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e Journal of Biological Sciences

ISSN: 2076-9946, EISSN: 2076-9954

Volume: 1, Issue 1 (December 2009) All rights reserved.

COMBATING CANCER WITH TUMOR IMMUNOTHERAPY


ASHISH RUNTHALA1, MURALI MONOHAR PANDEY2
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Lecturer, Biological Sciences, BITS, Pilani, Rajasthan, India 333031 Lecturer, Pharmacy Department, BITS, Pilani, Rajasthan, India 333031 E-mail: askamber100@gmail.com1 , mmpphd@gmail.com2

ABSTRACT Cancer is a worldwide health problem. Cancer immunotherapy can be studied in two categories: cancer vaccines and induction of immune response against specific antigens. For studying it in a better way to combat cancer, we should look it from the respective angles of immune systems components. For combating cancer, we should look into the roles of innate cells, antibodies, T cells, NK cells, macrophages, and at last the evasion of tumor responses. From all these concepts, we can land on many immunotherapy ways like Humoral or cellular immunotherapy, Passive antibody transfer, APC activity enhancement, and the important role of monoclonal antibodies, Dendritic cells, cytokines and interferons. Still moving further using a pool of these resources, we can combat cancer in a better way. Keywords: Cancer, Surveillance, Co-receptors, TATA, TSTA, CEA, AFP, ULBP, MIC, NKT, VEGF.

1. INTRODUCTION Cancer is the major health problem worldwide and one of the most important causes of morbidity and mortality in children and adults. Cancer arises from the uncontrolled proliferation and spread clones of transformed cells. From an immunological prospective, cancer cells can be viewed as altered self-cells that have escaped normal growth regulatory mechanisms. The concept of immune surveillance, which was proposed by Macfarlane Burnet in the 1950s, states that a physiologic function of the immune system is to recognize and destroy transformed cell clones before their growth into tumors and to kill tumors after their formation [1]. Cancer poses a unique trouble for the immune system because unlike an infection involving foreign cells, the problematic cells are self cells here. Now the immunity identifies and destroys such cells, but they can avoid their identification by many mechanisms like decreasing Class I MHC expression or by restraining expression of costimulators. It happens so because, co-stimulatory signals are as important as MHC expression because MHC mediated high affinity antigen detection cant occur without them. But how the cancer cells escape this strong identification process is still not fully understood [2]. For combating cancer, immunotherapy concept came in picture. It means any approach which manipulates a persons immune system to treat the diseases. Immunotherapy can prove to be an important tool to arrest cancer at an earlier stage. In general, these strategies range from therapeutic vaccines that manipulate a patients immune system de novo (active immunotherapy), to administration of biological reagents such as monoclonal antibodies, cytokines, or previously activated immune cells (passive immunotherapy) that deliver or modulate a specific patch of the systemic immune response [3]. In the entire system of a mature animal, cell renewal and cell death rates are balanced. It is so regulated that the any particular cell type number is always maintained. Occasionally, cancerous cells growth surpasses the constraint of normal growth rate, thereby producing a tumor, or neoplasm. A tumor incapable of indefinite growth and lacks the capability to invade the healthy surrounding tissues is termed benign. And, a tumor that keeps multiplying and becoming progressively more invasive is malignant. Such malignant cells show metastasis, i.e. they spread in the entire system through blood vessels (called angiogenesis) [4].

