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

Melanoma and the “Magic Bullet” (Monoclonal Antibodies)

Author:Jim Breitfeller

This 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 German scientist in the fields of hematology, immunology, and chemotherapy, and Nobel laureate. 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". He is credited with the first observation of the blood-brain barrier and the development of the first antibacterial drug in modern medicine.1

The Magic Bullet concept was base on selective targeting a disease with a toxin/agent to kill off the disease without effecting the rest of the body. Using this concept in 1909, he and 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” This proposed theory explaining the immune response in living cells.

The concept of a "magic bullet" was fully realized with the invention of monoclonal antibodies.

Today, we have a better understanding of our immune system but are still pushing back the 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 basic knowledge on the subject matter. In the late 1960’s, a book, was published by F.M. Burnet, “Cellular Immunology and Self and Not-Self” It proposed that Immune system could detect and destroy tumors, a cancer immunosurveillance.The key to a healthy immune system is that the immune system is able to recognize the body (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 the adjacent tissues, Dendritic Cells capture self and non-self antigens via specific receptors.

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Anything that can trigger this immune response is called an antigen. Antigens can be microbe , apart of a microbe, even a cell or tissue from transplant victims. Sometimes our immune system mistakes itself as non-self causing a response as an autoimmune response. Some examples of Autoimmune Diseases are Rheumatoid Arthritis, Multiple Sclerosis, and Lupus etc.1

Signals 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 recognizes an 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 have come into contact with a presentable antigen, they become activated into mature dendritic cells 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 with antigens derived from the pathogen, alongside non-antigen specific costimulatory signals.1

T-helper cells (also known as effector T cells or Th cells) are a sub-group of lymphocytes (a type of white blood cell or leukocyte) that play an important role in establishing and maximizing the capabilities of the immune system.1

A cytotoxic T cell (also known as TC, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell) belongs to a sub-group of T lymphocytes (a type of white blood cell) that are capable of inducing the death of infected cells or tumor cells; they kill cells that are infected with viruses (or other pathogens), or are otherwise damaged or dysfunctional.1

B cells are lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells). The principal functions of B cells are to make antibodies against antigens, perform the role of Antigen Presenting Cells (APCs) and eventually develop into memory B cells after activation by antigen interaction. B cells are an essential component of the adaptive immune 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 granules rich in histamine and heparin. It is best known for their role in allergy and hypersensitivity allergic reactions. Mast cells play an important protective role as well, being intimately involved in wound healing and defense against pathogens and processes that eliminate or prevent pathogenic problems1

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Eosinophil granulocytes, usually called are eosinophils, are white blood cells that are one of the immune system components responsible for combating infection. Along with mast cells, eosinophils also control mechanisms associated with allergy and asthma. They are granulocytes that develop during Haematopoiesis in the bone marrow before migrating into blood.1

Hematopoiesis

Source: Wikipedia

All immune cells begin as immature stem cells in the bone marrow. They respond to different cytokines and other signals to grow into specific immune cell types, such as T cells, B cells.

Cytokines

Components of the immune system communicate with one another by exchanging chemical

messengers called cytokines. These proteins are secreted by cells and act on other cells to

coordinate an appropriate immune response. Cytokines include a diverse assortment of interleukins, interferons, and growth factors.1

Some 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.

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T-Cell final development occurs in the Thymus. The thymus is a multi-lobed organ that is composed of a cortical and medullary area. During the T-Cell development, the cell moves through the different lobes due to the different microenvironments. Most of the cells that enter the thymus never make out alive to become a mature Naïve T-cell. A naive T cell or Th0 cell is a T cell that has differentiated in bone marrow, and successfully undergone the positive and negative processes of central selection in the thymus. In the Negative process, the T cell goes through a rigorous selection to self antigen tolarence before it is released into circulation. Once in circulation, the cells are able to respond to novel pathogens that the immune system has not yet encountered.1

The NaiveCD4 + the helper T cell, when activated, the Helper T –cell secretes mostly IL-2 which promotes growth and proliferation, and activation of T-cells, helper T cells and Natural Killer Cells. Once the helper cells mulitply, they start secreting other cytokines base on their costimulatory signals., concentration of antigen, and exposure to their microenviroment. This attracts more immune cells and the assault on the foreign invader begins.1

Regulatory T-cells (Tregs) (suppressor T cells) are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Regulatory T cells play an important role in preventing autoimmunity by suppressing the response of other T-cells to self- and other antigens. Several types of Tregs have been identified, including both CD4+ and CD8+ expressing subsets. One of the best characterized subsets, natural Tregs express both CD4 and high levels of CD25. Anergic cells can act as regulatory T cells by competing at the sites of antigen presentation and adsorbing out stimulatory cytokines such as IL-2. (Lack of IL-2/costimulation)1

III.Immunomogy History:In 1980, Dr. Steven A. Roesnberg and colleagues discovered novel novel method for killing metastatic cancer cells. They took lymphoid cells and exposed them to interluekin-2 (IL-2).These cells were able to lyse the tumor cells and kill them. The were a 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 were not shown to be effective in a randomized clinical trial when compared to IL-2 alone2

The Cells that were cultured from tumor infiltrating lymphocytes (TIL), had a better response to the tumors. We now come to know this therapy as Adaptive Cell Transfer Therapy.

