Melanoma and the magic bullet monoclonal antibodies 6-10-09

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My story of Melanoma Cancer

My story of Melanoma Cancer

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  • 1. Melanoma and the “Magic Bullet” (Monoclonal Antibodies)Author:Jim BreitfellerThis paper is dedicated to Leanne Schmall A patient who lost her battle with the Beast,Melanoma.I. Introduction:Paul Ehrlich - (March, 14 1854 –August, 20 1915)Dr. Ehrlich can be called the “Father of Modern Immunology”. He was a Germanscientist in the fields of hematology, immunology, and chemotherapy, and Nobellaureate. He is noted for his research in autoimmunity, calling it "horror autotoxicus".He coined the term "chemotherapy" and popularized the concept of a "magic bullet". Heis credited with the first observation of the blood-brain barrier and the development of thefirst antibacterial drug in modern medicine.1The Magic Bullet concept was base on selective targeting a disease with a toxin/agent tokill off the disease without effecting the rest of the body. Using this concept in 1909, heand his student came up with a treatment for Syphilis.One of his other works he is also famous for was called the “Side-Chain Theory” Thisproposed theory explaining the immune response in living cells.The concept of a "magic bullet" was fully realized with the invention of monoclonalantibodies.Today, we have a better understanding of our immune system but are still pushing backthe frontier in this area, as we try to decode the Mystery of Melanoma Cancer.II. The Immune System:Before we begin to talk about the treatment of Melanoma, we need to gain some basicknowledge on the subject matter. In the late 1960’s, a book, was published by F.M. Burnet, “Cellular Immunology andSelf and Not-Self” It proposed that Immune system could detect and destroytumors, a cancer immunosurveillance.The key to a healthy immune system is that the immune system is able to recognize thebody (Itself) from the (non-self) the foreign invaders, the cancer cells – tumors.In the absence of ongoing inflammatory and immune responses, Dendritic Cells (DC’s)patrol through the blood, the adjacent tissues, lymph and lymphoid organs.Dendritic cells (DCs) are immune cells and form part of our immune system. In theadjacent tissues, Dendritic Cells capture self and non-self antigens via specific receptors.Anything that can trigger this immune response is called an antigen. Antigens can bemicrobe , apart of a microbe, even a cell or tissue from transplant victims. Sometimesour immune system mistakes itself as non-self causing a response as an autoimmune 1
  • 2. response. Some examples of Autoimmune Diseases are Rheumatoid Arthritis,Multiple Sclerosis, and Lupus etc.1Signals from pathogens or pathogen-induced tissue damage, often referred to as "dangersignals", induce Dendritic cells to enter a developmental program, called "maturation",which transforms Dendritic cells (DCs )into efficient antigen-presenting cells (APCs)and T-cell activators. Danger signals are generated when receptors on DCs recognizesan encounter with bacteria, bacterial products viruses, viral products ,cytokines,molecules on T-cells (CD40L) and molecules derived from self cells (tumor cell lysates).These are Tumor cells that have been destroyed and broken into pieces. Once they havecome into contact with a presentable antigen, they become activated into mature dendriticcells and begin to migrate to the lymph node. Here they act as antigen-presenting cells:they activate T helper cells and T killer cells as well as B-cells by presenting them withantigens derived from the pathogen, alongside non-antigen specific costimulatorysignals.1T-helper cells (also known as effector T cells or Th cells) are a sub-group oflymphocytes (a type of white blood cell or leukocyte) that play an important role inestablishing and maximizing the capabilities of the immune system.1A cytotoxic T cell (also known as TC, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cellsor killer T cell) belongs to a sub-group of T lymphocytes (a type of white blood cell) thatare capable of inducing the death of infected cells or tumor cells; they kill cells that areinfected with viruses (or other pathogens), or are otherwise damaged or dysfunctional.1B cells are lymphocytes that play a large role in the humoral immune response (asopposed to the cell-mediated immune response, which is governed by T cells). Theprincipal functions of B cells are to make antibodies against antigens, perform the role ofAntigen Presenting Cells (APCs) and eventually develop into memory B cells afteractivation by antigen interaction. B cells are an essential component of the adaptiveimmune system. The adaptive immune system is composed of highly specialized,system.Mast cells/mastocyte is a reside in several types of tissues and contains many granulesrich in histamine and heparin. It is best known for their role in allergy andhypersensitivity allergic reactions. Mast cells play an important protective role as well,being intimately involved in wound healing and defense against pathogens and processesthat eliminate or prevent pathogenic problems1Eosinophil granulocytes, usually called are eosinophils, are white blood cells that areone of the immune system components responsible for combating infection. Along withmast cells, eosinophils also control mechanisms associated with allergy and asthma. Theyare granulocytes that develop during Haematopoiesis in the bone marrow beforemigrating into blood.1 2
  • 3. HematopoiesisSource: WikipediaAll immune cells begin as immature stem cells in the bone marrow. They respond todifferent cytokines and other signals to grow into specific immune cell types, such as Tcells, B cells.CytokinesComponents of the immune system communicate with one another by exchanging chemicalmessengers called cytokines. These proteins are secreted by cells and act on other cells tocoordinate an appropriate immune response. Cytokines include a diverse assortment ofinterleukins, interferons, and growth factors.1Some cytokines are chemical switches that turn certain immune cell types on and off.One cytokine, interleukin 2 (IL-2), triggers the immune system to produce T cells.T-Cell final development occurs in the Thymus. The thymus is a multi-lobed organ that iscomposed of a cortical and medullary area. During the T-Cell development, the cell movesthrough the different lobes due to the different microenvironments. Most of the cells thatenter the thymus never make out alive to become a mature Naïve T-cell. A naive T cellor Th0 cell is a T cell that has differentiated in bone marrow, and successfully undergonethe positive and negative processes of central selection in the thymus. In the Negativeprocess, the T cell goes through a rigorous selection to self antigen tolarence before it isreleased into circulation. Once in circulation, the cells are able to respond to novelpathogens that the immune system has not yet encountered.1 3
