immune response. The selection of a specific type of adjuvant is another challenge for cancer vaccine design and will be discussed later.The adjuvant used in this study was the outer membrane proteins (Omp) present on K. pneumoniae. Omp are capable of binding to tumor antigens and DC’s. The DC’s in turn uptake the omp-tumor antigen complex and present them to CD8+ T cells specific in attacking melanoma tumor cells, which was identified as a result of the vaccine (19). Similarly, Carr et al. synthesized a vaccine composed of the overexpressed antigen in many types of tumors, NeuGcGM3 with an outer membrane protein of N. meningitides known as Monotide ISA 51. All treated patients with stage III to stage IV breast cancer showed increase in antibody titers. The results suggest efficiency in DC recruitment and presentation of NeuGcGM3 on MHC class II proteins that induced CD4+ T cell response (2). Comparatively, heat shock proteins (HSP’s) bound to tumor associated antigens can elicit a potent immune response. Heat shock proteins are known as chaperone molecules because they transport antigens throughout the cell. It has been demonstrated that HSP’s bound to tumor associated antigens extracted from a tumor cell induce cancer immunity once injected back into the host (16). The reasoning behind the elicitation of such an effective immune response by HSP’s is currently being studied.

Similarly, cancer vaccines can be designed by the modification of DC’s in vitro. One method involves the fusion of DC’s with tumor cells. The logic behind this technique is relatively simple: direct fusion allows the DC’s to uptake tumor antigens and express them via MHC Class I and II molecules, thus stimulating a T cell response. (Beau, I couldn't remember exactly, but do DC's present both MHC molecules, or only MHC II molecules to stimulate T cell response??) Gong et al. showed breast tumor cell regression once introduced to a fused DC-human breast tumor cell in presence of isolated T-cells in vitro (6). Furthermore, using a slightly different fusion technique, He et al. ( You have used a lot of "et al." and names of scientists, you might want to ask Dr. Robson if it is better to leave scientists out of it and just say research states… Again, I don't know the format she wants very clearly) When you mention the authors name (et al) is that in your own words. If not try to write it in your own words used a fusion cell to elicit antibody and CTL responses to the tumor specific antigen, hCGβ. Knowingly, hCGβ is an antigen overexpressed in many common cancers such as colorectal, lung, pancreas, esophagus, breast, bladder, cervix, stomach, and prostate. He et al. coupled hCGβ with an anti-DC antibody known as B11. The coupled protein was then allowed to attach to the surface of DC’s in vitro activating antigen uptake and T cell response capable of producing anti-tumor effects in vivo (10). A more popular cancer vaccine design involves loading DC’s in vitro I think that in vitro goes in italic with tumor associated antigens. The general idea behind the process of creating this vaccine first involves extraction of DC’s from a patient with a particular cancer. The DC’s are then cultured and introduced to DNA, usually via viral vector, that codes for one or more tumor specific antigen. These genetically modified DC’s are then injected back into the patient where they travel to the secondary lymphoid tissue and present its tumor specific antigens to T-cells (21). An advantage to antigen loaded DC’s as a potential cancer vaccine is that pure uncontaminated antigens can be presented, while reducing other possible self antigen presentation that might be seen in genetically modified tumor vaccines as will be discussed later (5). To illustrate, Klein et al. compared the effects of genetically modified DC’s to genetically modified tumor cells in vitro. DC’s expressing the MAGE-1 gene associated w/melanoma cancer cells via adenovirus gene transfer was effective in reducing lung metastasis. Contrastingly, GM-CSF transduced tumor cells had no effect in reducing lung cell metastasis, although GM-CSF is (a) cytokine known for eliciting an antitumor immune response. Further results also showed DC’s modified with GM-CSF reduced lung cell metastasis (15). Likewise, Fong et al. used DC’s loaded with mouse prostatic acid phosphatase, a similar antigen present on human prostate cancer cells. Patients with metastatic prostate cancer were vaccinated with the autologous DC’s by i.v., i.d., or i.l. injection. All patients regardless of the type of injection showed increase in T cell response, noting a route-independent immune response (4). Finally, survivin is a good target tumor associated antigen when introduced with a cytokine adjuvant because it is overexpressed in breast, colorectal, and gastric cancer cells. Survivin protects these cancer cells from undergoing apoptosis and facilitates tumor angiogenesis. Idenoue et al. reported an increase in CTL’s in 100% of breast cancer patients, 83% of colorectal cancer patients, and 57% in gastric cancer patients after being injected with autologous survivin peptide pulsed DC’s (12). Because in vitro based DC cancer vaccines are personalized and require cell culture, monetary problems may reduce the chances for this type of cancer vaccine to become in effect in the future (21).