During those Trials, Rosenberg and colleagues saw a correlation between younger cultures and where the originals TILs were harvested. The younger, the better the response was with the patient. TILs that were harvested from the lymph node did not respond as much, indicating that specific location plays a role in the overall scheme of things. One must also note that Dr. Rosenberg’s Patients are a certain genotype (HLA-A2 positive).

In 1988, a research paper came out authored by Dr. Kyogo Itoh , Platsoucas,and Balch entitled: “Autologous Tumor Specific Cytotoxic Lymphocytes in the Infiltrate of Human Metastatic Melanomas”4 Activation by Interleukin 2 and Autologous Tumor Cells, and Involvement of the T Cell Receptor.

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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 TIL cell count increased to a maximum propagation in about 43 days. Tumors cells that were cultured with the TILs and the IL-2 were complete killed off. Lysing appeared five days into 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. These experiments and results lead to the Rosenberg’s trials.

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T Cells Mobilized Graphics

Adapted from Understanding the Immune System How It Works NIH Publication No. 03–5423

The T-cell is activated and secretes Cytokines

Antigen-Presenting cells

When a B cell or macrophage encounters an antigen

Cytokines trigger the Immune System to produce more T-cellsLike interluekin-2 (IL-2)

The T cells are Mobilized

Infected cells

vvv

vvv

Some T cells become Killer cells and track down infected body cells.

Some Cytokines attract immune cells & fresh macrophages, granulocytes and other lymphocytes to the site of the infection. And others direct and recruit once on the scene.

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In 1992, adoptive transfer of TILs in combination with IL-2 resulted in tumor regressions in 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 tumor escapes detection and destruction.

In a 1994 article entitled "Tolerance, Danger and the Extended Family", Dr. Polly Matzinger, layed out the idea that antigen-presenting cells (APC) respond to "danger signals" - most notably from cells undergoing injury. The immune system does not necessarily respond only to what is foreign but to anything that is dangerous. The danger model is based on the existence of the so-called second signal, in addition to the first signal directed at T-cells. The first signal comes from specific recognition of antigenic peptides 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 or tolerance.

Table 1.)

The main rules to generate an immune response or tolerant state

Cell Type Absence of Second Signal Second SignalNaïve T-cells undergo apoptosis only by Dendritic cells (DCs)

Memory T-cells undergo apoptosisall APCs, monocytes, macrophages, B-cells or DCs

B-cells undergo apoptosis only by memory/effector T-cells

Effector T and B-cells

perform functions after antigen recognition regardless

undergo apoptosis or revert to a resting state after a reasonably short period of time

Source: T-Cell Stimulation by Melanoma RNA-Pulsed Dendritic Cells3

The Danger Model is not universally accepted . Some immunologists, following Janeway's ideas (1989) believe that the immune response is based upon by evolutionarily forces of "pattern recognition receptors". So what happens if it is a combination of both of the therories?

By trying to manipulate the immune system into specifically recognizing and killing tumor cells, there is a thin line between breaking tolerance and inducing autoimmune disease can be easily crossed. This must be taken into account when protocols for Clinical Trials are formulated. Tumor cells have a number of other direct strategies for hiding from or fighting against an immune response. Most solid tumor cells are able to

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hide from T-cells because they grow out of reach of secondary lymphoid organs, so naive T-cells remain unaware of the tumor's existence. Tumors also secrete proteins/peptides that act as mediators between the tumor and the host (body) communicating in the tumor microenvironment. This somehow (may block the receptors or the signals) allowing the tumor to go undetected by the immune system.

IV. Lymphatic System of the Immune System

Source: http://cksinfo.com/medicine/anatomy/page2.html

The 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, of initiating adaptive immune responses. In addition to up-regulation of antigen-presenting and co-stimulatory molecules, maturation includes enhancing the

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ability of DCs to migrate out of the tissues and into secondary lymphoid organs, where interactions with T-cells take place.

IV. Immunosurveillance

Robert Schreiber’s group found evidence for immunosurveillance.