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Such cells grow extensively with increased blood flow to invade neighboring normal cells, thereby forming secondary tumors or metastases. But, the body evolved many ways to remove such cells. It is already known that both the cell mediated and humoral immune responses play a role in the tumor destruction. T cytotoxic lymphocytes has the potential to recognize altered self-antigen in context of MHC I [2]. Once such antigens are recognized, T cytotoxic lymphocytes divide into effector cells (cytotoxic T lymphocytes or CTLs), which then lyse the cancerous cells. But again, some cancers evolved a way to avoid such recognition in this fashion, by lowering the expression of MHC I or co-stimulatory signals. Fortunately, an additional set of lymphocyte called the natural killer cells (NK) has evolved in nature. They can explicitly target cells with reduced MHC I expression, by cytotoxic activity. They can also destroy tumor cells after being induced by innate immune system mediated antibodies, which have already recognized tumor cell surface antigen. NKs have CD16 receptors which bind to the Fc region of immunoglobulin, which is secreted by Blymphocytes as part of the humoral response. Immunoglobulins have antigen binding (Fab) and Fc (Constant) portions. The Fab region specifically binds to antigen and then the Fc region is meant for specified recognition by other cells, like NKs, Macrophages etc., which can then destroy the source cells which expressed antigens, thus mediating an effector response. But what is the need for immunotherapy? Even though the immune response to cancer is broad and very specialized, but it is clear from global high mortality rates from cancer, that the immunity is unable to combat cancer. Cancer immunotherapy can be branched into two categories: cancer vaccines and inducing immune response against specific antigens. The cancer vaccines stimulate immunity by using foreign antigens, which are analogous to cancer cell antigens. And the immune response against specific antigens provide micro-environment, conducive to the specialized recognition, presentation, and the destruction of specific antigens and the tumor cells from which they arose. Though cancer cant be cured completely, many immunotherapeutic methods are under trials, which increase the revival rates of cancer patients. Merging different techniques such as administering a vaccine and creating more favorable micro-environments have proved successful in regressing tumor. Tumor immunity show certain general features. Tumor cell antigens are recognized as foreign by the body. And, immunity fails mostly to combat tumor growth. The immune system can be activated by external stimuli to effectively kill tumor cells and eradicate tumors. Now there should be some way for tumor antigen identification. The first step in developing cancer immunotherapy is exploiting the immune system to trace molecules, which have potential to distinguish between cancer and normal cells. There are many tumor antigens, which can be the products of mutated oncogenes or tumor suppressor genes. Tumor antigens can be produced by mutation in cellular proto-oncogenes and tumor suppressor genes and may show some unknown functions like TATAs (Tumor associated transplantation antigens) and TSTAs (Tumor specific transplantation antigens). Tumor antigens are abnormal cellular proteins generally. Tumor antigens can be encoded by oncogenic viruses like EBV (Epstein- Barr virus), HPV (Human papiloma virus), SV40 (Simian virus), Papovirus, Adenovirus, Retroviruses (HTLV-1, Human T Lymphotrophic virus-1). These antigens can be oncofetal antigens, which are proteins expressed at high level on cancer cells and in normal fetal cells but not in adult tissues, e.g. CEA (Carcino embryonic antigen) and AFP (Alpha feto protein). Over-expressed mutated glycolipid and glycoprotein antigens like Gangliosides, Blood-group antigens, Mucins, can be treated as diagnostic markers for cancer therapy. Other than these they are certain differentiation antigens like CD10, CD20 which are normally present on cells of origin. So before targeting antigen, it is always advisable to check the presence of same antigen on other cells.

2. INDUCTION OF IMMUNE RESPONSE Other thing to be noted properly in immunotherapy is attempting the creation of micro-environment which can stimulate immune system in the best possible way. It is done by utilizing cytokines like interleukin (IL)-12, IL-2, by growing tumor antigen specific T lymphocytes in vitro, utilizing other stimulating factors and by transfection tumors with immunostimulatory genes in vivo [5]. Studies show that transferred allogeneic antigen specific T cells are best to treat the decreased hematologic malignancies. Self T cells can provide even more powerful immune response. The idea is to educate nave T cells against cancer antigen in vitro and re-infusing back to individual. Its merits are

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unfamiliar because we cant isolate and grow large number of APCs against specific antigens in vitro. APCs are advantageous for training T cells against specific antigen in thymus. Dendritic cells were tried for it but they failed due to their low multiplication potential. Cyclophosphamide showed anti tumor action for some tumors [4].When its injection is pooled with the adoptive immunotherapy, the results were dramatically increased. Another way is to transfect a super antigen gene, to stimulate a dominant response. Super antigen expression is thought to induce a better immune response. It is almost similar to virus vaccine method, though vector is injected locally in this case. It led to decreasing small tumors in 46% of the dogs. The injected tumors showed increased numbers of most T cell types and macrophages in the blood. These experimented dogs survived longer. Now, there can be the following Immune responses to Tumors.

2.1. THE INNATE IMMUNE SYSTEM Innate immunity responds to tumors because of constraints of cell transformation and interference in the prevailing microenvironment. Such signals can induce inflammation, activate effector cells to destroy tumors, and induce APCs to engulf tumor antigens and migrate to lymph nodes thereby triggering an adaptive response. Despite this, the tumors presence indicates that the developing cancer was able to avoid detection. Progressing tumors often evade the immune recognition by avoiding their recognition by immune cells, by being very poor immunogenic, by reduced expression of MHC or co-stimulator proteins, or by interrupting recognition by natural killer (NK) cells. Additionally, some tumors may also secrete Vascular Endothelial Growth Factor (VEGF) or IL-10 to obstruct the induction of DC. It is achieved by hindering expression of pro-inflammatory molecules through reduced expression of STAT3 protein. Thus, whenever a tumor is induced, tumor cells can escape elimination by down regulating the expression of targeted antigens, making tumor-reactive T cells ineffective or anergic to them. Sometimes, specific T cells are also deleted from the system making the system incompetent to recognize tumor cells. Despite these barriers, current immunotherapy methodologies are showing augmented potential for treating malignant diseases [6]. Innate Immune System does the job of alarming the host whenever it is breached by the foreign particles. It ultimately leads to the onset of effector mechanisms which lyses tumor cells or blocks angiogenesis for avoiding further tumor growth, by the production of cytokines. NK and T cells express receptors like NKG2D, which recognizes MHC class I chain related (MIC) or UL16 binding proteins (ULBP) up-regulated on tumor cells. Sometimes NK and T cells also express receptors with limited assortment to recognize tumors, through peptide or lipid antigens, presented in reference to MHC [7].