  • 4. The NaiveCD4 + the helper T cell, when activated, the Helper T –cell secretes mostlyIL-2 which promotes growth and proliferation, and activation of T-cells, helper T cellsand Natural Killer Cells. Once the helper cells mulitply, they start secreting othercytokines base on their costimulatory signals., concentration of antigen, and exposure totheir microenviroment. This attracts more immune cells and the assault on the foreigninvader begins.1Regulatory T-cells (Tregs) (suppressor T cells) are a specialized subpopulation of Tcells that act to suppress activation of the immune system and thereby maintain immunesystem homeostasis and tolerance to self-antigens. Regulatory T cells play an importantrole in preventing autoimmunity by suppressing the response of other T-cells to self- andother antigens. Several types of Tregs have been identified, including both CD4+ andCD8+ expressing subsets. One of the best characterized subsets, natural Tregs expressboth CD4 and high levels of CD25. Anergic cells can act as regulatory T cells bycompeting at the sites of antigen presentation and adsorbing out stimulatory cytokinessuch as IL-2. (Lack of IL-2/costimulation)1III.Immunomogy History:In 1980, Dr. Steven A. Roesnberg and colleagues discovered novel novel method forkilling metastatic cancer cells. They took lymphoid cells and exposed them tointerluekin-2 (IL-2).These cells were able to lyse the tumor cells and kill them. The werea different population than the Natural Killer cells. They coined the term “Lymphokine-activated killer cells” (LAK) for short. However, LAK cells with high dose IL-2 werenot shown to be effective in a randomized clinical trial when compared to IL-2 alone2The Cells that were cultured from tumor infiltrating lymphocytes (TIL), had a betterresponse to the tumors. We now come to know this therapy as Adaptive Cell TransferTherapy.During those Trials, Rosenberg and colleagues saw a correlation between youngercultures and where the originals TILs were harvested. The younger, the better theresponse was with the patient. TILs that were harvested from the lymph node did notrespond as much, indicating that specific location plays a role in the overall scheme ofthings. One must also note that Dr. Rosenberg’s Patients are a certain genotype (HLA-A2positive).In 1988, a research paper came out authored by Dr. Kyogo Itoh , Platsoucas,and Balchentitled: “Autologous Tumor Specific Cytotoxic Lymphocytes in the Infiltrate of HumanMetastatic Melanomas”4 Activation by Interleukin 2 and Autologous Tumor Cells, andInvolvement of the T Cell Receptor.In the report all twelve Metastatic Melanoma tumor cell suspensions activated by IL-2 ,TILs were present to a large degree. This confirmed Rosenberg’s theory earlier. The TILcell count increased to a maximum propagation in about 43 days. Tumors cells that werecultured with the TILs and the IL-2 were complete killed off. Lysing appeared five daysinto the experiment. The cytotoxic activity lasted for at least 59 days. In the control,without IL-2, the TILs eventually die off leaving the tumors cells enacted. Theseexperiments and results lead to the Rosenberg’s trials. 4
  • 5. T Cells Mobilized Graphics The T cells are Mobilized When a B cell or macrophage encounters an antigen Antigen-Presenting cells The T-cell is activated and secretes Cytokines Cytokines trigger the Immune Infected System to produce more T-cells cells Like interluekin-2 (IL-2) Some Cytokines attract immune cells & fresh macrophages, granulocytes Some T cells become and other lymphocytes to v Killer cells and track the site of the infection. And vv down infected body others direct and recruit vv cells. once on the scene. vAdapted from Understanding the Immune System How It Works NIH Publication No. 03–5423 5
  • 6. In 1992, adoptive transfer of TILs in combination with IL-2 resulted in tumor regressionsin approximately 30% of melanoma patients (Rosenberg SA), suggesting that theimmune system can play a critical role in the elimination of malignant cells.Now with a basic understanding of the immune system, we need to know how the tumorescapes detection and destruction.In a 1994 article entitled "Tolerance, Danger and the Extended Family", Dr. PollyMatzinger, layed out the idea that antigen-presenting cells (APC) respond to "dangersignals" - most notably from cells undergoing injury. The immune system does notnecessarily respond only to what is foreign but to anything that is dangerous. The dangermodel is based on the existence of the so-called second signal, in addition to the firstsignal directed at T-cells. The first signal comes from specific recognition of antigenicpeptides presented within MHC molecules (APC). The second signal is eithermediated through co-stimulatory molecules on APCs or delivered by T-helpercells. The presence or absence of the second signal determines immune responsiveness ortolerance.Table 1.) The main rules to generate an immune response or tolerant state Cell Type Absence of Second Signal Second Signal Naïve T-cells undergo apoptosis only by Dendritic cells (DCs) all APCs, monocytes, Memory T-cells undergo apoptosis macrophages, B-cells or DCs B-cells undergo apoptosis only by memory/effector T-cells undergo apoptosis or revert to a Effector T and B- perform functions after antigen resting state after a reasonably short cells recognition regardless period of time Source: T-Cell Stimulation by Melanoma RNA-Pulsed Dendritic Cells3The Danger Model is not universally accepted . Some immunologists, followingJaneways ideas (1989) believe that the immune response is based upon by evolutionarilyforces of "pattern recognition receptors". So what happens if it is a combination of bothof the therories?By trying to manipulate the immune system into specifically recognizing and killingtumor cells, there is a thin line between breaking tolerance and inducing autoimmunedisease can be easily crossed. This must be taken into account when protocols forClinical Trials are formulated. Tumor cells have a number of other direct strategies forhiding from or fighting against an immune response. Most solid tumor cells are able tohide from T-cells because they grow out of reach of secondary lymphoid organs, so naiveT-cells remain unaware of the tumors existence. Tumors also secrete proteins/peptidesthat act as mediators between the tumor and the host (body) communicating in the tumormicroenvironment. This somehow (may block the receptors or the signals) allowing thetumor to go undetected by the immune system. 6
  • 7. IV. Lymphatic System of the Immune SystemSource: http://cksinfo.com/medicine/anatomy/page2.htmlThe Dendritic cells (DCs) can be described as the most potent antigen presenting cells(APCs). They are also the only cells capable of activating naïve T-cells and, thereby, ofinitiating adaptive immune responses. In addition to up-regulation of antigen-presentingand co-stimulatory molecules, maturation includes enhancing the ability of DCs tomigrate out of the tissues and into secondary lymphoid organs, where interactions with T-cells take place. 7
  • 8. IV. ImmunosurveillanceRobert Schreiber’s group found evidence for immunosurveillance.The “three Es hypothesis” comes from this idea, and has been proposed by Schreiber andcolleagues in 2003.5 The idea is that immunosurveillance is one phase of a morecomprehensive process immunoediting which can be broken up into three componentphases: elimination, equilibrium and escape. In the elimination phase, immune cellsrecognize and eliminate the altered cells (Tumor Cells). Generally, this is sufficient.After, or simultaneous with, the elimination phase, is the equilibrium phase in whichthe tumor cells and the immune system exist in equilibrium of inaction: the tumor doesn’tgrow and the immune system doesn’t attack it. This can continue for years, and theindividual remains cancer free. Then, some tumors escape. This last phase, Escape Phaseoccurs when a tumor mutates sufficiently to evade elimination by the immune system andgrows out. The interesting thing that Schreiber and colleagues propose is that the tumorby the selective process over time during the elimination and equilibrium phases, evolvesuntil it can go undetected and escapes: that when immune system see the tumor cells, theimmune system does not get a danger signal and the tumor cells proliferate. The result iscancer. The Three Es of Cancer ImmunoeditingAdapted from Dr. Schreiber’s Hypothesis 8