The “three Es hypothesis” comes from this idea, and has been proposed by Schreiber and colleagues in 2003.5 The idea is that immunosurveillance is one phase of a more comprehensive process immunoediting which can be broken up into three component phases: elimination, equilibrium and escape. In the elimination phase, immune cells recognize and eliminate the altered cells (Tumor Cells). Generally, this is sufficient. After, or simultaneous with, the elimination phase, is the equilibrium phase in which the tumor cells and the immune system exist in equilibrium of inaction: the tumor doesn’t grow and the immune system doesn’t attack it. This can continue for years, and the individual remains cancer free. Then, some tumors escape. This last phase, Escape Phase occurs when a tumor mutates sufficiently to evade elimination by the immune system and grows out. The interesting thing that Schreiber and colleagues propose is that the tumor by the selective process over time during the elimination and equilibrium phases, evolves until it can go undetected and escapes: that when immune system see the tumor cells, the immune system does not get a danger signal and the tumor cells proliferate. The result is cancer.

The Three Es of Cancer Immunoediting

Adapted from Dr. Schreiber’s Hypothesis

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Recent improvements in the Researcher’s understanding of the Immune System such as the role of costimulatory T-Cells and Antigen presenting cells has led to the renewal of the developmental efforts in Immunology of Melanoma. Monoclonal Antibodies (mAbs). The theory has been around about 100 years. According to Dr. Ehrlich’s theory, the surface of white blood cells is covered with many side chains, or receptors, that form chemical links with the antigens they encounter. After binding of the specific antigen the cell is stimulated to produce more of the suitable type of receptor, which would then be shed into the blood stream as antibodies. Antibodies, also called immunoglobulins (Ig) are proteins that are found in blood or other bodily fluids of humans, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.Antibodies are produced by a kind of white blood cell called a B cell. 1

Source:http://users.path.ox.ac.uk/~scobbold/tig/ne w1/erlfg8.gif

The antibody that we are interested in is the Cytotoxic T lymphocyte-associated antigen.(CTLA-4). This antigen can inhibit T-cell responses and is involved in tolerance against self antigens. It was reported back in 1970 Bretscher and Cohn put forth the two-signal model of lymphocyte activation to explain self/nonself discrimination 6This model proposes that T-cell activation requires two independent signals. As the antigen interacts with the antibody receptor on the antigen-sensitive cell also known as the antigen presenting cell (APC) (Signal 1), it performs a conformational change which in turns paralyzes the whole cell. A new signal is now invoked (inductive signal) base on the new carrier of the antigen and antibody combined. (Signal 2) The T-cell activation not only requires the T–cell interacting with the antigen-MHC complex, but also the interaction of the CD28 receptor and the B7 as well. Once activated, the CD28 signaling leads to Interluekin-2 (IL-2) gene expression which helps promotes the immune system to propagate more T-cells.

The CD28 signaling activates the PI3 kinase and AKT pathway but they believe that there are no signal proteins involved according to Thompson and colleagues. They suggest that gycoloysis occurs along with energy metabolism causing the growth of the T-Cell.7

That 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-2.8

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Once the CD8+ T-cell is activated correctly, it can deliver a hit to the tumor cells if it can make its way passed the microenvironment of the tumor or tumors.

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 is an immune response that does not involve antibodies,but rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes (CTL), and the release of various cytokines in response to an antigen. The immune system historically, was separated into two branches: humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens.1

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Cytotoxic T lymphocytes (CTLs) appear to play key roles in the immunological destruction of many cancer cells. However T-helper cells are needed for the activation of tumor-destructive macrophages, NK cells and lymphokine-activated killer (LAK) cells.

Source: Immunologic Pathways

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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 phagocytes and microbes that infect non-phagocytic cells. It is most effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, and intracellular bacteria. It also plays a major role in transplant rejection.1”

Tumor Rejection

To cause the rejection of the tumors cells, The immune system must orchestrate a chain of events mediated by several types of Leukocytes including Dendtric cells (DC), Natuaral Killer cells (NK), CD4+ and CD8+ lymphocytes and others. This orchestration has many players in it including the T-Regs, sercreted cytokines, and Monoclonal antibodies (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-cell receptors (TCRs) are able to scan the Dendtric cells (DC’s) for antigen-MHC molecules. (ag-MHC) major histocompatability complex (class I or II). Based on the 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 only happens, it can lead to tolerance by means of fuctional paralysis of the (APCs) Antigen presenting cells (Anergy) or by the induction of clonal deletion (apoptosis). Anergic cells can act as regulatory T cells by competing at the sites of antigen presentation and adsorbing 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 and usually sends out a “danger signal” inflammation. The activated CD4+ T-cells can secrete many different cytokines including IL-4, IL-2 and activate the TH2 cells which are a subset of the CD4+ cells. The TH2 cells stimulate the B cells to mature into plasma cells that secrete antibodies. These antibodies that are produced are the cell-destructive kinds

