Tumor Immunology Introduction: Immune responses against tumor cells occur, in large part, due to expression of surface components of the malignant cell that are not expressed on the cell's normal counterpart that give rise to structures that are antigenic. Tumor-specific transplantation antigens "TSTA" = tumor antigens have been demonstrated for many tumors in a variety of animal species.

Tumor- antigens "T Ags" are shared by many tumors Tumor- antigens "T Ags" are shared by many tumors. Tumors may express both specific and shared antigens Demonstration of Tumor –specific Antigens of Chemically- induced Tumors: 1-Inject carcinogen into a mouse e.g Methyl cholanthrene -Excise tumor - Make preparation of tumor cells

11-inject prepared tumor cells into new mouse - Excise growing tumor - challenge with the same tumor - No tumor grows 111-Inject prepared tumor cells into new mouse -Challenge with different tumor -Tumor grows

Tumor immunology goals To elucidate the immunologic relationship between the host and the tumor. Use the immune response to tumors for diagnosis, prophylaxis, therapy. Tumor Ags Mechanisms that may lead to the appearance of immunogenic tumor Ags include: Mutation. Gene activation Clonal amplification Chromosomal deletion Chromosomal translocation

Some tumor Ags are unique to cancerous cells. Other tumor Ags are similar to normal cell Ags but are masked on normal cells, they may be over expressed on cancer cells e.g high levels of human epidermal growth factor receptor "HER" due to increased expression of the HER-2/neu-) oncogene as in certain breast and ovarian cancers. Some tumor Ags are present on embryonic cells but absent on normal adult cells "oncofetal Ags".

Tumor Ags may be products of mutated genes with hot spots for mutations. There is little or no cross-reactivity between physically induced tumors "e.g. those induced by U.V or X irradiation" . Carcinogens can cause clonal amplification of single cells due to transformation "genes possess mutation-sensitive hot spots". Tumor Ags are well demonstrated by virus-induced tumors "oncogenic viruses" which tend to show cross reactivity among tumors induced by the same virus. Categories of Tumor Ags

1- Normal cellular gene products: "expressed during embryogenesis. e 1- Normal cellular gene products: "expressed during embryogenesis. e. g" Oncofetal Antigens: a- Melanoma-associated Ag "MAGE". MAGE Ags are candidate tumor vaccine Ags, because their expression is shared by many melanomas. b- carcinoembryonic Ag "CEA". CEA is found primarily in serum of patients with cancers of the GIT, especially cancer of the colon. Elevated levels of CEA have also been detected in the circulation of patients with nonneoplastic diseases such as ulcerative colitis and pancreatitis.

c- -fetoprotein "FP".  FP is normally present at high concentrations in fetal and maternal serum but absent from serum of normal individuals. It is found in patients with hepatomas and testicular teratocarcinomas. 2- Mutant cellular gene products: There are several well characterized examples of tumor antigens derived from mutant cellular gene products.

In chronic myelogenous leukemia "CML" tyrosine kinase activity is increased which appears to be responsible for uncontrolled cell proliferation. Inhibitor of the genes "bcr/abl"- derived tyrosine kinase have shown promise for inducing complete hematologic responses in a sizable portion of patients with CML. Another example of a mutant cellular gene that is generated as a result of mutation is the mutant p53 protein. The p53 mutation generates common conformational changes in p53, a protein that normally acts as a suppressor of cellular growth.

Tumor immunity in vivo can be induced by vaccination against mutant P53 peptides if given with IL-12, because P53 is commonly expressed in cancer cells, T cells directed against normal P53 might preferentially destroy tumor cells. 3- Tumor Ags encoded by oncogenes Retroviral oncogenes have close relation with normal cellular genes called proto-oncogenes or c-onc. The gene products of protooncogenes have been identified as proteins with known functions in normal cells, including growth factor receptors and signal transducers.

The oncogene theory postulates that such-proto-oncogenes, when mutated or activated, display increased expression or inappropriate expression of mutated forms of their gene products, thereby contributing to neoplastic transformation and the development of cancer. Activation of oncogenes may be accomplished by chromosomal translocation, point mutation, gene amplification.