  • 9. Recent improvements in the Researcher’s understanding of the Immune System such asthe role of costimulatory T-Cells and Antigen presenting cells has led to the renewal ofthe developmental efforts in Immunology ofMelanoma. Monoclonal Antibodies (mAbs). Thetheory has been around about 100 years.According to Dr. Ehrlich’s theory, the surface ofwhite blood cells is covered with many side chains, orreceptors, that form chemical links with the antigensthey encounter. After binding of the specific antigenthe cell is stimulated to produce more of the suitabletype of receptor, which would then be shed into theblood stream as antibodies.Antibodies, also called immunoglobulins (Ig) areproteins that are found in blood or other bodily fluidsof humans, and are used by the immune system toidentify and neutralize foreign objects, such asbacteria and viruses.Antibodies are produced by akind of white blood cell called a B cell. 1 Source:http:// users.path.ox.ac.uk/~scobbold/tig/ne w1/erlfg8.gifThe antibody that we are interested in is the Cytotoxic T lymphocyte-associatedantigen.(CTLA-4). This antigen can inhibit T-cell responses and is involved intolerance against self antigens. It was reported back in 1970 Bretscher and Cohn putforth the two-signal model of lymphocyte activation to explain self/nonselfdiscrimination 6This model proposes that T-cell activation requires two independentsignals. As the antigen interacts with the antibody receptor on the antigen-sensitivecell also known as the antigen presenting cell (APC) (Signal 1), it performs aconformational change which in turns paralyzes the whole cell. A new signal is nowinvoked (inductive signal) base on the new carrier of the antigen and antibodycombined. (Signal 2) The T-cell activation not only requires the T–cell interacting withthe antigen-MHC complex, but also the interaction of the CD28 receptor and the B7 aswell. Once activated, the CD28 signaling leads to Interluekin-2 (IL-2) gene expressionwhich helps promotes the immune system to propagate more T-cells.The CD28 signaling activates the PI3 kinase and AKT pathway but they believe thatthere are no signal proteins involved according to Thompson and colleagues. Theysuggest that gycoloysis occurs along with energy metabolism causing the growth of theT-Cell.7That T-cell is the T-Helper cell.The AKT signaling can either lead to cell survival or programmed cell death calledApoptosis.It has been noted that CD4+ T-cells can be cross-prime CD8+ T-cells via IL-2.8Once the CD8+ T-cell is activated correctly, it can deliver a hit to the tumor cellsif it can make its way passed the microenvironment of the tumor or tumors. 9
  • 10. Activated T-Helper Cell (CD4+ T-cell)This T-cell activation can lead to Immune Response.Another type of response is call Cell-mediated immunity. Cell-mediated immunity isan immune response that does not involve antibodies,but rather involves the activation ofmacrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes(CTL), and the release of various cytokines in response to an antigen. The immunesystem historically, was separated into two branches: humoral immunity, for which theprotective function of immunization could be found in the humor (cell-free bodily fluidor serum) and cellular immunity, for which the protective function of immunization wasassociated with cells. CD4 cells or helper T cells provide protection against differentpathogens.1 10
  • 11. Cytotoxic T lymphocytes (CTLs) appear to play key roles in the immunologicaldestruction of many cancer cells. However T-helper cells are needed for the activation oftumor-destructive macrophages, NK cells and lymphokine-activated killer (LAK)cells.Source: Immunologic Pathways 11
  • 12. Cellular immunity protects the body by: 1. activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of foreign antigen on their surface, such as cancer cells displaying tumor antigens; 2. activating macrophages and natural killer cells, enabling them to destroy intracellular pathogens and 3. stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.1“Cell-mediated immunity is directed primarily at microbes that survive in phagocytesand microbes that infect non-phagocytic cells. It is most effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, andintracellular bacteria. It also plays a major role in transplant rejection.1”Tumor RejectionTo cause the rejection of the tumors cells, The immune system must orchestrate a chainof events mediated by several types of Leukocytes including Dendtric cells (DC),Natuaral Killer cells (NK), CD4+ and CD8+ lymphocytes and others. This orchestrationhas many players in it including the T-Regs, sercreted cytokines, and Monoclonalantibodies (mAb’s) and complexes. It is a delicate balance between self and non-self.At the Lymp Node drainage area, the lymphocytes (CD4+ and CD8+) with their T-cellreceptors (TCRs) are able to scan the Dendtric cells (DC’s) for antigen-MHCmolecules. (ag-MHC) major histocompatability complex (class I or II). Based onthe two signal model, a second signal from CD28 molecule is needed to activate the T-cells (T-lymphocytes). If communcation breaksdown, and the TCR signal onlyhappens, it can lead to tolerance by means of fuctional paralysis of the (APCs) Antigenpresenting cells (Anergy) or by the induction of clonal deletion (apoptosis). Anergiccells can act as regulatory T cells by competing at the sites of antigen presentation andadsorbing out stimulatory cytokines such as IL-2. This can halt the activation of the T-lymphocytes and no immune response is initiated.Once activated fully, the CD4+ T-cells can mobilize to where the event will take place andusually sends out a “danger signal” inflammation. The activated CD4+ T-cells can secretemany different cytokines including IL-4, IL-2 and activate the TH2 cells which are asubset of the CD4+ cells. The TH2 cells stimulate the B cells to mature into plasma cellsthat secrete antibodies. These antibodies that are produced are the cell-destructive kindsthat have anti-tumor behavior. The CD4+ can also cross-prime CD8+ T-cells in thepresence of IL-2 and are called (CTLs) Cytotoxic T Lymphocytes. Cross-priming isanother name for cross-presentation. The role of the CD8+ T cells is to monitor all thecells of the body, ready to destroy any that express foreign antigen fragments in theirclass I molecules. Three major events must occur to Activate CD8+ T cell mediatedresponse against melanoma. First, the T-cell receptor (TRC) must be triggered by a (or 12
  • 13. multiple) self antigen–derived peptide MHC class I complex12. This event dependsentirely on appropriate antigen presentation, which is most efficiently provided by maturedendritic cells . Once properly activated, may serve as tumor-specific effector Tcells .Second, simultaneously with T-cell receptor triggering, a distinct secondcostimulatory signal must be delivered, mediated by IL-2, B7-1, or B7-2, which engageIL-2 receptors and CD28 on the surface of the T cell, respectively . A source of thesecofactors for effective CD8+ T-cell stimulation can be provided by CD4+ T cells thatrelease critical amounts of IL-2, or by mature dendritic cells that display an increasedlevel of B7-1/B7-2 costimulatory molecules on their cell surfaces. Third, inflammatorycytokines, including IL-1, IL-6, IL-12, and IFN-γ provide a third signal that acts directlyon T cells, referred to as the “danger signal”. This signal was found to optimally activateTH1 differentiation and lead to clonal expansion of T cellsSome CD4+ T cells can develop into CTLs, but they can attack only those cell types (e.g.B cells, macrophages, dendritic cells) that express class II MHC molecules. Virtuallyevery cell in the body expresses class I MHC molecules, so CD8+ CTLs are not limitedin the targets they can attack. Cytotoxic T lymphocytes (CTLs) appear to play key rolesin the immunological destruction of many cancer cells. However T-helper cells areneeded for the activation of tumor-destructive macrophages, NK cells and lymphokine-activated killer (LAK) cells. If lymphocytes are cultured in the presence of Interleukin2, (IL-2) it results in the development of effector cells which are cytotoxic to tumorcells.12CTLs have cytoplasmic granules that contain the proteins perforin and granzymes.When the CTL binds to its target, the contents of the granules are discharged. A dozen ormore perforin molecules insert themselves into the plasma membrane of target cellsforming a pore that enables granzymes to enter the cell. Granzymes are serine proteases.The serine proteases are a family of enzymes that cut certain bonds in other proteins. It issimilar to what is in your laundry detergent. They are known as detergent enzymes. Theybreak the bond between the dirt and the fabric. By breaking up these proteins, they startdestroying the intracellular workings of the tumor cells.CTLs Binds to the tumor cells and discharges granules that contain the proteinsperforin and granzymes.Tumor Evading DetectionLack of costimulationMany Melanoma tumor cells do not have the B7 protein on their surface so this co-stimulatory second signal cannot take place. Theoretically, they should cause an immuneresponse but they do not stimulate an effective anti-tumor immune response. The firstsignal originates from the binding of the T cell receptor (TCR) to its antigen-MHC, and 13