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that have anti-tumor behavior. The CD4+ can also cross-prime CD8+ T-cells in the presence of IL-2 and are called (CTLs) Cytotoxic T Lymphocytes. Cross-priming is another name for cross-presentation. The role of the CD8+ T cells is to monitor all the cells of the body, ready to destroy any that express foreign antigen fragments in their class I molecules. Three major events must occur to Activate CD8+ T cell mediated response against melanoma. First, the T-cell receptor (TRC) must be triggered by a (or multiple) self antigen–derived peptide MHC class I complex12. This event depends entirely on appropriate antigen presentation, which is most efficiently provided by mature dendritic cells . Once properly activated, may serve as tumor-specific effector T cells .Second, simultaneously with T-cell receptor triggering, a distinct second costimulatory signal must be delivered, mediated by IL-2, B7-1, or B7-2, which engage IL-2 receptors and CD28 on the surface of the T cell, respectively . A source of these cofactors for effective CD8+ T-cell stimulation can be provided by CD4+ T cells that release critical amounts of IL-2, or by mature dendritic cells that display an increased level of B7-1/B7-2 costimulatory molecules on their cell surfaces. Third, inflammatory cytokines, including IL-1, IL-6, IL-12, and IFN-γ provide a third signal that acts directly on T cells, referred to as the “danger signal”. This signal was found to optimally activate TH1 differentiation and lead to clonal expansion of T cells

Some 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. Virtually every cell in the body expresses class I MHC molecules, so CD8+ CTLs are not limited in the targets they can attack. Cytotoxic T lymphocytes (CTLs) appear to play key roles in the immunological destruction of many cancer cells. However T-helper cells are needed for the activation of tumor-destructive macrophages, NK cells and lymphokine-activated killer (LAK) cells. If lymphocytes are cultured in the presence of Interleukin 2, (IL-2) it results in the development of effector cells which are cytotoxic to tumor cells.12

CTLs 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 or more perforin molecules insert themselves into the plasma membrane of target cells forming 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 is similar to what is in your laundry detergent. They are known as detergent enzymes. They break the bond between the dirt and the fabric. By breaking up these proteins, they start destroying the intracellular workings of the tumor cells.

CTLs Binds to the tumor cells and discharges granules that contain the proteins perforin and granzymes.

Tumor Evading Detection

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Lack of costimulation

Many 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 immune response but they do not stimulate an effective anti-tumor immune response. The first signal originates from the binding of the T cell receptor (TCR) to its antigen-MHC, and

provides the specificity of the interaction. Without this signal, the cell enters anergic state and can act as a T reg cell. Expression of B7 on the surface of a cell is the costimulatory signal necessary to allow for the cytolytic CD8+ T cell attack on the tumor. B7 display renders tumor cells capable of effective antigen presentation, leading to their eventual eradication.

Secretion of immunosuppressive cytokines

Another way tumors evade detection is by secretion of certain cytokines. They are low-molecular weight proteins that use their communication ability to regulate the immune response. Cytokines can act upon either the cells secreting them (autocrine) or on neighboring cells (paracrine) to generate activities in the targeted cells. This means they can act as light switches for on and off immune responses. For example, interleukin-2 activates a cell-mediated immune response, while interleukin-10 suppresses cell-mediated responses. Many types of cancer, including Melanoma, take advantage of this

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ability to down regulate this appropriate immune response to help extend their survival and proliferation. This causes cancer patients to fail in mounting a successful attack on the tumors. Immunosuppressive cytokines secreted by cancer cells include transforming growth factor-beta (TGF-beta), interleukin-10 (IL-10) and vascular endothelial growth 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 participating in both the innate and acquired immune response.TGF-beta inhibits the profilation T-cells, B cells, Natural killer cells (NK), and macrophages.TGF-beta also converts T-cells, which normally attack cancer with an inflammatory (immune) reaction, into regulatory (suppressor) T-cells, which turn off the inflammatory reaction. Another of TGF-beta's 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 of the processes necessary for CTL activation. Without this activation, there is no assault on the tumor cells from the CTLs. In addition to suppressing proliferation, TGF-beta has been shown to induce apoptosis (cell death) in B and T cells.

Another immunosuppressive cytokine is IL-10. It is capable of inhibiting the prodction of of pro-inflammatory cytokines like IFN-gamma, IL-2, and GM-CSF made by cells such as macrophages and T helper cells. IL-10 also displays potent abilities to suppress the antigen presentation capacity of antigen presenting cells. Secretion of IL-10 in the vicinity of a tumor can render the tumor totally insensitive to CTL-mediated lysis. It is most likely that the tumor’s microenvironment is altered enough to block or turn off the discharge granules that would lyses the tumor cell. However, it is also stimulatory towards 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 to the immune system.

Vascular endothelial growth factor (VEGF) is a cytokine that is produced by most tumors. This growth factor enables the tumor to expand vascularly when is in its growth phase. VEGF production can be induced in tumor cells that are not receiving enough oxygen.

