Immunological Control of Neoplasia - Chapter 1

Author: Christine Ichim

*Immunological Control of Neoplasia*

Christine V. Ichim and Thomas E. Ichim


Copyright © 1998 by Christine V Ichim and Thomas E Ichim

All rights reserved, no part of this work may be reproduced, stored in a retrieval system, or transmitted by any means, electronic,mechanical, photocopying or otherwise, without the prior written permission of Rapoport Publishing.

Rapoport Publishing
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ISBN # 0-9686617-0-X


*Introduction*

Immunological Control of Neoplasia is an interesting collection of facts and ideas related to the interactions between the immune system, host and their malignant cells. The authors of this book have lived with cancer for more than ten years. Thomas age 12 was faced with the diagnosis of chronic myelogenous leukemia (CML) in their mother. To deal with this, hes began a personal crusade to learn all about the disease with the aim of helping his mother and others with the diagnosis. This began with a school science project in which they demonstrated their mother’s leukemic cells to classmates to more formal studies both in libraries and laboratories.

This work is a synthesis of what Thomas learned about CML and immunotherapy in the treatment of cancer. The treatise ends with their hypothetical approach of how to treat CML. This book illustrates the passionate involvement of family, in patient care. Some of the ideas presented in this book may someday prove correct, while others, like many scientific hypotheses, are found to not work. However, the ideas presented here and brought forward to doctors challenge the medical and scientific community to think outside the walls in search of a cure. The importance of strong patient advocacy cannot be underestimated as we all try to improve treatment outcomes.

Mark D. Minden, M.D., Ph.D., FRCPC

*Chapter 1 Introduction to The Immunotherapy of Cancer*

Using the immune system to treat cancer is intellectually appealing due to the specificity, memory and efficacy of the immune response in combating traditional pathogens such as bacterial, parasitic or viral infections. Should an anticancer immune response be initiated, the patient could be spared the psychological impact of surgery and/or the toxic side effects of chemotherapy and radiation. The immunotherapy of cancer has been extensively attempted in various forms since the late nineteenth century because of its popular appeal to the patients and the medical community. Interestingly, cancer immunotherapy also has been one of the most controversial fields of science, to quote cancer researcher/reproductive immunologist Dr. David Clark of McMaster University, "I have witnessed the field of cancer immunology bring the rise and fall of many scientists" (David Clark, personal communication). At the core of the debate regarding whether cancer immunotherapy is of clinical value lies the question, is cancer really a part of nonself? The argument is that since cancer cells are derived from the host, although they are abnormal in certain parameters like increased proliferation or lack of differentiation, they are essentially still a part of the host and cannot be rejected by the immune system. The question thus arises, are there certain immunologically recognizable markers that are specific to cancer cells, and if these markers exist, then, can they be the targets of an immune response? If so, then why is there not a response mounted against cancer in patients that have already succumbed to it? Numerous scientists have investigated these questions for more than a century with opinions swinging on an almost cyclic basis. To appreciating the current knowledge of cancer, viewing tumour immunology from a historical perspective is important. In this chapter cancer immunotherapy will be examined from the first published report of successful immunotherapy by the New York physician William Coley in 1891(1), to the current gene therapeutic trials and DNA vaccinations.