  • 14. provides the specificity of the interaction. Without this signal, the cell enters anergic stateand can act as a T reg cell. Expression of B7 on the surface of a cell is the costimulatorysignal necessary to allow for the cytolytic CD8+ T cell attack on the tumor. B7 displayrenders tumor cells capable of effective antigen presentation, leading to their eventualeradication.Secretion of immunosuppressive cytokinesAnother way tumors evade detection is by secretion of certain cytokines. They are low-molecular weight proteins that use their communication ability to regulate the immuneresponse. Cytokines can act upon either the cells secreting them (autocrine) or onneighboring cells (paracrine) to generate activities in the targeted cells. This means theycan act as light switches for on and off immune responses. For example, interleukin-2activates a cell-mediated immune response, while interleukin-10 suppresses cell-mediated responses. Many types of cancer, including Melanoma, take advantage of thisability to down regulate this appropriate immune response to help extend their survivaland proliferation. This causes cancer patients to fail in mounting a successful attack onthe tumors. Immunosuppressive cytokines secreted by cancer cells include transforminggrowth factor-beta (TGF-beta), interleukin-10 (IL-10) and vascular endothelialgrowth factor (VEGF).TGF-beta is one of the most potent immunosuppressive cytokines characterized to date.It is capable of affecting the proliferation, activation and differentiation of cells 14
  • 15. participating in both the innate and acquired immune response.TGF-beta inhibits theprofilation T-cells, B cells, Natural killer cells (NK), and macrophages.TGF-beta alsoconverts T-cells, which normally attack cancer with an inflammatory (immune) reaction,into regulatory (suppressor) T-cells, which turn off the inflammatory reaction. Another ofTGF-betas affect is on cytotoxic T lymphocytes (CTLs) This is very important for anti-tumor immunity because of their cytotoxic effects. TGF-beta down-regulates many ofthe processes necessary for CTL activation. Without this activation, there is no assault onthe tumor cells from the CTLs. In addition to suppressing proliferation, TGF-beta hasbeen shown to induce apoptosis (cell death) in B and T cells.Another immunosuppressive cytokine is IL-10. It is capable of inhibiting the prodction ofof pro-inflammatory cytokines like IFN-gamma, IL-2, and GM-CSF made by cells suchas macrophages and T helper cells. IL-10 also displays potent abilities to suppress theantigen presentation capacity of antigen presenting cells. Secretion of IL-10 in thevicinity of a tumor can render the tumor totally insensitive to CTL-mediated lysis. It ismost likely that the tumor’s microenvironment is altered enough to block or turn off thedischarge granules that would lyses the tumor cell. However, it is also stimulatorytowards certain T cells, mast cells and B cells. It enhances B cell survival, proliferation,and antibody production. As you can see, IL-10 has many rolls to play when it come tothe immune system. Vascular endothelial growth factor (VEGF) is a cytokine that is produced by mosttumors. This growth factor enables the tumor to expand vascularly when is in its growthphase. VEGF production can be induced in tumor cells that are not receiving enoughoxygen.Regulatory T-cells (Tregs) (suppressor T cells) are a specialized subpopulation of Tcells that act to suppress activation of the immune system and thereby maintain immunesystem homeostasis and tolerance to self-antigens.1Tumor Growth kineticsThe cell cycle has four stages: 1. G1 phase when the cell increases in size and gets ready to replicate its DNA. 2. S phase when the cell synthesizes or copies its chromosomes 3. G2 phase in which the cell prepares to divide 4. M phase when mitosis occurs.When the various growth inhibitory proteins and checkpoint controls which regulate thiscycle become disabled due to mutations characteristic of cancerous cells, the cell cycle isno longer under tight regulation. Tumor cells are capable of proliferating so quickly thatthe immune response is not fast enough to keep their growth in check. The growth of the 15
  • 16. tumor cells outpaces the immune response and escape the detection of the immunesystem. Lack of cell cycle controls leads to excessive proliferation of tumor cells.In February 2003, a National Cancer Institute Clinical Trial (NCT00058279)“Monoclonal Antibody Therapy and Interluekin-2 in treating Patients with MetastaticMelanoma” was started with Dr. Rosenberg at the forefront. They were hoping thatthey would get synergetic outcome based on the two single therapies.Method: Thirty-six patients received anti-CTLA-4 antibodies every three weeks. Thedoses were as follows: three patients per cohort received the following.(0.1, 0.3, 1.0,and 2.0 mg/kg.) the other twenty-four patients received 3 mg/kg. All patients receivedIL-2 Therapy (720000 IU/kg) every 8 hours to a maximum of 15 doses. Results: 8 of 36 had object response (22%)3 of 36 had complete response (CR) (8%)5 of 36 had a grade3/4 autoimmune toxicity due to the Anti-CTLA-4 administration(14%)Conclusion: Base on this trial, there was no evidence to support a synergic effect of theCLTA-4 Blockage plus the IL-2 addition because alone as a single agent; the 22percent objective response rate was as expected. The one thing that came out of the trialwas that there was a durable cancer regression from the treatment.Results were in disagreement with research that was done in 2005 with Human CTLA-4 knock-in mice by Dr. Lute and colleagues. “Therefore, it is likely that even the mostefficient anti-CTLA-4 antibody will need to be used in combination with other reagentsin order to achieve complete rejection of established tumors.8”Base on clinical trials in 2008, Anti-CTLA-4 Blockage did not show a better immune responsethen the FDA approved Dacarbazine.9 As a single agent, this was quite disappointing to theresearchers and the melanoma patients alike. One good thing that came out of the trialswas that the complete responders had a more durable response.The Orchestration of an Immune Response UnrehearsedIn 2006, after two fail attempts (Interferon and Dacarbazine with Patrin) to stop theprogression of my melanoma, I was able try CTLA-4 Blockage. It was one of my firstchoices, but due to protocol, I had to try the FDA approved therapy first. I had researchedthis monoclonal antibody. On 10-24-2005 when I was first diagnosed with melanoma, Icontacted Dr. Luis H. Camacho who was currently at MD Anderson.Subject: Paper on Antitumor Activity 16
