6.10 Immunotherapy

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Another strategy that can be used to treat malignant tumors is immunotherapy. Immunotherapy is a treatment which takes advantage of an already existing and functional and specific immune system and enhances its response tumors (1). Our immune system normally identifies specific molecular markers known as antigens on foreign infectious agents as well as on one’s own cells that have been infected by foreign agents, and seeks to neutralize and destroy these infectious particles (2). There are two common types of specific, adaptive immune responses: 1) humoral immune response and 2) cellular response outlined below. 


The importance of immunotherapy in personalized cancer medicine can be attributed to the specififcity of the immune system itself. Thereotically, enhancing the immune response to a particular tumor, would allow us to give general drugs to the the immune system, which is similar to all beings, such that it elicits a specific immune response which will vary in every different cancer case; as all cancers pocess a heterogeneity property that distinguishes each from another. In this sense, given that science is one day able to reach this point, it will essentially allow the immune system to do 'the work for you' by creating a personalized anti tumour response with a general immune enhancement drug. In this section we discuss the properties of the immune system and the means by which we can manipulate it to fight cancer for us. 



Humoral Immune Response


The humoral immune response, which generates extracellular substances such as antibodies, can be used to neutralize or opsonize pathogens through binding to surface antigens (2). Antibodies are produced by B cells upon recognition of particular antigens through interactions with their B cell receptor. Once B cells recognize foreign antigens, they are activated by T helper cells,  and can differentiate into two types of cells: plasma cells or memory cells (2). Plasma cells actively secrete antibodies throughout the body, in order to neutralize pathogens. Memory B cells on the other hand, retain their specificity for the same foreign antigen, but don’t secrete antibodies; instead they are activated upon subsequent exposures to the foreign antigen (ie. a second infection) and stimulate a quicker humoral response (2).



Cellular Response


The second type of adaptive immune response is the cellular response, which works to deal with infectious agents (ie. viruses) that have entered into cells. Antigen presenting cells present antigens derived from these foreign agents on cell surface molecules known as the major histocompatibility complex (MHC) (2). These antigens are presented to T cells through interaction with their T cell receptors. There are three main types of T cells. Cytotoxic T cells (CTLs) recognize antigens presented on MHC class I, which are ubiquitously expressed on cells throughout the body, and function to kill the cell via action of perforin and granzymes (2). T helper cells recognize antigens presented on MHC class II, which are only expressed on antigen presenting cells, and function to activate the humoral response by stimulating antibody producing B cells, or they can aid in activating cytotoxic T cells. Treg cells meanwhile suppress the actions of CTLs in order to prevent an autoimmune response. However, the actions of Treg cells can promote survival of cancer cells by suppressing CTLs that are normally able to destroy them (3).



Immune surveillance represents another hurdle to tumor growth in humans


Evidence has been shown that our immune system has a critical role in combatting cancer (3). The growth of tumors can be suppressed by activating anti-tumor adaptive responses (3). This can be accomplished through the activity of anti-tumor cytotoxic T cells, which can induce FAS, perforin, or cytokine pathways to leading to destruction of tumor cells (3). Additionally, the anti-tumor response can be dependent on antibodies through cell-mediated toxicity, as well as by lysis induced by activation of the complement pathway (3). The complement pathway is activated by foreign antigens or immune complexes, which can be found in malignant tumors, and contains several components which form a membrane attack complex to lyse cells (3). Many tumors arise during organ transplantation processes, as a result of viral infections (2). Yet, even in populations with a high rate of viral infections, the proportion of viral-induced malignancies is low, as seen in the case of Ebola virus infections (2). One mechanism suggests that this is because our immune system is primed to protect us against viral infections, whether they induce cancer or not (2).  Another mechanism suggests that the immune system normally identifies and eliminates virus-transformed cells in those who are not immunocompromised (2). Also, it has been found that human tumors contain a significant amount of lymphocytes. An explanation for this observation is that our immune system deploys these lymphocytes for the purpose of eliminating cancerous cells (2).


However, our immune system is sometimes inadequate to deal with cancer cells, for instance if one is immunocompromised. This is where immunotherapy comes into play. Immunotherapy against cancer makes use of both types of specific, adaptive immunological defense strategies described above.



Humoral Immunotherapy


Anti-tumor activity can be generated using humoral responses either by providing in vitro-produced antibodies or by augmenting in vivo B cell responses (1). The first method is achieved through the passive transfer of antibodies. Mouse monoclonal antibodies (mAbs) have been experimented in humans to target the cell surface receptor IL-2, which is expressed by many T cell leukemia and lymphoma cells (1). Other cancer cell targets include CD20, which is targeted by the mAb rituximab, and when combined with chemotherapy, causes a decrease in the signs and symptoms of B cell lymphomas (1). mAbs can also be used to block cell growth and survival by blocking survival and growth signals, an example of which is the blocking of the epidermal growth factor receptor in epidermal tumors (1). These antibodies can also induce signals leading to apoptosis through binding certain receptors (1). This method can also include transfer of antibodies that have radionuclides attached that can bind tumor cells and cause DNA damage leading to cell death, or they can stimulate various receptors on effector cells such as CD3 on cytotoxic T cells (1).  A problem with passive antibody transfer, which needs to be addressed, is the highly toxic effect on regular cells and tissues.