2.2 EXPLOITING INNATE IMMUNITY IN COMBATING TUMOR NK, NKT and T cells express interferon (IFN) once they are activated. IFN leads to the lysis of tumor cells and provoke other components of innate immunity like DCs, which then generate IFN after being activated. This feedback pathway is vital for increasing the vulnerability of IL-12 dependent mice towards induced tumors, as is often clear with the increased clonal expansion of NK and T cells for combating tumor. Administration of IL-12 in murine appeared promising in murine copies, but was later found undesirable for human usage because of possible liver toxicity. A substitute for stimulating innate immunity is to stimulate NK cells in vitro by culturing them with IL-12 and then administering back to patients simply or with IL-2, in large number. Such advancement has yielded the marked antitumor activity. Nevertheless such an approach has leads to the development of severe toxicity in the system, because of the expression of Tumor Necrosis Factor (TNF) by stimulated NK cells. The careful in vivo stimulation of NKT cells either by onset of glycolipid alpha-galactosylceramide or by administering bisphosphate compounds shows really interesting results in initial trials. Presently systemic toxicity restricts such an approach in treating tumors, where there is a limited blood flow. Thus, extensive usage of this method still requires cytokine activity specifically targeting the tumor.

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2.3. INNATE CELLS AS INITIATORS OF THE ADAPTIVE IMMUNE RESPONSE Initially adjuvants were administered directly into solid tumors to stimulate inflammation and recruit immune effector cells. This approach is still common for superficial bladder carcinomas. However, many a times this strategy becomes ineffective by producing transforming growth factor (TGF)- like proteins to prevent activation of the immune response. Cellular and molecular events suggest that tumor immunity can be stimulated by providing pro-inflammatory cytokines and/or chemokines at the respective sites of solid tumors. We still dont know, how to harness chemoattractants and activators, but expression of molecules like secondary lymphoid tissue chemokine, LIGHT in tumor sites can make the microenvironments around tumor highly immunogenic. An alternative approach could be to provide a ligand for the receptor tyrosine-kinase Flt-3, expressed by hematopoietic precursor cells. Cancer treatment with soluble Flt-3 ligand has induced large increases in circulating DCs, but these were largely failed to augment immune responses. Thus, DCs still require activation signals to induce adaptive responses.

2.4. FUNCTION OF T LYMPHOCYTES T cell activation depends on the expression of TAAs by APCs. But only DCs can prime naive T cells, stimulating their differentiation into effector cells. This property could potentially make DCs ideal agents for promoting TAAspecific immunity. However, the inability to cultivate them in large numbers made it a failure. The DCs differentiation inducing techniques development can revolutionize this study [6], [8]-[10]. 2.5. ANTIBODIES TASK Antibodies may activate complement system or they can also stimulate cell mediated cytotoxicity, in which Fc receptor-bearing macrophages or NK cells mediate the killing.

2.6. ROLE OF NK CELLS NK cells may destroy many types of tumor cells, especially cells which reduce class I MHC expression and escape killing by CTLs. Their activity is enhanced by cytokines, including interferon and interleukins (IL-2 and IL-12), and this anti tumor effect is due to stimulation of NK cell activity. These NK cells, called lymphokine- activated killer (LAK) cells, are derived by culturing peripheral blood cells or TILs from tumor patients with high IL-2 or IL-12 dose.

2.7. ROLE OF MACROPHAGES T cells can be activated by macrophages and produce INF-. They destroy tumor cells by release of lysozymes, reactive oxygen intermediates and nitric oxide (NO). Activated macrophages also produce the cytokine TNF, which act as an agent that kills tumors by thrombosis mainly.

2.8. EVASION OF TUMOR RESPONSES MHC I expression may be down regulated on tumor cells to avoid their recognition by CTLs. Tumors may loose expression of antigens that elicit immune responses. Tumors dont express co stimulators or class II MHC molecules, and fail to induce CTLs. Tumor antigen may also induce specific immunologic tolerance.

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2.9. IMMUNOTHERAPY FOR TUMORS The immunotherapy approach as cancer remedy has raised a great interest all throughout. It is because the current cancer therapies rely on drugs that kill dividing cells or block cell division, and these treatments have many side effects on wild type cells, thereby increasing the morbidity and mortality rates. Immune responses are specific and thus it has the potential of being the most tumor-specific treatment. Immunotherapy strengthens the weak host immune response to the tumors (active immunity). Now look on the following issues further [11].