Examples of proto-oncogenes which are activated include c-myc, c-abl, c-k-ras ,they are associated with Burkitt lymphoma,CML, and lung-colon carcinomas respectively. Animal studies have shown that tumors induced by oncogenic viruses exhibit extensive immunologic cross-reactivity. This is because any particular oncogenic virus induces the expression of the same antigens in a tumor, regardless of the tissue of origin or the animal species. There is considerable evidence that several human cancers are caused by viruses including Epstein-Barr virus "EBV", human T cell leukemia virus human papilloma virus and HBV.

Immunologic Factors Influencing the Incidence of Cancer The term immune surveillance was coined to describe the concept of immunologic resistance against the development of cancer. Patients with immunodeficiency diseases are usually susceptible to viral infections and certain malignant neoplasms. .

Tumor cells, like those induced by viruses, are sensitive to immunologic destruction. Nevertheless, the natural development of immunologic responses usually fails to prevent cancers from developing. It is hoped that the successful manipulation of such responses "e.g vaccination with tumor antigens" will serve as a viable option for the prevention or treatment of cancer in the future.

Effector Mecahnisms in Tumor Immunology Adaptive and innate immune responses play important roles in the relationship between the host and the tumor. Immune effector mechanisms that are potentially capable of destroying tumors in vitro include Antibodies, T cells, NK cells, LAK cells, Macrophages and cytokines.

In general, destruction of tumor cells by these mechanisms is more efficient in the case of dispersed tumors "i.e when the target tumor cells are in single-cell suspension" than in the case of solid tumors, probably because dispersed cells are more accessible to the immune system. 1- B cell Responses to Tumors Both IgM and IgG antibodies have been shown to destroy tumor cells in vitro in the presence of complement. Studies conducted with mice indicate that antitumor antibodies are effective in destroying some leukemia and lymphoma cells and in reducing metastases in several other tumor systems.

Other studies, show that the same antibodies are ineffective in destroying the cells of the same tumor in a solid form. 2- Destruction of Tumor cells by opsonization and phagocytosis Destruction of tumor cells by phagocytic cells has been demonstrated in vitro in presence of antitumor immune serum and complement. 3- Antibody- mediated loss of Adhesive Properties of Tumor Cells It appears that metastatic activity of certain kinds of tumors requires the adhesion of the tumor cells to each other and to the surrounding tissue. Antibodies directed against tumor cell surfaces may interfere with the adhesive properties of the tumor cells (done in vitro).

4- Cell-Mediated Responses Direct destruction of tumor cells by cytotoxic T lymphocytes has been demonstrated in vitro. Tumor-specific TC are responsible for destruction of virally-induced tumors in vivo. Antibody-Dependent, cell-mediated cytotoxicity "ADCC". This process involves: binding of tumor specific antibodies to tumor cell surface. interaction of phagocytes possessing FC receptors for antibody attached to tumor cells. destruction of tumor cells by substances released from phagocytes.

C. Destruction of tumor by Natural killer "NK", Natural Killer/T "NKT" and lymphokine-activated killer cells "LAK" NK cells can kill certain tumor cells without prior sensitization and without MHC restriction. NK cells have receptors for the FC region of IgG and can participate in ADCC. NK cells secrete TNF- which induces hemorrhage and tumor necrosis. LAK cells are tumor-specific killer cells obtained from the same patient. The cells are grown in vitro in the presence of IL-2 and transferred back to the same patient. They constitute a heterogenous population of lymphocytes which include NK cells.

D. Destruction of tumor cells by activated macrophages and neutrophils Macrophages are activated by IFN- produced by activated T CD4+ T lymphocytes which are tumor specific, they release IFN- after activation by tumor antigen. Activated macrophages are cytotoxic to microorganisms, tumor cells by releasing lysosomal enzymes and TNF-.

E. Cytokines They can either stimulate or inhibit the growth of premalignant or malignant cells. Production of tumor growth factor- "TGF-" by some tumor cells promotes tumor growth due to the angiogenic properties of this cytokine.