  • 17. “Luis Camacho, My name is Jim Breitfeller and I have recently been diagnosed withmelanoma will need some sort of Ontological therapy after my surgery. I ran across anabstract of yours (Antitumor activity in Melanoma and anti-self responses in Phase 1trials with the anti-Cyctotoxic T Lymphocyte-Associated Antigen 4 MonoclonalAntibody CP-675,206) in the Journal of Clinical Oncology. Is it possible to get a copy ofyour paper? It can be emailed to the address below.”Camacho response:Dear James,Thank you for your note. The CTLA4 antibodies in melanoma are currently underdevelopment and completing the approval process with the FDA (Phase II and Phase III).The overall response rates in my mind will be near 20-30% with a good number ofpatients attaining long term remissions. However, none of the programs are currentlyoriented to patients rendered NED (Stage III or IV). They are in fact for patients withadvanced disease. From your brief introduction, I think your best options are to obtain anHLA typification and go for an adjuvant trial.Please feel free to page me if you need further information. Pager is 713.404-5319Best,LuisCP-675,206, a novel monoclonal antibody, enlists the immune system to fight advancedmelanomaSome Positive Test results of the CTLA-4Early testing of an experimental human monoclonal antibody showed a striking benefit inpatients with advanced melanoma, say researchers at The University of Texas M. D.Anderson Cancer Center, who presented their findings at the annual meeting of theAmerican Society of Clinical Oncology. Of 39 patients given a single injection of CP-675,206 (known as CP-675), tumors disappeared in three patients, shrunk in a fourthpatient, and cancer stopped growing in five other patients. These responses haveremained since their initial treatment, which ranged from 13 to 28 months ago.Most of the patients in the trial had advanced melanoma, which has a median survival ofless than a year, says the studys principal investigator, Luis Camacho, M.D., MPH,assistant professor in the Department of Melanoma Medical Oncology."We were very pleasantly surprised to find such objective antitumor responses in a PhaseI clinical trial, which is designed to find the ideal dose and to look for side effects," saysCamacho. "These results are very early, but they are encouraging to us because there areno good agents available to treat melanoma once it has spread."Source: Laura Sussman from (ASCO) American Society of Clinical Oncology 17
  • 18. At the time of the request, I was not at the correct stage but I knew that this might be thepath of the future. I did contact him and we discussed my options at that time. I was justlearning the ropes.On 9/3/06 I contacted Dr. Rosenberg just in case I needed a back up plan if the CTLA-4blockage did not work. At that time I did not know I was the wrong HLA-02 type forRosenberg’s trials.“I am Contacting Dr. Steven A. Rosenberg at the National Cancer Institute in Bethesda,Maryland.He is the lead the researcher on the Gene Therapy Trials.Log onto the CBS website for the story!!!!!!http://www.cbsnews.com/stories/2006/08/31/health/main1955526.shtmlThe research team recently applied to the Food and Drug Administration (FDA) to try thenew cells in about 100 patients. The FDA is expected to respond to the request by mid-September.Dr. Rosenberg, I just got the news of your Gene Therapy Experiments. The initial resultslook somewhat promising. I applauded you and your team for making great strides in thecure for melanoma cancer.I am a cancer patient (48 yrs. old) under the care of Dr. John Kirkwood at the HillmanCancer Center at the University of Pittsburgh. I have gone through a wide incision, lymphnodes removal, Interferon therapy, and Dacarbazine therapy without success. I ampresently on track to start a clinical trial with CTLA-4 monoclonal antibodies September13, 2006. I have some tumors on my right side of my back and some in each lobe of mylungs. I would like to be considered for your next round of Gene Therapy in the comingmonths if I have no response to the CTLA-4 treatment. Please let me know if you wouldneed a copy of my medical records to date.Thanks again for the great work you are doing and I hope to hear from you in the nearfuture.Best Regards,Jim BreitfellerOn 9/5/06 I received a call from Dr. Rosenberg’s office this morning while I was at Dr.Marino’s office. Kathy Morton (Research Nurse) contacted me by phone and asked a fewquestions about my health. She went on to say if I go with the CTLA-4 therapy, it wouldtake about 2 months to washout before I could try the Gene Therapy. They would alsohave to do a colon biopsy to check the colon for any adverse conditions from theCTLA_4. She then gave me her direct phone number if I want to pursue the gene therapyat a later date. 18
  • 19. So, on 9/13/06 (day 1)I had my first and only infusion of anti-CTLA-4 monoclonalantibodies. A dose of 15 mg/kg on Day 1. This was done as an outpatient procedure.Anti-CTLA4 monoclonal antibodies block the ability of CTLA4 to down-regulate T cellproliferation. The theory behind this therapy is that by decreasing the inhibitory signal,there will be a subsequent increase in the number of activated T-cells available, toimprove the ability of the T-cells to recognize melanoma cells as non-self.Before we can go any further, we need to know the clinical pharmacokinetics (pk)of anti-CTLA-4 monoclonal antibodies. Base on published papers, the predicted half-live of theantibody is around 3 weeks.11 This means your body will eliminate half the dose that wasinfused in you in about 21days. So, in 42 days or there about, the drug concentration inmy system is about 3.75 mg/Kg.I started my CTLA-4 treatment at 9:15 am at 100 ml/hr and I had 500 mls hanging on myrack (Miss Daisy). I call the rack Miss Daisy because I have to take it with me where everI go which includes the bathroom. I am driving Miss Daisy!! This will take us to 3:15 pmand then they draw blood for a pk study an hour later. So, we won’t get out until about4:30 pm and home until 10:00 pm.Day 7 -9/19/06 “Along with the fatigue, my muscles ache like they have lactic acid inthem”. Is this an indication of something? All immune cells begin as immature stemcells in the bone marrow.Day 15 -9/27/06 about half the CTLA-4 antibodies are depleted. It appears that theCTLA-4 has stimulated my immune system. In the pass week, I noticed that there wasredness around the area where my tumors are located. Also it is becoming quitetender in that area. This is Great news!!!!! It appears that the treatment my have kickstarted my immune system. The only way we will know for sure is another CT scan. Thatis not scheduled until November 23rd.I sure hope this isn’t a false positive. Anyway, they gave me an antibiotic just in case it isan infection.This inflammatory response provides a third signal that acts directly on T cells, referredto as the “danger signal”. “This signal was found to optimally activate TH1 differentiationand lead to clonal expansion of T cells12.With this clonal expansion of the T cells and the secretion of IL-2, The Immune system isgearing up to make an assault on the foreign invaders, the tumors.In 1988, a paper was published Autologous Tumor Specific Cytotoxic Lymphocytes inthe Infiltrate of Human Metastatic Melanomas Activation by Interleukin 2 andAutologous Tumor Cells, and Involvement of the T Cell Receptor by Itoh andColleagues.4 In their studies, they propagated (TILs) Tumor infiltrate lymphocytes cellsfrom 12 Metastatic Melanoma patients. They preformed kinetic growth studies in IL-2and even broke it down three Surface markers (CD3,CD4 and CD8). The results are asfollows:The average maximum propagation was 43 days. (N=12)The average maximum propagation for (lung, Axilla) was 40 days (n=3)The average maximum propagation for (CD3) was 78 +/- 11 days (n=12)The average maximum propagation for (CD4) was 33 +/- 10 days (n=12) 19