Pursuing this these further(this phrase seems unnecessary) , cancer vaccines can be made by genetically modifying tumor cells as mentioned earlier. One way this can be done is through cytokine-tumor modification. Tumor cells are known to release immunosuppressive cytokines, which can explain tumor immune escape. To counter this action, Wakimoto et al. synthesized a vaccine that consisted of irradiated tumor cells with genes coding for cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Previous studies showed that GM-CSF was the most powerful cytokine in antitumor immunity, which works by recruiting DC’s and enhancing its functionality. GM-CSF in combination with IL-4 is supposed to recruit DC’s to tumor sites in the most efficient manner. Results from this study revealed normal tumor cells that were injected four days after the administration of the gene-modified tumor vaccine were rejected in mice. Moreover, this cancer vaccine apparently permeated through the blood-brain barrier, reaching the CNS and effectively rejected brain tumor cells (26). Additionally, immunostimmulatory cytokines can be injected into tumor cells via viral vector to initiate an immune response. Studies have shown an increase in tumor suppression in mice by injecting an adenovirus coding for cytokines IL-2 and IFN-β (17). Although cytokines IL-2 and IFN-β are known toxins at a high enough concentration, by injecting them locally into a tumor cell reduces the toxicity effect. As a result, cancer vaccines are currently being tested on kidney cancer and melanoma (17,21). As well, according to recent studies, IL-12 stimulates an antitumor effect with dose dependant toxicity in humans. Hara et al. transduced IL-12 along with IL-18 ( a cytokine known to induce IFN-γ, which recruits NK cells) in renal carcinoma cells in mice, which resulted in tumor suppression by increase activity of CTL’s and NK cells. Results also suggested inhibition of the antitumor effect by CD4+ TH2 cells, which are involved in antibody production (9). Although many studies have reproduced this effect, TH2 cells have been shown to induce an antitumor effect in B cell lymphoma, breast, and colon cancer in humans (3). Toda et al. uses IL-12 gene modified mice subcutaneous tumor cells with the addition of a cell suicide gene HSV TK/GCV via HSV vector. The main reason for the suicide gene is to kill any cell with the cellular protein TK, while IL-12 recruits the immune cells. Positive antitumor results were obtained, however this vaccine is being tested further due to harmful cell suicide effects (25).

In addition, genes coding for tumor specific antigens and/or adhesion molecules, in concert with cytokines, can be injected into tumor cells in order to cause an immune response (21). For example, Schirrmacher et al. used a Newcastle Disease virus (NDV) with genes coding for two adhesion molecules to inject tumor cells in vivo. The adhesion molecules allow for CTL attachment, resulting in tumor necrosis. This vaccine design was shown to increase survival rates of stage II malignant melanoma patients by a 4-fold factor (24). Additionally, Gulley et al design a genetically modified tumor vaccine that targets prostate cancer. Prostate cancer affects over 200,000 men each year and is the most common noncutaneous cancer among men in the United States (8). The targeted antigen is known as PSA, which is only found in prostatic epithelial cells. PSA genes were transduced into a poxvirus, which was then injected into tumor cells of 17 prostate cancer patients. Along with radiotherapy, 13/17 patients showed increase in PSA-specific T cells (8). On the contrary, many studies have shown negative results in CTL induction by these vaccines; however, current research is being directed towards histones. Savage et al. identified a rise in CD8+ T cell response that reacted with the histone H4 peptide. The H4 histone peptide is seen in prostate cancer in mice; however, histones (histone peptides) have never been classified as a type of antigen because they are not well presented on MHC class I molecules (22). Ongoing research is still being conducted on histone gene injection into tumor cells and its effects on antitumor immunity.