2.9.1 HUMORAL IMMUNOTHERAPY B cell activation produces antibodies to trap the tumor antigens and initiate complement system for cell lysis, bridge NK cells or macrophages to the tumor for antibody-dependent cell mediated cytotoxicity (ADCC). They can also interfere with tumor growth by blocking the signals for survival or apoptosis. They can also augment immunity by assisting uptake and expression of cancerous agglutinogens of APCs. Therefore, B cell response is facilitated in vivo or by introducing in vitro generated antibodies, having capability to encourage antitumor activity [12]-[14]. The monoclonal antibodies (MAbs) targeted against the IL-2 receptor [15], expressed by many T cell leukemias, lymphomas, and as by T cells, have proved their role in antibody transfer in cancer therapy for humans. However, this has been underestimated. The rituxiMAb drug binds CD20 and, if given with no chemotherapy, can be effective in reduction in B cell lymphomas patients, largely by ADCC. Some MAbs alone can mediate antitumor activity by inducing apoptosis. The antitumor action of MAbs can be improved by affixing radioisotopes, drugs, or recombinant antibodies which have the specific potential to bind cancerous cells and induce CD3 or Fc receptors. Improving antitumor activity without inducing toxicity to normal tissues is also a challenge.

2.9.2 CELLULAR IMMUNOTHERAPY T cells express antigen receptors, which with the reference of MHC can recognize tumor antigens, which can be over-expressed self antigen, mutation, or abnormal expression of developmental tissue specific antigen. For showing antitumor activity, T cells must be stimulated by APCs along with other necessary secondary signals through costimulators. These activated T cells then leads to the expression of cytokines, like IFN and TNF which ultimately seize malignant development by preventing angiogenesis, lysing tumor cells with the help of perforins and/or Fas. Thus if we identify tumor antigens, we can provide them in immunogenic conformations to induce responses.

2.9.3 ADOPTIVE THERAPY High-dose chemoradiotherapy with an allogeneic hematopoietic stem cell transplant (HSCT) has become standard therapy for many hematologic malignancies. But one associated problem here is graft-versus-host disease (GVHD), due to allogeneic donors T cells injuring the foreign but ordinary host tissues. However, malignant cells surviving chemoradiotherapy are also host cells, and patients with GVHD have lower relapse rates from an associated graft versus tumor (GVT) result. T cells mediate this antitumor activity. Ongoing hunt to target conserved epitopes permit specific targeting of just cancerous cells [16]. Another option instead of to using allogeneic T cells is to mediate antitumor responses. It can be done by isolating autologous tumor-reactive T cells, then culturing them in vitro, and then reinfusing the cells back to the patient. This approach avoids many obstacles to generate an adequate response in vivo, as the APCs and their microenvironment can be more precisely controlled in vitro. However, this strategy requires extensive manipulation of T cells in vitro with retention of specificity and function, such that after infusion they can survive and target the tumor cells. Initial therapies used tumor-infiltrating lymphocytes as augmented tumor-reactive cells, but they can arise from circulating blood lymphocytes. Best methods for stimulating T cells in vitro against specific antigen are still getting developed. Cytokines are also supplemented during cell culture to support lymphocyte culture. Thus it has been

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possible to expand tumor-reactive specific T cells in vitro, thereby infusing them to reach in vivo frequencies beyond that attainable with current vaccine treatments. It mediates complete elimination of large disseminated tumor masses. It helps in achieving high in vivo frequencies of tumor-reactive effector cells. Analogous therapy trials for cytomegalovirus and Epstein-Barr virus infection in immunosuppressed hosts have shown increased in vivo persistence of CD8 effector T cells along with helper T cells. Such CD4+ T cells lead to increased cytokine production and APC activation, which thus improves the quality and quantity of the CD8+ cells responses against specific tumors. However, unlike CD4 and CD8 responses induced by viruses, characterizing tumor reactive CD4 T cells specificity has been more difficult than with CD8 responses. Thus, CD4 help is mandatory to transfer tumor reactive CD8 cells as surrogate exogenous cytokines [17], [18]. Infusion of T cell clones, instead of polyclonal Tcell lines, emphasizes superiority of adoptive therapy as it is provides specificity, avidity, and effector functions of infused cells. This facilitates analyzing needs for efficacy, toxicitys basis, and thereby improved therapies. CD8 T cell clones infusion has been shown to be effective for preventing viral infections and malignant diseases. These studies demonstrated that nontoxic IL-2 doses are sufficient to promote the in vivo persistence and antitumor activity of CD8 T cells. Another way to augment activity and survival of infused cells is to use endogenous homeostatic mechanisms that restore lymphocyte numbers after lymphopenia. The mechanisms of IL-7 and IL-15 production are not entirely known, but premeditated depletion of lumphocytes in patients before T cell transfer can result in the extensive proliferation of infused T cells, creating an in vivo collection dominated by the desired effector population. This strategy is under clinical evaluation, and it may form a micro-environment more beneficial to mediate an antitumor effect. Integrating vectors can be used to genetically modify T cells before infusion to enhance tumor recognition, cell survival, migration, or effector functions artificially. For e.g. Recognition of tumor by T cell is due to expression of high-affinity transmembrane receptors with the epitopic recognition structure of an antibody and the intracellular signaling domain of a T cell receptor. This recognition of tumor antigens by T cells in MHC-independent like MAbs but utilize effector mechanisms of a T cell. Alternatively, T cell receptor genes can be released from effective T cell clones. This is safe in therapy and when inserted into T cells from other patients with tumors that express the same tumor antigen and MHC-restricting allele, the need to isolate tumor-reactive effector cells in each individual patient can be overcome. Infused cell survival can also be enhanced by introducing chimeric cytokine receptors that use cytokines such as GMCSF, produced by effector CD8 T cells after target recognition as regulated autocrine growth signals. Another strategy is to introduce new structures or functions to T cells to disrupt signaling pathways that normally serve to reduce responses such as those through Cbl-b, an adapter protein that negatively regulates T cell receptor signal strength. Preliminary studies in which dominant-negative Cbl-b proteins are expressed or Cbl-b expression is reduced through siRNA approaches suggest that increasing signal strength can both reduce the T cell activation threshold and restore IL-2 production to effector CD8 T cells. Many other targets for such strategies have been studied in knockout mice, suggesting the possibility of designing T cells capable of circumventing many obstacles posed by tumors.