In the eighteenth century clinical observations were recorded of neoplasia entering remission when patients developed bacterial infections (reviewed in ref 2). With this knowledge in mind, and knowing that other bacterial treatments of sarcomas were previously attempted (3-5), Coley began routinely using certain bacteria for treatment of neoplasia (Coley's work is reviwed in reference 6). The initial observation that sparked Coley's interest was that unlike his first patient, a young who died of a sarcoma in her arm, a different patient with a similar type of sarcoma in the neck had lived for seven years after diagnosis with no detectable signs of cancer. The latter patient was reported to have had repeated encounters with bacterial infections. Coley noted that in the medical literature, the bacterial infection most associated with sarcoma remission was erysipelas, a streptococcal infection of the skin (7). Thus, Coley induced such an infection in an inoperable late stage neck sarcoma patient by injecting him with streptococcal broth cultures near the tumour (8). This led to the eradication of a "hen egg”-sized tumour within ten days, and resulted in patient survival for eight years, after which he died of tumour relapse (9). Coley’s vaccine preparation was altered after administering it to his first patient since the artificially induced erysipelas produced severe side effects including uncontrollable fever. The first modification replaced the live culture with heat-killed streptococci, but this was not effective at inducing erysipelas or remission of cancer (6). Coley then decided to use a mixture of heat-killed serratia marcescens and heat-killed streptococci, this combination was chosen since the virulence of streptococci could be augmented by coinjection with dead serratia marcescens in the rabbit (10). The combination of the two heat-killed bacteria added a great clinical benefit to Coley's therapy since the usage of heat killed bacteria would spare the patient of the unpredictable adverse effects associated with the injection of live pathogens. The combination of heat-killed serratia marcencens and heat-killed streptococci became known as "Coley's Toxins.” Coley continued treatment of patients with this bacterial preparation until his death in 1936. Cure rates achieved for soft tissue sarcoma using Coley's toxin was more than 10%, while greater numbers were induced into temporary remission (6). It should be noted that, similar to modern day treatment of neoplasia with IFN-( and TNF " (11), Coley's toxin was effective mainly in soft tissue sarcomas (12). After Coley's death, interest in his therapy diminished because chemotherapy and radiotherapy proved to be more effective. Also, the latter’s efficacy was not limited to soft tissue sarcomas—a rare type of neoplasm that represents about 1% of all cancers diagnosed annually in the United States (13). Interest in Coley's work reemerged with the work of Johnston and Novales in 1962 (14) and continues with the work of Coley's daughter at the CHIPSA Institute in Mexico (15) and the clinical trials of Dr. Havas at Temple University in Philadelphia (16). Although today Coley's toxins are largely disregarded as alternative medicine, it is stunning that the sophisticated cytokine-therapy protocols currently used, are not even as effective as the brute approach used by an eclectic clinician one hundred years ago! (Compare Coley's 10% cure rate with success achieved in references 17 and 18). Besides clinical contributions, the work of Coley had an important impact on the way cancer was perceived and it laid the conceptual groundwork for future theories regarding immune control of cancer. One important outcome of Coley's work was that it led scientists to search for the mechanisms by which endotoxin containing bacteria induced an antitumour response. Work along this train of thought led Carswell et al to discover the serum factor in endotoxin-administered mice that causes tumour necrosis in 1975. This 43 kilodalton protein was named tumour necrosis factor (TNF) (19) and is a very important hormone-like substance called a cytokine. TNF is involved in the regulation of diverse biological activities such as blood pressure (20-23), immune response (24-27), sleeping patterns (28-30) and control of body temperature (31-33).

Quite independent of Coley's work, on another front were the tumour vaccine approaches in the early 1900s. These experiments excised a tumour from a certain mouse breed, transplanted it to another breed and noticed that the tumour would get rejected. Initially this came as a surprise because it implied that an immune response against tumours could be elicited (34). Two groups later showed that injection with dead tumour cells in a tumour free animal would increase the animal's resistance to subsequent tumour challenge (35,36). Although much excitement was generated by these studies, it was soon discovered that the reason for rejection and the subsequent "immunity" to the tumours was not a result of the immunogenic properties inherent to the tumour, but because it was a transplant across allogeneic barriers. This conclusion was reached in a review of the tumour immunology literature by Woglom in 1929 (37). Although the field of tumour immunology seemed defeated from a basic science perspective, the advent of inbred mice brought about new experimental possibilities for testing theories of tumour immunogenicity. In 1943 Ludwig Gross demonstrated in the C3H inbred mouse strain that rejection of tumour tissue would occur when transplanting a chemically induced sarcoma to a genetically identical mouse, in contrast to noncancerous tissue that would not get rejected (38). These tumour rejection experiments were repeated with similar results by several groups including Prehn and Main (39), Klein et al (40), and Old et al (41). Rejection of cancerous tissue in a syngeneic graft suggested the existence of tumour specific antigens that could be used as therapeutic targets. This sparked a renewed interest in tumour immunology marked by many groups attempting to develop the "cancer vaccine.”