TNF and IFN have antitumor effects as they upregulate MHC class I and Class II molecules on some tumor cells. Decreased expression of these MHC determinants allows tumor cells to evade the actions of cytokine upregulation of MHC molecules thereby facilitates important cell-mediated effector mechanisms. Transfection of tumor cells with genes coding for cytokines IL-1, IL-7, IFN- followed by transfer of such cells into tumor-bearing mice has been shown to significantly inhibit the growth of tumors.

Limitation of the Effectiveness of the Immune Response Against Tumors There is no question that an immune response can be induced against tumors. Why, then, in spite of the immune response, does the tumor continue to grow in the host? Several possible mechanisms may be operational either alone or in combination with each other as shown below: Mechanisms of Tumor Escape from Immunologic Destruction A- Tumor-related Failure of tumor to provide a suitable target

lack of antigenic epitope(tumor antigen) Lack of MHC classI molecules Deficient antigen processing Antigenic modulation Antigenic masking of the tumor Resistance of tumor cells to tumoricidal effector pathway Failure of tumor to induce an effective immune response

lack of antigenic epitope Decreased MHC or tumor antigen expression by the tumor Lack of co-stimulatory signal Production of inhibitory substances by the tumor,e.g cytokines Shedding of tumor antigen and tolerance induction Induction of T cell signaling defects by tumor burden B Host –related

Failure of the host to respond to an antigenic tumor *Immune suppression or deficiency of host including apoptosis and signaling defects of T cells due to carcinogen( physical ,chemical) infections or age * Deficient presentation of tumor antigens by host APCs * Failure of host effectors to reach the tumor e.g stromal barrier * Failure of host to kill variant tumor cells because of immunodominant antigens on parental tumor cells

Immunodiagnosis Immunodiagnosis of tumors may be performed to achieve two separate goals: To detect Ags specific to tumor cells. Assessment of the host's immune response to the tumor. Tumor cells may express cytoplasmic, cell-surface or secreted products that are different in nature and/or quantity from those produced by their normal counterparts. Because of the generally weak antigenicity of the tumor-specific markers, such differences have generally been demonstrated by the use of antibodies produced in xenogeneic animals.

The use of mouse monoclonal antibodies has greatly enhanced the specificity of immunodiagnosis of human tumor cells and their products. Some of the most widely used and reliable immunodiagnostic procedures for the detection of malignancies are described below: Detection of myeloma proteins produced by plasma cell tumors Light chains of immunoglobulins are found in urine "Bence-Jones Proteins". This is an indication of plasma cell tumors.

2. Detection of Alpha Fetoprotein "FP" This protein is produced by fetal liver cells and found in fetal serum. After birth, the level of FP falls to 20ng/ml. Levels of FP are elevated in patients with liver cancer as well as ovarian and testicular embryonal and noncancerous hepatic disorders such as cirrhosis and hepatitis. Serum FP concentrations of 500-1000 ng/ml generally indicate the presence of tumor that is producing FP and monitoring FP levels determines regression or progression of the tumor.

3. Carcinoembryonic Antigen "CEA" A glycoprotein produced normally by cells that line the gastrointestinal tract, in particular the colon. If the cells become malignant, CEA is released into the blood. Concentrations of CEA exceeding 2.5ng/ml indicate malignancy. High levels in blood may be due to noncancerous diseases such as cirrhosis of the liver and inflammatory diseases of the intestinal tract and lung.

4. Prostate-Specific Antigen "PSA" It is a glycoprotein located in ductal epithelial cells of the prostate gland. Levels above 8-10 ng/ml in blood suggest prostate cancer. 5. Cancer Antigen 125 (CA-125) Used for diagnosing and monitoring therapy for ovarian cancer. Circulating levels of CA-125 also increase during peritoneal inflammatory processes. 6. Other Markers B 72.3 is a monoclonal antibody that recognizes all carcinomas in humans "Pancarcinoma antigen".

Immunotherapy Numerous attempts have been made to treat cancers in animals and humans by immunological means. To date, immunotherapy has not been proved to be an effective treatment of cancer, either when used as the sole treatment or as an adjunct to other forms of therapy such as chemotherapy, radiotherapy, or surgery. Currently, a wide range of strategies in experimental immunotherapy of tumors are in use .