2.9.5 ENHANCEMENT OF APC ACTIVITY GM-CSF cultured Mouse DCs incubated with tumor fragments, when reinfused into mice, have been shown to activate both tumor antigen specific TH cells and CTLs. These mice displayed tumor immunity when infused with live tumor cells. These experiments aimed at APCs clonal expansion, to activate TH or CTLs specific for tumor antigens. One approach here is to transfer tumor cells encoding GM-CSF gene. After re-infusion into the patients, they will secrete GM-CSF, enhancing the differentiation and activation of host APCs, especially dendritic cells [9]. As these DCs accumulate around the tumor cells, GM-CSF secreted by the tumor cells will enhance the presentation of tumor antigens to TH and CTL cells.

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Another way to increase number of DCs is to culture them from peripheral-blood progenitor cells in the presence of GM-CSF, TNF- and IL-4 .These three cytokines induce the progression in the population of dendritic cells [19]. A number of adjutants, like attenuated strains of Mycobacterium bovis called bacillus Chalmette Guerin (BCG) and Corynebacterium parvuum, have been used to boost tumor immunity .These adjutants activate macrophage, inducing cytokines expression , class II MHC molecules and the B-7 co-stimulators. These macrophages are better activators of TH cells, resulting in global increase in both humoral and cell mediated responses.

2.9.6 DENDRITIC CELL-BASED IMMUNOTHERAPY The DC-based immunotherapy against cancer varies in several aspects. Key variables include the choice of TAAs, like defined species or heterogeneous pool of different macromolecules; introduction technique of TAAs into DCs; and DCs preparation. Heiser et al. fixed a simple approach, by introducing a single mRNA species into immature DCs, though it is reflects the alternative variables. The TAAs used here are mutated gene products, such as mutant RAS, p53, or normal self-antigens which are over expressed or ectopically expressed in tumors. TAAs used as cell surface antigens may result in undesired autoimmune responses. Thus, in melanomas, TAAs might include proteins expressed in skin and retina, and DC-mediated antimelanoma immunity can also depigment the skin, leading to vitiligo, or may impair visual acuity. Such complications have put doubt on vaccinating against TAAs that are selfantigens, particularly if those self-antigens occur normally. The choice of prostate-specific antigen (PSA), used by Heiser et al., could avoid this problem, since it is expressed primarily in a dispensable organ, the prostate. Initially TAA presentation in DCs was driven by application of synthetic peptides. As peptides are easily made, other strategies may be advantageous, since they target either CD8+ or CD4+ T cells, rather than both together [19]. The peptide use also limits the expressed epitopes. Here MHC haplotypes can show potential for clinical applications, because of its diversity in the human population. A prior knowledge regarding protective epitopes is also required, a setback which can be avoided by exposing DCs to more heterogenous proteins or nucleic acids. DCs can also be exposed to tumor lysates or apoptotic bodies, thereby allowing presentation of a wide era of TAAs. Ideally, it may be the result of an autologous tumor, ensuring including individual TAAs. Another advancement of transfecting DCs with recombinant DNA vectors encoding TAAs can obviously be effective, but its use is coupled with its related safety issues. Heiser et al. suggested mRNA transfection as a reply to many TAA expression problems. mRNA may be derived from tiny amounts of autologous tumor. A given tumors RNAs are expected to encode the relevant TAAs. Application of mRNA to DCs allows activation of both CD4+ and CD8+ T cells. Heiser et al. tested this strategy by transfecting DCs with a single mRNA, encoding PSA. But it was noted that an even general approach was feasible. For instance, liposomes could be used to deliver mRNA to cells, or we can stabilize mRNA by adding flanking sequences. The cells potential for TAA processing and presentation can be improved by inducing expression of accessory molecules. These improvements can be used to immunize against poorly immunogenic TAAs. Another necessary factor in immunotherapy is source and differentiation status of the DCs. Heiser et al. used immature monocyte-derived DCs which are already proven the best in human trials. CD34+ cell derived immature DCs are inferior to these cells. DCs stage while using and the allied features is a cynical situation, although matured DCs may be superior [20], [21].