The cancer vaccine is not aimed at inducing prophylaxis but to stimulate the otherwise dormant antitumour immune responses of the host (42). This response is referred to as "dormant" since an appropriate response would have eradicated the tumor. This dormancy must be overcome through "educating" the immune system that the tumour is part of nonself (or danger) and therefore needs to be eliminated. One way to educate the immune response to view the tumour as danger is to inject dead tumour cells into a site that is different from the site in which the tumour developed. Such an ectopic injection will present tumour antigens to the immune system in an anatomical location that is free from the local immunosuppressive effect of the growing tumour. To increase the probability of inducing an immune response, the dead cancer cells should be co-injected with an adjuvant, analogous to the way that bacterial vaccines provide the most protection when co-injected with adjuvant. Although multitudes of these experiments were successful in animal s, when the "cancer vaccine" was tried in humans, very little, if any increase in patient survival was reported (reviewed in reference 43). The failure of this protocol was blamed in part on the unnaturalness of the s used (44,45). Cancer s were easily curable in mice and other small animals since the tumour cells used were highly immunogenic, this did not represent tumour cells found in the human population (46). For example, a type of tumour used to induce immunity was the methylcholanthrene (MCA)-initiated neoplasm. MCA is a powerful carcinogen that induces neoplasia several weeks post administration (47). This type of cancer is not comparable to human cancers since one rarely develops cancer after a single large exposure to a carcinogen. The human cancer situation is a much more latent process that allows for the accumulation of several mutations over time (48-50). The significance of these mutations is that they allow for a variety of host evasion mechanisms to develop in the cancer, based on survival of the fittest cancer cell. The chemically induced cancer contains fewer mutations and has not undergone a natural selection process based on its immune evasion mechanisms. A demonstration of the immunogenicity of MCA induced cancers compared to spontaneously occurring cancers is seen when irradiated cells of each tumour are injected into syngeneic mice followed by a challenge with a live tumour inoculum of the same tumour used to vaccinate. Mice vaccinated with the immunogenic MCA tumours reject the inoculom whereas mice immunized with the nonimmunogenic spontaneous tumours succumb to neoplastic growth (51,52).

Another explanation for the clinical failure of the tumour vaccine is the immunosuppression that exists in patients entering clinical trials. Since many of them are in the terminal phase of their respective neoplasm, they are likely to possess very weakened immune function (53-58). In fact, anergy to a variety of antigens has been shown in several end-stage cancer patients (59-61). In addition to the immune suppression induced by the tumor, immunotherapy patients are routinely administered chemotherapy to reduce the initial tumour load; this further contributes to the state of immune suppression (62-64). Immunizing with tumour antigens when the patient is immunosuppressed may not only be ineffective, but even detrimental since it can lead to activation of a cancer promoting immune response. This response has been described by Prehn et al. Its existence has been substantiated by the T cell deficient mouse, which lacks a T-cell dependent immune system, and has a decreased rate of proliferation of transplanted sarcomas compared to control mice (65-68).

Although interest in cancer immunotherapy waned in the 1980s because of clinical inefficiency (69-72), several groups have pursued the research in the belief that cancer cells can elicit an immune response but are subverted by immune evasion mechanisms. This school of thought combined with the discovery and cloning of melanoma specific antigens by Boon's group in 1991 (73) led to a renewed interest in cancer immunotherapy. The premiss heading this new movement is that cancer cells are antigenic but not immunogenic (74). This wave of interest in tumour immunology might be standing on more solid scientific ground than the preceding surges since today molecular evidence for all antigens described is available, in contrast to the crude preparations used in the previous years. Additionally, there exists much stronger knowledge of immunology and immunomanipulation then there ever has. For example, we now know at the molecular level agents involved in the initiation, effector, and resolution stages of the immune response and we can alter any of these stages of response in different animal s.

The new interest in cancer immunotherapy has led to the development of unique methods to immunomodulate the patient. These methods, including the adoptive transfer of immune cells and inoculation with cytokine cDNA-transfected irradiated tumor cells will be described below.