The second method involves enhancing in vivo B cell responses. This can be readily accomplished by immunizing patients with immunogenic adjuvants or conjugated antigens, which can stimulate T helper cell responses (1). The antibodies produced from priming by these types of antigens have been shown to elicit anti-tumor response as well as longer periods of decreased signs and symptoms of cancer cells, such as those part of B cell lymphoma (1). Theoretical strategies have also been suggested for enhancing normal B cell responses. These include ligating B cell stimulatory molecules such as CD40 or introducing the cytokine IL-4 to induce B cell proliferation (1). However, these strategies have the potential of inducing autoimmune antibodies, which react against self, creating a very dangerous situation (1). Thus, humoral immunotheraphy will predominantly continue to consist of passive transfer of antibodies.  



Cellular Immunotherapy


Cellular immunity involves the immune cells actively assaulting the tumor cells that can be detected. The ideal cellular response required to eliminate a tumor mass and prevent relapse is the active killing of tumor cells by Cytotoxic T lymphocytes (CTLs) along with active survielance by memory CTLs in case of ressurgence of quiescent cancer cells. This response is multi-partite and involves CTLs that physically migrate to the tumor site to conduct offensive operations resulting in tumor death, and supporting cells such as Th1 polarized T helper cells that do not kill directly but are nonetheless essential. The origins of the response must also be detected and relayed by bone-marrow derived antigen presenting cells (APCs) such as dendritic cells, to seed a cellular response. However, neomorphic mutations and/or abberant epigenetic regulation arising from genomic instability in the tumor cells have been observed to interfere or block nearly every mechanisim and step involved in generating a cellular response. Therapies developed also enhance one or more of these same mechanisims to offset tumor interference and kill the tumor; we will talk about 2 main approaches used and mention a few other treatments in active research (1,4,5,6). 


Adoptive T cell Therapy has been the most direct approach aimed at achieving the holy grail of anti-cancer treatment, active and memory CTLs. As the name implies, treatment requires isolation of T cells from the patient, treating them in a way then re-introducing them into the patient to combat tumor growth (1,4). Perhaps one of the greatest discoveries in the field of cancer immunology is that tumor cells are infact riddled with immune cells. Immune cells of all types are enriched in tumors, of which the most interesting are the Tumor-infiltrating Lymphocytes (TILs). TILs can be extracted from a tumor biopsy, grown in cultures containing T cell growth stimuli such as anti CD3 antibody and Il-2, and re-introduced into the host (1). The advantages of such a therapy are elimination of possible graft versus host disease observed in allogenic T cell adoption, and fairly low toxicity. Adverse reactions include the accidental activation of auto-reactive T cells that when inserted back into the host, cause adverse autoimmune reactions.



Adoptive Dendritic cell Therapy aims at creating a favorable environment for T cell activation. Host DCs are purified from peripheral blood and grown in vitro. The cells are then incubated with lysed tumor cells removed in a biopsy (4, 6). This allows the DCs to pick up and present on MHCI (via cross presentation) and MHCII the tumor antigen. Alternatively, DCs can be transduced with tumor antigen genes and thus cause expression of the endogenous tumor antigen on MHCI (4). The DCs are then reinserted into the host where they can activate tumor specific T cells and allow the formation of a cellular response. This method allows for reduction in potential adverse autoimmune reactions, but is more indirect than direct activation of T cells and may be less efficacious. Other concerns include the fear that the DCs are being driven into a state that is too mature, and is only reached upon arrival of the DC in the lymph node. These overly-mature DCs are less effective in migrating and may die off at the site of injection (4). 


The final class of cellular immunity therapies consist of molecular treatments aimed at generating a cellular response without directly handling ex vivo cells. In this class any one of the pathways may be targeted for treatment: activation by DCs, T helper response, and active CTL killing. In a study by Phan et. al. on metastatic melanoma patients, anti-CTLA-4 antibody was administered in the hopes of achieving a more rigorous T cell response (5). Cytotoxic T-Lymphocyte Antigen 4, CTLA-4, is an inhibitory receptor on T cells that prevents costimulatory signals sent by ligation of CD28 receptor on T cells with the costiumlatory molecules CD80/86 on APCs. While regression was observed in 3 of 14 patients (21%), 6 patients (43%) were observed to develop various autoimmune diseases including dermatitis, enterocolitis, hepatitis, and hypophysitis (5). 