2.9.7 CYTOKINE THERAPY Early cancer immunotherapy trials were aimed at IL-2 or additional T cellactivating cytokines that induce clonal T cell expansion against TAAs. These cells were expected to permeate tumor leading to destruction of specific tumor cells. Attempts to enhance antitumor immunity yielded tumor regression sometimes, but they got failed to stop cancer significantly. Some cytokines used in cancer immunotherapy are:-

IL-2: It is marketed by Chiron as Prokeukin. When administered in high doses, it yields an increased number of blood lymphocytes, NK cells, LAK and NK activity, and increased serum levels of TNF, IL-1, and INF-y. However, there are toxic side effects due to high dosage, because of cytokine over-production after being stimulated by IL-2.

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TNF: TNF shows anti tumor effects, though it yields extremely toxic effects. Local TNF injection can treat limb sarcomas and in such cases, there is a decrease in venous blood returning from limbs to decrease systemic effects. INF-: It has an apoptotic effect on cells, by increasing lytic ability of NK cells, and expression of class I MHC molecules on various cell types. INF-y: It increases MHC expression and thus activates macrophages and NK cells. GM-CSF and G-CSF: They are used in cancer treatment to shorten the neturopenic time period of a patient during chemotherapy or an autologous bone marrow transplant. These cytokines stimulate the maturation of granulocyte precursors [22].

2.9.8. INTERFERON Interferons belong to group of cytokines. They are produced naturally by white blood cells in the body (or in the laboratory) against infection, inflammation, or stimulation. They are used for treating viral diseases, like hepatitis B. Interferon-alpha showed an antitumor effect initially, by slowing tumor growth and by activating immune system. It is FDA approved common method for treating cancers, like multiple myeloma, chronic myelogenous leukemia and hairy cell leukemia. Interferon-beta and interferon-gamma are also examined. Other cytokines with antitumor activity include interleukins and TNF. IL-2 is a common treatment for kidney cancer and melanoma. Some of their side-effects include malaise and flu-like syndromes. At higher dosages, their side effects are greatly amplified [19].

2.9.9. MONOCLONAL ANTIBODY THERAPY The immune system is a defending body against antigens. One of its important arms constitutes antibodies. The immune systems B lymphocytes produce antibodies to target cell surface antigens. Such antigens can be targeted by antibodies or immune cells. Antibodies have a promising role in the cancer treatment. Monoclonal antibodies (MAbs) act on cancer cells in the similar fashion like natural antibodies, by tracing and binding the target cells. They then amplify immune response through up-regulation of other cells. They are very specific against an antigen. They are classified as Biological Response Modifiers, as they affect the immune system specifically. Hence hey are considered in immunotherapy unlike chemotherapy by drugs. Now, MAbs are made by infusing human cancerous cells in mice. Such antibody producing clone of cells are selected and fused with immortal cell lines like HeLa cells, to develop constitutively growing cells, which can produce antibodies. Such fused cells are hybrid and such antibodies are known as Monoclonal Antibodies. This technique is known as Hybridoma Technology. Such cells are nothing but factories which can produce pure antibodies endlessly. MAbs augment the patients immune response against tumor. Though this treatment has shown some potential in targeting lymphoma, but is still not perfect, as sometimes the infused antibodies dont produce or block a normal response. Cancerous cells also sometimes shed their specific antigens, their by being most likely avoided by the immune response of the system. Therefore, MAbs combined with another form of therapy have also been tried, for increased effectiveness [23], [24]. For e.g. MAbs in combination with chemotherapeutic agent have been attempted, which then attack the cancerous cells in two ways: Chemotherapeutic chemical attack and MAb mediated Immune response. This chemotherapy can be more useful when the target cells are attenuated by MAbs. In most such practices, monoclonal antibodies are linked with radioactive substance like radioactive iodine, which targets and destroys the cancerous cells. This therapy makes the tumor cells, acquire a larger proportion of radiation and thus the normal tissue is secured. Such Radio-labeled antibodies are mostly useful in diagnosis of specific tumors [19]. MAbs linked with other types of Biological Response Modifiers (BRMs) or toxins are still in trials. When such antibodies are fastened onto cancerous cells, they let such BRMs free for acting onto tumors. Such an approach can help destroy cancerous cells in the bone marrow of a patient itself, even before an autologous transplantation of bone marrow. This is a methodology, where the bone marrow of a patient is extracted, stored and later given back to the patient again, after treating him with high-dose chemotherapy and /or radiotherapy.