  • 20. The average maximum propagation for CD4 (lung, Axilla) was 26 days (n=3)The average maximum propagation for (CD8) was 49 +/- 17 days (n=12)The average maximum propagation for CD8 (lung, Axilla) was 57 days (n=3)Base on the above data, it would take about 49 days for my activated T cells to reachmaximum propagation.Day 29- 10/11/06, A couple of days ago, Dee noticed two new growths on my back. Iwas hoping for the best. Anyway, we got confirmation from the Hillman Center that it is2 new tumors growing. This really stinks. I think it is time to take out the “Weed beGone”. This is not what I was hoping to hear. It was decided that the CTLA-4 blockagetherapy was to be terminated. My CD4+T cells were just about at maximumpropagation.Dr. Kirkwood, decided that the next line of defense would be Interleukin-2 (IL-2).Results of early PROLEUKIN® IL-2 Clinical TrialsYear received FDA Approval 1998Number of Patients 270 patientsNumber of Trials 8Response In 16% of the patients, tumors shrank or disappeared as a result ofPROLEUKIN® IL-2 therapy.In 6% of the patients, the tumors disappeared completely.Results From these trials, it was determined that a patient whose tumors completelydisappeared from the treatment remained cancer-free for a median of 4.9 years.I needed to washout the CTLA-4 blockage and have some test run before I would beaccepted into the next trial. We know from the PK studies that it would takeapproximately 150 days to eliminate the antibodies from my system.Day 43-10/25/06, I got the results back from the Scans and it wasn’t good. The cancer isspreading in my lungs quite rapidly according to the CT scans. There are now over 40+nodules ranging from 15 mm down to < 5 mm. No wonder I been having shortness ofbreath. I thought it was my lack of exercise. Dr. Pandya gives my prognosis a poor rating.I guess I will have to sit in the corner. (CTLA-4 Antibodies are gone from my body.)The cancer has made its way to the “Escape Phase” and is now out of control. This isalso the average maximum propagation time of the cultured T-cells.Day 50- 11/1/06, the first cycle of High dose Interleukin-2 (IL-2). It just so happen tobe the maximum propagation of the CD8+ T cells. All of the anti-CTLA-4 is washedout. We also, most likely have the most CD4+ T reg cells. These are the cells that helpregulate the immune response so it doesn’t go into overdrive and cause an autoimmuneresponse.If we reset the clock for the second therapy (LI-2), then we can follow theactivation of the CD8+ T-cells. My body has become a big 20
  • 21. Erlenmeyer flask. Erlenmeyer flasks are used in microbiology for the preparation ofmicrobial cultures.So on day 50- 11/1/06, we innocuated my body with IL-2 – a growth factor. So based onthe Itoh study, I should be activating the CD8+ T-cells into a mature state (TILs andLAK cells.) It should take roughly 50 days they would be at there maximum growthphase.In Itoh’s study the cultures were supplemented every 5 days by replacing half thecultured medium with fresh medium containing (IL-2) as one of the supplements. MyIL-2 additions were every 21days. (600,000IU/kg for high dose IL-2)On 78th day 11/29/06, the second cycle of IL-2 was administered. I t was pushed back aweek due to the Thanksgiving Holiday. I completed 8 doses which is the average thatpatients can withstand.On day 93 12/14/06, I have another CT scan. I am trying to recover between cycles.On day 98 12/19/06 we got the CT Scan Results: What a Christmas Present!!!!!! Thetumors were shrinking!!!!!!!Melissa’s Note:Im Christmas shopping.....but Heather called me with the results....IAM SOOOOO HAPPPY FOR YOU!!!!!!!!!!!!!YIPPPPPEEEEEE!!!!!!!Hope you have a wonderful holiday, and Ill see you soon :) :) :) :)MelissaAs you can see, the timing and the players of this Orchestration all fell into place. Asingle Bullet of Monoclonal antibodies started a chain reaction with a whole sequence ofevents which lead to the restarting of my immune system. Without that bullet, therewould have been no "Danger Signal""Melanoma and the Magic Bullet (Monoclonal Antibodies)" 21
  • 22. So now we know what had transpired with the therapy, we need to know how and why ithappened. I will try to decipher and or postulate each step of the therapy.First, how did I get the right antigen to be presented on the Antigen Presenting Cell(APC)? There are three types of Antigen Presenting Cells: • Macrophages • Dendritic Cells • B CellsWe will focus our attention on the Dendritic cells (DCs ) because I postulate that thesecells played a major roll in help generating an immune response. Induced Dendritic cellsgo through a developental program call maturation, which transforms them into efficientantigen-presenting cells (APCs) and T-cell activators. They are the most potent of thethree APCs.So what really happened? Well, Dr. Kirkwood started me out on Dacarbazine withPaTrin-2. Dacarbazine is a chemotherapy agent, approved by the FDA for fightingMelanoma. Dacarbazine alkylates and cross-links DNA during the phases of the cellcycle, resulting in disruption of DNA function, causing cell cycle arrest, and apoptosis.17The only problem is that the Melanoma Cells overexpresses this enzyme called MGMT.Proteins known as DNA repair enzymes are present in cells to target damaged DNA andreverse the modifications caused by alkylating agents. One such enzyme ismethylguanine methyltransferase (MGMT). MGMT directly reverses the chemicalmodification guanine, one of the four building blocks of DNA, allowing normalreplication to take place.The DNA-repair enzyme MGMT is a key factor in resistance to alkylating agents. Thisis one reason why the Dacarbazine therapy doesn’t have a very successful response rate.The MGMT enzyme repairs what the dacarbazine cross-links. So, PaTrin-2 was added tothe trial. This drug is known to inactivate the MGMT activity. By inactivating theMGMT enzyme, it makes the tumors cells more susceptible to the chemotherapy. 22
  • 23. This therapy was able to get the tumors cells to shed some antigenic Protein which Itheorize and was used as the presenting antigen. This made the antigen “tumor-specific.” Base on a paper by Dr. Olivera J.Finn called Cancer Immunology published in the NewEngland Journal of Medicine in June 19, 2008, there are three ways for self antigens tobecome Tumor Antigens: 1. Mutation 2. over expression 3. Post-translational ModificationI postulate that some failure of the tumor cells to repair the DNA damage cause by theDacarbazine in the present of PaTrin-2 resulted in a mutation causing the cancer cells toshed an antigenic peptide. But I was still missing a signal or signals to activate myimmune system.The second step was the inoculation of the (CTLA-4 Monoclonal antibody) mAb. Thedose was given at 15 mg/Kg. This was the highest dose given based on thepharmokenetics We know from the theory, we need to engage the B7 receptor. Theresearch states that the anti-CTLA-4 has a higher binding affinity for the B7 receptor. Soit will react first.Pinpointing when T cell costimulatory receptor CTLA-4 is engaged is important becausewithout this blockage, the T-cell will not stay activated.Dr. James Allison and colleagues in the late 1990’s did some studies with mice. In themouse model, the anti-CTLA-4 blockage caused an autoimmune response which wasdiabetes in the mice.13 Before I go any further, I must make a note of caution. That is notall immune responses in mice models crossover to the human model, but the models areusually a good predictor. So with that said, In the research paper “Pinpointing when theT-cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T-cells”,it took about 12 days to see a response to the Anti-CTLA-4 mAb in the mice. I had aninflammatory and it was noticed at 15 days after the induction of the antibodies. Recent advance in autoimmunity research reveals that the innate immune system is ableto recognize self-targets and initiate inflammatory response in a similar way as withpathogens. Accordingly, alterations in cell morphology are recognized by the innateimmune system resulting in an acute inflammatory response (Carroll and Holers,2005). 23