Adoptive transfer of activated immune cells

Many studies show that tumours protect themselves from immune mediated destruction through the secretion of immunosuppressive factors (75-83). These tumour-secreted substances will be described in more detail in chapter 3, for now we will simply call them immunosuppressants. Stimulating immune cells ex vivo is much easier than in vivo, since in the former they are maintained in the absence of tumour-secreted immunosuppressive factors, thus making activation easier. Some researchers have tried to activate host immune cells in vivo by “flooding” the patient with immunomodulators. However, these protocols were problematic because the large quantities of cytokines needed to achieve proper activation would induce toxicity. A well-known example is the interleukin-2 (IL-2) trials in which the dosage needed to attain immune activation was so high that many patients had to cease therapy due to toxicities (84-86). Several groups are presently attempting to lower IL-2 toxicity by co-administering it with various agents including dexamethasone (87), pentoxifylline (88), indomethacin (89) and melatonin (90). Results are optimistic but too preliminary to draw conclusions. Stimulating the immune cells ex vivo is less toxic than systemic immunomodulation and is also less expensive since a smaller amount of immunostimulant is used.

Ex vivo stimulation approaches began in mouse studies where immunity to tumours could be passed from a resistant mouse to a control mouse through the transfer of splenocytes (91-94). Further analysis revealed that for the optimal transfer of protection CD4 and CD8 T cells had to be transferred together (95, 96). These mouse studies however, were not truly ex vivo experiments since stimulation of the lymphocytes occurred inside the mice. The question was "can we activate immune cells nonspecifically ex vivo?" or even better, "can we activate ex vivo T cells specific to the cancer?" The latter approach is superior to the former since nonspecific ex vivo activation can result in reactivation of autoreactive immune cells that can trigger autoimmunity. Early attempts did in fact activate patient cells nonspecifically by taking out leukocytes, adding the polyclonal T cell mitogen PHA ex vivo and then reinfusing the activated cells back into the host. The success of this therapy was not superior to conventional treatments such as chemotherapy or radiation and therefore it was abandoned (97-99).

The other ex vivo stimulation approach had a stronger success rate. This approach involved purifying peripheral blood mononuclear cells (i.e., lymphocytes), activating them with the immunostimulant interleukin 2, and reinfusing the activated lymphocytes back into the autologous patient. In contrast to the first approach that aimed at nonspecifically activating T cells, this approach activated natural killer (NK) cells and a subset of T-cells with NK-like ability called lymphokine activated killer (LAK) cells. (100-102). These cells possess the ability to kill tumour cells expressing abnormal levels of MHC (103), and target cells not expressing empty MHC class 1 (RG Miller, personal communication).

Another immunotherapeutic approach that activates immune cells ex vivo involves using tumour infiltrating lymphocytes (TIL) against solid tumours. TILs have been noticed in a variety of tumours and are correlated with a favorable prognosis in certain cancers including liver carcinoma (104), melanoma (105, 106), bladder cancer (107), and ovarian cancer (108). It is the belief of many tumour immunologists that TILs infiltrate tumours to induce their eradication, however, this does not occur in vivo because tumour-secreted immunosuppressive factors inhibit immune activation. TIL therapy involves surgically extricating a tumor mass, separating the TILs from the tumour cells on a density gradient, expanding the lymphocytes in immunostimulatory in vitro conditions and reinfusing the activated killer cells back into the patient (109, 110). Mouse s contrasting the antitumour efficacy of TIL therapy to LAK therapy showed that TIL therapy had approximately a one hundred fold greater tumoricidal effect (111,112). A possible reason why TILs had an augmented tumor eradicating effect is that this therapy activates only lymphocytes that have recognized the tumor and are reacting to it. This is in contrast to LAK therapy that activates a plethora of cells, of which only a fraction are specific to the tumor. In the clinic, results using TIL have been fair, with reproducible responses in approximately 20% of melanoma patients (113). A means of augmenting the efficacy of TILs is to enhance their killing potential by transfecting them with cDNA to TNF (114). Results from the clinic on this exciting modality have not yet been published.


Cytokine Gene Therapy

One of the greatest advances of modern day immunology is the understanding of the molecules that regulate the immune response. Cytokines, a family of intercellular messenger proteins, are important immunoregulators. The cytokines that have been shown to contribute to a cell mediated response are generally categorized as Th1-like, these include IL-2, IFN-(, TNF-", IL-12 and IL-15. Conversely, cytokines belonging to the Th2-like family are believed to favor an antibody mediated immune response, these include IL-4, TGF-B, IL-10 and IL-6 (124-125). A protective response against cancer is thought to be cellular since components of the cell mediated immune system such as NK cells and cytotoxic T cells have been shown to eradicate tumours in vitro and in vivo. Antibody responses to tumours are generally nonprotective and may contribute to tumour progression by inhibiting the cell mediated antitumour response (116). The enhancement of tumour growth by noncytotoxic antibodies is called "immunological enhancement." This phenomenon was first described in the 1907 by Flexner and Jobling (126) who showed that injection with dead autologous tumour cells enhanced the growth of preexisting tumours. The term, immunological enhancement, was popularized by Kaliss in the 1950s who conducted many studies investigating this phenomenon (127-131). Therefore, generally, Th1-like cytokines mediate an antitumor response, whereas Th2-like cytokines allow for tumor progression (115-123). However, these concepts are generalizations and many exceptions exist to this paradigm.