The next type of cancer vaccine involves synthesis of carbohydrate/lipid based molecules. Synthetic carbohydrate and lipid based molecules offer a promising future for cancer vaccines. Other cancer vaccines involve extracting certain antigens/antigen genes from tumor cells, which in itself has little yield and is quite costly (13). On the other hand antigens can be synthesized from simple, cheap organic molecules that almost mimic the exact antigen that is specific or overexpressed in tumor cells. Keding and Danishefsky designed a synthetic vaccine targeting prostate cancer specific antigens, Tn, TF, STn, Lewisy, and Globo-H. All five antigen subunits were synthesized into a linear compound with KLH attached as an antigen carrier and adjuvant (13). Although the potency of this specific multifaceted cancer was not tested in this study, future experiments are being done pre-clinically. On the same note, many studies have shown that carbohydrate-based vaccines against cancer do initiate an immune response. To illustrate, Kudryashov et al. synthesized the Lewisy (Ley) antigen with KLH. Ley is overexpressed on ovary, pancreas (pancreatic), prostate, breast, colon, and non-small cell lung cancer cells, making it a potent target antigen. According to the results, the Ley carbohydrate vaccine was successful in Ley-specific antibody production in mice (31). Another example demonstrates the effectiveness of carbohydrate based cancer vaccines. Ragupathi et al. were able to synthetically construct two carbohydrate structures, each bearing three tumor specific antigens. Both structures, one containing a Globo-H- Ley-Tn complex and the other containing a TF- Ley-Tn complex, both produced an increased amount of antibodies in vitro with human tumor cell lines. It was concluded that the increased amount of different antigens in one carbohydrate complex created a more diverse and stronger immune response to the specific antigens (38). Similarly, synthetic lipid based cancer vaccines can be used to activate DC’s. Jackson et al. used the idea of synthesizing a lipid-peptide based vaccine to target Toll-like receptor 2 (TLR-2) on DC’s to activate maturation. The results clarified that the lipid moiety increased an immune response to the specific peptide present in the vaccine (40). The biological effectiveness and ease of construction of totally synthetic based vaccines makes them a highly attractive choice for future use (40).

It has also been observed that immunotherapeutic cancer vaccines work more effectively when another cancer treatment technique is applied. Surgical resection can increase the survival rate of metastatic melanoma patients from a five year survival rate of 6% to nearly 20%. Moreover, when Canvaxin, a immunotherapeutic cancer vaccine, was applied after surgical resection, the 5 year survival rate increase 2-fold as opposed to surgery alone (11). Additionally, results from the study by Antonia et al., as described previously, showed an increase immune response when chemotherapy was applied after the injection of a genetically modified DC vaccine (1). Finally, radiotherapy has been proven effective in increasing anticancer immunity with an immunotherapeutic cancer vaccine. For example, increase in PSA specific CTL’s were observed in patients who were treated with a genetically-modified tumor vaccine after radiotherapy as opposed to the cancer vaccine alone (8).

Lastly, cancer vaccines can be used to block cancer immunosuppressive molecules and receptors. One molecule known to suppress an immune response is CTLA-4. CTLA-4 is an immunoregulatory molecule up regulated on T cells two or three days after T cell activation. CTLA-4 binds to costimulatory molecules (B7-1,2) on DC’s, which interferes with IL-2 production and deactivates T cells. Although CTLA-4 inhibits some effective immune responses, it is essential for the tolerance of self antigens (34). In normal T cell activation, CD28+ molecules on T cells attach to the costimulatory molecules on DC’s, activating an effective immune response. To counteract this mechanism, monoclonal antibody therapy can be used to block the CTLA-4 molecule from binding to the costimulatory molecules (21,27). For instance, anti-CTLA-4 antibody (MDX-010) was used with a vaccination of the gp100 melanoma associated antigen, resulting in an increase in T cell response in all seven patients tested while 3/7 experienced tumor necrosis (34). Also, the antigen epidermal growth factor-R (EGF-R), which is overexpressed in breast and epithelial cancer cells, has been discovered as a good target to slow down the rate of metastasis and tumor invasiveness (7). An administration of an anti-EGF-R antibody with an adjuvant resulted in a successful immune response in 60% of patients tested (7). Currently, there are over 20 approved monoclonal antibodies used as a cancer vaccine to block tumor growth proteins and receptors.