Regulatory T-cells (Tregs) (suppressor T cells) are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens.1

Tumor Growth kinetics

The 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

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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 this cycle become disabled due to mutations characteristic of cancerous cells, the cell cycle is no longer under tight regulation. Tumor cells are capable of proliferating so quickly that the immune response is not fast enough to keep their growth in check. The growth of the tumor cells outpaces the immune response and escape the detection of the immune system. 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 Metastatic Melanoma” was started with Dr. Rosenberg at the forefront. They were hoping that they would get synergetic outcome based on the two single therapies.

Method: Thirty-six patients received anti-CTLA-4 antibodies every three weeks. The doses 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 received IL-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 the CLTA-4 Blockage plus the IL-2 addition because alone as a single agent; the 22 percent objective response rate was as expected. The one thing that came out of the trial was 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 most efficient anti-CTLA-4 antibody will need to be used in combination with other reagents in 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 response then the FDA approved Dacarbazine.9 As a single agent, this was quite disappointing to the researchers and the melanoma patients alike. One good thing that came out of the trials was that the complete responders had a more durable response.

The Orchestration of an Immune Response Unrehearsed

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In 2006, after two fail attempts (Interferon and Dacarbazine with Patrin) to stop the progression of my melanoma, I was able try CTLA-4 Blockage. It was one of my first choices, but due to protocol, I had to try the FDA approved therapy first. I had researched this monoclonal antibody. On 10-24-2005 when I was first diagnosed with melanoma, I contacted Dr. Luis H. Camacho who was currently at MD Anderson.

Subject: Paper on Antitumor Activity

“Luis Camacho, My name is Jim Breitfeller and I have recently been diagnosed with melanoma will need some sort of Ontological therapy after my surgery. I ran across an abstract of yours (Antitumor activity in Melanoma and anti-self responses in Phase 1 trials with the anti-Cyctotoxic T Lymphocyte-Associated Antigen 4 Monoclonal Antibody CP-675,206) in the Journal of Clinical Oncology. Is it possible to get a copy of your 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 under development 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 of patients attaining long term remissions. However, none of the programs are currently oriented to patients rendered NED (Stage III or IV). They are in fact for patients with advanced disease.  From your brief introduction, I think your best options are to obtain an HLA typification and go for an adjuvant trial.

Please feel free to page me if you need further information. Pager is 713.404-5319 Best,

Luis

CP-675,206, a novel monoclonal antibody, enlists the immune system to fight advanced melanoma

Some Positive Test results of the CTLA-4

Early testing of an experimental human monoclonal antibody showed a striking benefit in patients with advanced melanoma, say researchers at The University of Texas M. D. Anderson Cancer Center, who presented their findings at the annual meeting of the American 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 fourth patient, and cancer stopped growing in five other patients. These responses have remained 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 of less than a year, says the study's principal investigator, Luis Camacho, M.D., MPH, assistant professor in the Department of Melanoma Medical Oncology.

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"We were very pleasantly surprised to find such objective antitumor responses in a Phase I clinical trial, which is designed to find the ideal dose and to look for side effects," says Camacho. "These results are very early, but they are encouraging to us because there are no good agents available to treat melanoma once it has spread."

Source: Laura Sussman from (ASCO) American Society of Clinical Oncology

At the time of the request, I was not at the correct stage but I knew that this might be the path of the future. I did contact him and we discussed my options at that time. I was just learning the ropes.

On 9/3/06 I contacted Dr. Rosenberg just in case I needed a back up plan if the CTLA-4 blockage did not work. At that time I did not know I was the wrong HLA-02 type for Rosenberg’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.shtml

The research team recently applied to the Food and Drug Administration (FDA) to try the new 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 results look somewhat promising. I applauded you and your team for making great strides in the cure for melanoma cancer.

I am a cancer patient (48 yrs. old) under the care of Dr. John Kirkwood at the Hillman Cancer Center at the University of Pittsburgh. I have gone through a wide incision, lymph nodes removal, Interferon therapy, and Dacarbazine therapy without success. I am presently on track to start a clinical trial with CTLA-4 monoclonal antibodies September 13, 2006. I have some tumors on my right side of my back and some in each lobe of my lungs. I would like to be considered for your next round of Gene Therapy in the coming months if I have no response to the CTLA-4 treatment. Please let me know if you would need 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 near future.

Best Regards,Jim Breitfeller

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On 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 few questions about my health. She went on to say if I go with the CTLA-4 therapy, it would take about 2 months to washout before I could try the Gene Therapy. They would also have to do a colon biopsy to check the colon for any adverse conditions from the CTLA_4. She then gave me her direct phone number if I want to pursue the gene therapy at a later date.