Administering cytokines systemically to alter the immune response of the patient has yielded poor results in the clinic since high doses are needed to compensate for the short half life of many cytokines in vivo (132, 133). One of the reasons previous tumour immunization studies have failed was because the profile of the immune responses generated was not protective (43). Thus, it was a great achievement when genetic engineering technology allowed cytokine genes to be transfected into tumour cells. This allows for cytokines to be delivered to the actual microenvironment of the tumour, sparing the patient of toxicities associated with systemic cytokine administration.

Vaccination with tumor cells transfected with cytokine cDNA is based on the principle that tumour cells are antigenic but not immunogenic (74). This implies that tumour-specific markers do exist, however, they cannot be recognized by the immune system and thus fail to initiate an immune response. Transfecting tumour cells with cytokine genes increases the immunogenicity of the transfected cells so that they are readily rejected. Once the transfected cells are rejected, the immune response starts to recognize the untransfected cells as foreign and begins eliminating them. The injection of transfected cells acts as an initiation, or a breaking of tolerance, to "awaken" the immune system into "seeing" that cancer cells are not part of self (134). Analogously, breaking of tolerance is seen when autoimmunity is induced by immunization of guinea pigs with myelin basic protein plus adjuvant. The guinea pig is tolerant to myelin in the same way the host is usually tolerant to the tumour antigens. When myelin is coinjected with adjuvant, the host recognizes myelin as an immunogen and quickly rejects it. This occurs because adjuvant nonspecifically stimulates an immune response to the antigens that it is simultaneously injected with. The same T cells that rejected the myelin injected with adjuvant then start to attack the myelin in the nervous system causing experimental allergic encephlomylitis (EAE), a disease resembling multiple sclerosis (135,136). One may ask, "is it not more economical to inject irradiated tumour cells with adjuvant than transfect them with cytokines?” While this may be more economical, this approach is not feasible in the clinic because adjuvant mixtures are too toxic. The most effective adjuvant, Freund's complete, is too toxic to be used in humans; while the adjuvant that is less toxic, Freund's incomplete, lacks the potency of Freund's complete and is not useful for tumour immunization (137).


An advantage of transfecting tumours with cytokine genes is that it allows for a greater degree of specificity than coadministering them with adjuvant. For example, transfecting melanoma cells with IFN ( can stimulate a cytotoxic T cell response (138-140). A reason for this may be that tumor cells treated with IFN-( increase expression of MHC class 1 molecules (141), MHC class 2 molecules (142,143) and costimulatory molecules such as B-7 (144,145). A strong T cytotoxic response is desirable in some tumours, whereas in others an initial macrophage response is protective. To attract macrophages to kill the transfected tumour, macrophage stimulatory cytokines are transfected, for example the macrophage chemoattractant protein (146), or GM-CSF (147). Further, if the protective response is one initiated by NK cells, then making the tumour immunogenic from the perspective of NK cells is important. This has been done by transfecting tumour cells with interleukin-10 (148-150). While interleukin-10 is an inhibitor of the Tc cell response (151-154), it also reduces surface expression of MHC class 1 and 2 on tumour cells (155). This is postulated to make the tumor cells susceptible to NK lysis since MHC molecules send inhibitory signals to NK cells (103).

Chapter 1 - Introduction to The Immunotherapy of Cancer
"Chapter 2 - Components of Immune Response":http://www.stemcellpatents.com/journal-show-42
"Chapter 3 - Immune Suppression in Cancer":http://www.stemcellpatents.com/journal-show-43
"Chapter 4 - Immune Interactions with Chronic Myeloid Leukemia (CML)":http://www.stemcellpatents.com/journal-show-44

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