2. Tumor specific monoclonal antibodies e.g anti-CD20 for the treatment of certain lymphomas. Anti Her2/neu-1 for the treatment of certain patients with breast and ovarian cancers. These antibodies can mediate cytolysis either by engaging NK cells via FC receptors "ADCC" or by complement activation. 3. Attachment of toxins such as ricin or radioactive isotopes to tumor-specific antibodies which are delivered specifically to the tumor cells for direct killing.

4. Xenogeneic antibodies "e 4. Xenogeneic antibodies "e.g mouse anti-human monoclonal antibodies" have been molecularly engineered using recombinant DNA technology to humanize their constant regions. 5. Bispecific antibody constructs designed to bring immune effector cells into contact with tumor cells and to simultaneously stimulate the cytotoxic activity of effector cells. Examples include antibodies that recognize unique tumor antigens and IgG FC receptors "CD16" to activate NK cells. Similarly, bispecific antibodies constructs containing Fabs specific for tumor antigens and CD3 have also been studied.

6. Recombinant fusion proteins consisting of antitumor antibodies and cytokines "immunocytokines" such fusion proteins concentrate cytokine-mediated immune effector functions at the tumor site. 7. Cytotoxic T lymphocytes "CTLs" can be activated against tumor antigens by tumor cells rendered immunologic by expression of either costimulatory molecules such as CD80/CD86 or cytokines. A highly effective method for stimulating tumor-specific CTLs involves the presentation of MHC class I tumor antigen peptides by dendritic cells.

These highly efficient APCs normally express high levels of cell surface costimulatory molecules, therefore enhancing their ability to present tumor antigens to effector T cells. Dendritic cells are either directly loaded with peptides or exposed to tumor cell lysates, tumor proteins, or they are transfected with tumor-derived cDNA in an expression vector. They are then transferred to the tumor-bearing host in hope of activating cytotoxic T cells to kill the tumor cells.

8. Tumor antigens that have been molecularly characterized, active vaccination using recombinant vaccines have been developed using vaccinia, Listeria or virus-like particles. Active immunization has also been studied by injection of naked DNA plasmid constructs "DNA vaccine" with the goal of having the unique tumor antigen encoded and expressed by muscle cells. In some cases, the genes encoding cytokines "e.g GM-CSF, IL-2, IL-12" are also introduced to improve the presentation of the tumor antigen by dendritic cells at the site of injection.  

9. Augmentation of specific anticancer immunity, using nonspecific enhancement of the immune response, e.g stimulation of macrophages using Baccille Calmette-Guerin "BCG" or Corynebacterium Parvum. One example is the use of BCG for treating patients with residual superficial urinary bladder cancer. Repeated instillation of live Mycobacteria into the bladder by way of catheter after surgery has become the treatment of choice for superficial bladder cancer.

Synthetic molecules have been used e Synthetic molecules have been used e.g pyran copolymer, Maleic anhydride divinyl ether, polyinosinic –polycytidylic acid pyrimidines. They induce IFN production. Many herbal extracts have been used to stimulate the immune response e.g Echinacea, ginseng. 10. Trials are also in progress on the effects of various cytokines "IFN, IFN, IFN, IL-1, IL-2, IL-4, IL-5, IL-12, TNF, and others" either singly or in combination on tumor regression.

11. LAK cells and TILs have also been applied to the treatment of cancer with variable results. LAK cells are produced in vitro by cultivation of the patient's own peripheral lymphocytes with IL-2. Upon reinfusion into the patient, dramatic improvement has been reported in a number of cases. - A documented success has also been reported using TIL transferred to patients with melanoma. These lymphocytes, removed from a tumor biopsy, expanded in vitro with IL-2, and given back to the tumor-bearing individual, have an antitumor activity many times higher than LAK cells, thus less is needed for therapy.

Tumor Immunoprophylaxis Immunization against oncogenic viruses, it has been successful in chickens. Immunization against transplantable animal tumors, using as immunogens: sublethal doses of liver tumor cells. tumor cells in which replication has been blocked. enzymatically modified surface membrane tumor cells. extracts of Ags from the surface of tumor cells. Despite successes in the protection of experimental animals, the efficacy of immunoprophylaxis for protection of humans and animals against spontaneous tumors has not been sufficiently evaluated.