So, on 9/13/06 (day 1)I had my first and only infusion of anti-CTLA-4 monoclonal antibodies. 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 cell proliferation. 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, to improve 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 the antibody is around 3 weeks.11 This means your body will eliminate half the dose that was infused in you in about 21days. So, in 42 days or there about, the drug concentration in my 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 my rack (Miss Daisy). I call the rack Miss Daisy because I have to take it with me where ever I go which includes the bathroom. I am driving Miss Daisy!! This will take us to 3:15 pm and then they draw blood for a pk study an hour later. So, we won’t get out until about 4:30 pm and home until 10:00 pm.

Day 7 -9/19/06 “Along with the fatigue, my muscles ache like they have lactic acid in them”. Is this an indication of something? All immune cells begin as immature stem cells in the bone marrow.

Day 15 -9/27/06 about half the CTLA-4 antibodies are depleted. It appears that the CTLA-4 has stimulated my immune system. In the pass week, I noticed that there was redness around the area where my tumors are located. Also it is becoming quite tender in that area. This is Great news!!!!! It appears that the treatment my have kick started my immune system. The only way we will know for sure is another CT scan. That is not scheduled until November 23rd.I sure hope this isn’t a false positive. Anyway, they gave me an antibiotic just in case it is an infection.This inflammatory response provides a third signal that acts directly on T cells, referred to as the “danger signal”. “This signal was found to optimally activate TH1 differentiation and lead to clonal expansion of T cells12.

With this clonal expansion of the T cells and the secretion of IL-2, The Immune system is gearing up to make an assault on the foreign invaders, the tumors.

In 1988, a paper was published Autologous Tumor Specific Cytotoxic Lymphocytes in the Infiltrate of Human Metastatic Melanomas Activation by Interleukin 2 and Autologous Tumor Cells, and Involvement of the T Cell Receptor by Itoh and

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Colleagues.4 In their studies, they propagated (TILs) Tumor infiltrate lymphocytes cells from 12 Metastatic Melanoma patients. They preformed kinetic growth studies in IL-2 and even broke it down three Surface markers (CD3,CD4 and CD8). The results are as follows: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)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 reach maximum 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 is 2 new tumors growing. This really stinks. I think it is time to take out the “Weed be Gone”. This is not what I was hoping to hear. It was decided that the CTLA-4 blockage therapy was to be terminated. My CD4+T cells were just about at maximum propagation.

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 of PROLEUKIN® IL-2 therapy.

In 6% of the patients, the tumors disappeared completely.Results From these trials, it was determined that a patient whose tumors completely disappeared 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 be accepted into the next trial. We know from the PK studies that it would take approximately 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 is spreading 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 of breath. 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 is also the average maximum propagation time of the cultured T-cells.

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Day 50- 11/1/06, the first cycle of High dose Interleukin-2 (IL-2). It just so happen to be the maximum propagation of the CD8+ T cells. All of the anti-CTLA-4 is washed out. We also, most likely have the most CD4+ T reg cells. These are the cells that help regulate the immune response so it doesn’t go into overdrive and cause an autoimmune response.

If we reset the clock for the second therapy (LI-2), then we can follow the activation of the CD8+ T-cells. My body has become a big Erlenmeyer flask. Erlenmeyer flasks are used in microbiology for the preparation of microbial cultures.

So on day 50- 11/1/06, we innocuated my body with IL-2 – a growth factor. So based on the Itoh study, I should be activating the CD8+ T-cells into a mature state (TILs and LAK cells.) It should take roughly 50 days they would be at there maximum growth phase.

In Itoh’s study the cultures were supplemented every 5 days by replacing half the cultured medium with fresh medium containing (IL-2) as one of the supplements. My IL-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 a week due to the Thanksgiving Holiday. I completed 8 doses which is the average that patients 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!!!!!! The tumors were shrinking!!!!!!!

Melissa’s Note:

I'm Christmas shopping.....but Heather called me with the results....I AM SOOOOO HAPPPY FOR YOU!!!!!!!!!!!!! YIPPPPPEEEEEE!!!!!!! Hope you have a wonderful holiday, and I'll see you soon :) :) :) :) Melissa

As you can see, the timing and the players of this Orchestration all fell into place. A single Bullet of Monoclonal antibodies started a chain reaction with a whole sequence of events which lead to the restarting of my immune system. Without that bullet, there would have been no "Danger Signal"

"Melanoma and the Magic Bullet (Monoclonal Antibodies)"

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So now we know what had transpired with the therapy, we need to know how and why it happened. 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 Cells

We will focus our attention on the Dendritic cells (DCs ) because I postulate that these cells played a major roll in help generating an immune response. Induced Dendritic cells go through a developental program call maturation, which transforms them into efficient antigen-presenting cells (APCs) and T-cell activators. They are the most potent of the three APCs. So what really happened? Well, Dr. Kirkwood started me out on Dacarbazine with PaTrin-2. Dacarbazine is a chemotherapy agent, approved by the FDA for fighting Melanoma. Dacarbazine alkylates and cross-links DNA during the phases of the cell cycle, resulting in disruption of DNA function, causing cell cycle arrest, and apoptosis.17 The 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 and reverse the modifications caused by alkylating agents. One such enzyme is methylguanine methyltransferase (MGMT). MGMT directly reverses the chemical

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modification guanine, one of the four building blocks of DNA, allowing normal replication to take place.