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2.9.10. CANCER VACCINES Cancer Vaccines are based on the fact that when antigens, which are almost identical to the tumor antigens on cancerous cells and at the same time immunogenic, are injected in a system, they develop a specialized immune response in the system. Such a response targeted against tumor antigens, can wipe out the infected antigen from the system. This response is also capable of producing specific antibodies, stimulating B and T cells, which are capable of destroying the tumor cells. The source of such antigens is often the persons own cancer cells. Such cancer vaccines are also based on the usage of recombinant virus, which leads to the expression of antigens and costimulatory signals, once being injected into the tumor cells. In both these developed responses, the focus is the creation of antigens to elicit an immune response, which develops specific memory B and T cells against antigens, thereby upsetting the future growth of tumor. Many such cancer vaccine prototypes have been produced till now. Some utilize the cancer cells of patients while others utilize recombinant virus. The hapten can solve a common problem, which arises when a cancer vaccine is developed. This is normally termed as the Antigen Tolerance problem. It is because a cancer cell develops within the organism. So, it typically possesses few molecules which are foreign. Therefore, many tumor cells inhibit the presentation of such foreign antigens, thereby avoiding their recognition in the system. The hapten is antigenic in nature, is recognized as foreign and thus initiates an immune response. This hapten thus makes the cancer cells recognition process prevalent which then even leads to the recognition of altered self-antigens, after the processing of foreign particles by macrophages. It thus elicits a massive immune response, which is skilled at killing the tumor. In such treatment process, tumor cells are selected from a patient and and the hapten is the tagged with them. Such a complex is then injected back into the patient with Bacillus-Calmette-Guerin (BCG) adjuvant, with an initial dosage of cyclophosphamide. Such a vaccine treatment is able to increment survival rate of patients by more than fifty percent. Its testing is still on and is showing promise for other types of cancer as well. Another type of vaccine is based on the usage of recombinant vaccines. In these vaccines, viral particles are designed to target cancer cells, which code for specialized antigens (Superantigens). These vaccines permeate the cancer cells, and leads to the expression of these superantigens. Such antigens are selected based on their ability to induce an effective immune response. Viral construction to act as vaccine against murine tumors is achieved. Oxford England researchers have created a recombinant virus, coding for E.coli Beta-galactosidase, Cytokine IL-12 and costimulatory B-7 molecule. IL-12 has the advantage that it can induce NK cell based immune response, which is vital for transiting T cells into specialized Cytotoxic T lymphocytes. B-7 is a co-stimulator and is found on most APCs. B-7 expression is vital for the recognition of an antigen by such stimulated T lymphocytes. Such viral particle is then injected into mice, already infected with murine colon carcinoma cells. Such viruses have been created in a large variety, where some codes for IL-12, some code for B-7 only and a negative control group also, which received no specific viral injections. External IL-12 was also injected in some mice strains under trials, where viral infection was already prevailing. The mice, which received both the vaccines coding for B-7 and IL-12 and also received an exogenous supply of IL-12 showed the greatest survival rate of patients and regression rate tumors. Such an experiment clearly highlights the importance of co-stimulators in demolishing cancerous cells [25], [26].

2.9.10.1. SPECIALIZED CANCER VACCINES AGAINST SPECIFIC PATIENT The patients immune response, also known as immune surveillance, clearly fails in treating tumors. Therefore, the important aim of cancer vaccines is to elicit a more efficient immune response in the patients. Many such approaches have been tried.

2.9.10.2. DENDRITIC CELL BASED VACCINES Dendritic cells are the most potent APCs. They swallow up the antigens, process them into small peptides and then express them to T cells, in reference to specific MHC. So, to make dendritic cell based vaccines, this approach can be clearly stated as follows: Dendritic cells are first retrieved from the patient.

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These cells are then represented in vitro to specific tumor antigens of the patient. Such antigens are specifically tyrosinase on melanocytes, Prostratic acid phosphatase (PAP) on prostrate cells. Such antigens can be fused with stimulatory molecules like granulocyte-macrophage colony-stimulating factor (GMCSF). These dendritic cells are then injected back to the patient. Such cells then elicit a strong immune T cell based response. Such an approach has delivered a strong promise against melanoma, prostate cancer and lymphoma.

2.9.10.3. PATIENT-SPECIFIC TUMOR ANTIGEN VACCINES This study is still in the process. These vaccines are based on the antigens taken from the patients specific tumor cells. Such an approach can be broadly described in the following steps: Select the specific tumor cells from a specific patient. Design their complex with best possible adjuvants. Inject the complex back to the patient. Such vaccines are still in clinical trials for use against chronic myelogenous leukemia (CML)

2.9.10.4. VACCINES BASED ON SPECIFIC TUMOR-ANTIGENS Such vaccines are often used to develop a specific immune response in patients with specific antigens, which are mostly expressed by that specific tumor-type, but not by other normal cells. Such vaccines are utilized as a complex with adjuvant which can enhance an immune response to the greatest possible extent.