Although many studies have revealed a variety of effective cancer vaccines in vivo and in vitro, there are still many obstacles in creating a totally effective vaccine. One challenge involves the eluding host vs. graft disease.(I would suggest a providing a short definition for host vs graft along with the follow explanation) Because only a small number of antigens discovered are specific to tumor cells, many antigens involved with antitumor immunity involve self antigens. Usually, T cells that are reactive to self antigens are eliminated in the thymus, however, for most antitumor vaccines to work, some of these T cells need to escape central tolerance in the thymus. These T cells are activated as soon as an antigen, whether self or non-self, is presented for example on DC’s with immune activating signals (21). As a result, the T cells become activated, which will initiate an immune response not only against the specific tumor cells, but also against normal cells containing the specific antigen. Autoimmune reactions have been seen in many murine models as well as clinical trials (3). Additionally, uncontrolled immune responses will cause autoimmunity. If cytokines and CTL’s are not regulated, they will over saturate the lymphoid organs and trigger autoimmunity (21). Another challenge involves choosing the most effective adjuvant. As of 2003, there have only been two adjuvants approved for clinical use, alum and squalene-oil-water emulsion (3). However, many pre-clinical and clinical studies use adjuvants like various cytokines such as GM-CSF, IL-12, IL-2 and IL-4 and bacterial components consisting of LPS, lipid A, and unmethylated CpG dinucleotides to induce an immune response. Chiefly, there are two classes of adjuvants that can be used to control the pathway in which the antigen can be presented. Bacterial products are mainly used to activate CTL’s, which involves antigen presentation via MHC class I molecules (3). Also, antigens can also be directed into the cytoplasm allowing presentation via MHC class I molecules. Most vaccines consisting of all types of antigens are effective in presentation via MHC class II molecules (3). Based on the desired immune response, adjuvants play an important part in cancer vaccines. Moreover, patients with cancer are usually known to be of old age (65-80 years), meaning they no longer produce naive T cells. The immune response for older patients requires recognition of a particular antigen through memory T cells, which may or may not be the most effective (3, 27). Overall, primary immune responses in older patients are deficient. To counter this problem, many efforts have been made to compromise old age problems. For instance, activation of CD137, a co-stimulatory molecule, was shown to increase T cell responses in old mice. Additionally, unmethylated CpG-DNA was shown to effectively enhance cellular and humoral immunity in old mice (3). However, many obstacles still exist in tumor immunity in old patients. Furthermore, maintenance and identification of long term memory cells is yet another challenge facing a cancer vaccine design. In order to acquire a large pool of memory cells, a strong primary immune response is necessary; however, few studies were able to acquire strong enough immune responses to obtain memory cells. To emphasize, maintenance of longevity of memory cells is not known for cancer immunity. On top of that, long-term memory cells may not even exist in chronic diseases like cancer because tumor specific T-cells are introduced to tumor antigens constantly, making a boost of antigens through a vaccine unable to trigger long term memory (3). This causes many concerns because memory cells are essential to all prophylactic and therapeutic vaccines. Most importantly, circumventing tumor immune evasion remains the biggest barrier in creating a tailor-made cancer vaccine. Tumor cells are known to dodge the immune system by inhibiting expression of tumor antigens, interfering with activation of DC’s, and hindering the activation of T cells (21). Most tumor cells have defective TAP transporter expression, thus decreasing the amount of MHC molecules present on the tumor surface, resulting in poor antigen expression (21). Additionally, tumor cells lack costimulatory molecules, making T cells in general ineffective. Both decrease in antigen presentation and lack of costimulatory molecules result in tumor immune escape. Moreover, tumors recruit immature DC’s allowing them to present tumor antigens while the DC remains inactivated, thus allowing tumor cells to be tolerated by the immune system (21). Also, tumors secrete anti-inflammatory cytokines such as IL-10 and TGF-β to reject effector T cell function. An anti-TGF-β antibody in conjunction with IL-2 revealed an increase immune response in animal models (23). Finally, in many patients with cancer, an increase in regulatory T cells (Tregs) is observed (28). Tregs are CD4+ T cells with a CD25+ receptor attached. These Tregs are known to suppress immunity against cancer because they inhibit the ability of CTL’s to react with antigens. Although it may seem obvious to inhibit Tregs, they are essential in suppressing autoimmunity (21,28).**(I suggest dividing this paragraph into at least two

To conclude (i don't think "to conclude" is needed), there are many different types of alleged cancer vaccines shown to not only increase immune responses, but to suppress tumor cells. Many of these vaccines are based upon increasing immune recognition of specific tumor antigens. However, the diversity of all the different antigens present on all the different cancer cells makes it fairly difficult to design one vaccine against all cancers. One approach would be to keep testing out different vaccines on different tumor antigens with different adjuvants in hopes of one day creating the perfect combination that will elude all hurdles to antitumor immunity. Another approach may be to create a totally different type of vaccine that looks more into cancer prevention. In any manner, more studies need to be conducted clinically in order to one day find the vaccines that will treat and possibly prevent cancer.

Beau- Your paper has a good transition. I think that possibly there are some parts that you are trying like to explain several things at the same time and it can confused the reader a little bit. Also you should ask Dr. Robson about having to many sentences with the authors name (et al.) Besides all that your paper is very good.. :)
-Beau, I'm not sure what Dr. Robson is looking for, but maybe a little background on how a normal cell becomes a tumor cell (its life cycle(problems with cell cycle), how it spreads, etc.) at the beginning might help the reader jump into your paper. Great research, some comments are in the text for you to look at. Ryan

-Beau- Very well written paper. Each paragraph has a nice transition. I wonder if maybe more of a thesis could be added to the opening paragraph; something that would introduce that your are going to discuss in your paper to help the reader. **
Beau, agreed-your paper is very well written. I would only suggest breaking down some of your larger paragraphs, keep the reader interested.**

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