The DNA-repair enzyme MGMT is a key factor in resistance to alkylating agents. This is 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 to the trial. This drug is known to inactivate the MGMT activity. By inactivating the MGMT enzyme, it makes the tumors cells more susceptible to the chemotherapy.

This therapy was able to get the tumors cells to shed some antigenic Protein which I theorize 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 New England Journal of Medicine in June 19, 2008, there are three ways for self antigens to become Tumor Antigens:

1. Mutation2. over expression3. Post-translational Modification

I postulate that some failure of the tumor cells to repair the DNA damage cause by the Dacarbazine in the present of PaTrin-2 resulted in a mutation causing the cancer cells to shed an antigenic peptide. But I was still missing a signal or signals to activate my immune system.

The second step was the inoculation of the (CTLA-4 Monoclonal antibody) mAb. The dose was given at 15 mg/Kg. This was the highest dose given based on the pharmokenetics We know from the theory, we need to engage the B7 receptor. The research states that the anti-CTLA-4 has a higher binding affinity for the B7 receptor. So it will react first.

Pinpointing when T cell costimulatory receptor CTLA-4 is engaged is important because without 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 the mouse model, the anti-CTLA-4 blockage caused an autoimmune response which was diabetes in the mice.13 Before I go any further, I must make a note of caution. That is not all immune responses in mice models crossover to the human model, but the models are usually a good predictor. So with that said, In the research paper “Pinpointing when the T-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 an inflammatory 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 able to recognize self-targets and initiate inflammatory response in a similar way as with pathogens. Accordingly, alterations in cell morphology are recognized by the innate immune system resulting in an acute inflammatory response (Carroll and Holers,

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2005).

Well in my therapy, an inflammatory response was noted on day 15. This suggests that the 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-protective immunity 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 event depends entirely on appropriate antigen presentation, which is most efficiently provided by 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 signal must be delivered, mediated by IL-2, B7-1, or B7-2, which engage IL-2 receptors and CD28 on the surface of the T cell, respectively (17). A source of these cofactors for effective CD8+ T-cell stimulation can be provided by CD4+ T cells that release critical amounts of IL-2, or by mature dendritic cells that display an increased level of B7-1/B7-2 costimulatory molecules on their cell surfaces. Third, inflammatory cytokines, including IL-1, IL-6, IL-12, and IFN-γ provide a third signal that acts directly on T cells, referred to as the “danger signal”. This signal was found to optimally activate TH1 differentiation and lead to clonal expansion of T cells12.

Once the CD4+ T-cells are activated by the blockage of the CTLA-4 receptor, They start secreting 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 of Cytokine (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 the T-Regulatory foxp3 (CD4+ CD25+ FoxP3). It is postulated that the CTLA-4 that is expressed by the Treg is critical for the suppression of the immune responses by affecting the potency of the antigen-presenting cells to activate other T-cells.14 These Tregs even though they are small in number (Only about 5% of the total T-cells) can suppress the immune 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). The Tregs are most likely have multiple suppressive mechanisms and each one activated based on the microenvironment and the context of the immune response. One of those mechanisms by which the treg could mediate suppression is by secretion of a suppressive cytokine like IL-10. Thus, The CTLA-4 is an important key molecular target for controlling the Treg-suppressive function.

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So base on our knowledge now, we have three ways to tip the balance towards an immune 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 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.15

3. 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.

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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-2.16

This 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 would be used for proliferation of the T-regs to maintain that ratio for homeostasis. By depleting or blocking the functionality of the T-regs, one can push the equilibrium of the immune system in favor of an immune response.

We don’t want to eliminate the CD4+ T-cells altogether, because they are essential for the 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 a class II histocompatibility molecule. CD4+ T cells are essential for both the cell-mediated and antibody-mediated branches of the immune system:

cell-mediated immunity

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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 and recent reports, IL-2 is not needed for developmental CD4+ CD25+ Treg cells in the thymus but does play an important role in the maintenance and function in the peripheral.18 Peripheral is defines as secondary system outside the bone marrow and thymus. It entails the site of antigen, immune system interaction. IL-2 is required for the peripheral generation of Tregs based Abbas’s and colleagues research.19 IL-2 prevents the spontaneous apoptosis of the CD4+ CD25+ Treg cells. It has been reported that patients with multiple advance-stage tumors have elevated levels of Tregs within the tumor microenviroment.20 Interluekin-2 is the survival factor for CD4+ CD25+ Treg cells.21 If the addition of IL-2 is on or before the maximum propagation of the CD4+ T cells, the Tregs population can increase 5-fold in a 96 hour period based on certain growth mediums. By controlling the addition of the endogenous IL-2, one has a knob to turn and can lead to the control of the expansion of the Tregs. When you combined this control with the anti-CTLA-4 blockage, you can shift the balance of the immune response.