  • 24. Well in my therapy, an inflammatory response was noted on day 15. This suggests thatthe costimulatory receptor was fully engage to cause an inflammatory response, The“Danger Signal”“Three major events must occur to induce CD8+ T cell–mediated, tumor-protectiveimmunity against syngeneic melanoma. First, the T-cell receptor must be triggered by a(or multiple) self antigen–derived peptide MHC class I complex . Therefore, this eventdepends entirely on appropriate antigen presentation, which is most efficiently providedby mature dendritic cells. Peripherally tolerant or “ignorant” self-reactive T-cell clones,once properly activated, may serve as tumor-specific effector T cells .Second,simultaneously with T-cell receptor triggering, a distinct second costimulatory signalmust be delivered, mediated by IL-2, B7-1, or B7-2, which engage IL-2 receptors andCD28 on the surface of the T cell, respectively (17). A source of these cofactors foreffective CD8+ T-cell stimulation can be provided by CD4+ T cells that release criticalamounts of IL-2, or by mature dendritic cells that display an increased level of B7-1/B7-2costimulatory molecules on their cell surfaces. Third, inflammatory cytokines, includingIL-1, IL-6, IL-12, and IFN-γ provide a third signal that acts directly on T cells, referred toas the “danger signal”. This signal was found to optimally activate TH1 differentiationand lead to clonal expansion of T cells12.Once the CD4+ T-cells are activated by the blockage of the CTLA-4 receptor, They startsecreting cytokines in the first 24 hours. Interleukin -2 (IL-2 ) is the first to be secreted.This secretion of IL-2 promotes the CD4+ T-cells to proliferate. This critical amount ofCytokine (IL-2) not only helps the CD4+ T-cells, but also need to help develop the CD8+T-cells into (CTL) Cytotoxic T lymphocytes.With this clonal expansion of the CD4+ T-cells also generates more subsets including theT-Regulatory foxp3 (CD4+ CD25+ FoxP3). It is postulated that the CTLA-4 that isexpressed by the Treg is critical for the suppression of the immune responses by affectingthe potency of the antigen-presenting cells to activate other T-cells.14 These Tregs eventhough they are small in number (Only about 5% of the total T-cells) can suppress theimmune system response. This suppression is mediated by the CTLA-4 dependent down-regulation of the B7-1 and B7-2 receptors on the antigen presenting cell (APC). TheTregs are most likely have multiple suppressive mechanisms and each one activatedbased on the microenvironment and the context of the immune response. One of thosemechanisms by which the treg could mediate suppression is by secretion of a suppressivecytokine like IL-10. Thus, The CTLA-4 is an important key molecular target forcontrolling the Treg-suppressive function. So base on our knowledge now, we have three ways to tip the balance towards animmune response: 1. If you limit the expansion of the of the CD4+ T-cells, you can also limit the Treg expansion. This can be done by the timing of the addition of the interleukin-2 24
  • 25. after the T-cell is activated. By limiting the IL-2 concentration during expansion, you deplete the IL-2 in the microenvironment which is needed for the proliferation of the Tregs.2. Using anti-CTLA-4 Blockage not only restores the TCR-driven T-cell proliferative potential, but also confers the lymphocyte resistance to the Tregs.153. Lymphodepletion of the Treg cells and others can tip the balance toward an immune response like in Dr. Rosenberg’s (ACT) Adoptive Cell Transfer Therapy. 25
  • 26. Another mechanism that was postulated in 2006 by Almeida and colleagues was that the T-regs were indexed to the number of activated CD4+ T-cells that were secreting IL-2.16This meant that there was proportion/balance between the Tregs and the activated CD4+T-cell. This showed if there was an increase in activated T-cells, the IL-2 produced wouldbe used for proliferation of the T-regs to maintain that ratio for homeostasis. By depletingor blocking the functionality of the T-regs, one can push the equilibrium of the immunesystem in favor of an immune response.We don’t want to eliminate the CD4+ T-cells altogether, because they are essential forthe maintenance, functionality and proliferation of the B cells and that also help cross-prime the CD8+ T-cells. CD4+ T cellsCD4+ T cells bind an epitope consisting of an antigen fragment lying in the groove of aclass II histocompatibility molecule. CD4+ T cells are essential for both the cell-mediatedand antibody-mediated branches of the immune system: • cell-mediated immunity These CD4+ cells bind to antigen presented by antigen-presenting cells (APCs) like phagocytic macrophages and dendritic cells. The T cells then release Cytokines that attract other cells to the area. The result is inflammation and the accumulation of cells and molecules that attempt to wall off and destroy the antigenic material (an abscess is one example; the rash following exposure to poison ivy is another). • antibody-mediated immunity These CD4+ cells, called helper T cells, bind to antigen presented by B cells. The result is the development of clones of plasma cells secreting antibodies against the antigenic material.So, where does Interluekin-2 (IL-2) come into play? According to Byung-Scok et al andrecent reports, IL-2 is not needed for developmental CD4+ CD25+ Treg cells in the thymusbut does play an important role in the maintenance and function in the peripheral.18Peripheral is defines as secondary system outside the bone marrow and thymus. It entails thesite of antigen, immune system interaction. IL-2 is required for the peripheral generation ofTregs based Abbas’s and colleagues research.19 IL-2 prevents the spontaneous apoptosis ofthe CD4+ CD25+ Treg cells. It has been reported that patients with multiple advance-stagetumors have elevated levels of Tregs within the tumor microenviroment.20 Interluekin-2 is thesurvival factor for CD4+ CD25+ Treg cells.21 If the addition of IL-2 is on or before themaximum propagation of the CD4+ T cells, the Tregs population can increase 5-fold in a 96hour period based on certain growth mediums. By controlling the addition of the endogenousIL-2, one has a knob to turn and can lead to the control of the expansion of the Tregs. Whenyou combined this control with the anti-CTLA-4 blockage, you can shift the balance of theimmune response. 26
  • 27. Now here is the catch. The maintenance and function of the CD8+ T-cells require CD4+ cellswhich secrete IL-2. So we don’t want to deplete the CD4+ cells, we want to control theexpansion of the Tregs which are a subset of the CD4+ cells. It has been postulated by someresearchers that the Anti-CTLA-4 blockage also suppresses the Treg function in a differentmechanism. By using IL-2 as the rate limiting factor, we can suppress the CD4+ CD25+ Tregcell expansion by controlling the concentration and timing of the Inerluekin-2 at the tumormicroenvironment.The Interluekin-2 plays another role in this Melanoma Maze. In a study by Janas et al, Il-2increases the expressions of the perforin and granzyme A, B and C genes in the CD8+ T-cells.This increase expression causes the CD8+ T-cells to mature into Cytoxic T Lymphocytes(CTLs). The exogenous IL-2 is required for the granzyme proteins. As stated previously, 27
  • 28. CTLs have cytoplasmic granules that contain the proteins perforin and granzymes. A dozenor more perforin molecules insert themselves into the plasma membrane of target cellsforming a pore that enables granzymes to enter the cell. Once in the tumor cell, theseenzymes are able to breakup (lyse) the cell and destroy it. This is the beginning of the end forthe cancer cells. The tumors begin to shrink and the rest is history, “An Inmmune ResponseUnrehearsed.”On the other hand, prolong therapy with Il-2 can result in causing apoptotic death of thetumor- specific CD8+ T-cells.23Clearly in a clinical setting, timing, dose, and exposure to these drugs play a major roll inthe immunotherapy, and can have dramatic effects on the outcome. All it takes is thatone magic bullet to start the immune reaction. 28
  • 29. 29
  • 30. The Orchestration of an Immune Response Unrehearsed 30
  • 31. Acknoledgements:I like to thank my wife Dee for putting up with me during the research and writing of thispaper. I would also like to recognize Leanne Schmall, who is with us today in spirit. Itwas her journey that inspired me on writing this paper.We will miss her very much andshe will not be forgotten. The Melanoma Warroir . 31