A much more successful story is Imiquimod. Imiquimod is a small molecule agonist of TLR-7. Ligation of Imiquimod to TLR-7 has been shown to cause DCs to mature and secrete the inflammatory cytokines interferon alpha (IFN-alpha), interleuking 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) (7). The mechanism of action then involves the activated DCs around the tumor cells to take up the tumor antigen and migrate to the lymph node, and there they launch an anti-tumor cellular response. Imiquimod is applied as a topical cream used to treat skin cancers, but has recently been used in vaginal intraepithelial neoplasias (8). Imiquimod has been proven to be highly effective in some incidents of cancer such as basal cell carcinomas, in which Imiquimod has been found to have a 100% success rate if treatment was conducted twice daily (8). The adverse effects of Imiquimod are the result of the dermal inflammation that follows treatment. The dermatitis frequently causes bloody lesions on the skin that may cause scarring (8). This has caused some patients to believe their condition is worsening and be reluctant to re-apply Imiquimod. 


Caveats of Adoptive Cell Therapy: The Tumor Escape Hypothesis


For quite some time there have been ongoing debates on whether adoptive cell therapy actually causes more good than harm to the patients. Recent clinical follow up studies on patients who have undergone adoptive cell therapy show that most of these patients relapse with highly aggressive metastases that are unresponsive to most therapies available today (11). These patients ultimately die due to incurable wildly aggressive metastases. The reason behind these aggressive relapses can be explained using the tumor escape hypothesis (10, 11).


Today, the role of the immune system in shaping the immunogenicity of tumors and preventing tumor formation has been unequivocally established. Experiments on immunodeficient mice show that tumors arising in these mice tend to be more immunogenic than tumors arising in immunocompetent mice (10). The reason behind this observation is that recognition of tumor antigens by the immune system drives the depletion of most immunogenic tumor cells from a tumor, thus, selecting for tumor cells which are non-immunogenic (12).


The tumor heterogeneity model should be kept in mind when thinking about the effects of the immune system on solid tumors. The cellular composition of solid tumors is made up of cells that are immunogenic and cells that are non-immunogenic. The degree to which a tumor undergoes immunoediting is dependent on the degree of immunocompetence of the host. The immune escape model (also known as “immunoediting”) states that a solid tumor will be ‘edited’ by an individual’s immune system in a manner that eliminates all immunogenic tumor cells from the population, selecting for ‘escape mutants’ which can proliferate uncontrollably in the presence of a robust immune system. Escape mutants typically make up a very small percentage of a tumor. These mutants are characterized by their lack of expression of tumor-associated antigens which makes them, by definition, non-immunogenic. These cells will typically express, or overexpress, self-antigens which allow them to proliferate uncontrollably. However, it is worth mentioning that tumor cells expressing tumor-associated antigens will proliferate preferentially to these escape mutants, and it is for this reason that these mutants are in such small numbers in a tumor containing cells that are expressing tumor-associated antigens. The tumor escape hypothesis can be divided into three phases: elimination, equilibrium and escape (10).


The elimination phase of the immune escape model is characterized by a robust immune response against most cells present in the tumor. During this phase expanded clonal populations of B cells, CD4+ T helper cells and CD8+ cytotoxic T cells act to deplete the tumor of the cells that they have strong specificity towards (10). Each clone is generally specific to one tumor-associated antigen.


The equilibrium phase is characterized by a dormant state where the tumor is not proliferating at high rates and the immune response against the tumor is not robust. Essentially tumor cell proliferation is kept at bay by the robust immune response, but the tumor is not completely cleared (10). During this time the immune system prevents the proliferation of tumor cells expressing tumor-associated antigens while remaining ineffective at mitigating the expansion of the escape mutant population (10).


The escape phase is defined by the rapid outgrowth and proliferation of escape mutants which cannot be sequestered by the immune system. These tumors are highly aggressive and are generally resistant to most common treatments (10). Typical characteristics of escape mutants is reduced MHC class I expression, which conveys their reduced immunogenicity (10, 11). In addition to reduced antigen expression, these escape mutants also express immunoregulatory molecules such as IDO, PD-L1 CTLA-4 and Tim3, which act to inhibit the immune response against them (10).


Targeted immunotherapies based on the immune escape model


Since adoptive cell transfer often results in relapses of highly aggressive metastases, numerous approaches have been under development in order to minimize the escape mutant characteristics of tumors. Approaches that involve combinations that target multiple pathways may prove synergistic and are often capable of generating a systemically effective memory response.


One example of this is the recent clinical combination of Ipilimumab and Nivolumab, which are anti-CTLA-4 and anti PD-1 monoclonal antibodies, respectively (10). PD-1 and CTLA-4 are surface receptors located on immune cells. Ligands expressed on the surface of tumor cells bind to these receptors and induce apoptosis and contact-dependent cell inhibition in the target immune cells, respectively (10).


The ultimate goal of this treatment is to make the immune cells themselves unresponsive to the evasion mechanisms of the tumor that were selected by the immune system initially. Therefore, this treatment allows the further clearance of immune escape mutants from the system.


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