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Now here is the catch. The maintenance and function of the CD8+ T-cells require CD4+ cells which secrete IL-2. So we don’t want to deplete the CD4+ cells, we want to control the expansion of the Tregs which are a subset of the CD4+ cells. It has been postulated by some researchers that the Anti-CTLA-4 blockage also suppresses the Treg function in a different mechanism. By using IL-2 as the rate limiting factor, we can suppress the CD4+ CD25+ Treg cell expansion by controlling the concentration and timing of the Inerluekin-2 at the tumor microenvironment.

The Interluekin-2 plays another role in this Melanoma Maze. In a study by Janas et al, Il-2 increases 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,

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CTLs have cytoplasmic granules that contain the proteins perforin and granzymes. A dozen or more perforin molecules insert themselves into the plasma membrane of target cells forming a pore that enables granzymes to enter the cell. Once in the tumor cell, these enzymes are able to breakup (lyse) the cell and destroy it. This is the beginning of the end for the cancer cells. The tumors begin to shrink and the rest is history, “An Inmmune Response Unrehearsed.”

On the other hand, prolong therapy with Il-2 can result in causing apoptotic death of the tumor- specific CD8+ T-cells.23

Clearly in a clinical setting, timing, dose, and exposure to these drugs play a major roll in the immunotherapy, and can have dramatic effects on the outcome. All it takes is that one magic bullet to start the immune reaction.

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The Orchestration of an Immune Response Unrehearsed

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Acknoledgements:

I like to thank my wife Dee for putting up with me during the research and writing of this paper. I would also like to recognize Leanne Schmall, who is with us today in spirit. It was her journey that inspired me on writing this paper.We will miss her very much and she will not be forgotten. The Melanoma Warroir .

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References

1. Definitions source: Wikipedia

2. 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 2004

4. Itoh, K; Platsoucas, CD; Balch, CMAutologous Tumor Specific Cytotoxic Lymphocytes in the Infiltrate of Human Metastatic Melanomas Activation by Interleukin 2 and Autologous Tumor Cells, and Involvement of the T Cell Receptor [published J . Exp. MED. The Rockefeller University Press. 1988 Oct 1; Vol 168 October 1988 1419-1441http://jem.rupress.org/cgi/reprint/168/4/1419.pdf

5. Gavin P. Dunn,1 Lloyd J. Old, and Robert D. Schreiber1 The Three Es of Cancer Immunoediting1Department of Pathology and Immunology, Center for Immunology, Washington University 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: 1042-1049

http://www.sciencemag.org/cgi/content/abstract/169/3950/1042?ijkey=6f8a6a0551fdb21d9b76190c36e697d126779171&keytype2=tf_ipsecsha

7. Winoto, ALecture 13: T cell activation and signalinghttp://mcb.berkeley.edu/courses/mcb250/lecture13.pdf

8. 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 Liu

Human CTLA4 knock-in mice unravel the quantitative link between tumor immunity 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

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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 Liu

10. 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 Primates

http://www.aapsj.org/abstracts/NBC_2008/NBC08-000658.PDF

12. 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#B13

13. 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.full

14. 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

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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, France

16. 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: [email protected] and [email protected]

17. Vincent A. Barvaux 1 , Paul Lorigan 2 , Malcolm Ranson 2 , Amanda M. Gillum 3 , R. Stanley McElhinney 4 , T. Brian H. McMurry 4 andGeoffrey P. Margison 1 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

repair by O6-alkylguanine-DNA alkyltransferase; Molecular Cancer

Therapeutics October 1, 2004 3, 1215 http://mct.aacrjournals.org/content/3/10/1215.full

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18. Byung-Seok Kim, Young-Jun Park, Chang-Yuil Kang;Laboratory of Immunology; Institute of Pharmaceutical Sciences, Seoul National University,Seoul,Korea

19. 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, 2005

20. Gajewski T F, Chesney J, Curiel T,Emerging Strategies in Regulatory T-cell Immunotherapies;Clinical Advances in Hematology Oncology Jan 2009

21. 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; 5287-5296

22. 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, Australia

23. Shrikant P, Mescher MF;Opposing Effects of IL-2 in Tumor Immunology: Promoting CD8+ T cellGrowth and Inducing Apoptosis;Journal of Immunology 2002 169: 1753-1759

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