  • 32. References1. Definitions source: Wikipedia2. Rosenberg, SA; Lotze, MT; Yang, JC; Topalian, SL; Chang, AE; Schwartzentruber, DJ; Aebersold, P; Leitman, S; Linehan, WM; Seipp, CA. Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer [published erratum appears in J Natl Cancer Inst 1993 Jul 7;85(13):1091]. J Natl Cancer Inst. 1993; 85:622–632.3. Javorović, MiranT-Cell Stimulation by Melanoma RNA-Pulsed Dendritic CellsThesis ; Jan. 15 20044. Itoh, K; Platsoucas, CD; Balch, CMAutologous Tumor Specific Cytotoxic Lymphocytes in the Infiltrate ofHuman Metastatic Melanomas Activation by Interleukin 2 and AutologousTumor Cells, and Involvement of the T Cell Receptor [published J . Exp.MED. The Rockefeller University Press. 1988 Oct 1; Vol 168 October 19881419-1441http://jem.rupress.org/cgi/reprint/168/4/1419.pdf5. Gavin P. Dunn,1 Lloyd J. Old, and Robert D. Schreiber1The Three Es of Cancer Immunoediting1 Department of Pathology and Immunology, Center for Immunology, WashingtonUniversity School of Medicine, St. Louis, Missouri 63110;Annual Review of Immunology, April 2004,Vol.22. Pages 329-360(doi: 10.1146/annurey.immunol.22.012703.104803)6. Bretscher, P., and M. Cohn. 1970. A theory of self-nonself discrimination. Science 169: 1042-1049http://www.sciencemag.org/cgi/content/abstract/169/3950/1042?ijkey=6f8a6a0551fdb21d9b76190c36e697d126779171&keytype2=tf_ipsecsha7. Winoto, ALecture 13: T cell activation and signalinghttp://mcb.berkeley.edu/courses/mcb250/lecture13.pdf8. Kenneth D. Lute, Kenneth F. May, Jr, Ping Lu, Huiming Zhang, Ergun Kocak, Bedrick Mosinger, Christopher Wolford, Gary Phillips, Michael A. Caligiuri, Pan Zheng, and Yang LiuHuman CTLA4 knock-in mice unravel the quantitative link between tumorimmunity and autoimmunity induced by anti–CTLA-4 antibodiesBlood. 2005 November 1; 106(9): 3127–3133. Prepublished online 2005 July 21. doi:10.1182/blood-2005-06-2298http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1895337 32
  • 33. 9. Kenneth D. Lute, Kenneth F. May, Jr, Ping Lu, Huiming Zhang, Ergun Kocak, Bedrick Mosinger, Christopher Wolford, Gary Phillips, Michael A. Caligiuri, Pan Zheng, and Yang Liu10. Ribas, A. Hauschild, R. Kefford, C. J. Punt, J. B. Haanen, M. Marmol, C. Garbe, J. Gomez-Navarro, D. Pavlov, M. Marshall;Phase III, open-label, randomized, comparative study of tremelimumab (CP-675,206) and chemotherapy (temozolomide [TMZ] or dacarbazine [DTIC]) in patients with advanced melanoma; J Clin Oncol 26: 2008 (May 20 suppl; abstr LBA9011)11. H. F. Wang1, J. M. Lovering1, R. M. Shepard1, D. Zhang2, T. A. Smolarek1, J. W. Findlay3 1Pfizer Inc, 2FDA, 3Gilead Sciences Inc; Pharmacokinetics of Tremelimumab, a Cytotoxic T Lymphocyte-Associated Antigen 4 (Ctla4) Blocking Monoclonal Antibody, in Nonhuman Primateshttp://www.aapsj.org/abstracts/NBC_2008/NBC08-000658.PDF12. Holger N. Lode,1 Rong Xiang,1 Ursula Pertl,1 Elisabeth Förster,2 Stephen P. Schoenberger,3 Stephen D. Gillies,4 and Ralph A. Reisfeld1; 1The Scripps Research Institute, Department of Immunology, La Jolla, California, USA2University Children’s Hospital Vienna, Vienna, Austria3La Jolla Institute for Allergy and Immunology, Division of Immune Regulation, San Diego, California, USA4Lexigen Pharmaceuticals Corp., Lexington, Massachusetts, USA Melanoma immunotherapy by targeted IL-2 depends on CD4+ T-cell help mediated by CD40/CD40L interaction; J Clin Invest. 2000 June 1; 105(11): 1623–1630. doi: 10.1172/JCI9177 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=300854#B13#B1313. James P. Allison, Fred Lühder, Cynthia Chambers, Christophe Benoist, and Diane Mathis ;Pinpointing when the T-cell costimulatory receptor CTLA- 4 must be engaged to dampen diabetogenic T-cells. PNAS 2000 97:12204- 12209; published online before print October 17, 2000 http://www.pnas.org/content/97/22/12204.full14. Kajsa Wing,1* Yasushi Onishi,1,2 Paz Prieto-Martin,1 Tomoyuki Yamaguchi,1 Makoto Miyara,1 Zoltan Fehervari,1 Takashi Nomura,1 Shimon Sakaguchi1,3,4 1; CTLA-4 Control over Foxp3+ Regulatory T Cell Function; Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan. 2Department of Rheumatology and Haematology, Tohoku University Graduate School of Medicine, Sendai 980- 8574, Japan. 3Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. 4Laboratory of 33
  • 34. Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita 565-0871, Japan.15. Cedric Menard,1,2 Francois Ghiringhelli,1,4,5 Stephan Roux,1,2 Nathalie Chaput,1,2 ChristineMateus,3 Ursula Grohmann,6 Sophie Caillat-Zucman,7 Laurence Zitvogel,1,2 and Caroline Robert1,3; CTLA-4 Blockade Confers Lymphocyte Resistance to Regulatory T-Cells in Advanced Melanoma: Surrogate Marker of Efficacy of Tremelimumab? ;Clin Cancer Res 2008;14(16) August 15, 2008; Authors’Affiliations: 1Center of Clinical Investigations, CBT507, 2Institut National de la Sante et de la Recherche Medicale U805, and 3Department of Medicine, Dermatology Unit, Institut Gustave Roussy, Villejuif, France; 4Department of Medicine, Centre Georges Francois Leclerc, 5CRI Institut National de la Sante et de la Recherche Medicale 866, Faculte¤ de Me¤decine, Dijon, France; 6Department of Experimental Medicine, University of Perugia, Perugia, Italy; and 7Institut National de la Sante et de la Recherche Medicale U561, Hospital St. Vincent de Paul, Paris, France16. Afonso R. M. Almeida,2,3 Bruno Zaragoza, and Antonio A. Freitas3; Indexation as a Novel Mechanism of Lymphocyte Homeostasis: The Number of CD4_CD25_ Regulatory T Cells Is Indexed to the Number of IL-2-Producing Cells1; The Journal of Immunology, 2006, 177: 192–200 1 This work was supported by the Ligue Nationale Contre le Cancer, Association pour la Recherche Contre le Cancer, Agence Nationale de la Recherche Contre le SIDA, Association Franc¸aise des Myopathies, Centre National de la Recherche Scientifique, and the Institut Pasteur. A.R.M.A. was supported by the Fundac¸ao para a Ciencia e Tecnologia, Lisboa, Portugal. 2 Current address: Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH- 6500 Bellinzona, Switzerland. 3 Address correspondence and reprint requests to Dr. Antonio A. Freitas, Lymphocyte Population Biology Unit, Unite´ de Recherche Associae´, Centre National de la Recherche Scientifique 1961, Institut Pasteur, 28 Rue du Dr. Roux, 75015 Paris, France or Dr. Afonso R. M. Almeida at the current address: Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500, Bellinzona, Switzerland. E-mail addresses: afreitas@pasteur.fr and almeida@irb.unisi.ch17. Vincent A. Barvaux1, Paul Lorigan2, Malcolm Ranson2, Amanda M. Gillum3, R. Stanley McElhinney4, T. Brian H. McMurry4 andGeoffrey P. Margison1 1Paterson Institute for Cancer Research, Manchester, United Kingdom; 2Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom; 3Genta Inc., Berkeley Heights, New Jersey; and 4Trinity College, Dublin, Ireland Sensitization of a human ovarian cancer cell line to temozolomide by simultaneous attenuation of the Bcl-2 antiapoptotic protein and DNA 6 repair by O -alkylguanine-DNA alkyltransferase; Molecular Cancer Therapeutics October 1, 2004 3, 1215 http://mct.aacrjournals.org/content/3/10/1215.full 34
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  • 36. 18. Byung-Seok Kim, Young-Jun Park, Chang-Yuil Kang; Laboratory of Immunology; Institute of Pharmaceutical Sciences, Seoul National University,Seoul,Korea19. Knoechel B, Lohr J, Kahn E, Bluestone JA, Abbas AK; Sequential Development of Interluekin-2 dependent effector and regulatory T cells in response to endogenous systematic antigen;J Exp Med 202:1375-1386, 200520. Gajewski T F, Chesney J, Curiel T, Emerging Strategies in Regulatory T-cell Immunotherapies; Clinical Advances in Hematology Oncology Jan 200921. Bensigner S J, Walsh P T, Zhang J, Carroll M, Parsons R, Rathmell J, Thompson C B, Burchill M A, Farras M A,Turka L A; Distinct IL-2 Receptor Signaling Pattern in CD4+ CD25+ T Regulatory cells; Journal of Immunology,2004,172; 5287-529622. Janas M L, Groves P, Kienzle N, Kelso A; IL-2 Regulates Perfin and Granzyme Gene Expression in CD8+ T cells Independently of its Effects on Survival and Proliferation: Cooperative Research Center for Vaccine Technology and Queensland Institute of Medical Research, Brisbane, Australia23. Shrikant P, Mescher MF; Opposing Effects of IL-2 in Tumor Immunology: Promoting CD8+ T cell Growth and Inducing Apoptosis; Journal of Immunology 2002 169: 1753-1759 35