2.9.11. GENETIC POLYMORPHISM AND TUMOR IMMUNOTHERAPY Genetic polymorphisms are natural variations in the genomic DNA sequence present in more than 1% of the population. Genetic polymorphisms in therapeutic targets and metabolic pathways can significantly impact both drug efficacy and toxicity. In cancer treatment the resistance of some tumors to chemotherapy is a prime example of phmarmacogenetic alterations affecting clinical outcome. Significant heterogeneity of responses also appears to be associated with other approaches to cancer therapy, including the newer immunotherapeutic strategies that employ dendritic cell (DC), T cell, antibody or chemokine/ cytokine administration or functions. The identification of relevant genetic polymorphisms for a particular patient offers a powerful tool for improved prognostication and selection of therapy. An early example of such efficacy pharmoco-genetics is the use of DAKA HercepTest, a semi-quantitative assay for over expression of Her2/neu protein in breast tumor tissue, so as to predict those patients likely to respond to Herceptin immunotherapy. A focus of newer approaches to cancer immunotherapy is the enhancement and modulation of DC functions. This includes: i) promoting the growth and differentiation of DCs (e.g. incorporation of DC growth and/or activation factors into a vaccine), ii) promoting and enhancing T cell activation by DCs (e.g. via the activation of signal 1 and signal 2, and/or blockade of inhibitory signals) and iii) exploiting the antigen-presenting function of DCs (e.g. DCs pulsed with synthetic peptide, transduced with genes derived from relevant viral and tumor antigens or fused with tumor cells). These approaches characteristically enhance the efficiency and context of immune processing and presentation of tumor antigens with the intent of generating cancer-specific immune responses. DCs can express high levels of antigen presenting molecules, such as major histocompatibility complexes (MHC) class I and MHC class II, costimulatory molecules including CD80, CD86 and CD40 etc, adhesion molecules, and various of levels of chemokine receptors, toll-like receptors (TLRs) and produce a variety of cytokines, especially IL-12 and proinflammatory cytokines (IL-1, IL-6, TNF-(, IFN-( and (). These immune regulatory molecules and cytokines play a critical role in DC-associated immunotherapy against cancers. Numerous genetic polymorphisms within these immune regulatory molecules have been identified and they may profoundly influence the efficacy and toxicity of immunotherapies against cancer [27].

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3. CONCLUSION

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As the systems immunity cant recognize and annihilate tumor cells, these complex mechanisms often fail in developing a wide immune response, which can inhibit the malignancy and control the cancerous growth. This tumor growth can normally evade even a humans specialized complex immune system. Many immunotherapy methodologies have been attempted till date to identify and demolish specific tumors. Specialized cancer vaccine and therapeutic methods are the two prime goals of immunotherapy, which can be specifically utilized to target the bodys environment for achieving the best results. Cancers generally show many properties, making their recognition very difficult by the immune system. If we are able to successfully understand and utilize such properties, we can devise a general cure for targeting diverse cancer types. The presently increasing mortality rates around the globe definitely show that such a general cure for cancers will be of great help to mankind. Human genome sequencing, growing databases of SNPs and other polymorphisms together with a greater understanding of the key regulatory and effector molecules of the immune system provide the tools to identify correlations between polymorphism and the outcome of, or adverse reactions to cancer immunotherapy. In the near future, technologies such as quantitive PCR, pyrosequencing and microarray chips will enable rapid and high throughput screening of individual patients for particular polymorphisms of prognostic significance or importance to selection of treatment, including immunotherapeutic approaches for cancer.

4. FUTURE DIRECTIONS It was not long ago that most scientists questioned whether the human immune system was capable of recognizing spontaneously arising tumors and whether immune therapy could ever become a meaningful treatment for human malignancy. The answers are a resounding and unequivocating Yes! The administrations of cytokines and MAbs have already become components of some standard cancer treatment regimens. Many vaccines have advanced through preliminary testing to efficacy trials, and T cell therapy is being explored with new sophistication in many disease settings. As our understanding increases of the requirements for immune cell activation, homing, and accumulation at tumor sites, and for disrupting the regulatory mechanisms that limit responses, the ability to direct a coordinated and effective attack against tumors engaging multiple components of the immune system should evolve in parallel. Clinical trials of passive transfer of large numbers of tumor-reactive T cells or MAbs should not only help define the nature and magnitude of responses that will be necessary to achieve by stimulating endogenous responses, but will offer the possibility to genetically or chemically engineer immune responses with functional capacities beyond what can be elicited from the normal immune system. Despite Niels Bohrs caution that prediction is very difficult, especially about the future, we confidently predict that immunotherapy will become an increasingly essential component of future cancer therapies.

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