Prophylactic vaccines against cancers of non-infectious origin: a dream or a real possibility?

The dramatic progress in tumour biology and immunology in the past several years has opened new avenues for the treatment and prevention of cancer. One of the great contributions of the immunotherapeutic approaches is an increasing understanding of the immunology of cancer, which is, gradually creating conditions for the development of prophylactic anti-cancer vaccines. Efficient vaccines have been developed and employed for the prophylaxis of two frequent cancers of viral origin, namely cervical cancer and liver cancer. The new knowledge on the interactions between the immune system and the malignant tumors seems to provide means for the development of prophylactic vaccines against cancers developing due to the mutations in the proto-oncogenes converting their products into oncoproteins. According to the present estimates, these cancers form a great majority of human malignancies. Recent evidence has indicated that the immune system recognizes such mutated proteins, and that the development of cancer is due to the failure of the immune system to eliminate neoplastic cells. Followingly, it can be expected that inducing immunity against the mutated epitopes will increase the capacity of the body to deal with the initiated precancerous cells. In the present paper this hypothesis is primarily discussed in the relationship with colorectal cancer (CRC), which seems to be a well-fitting candidate for prophylactic vaccination. CRC is the third most frequent malignancy and the fourth most common cause of cancer mortality. Mutations of two proto-oncogenes, namely RAS and RAF, are involved in the majority of CRC cases and, in addition, they are shared with other human malignancies. Therefore, the strategy to be used for prophylaxis of CRC is discussed together with several other frequent human cancers, namely lung cancer, pancreatic duct cancer and melanoma. The prophylactic vaccines proposed are aimed at the reduction of the incidence of these and, to a lesser extent, some other cancers.

Intracellular Cytokines Produced by Stimulated CD3+ Cells from Chronic Myeloid Leukemia Patients.

Our work examined the production of intracellular interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-4 by in vitro stimulated CD3+ cells from 38 chronic myeloid leukemia (CML) patients. At the time of diagnosis the percentages of cells producing INF-γ, TNF-α, and IL-2 were strongly suppressed compared to those in healthy control subjects. Hematological remission achieved through treatment with tyrosine-kinase inhibitors was associated with a highly significant increase in the ratio of cells producing all 4 cytokines. The percentages of CD3+ cells producing cytokines were dependent on age, more so in CML patients than in healthy controls, and they negatively correlated with the number of leukocytes. Patients with an optimal therapy outcome possessed higher percentages of cytokine-producing CD3+ cells at diagnosis than those with nonoptimal outcomes. This difference was statistically significant in the case of INF-γ-producing cells, and it was on the brink of significance in the case of IL-2-producing cells.

Predictive value of serum cytokine levels in chronic myeloid leukemia patients.

Serum samples taken at diagnosis in 28 chronic myeloid leukemia patients were tested for the presence of 20 cytokines by a magnetic bead-based Bio-plex immunoassay. According to complete cytogenetic remission achieved at 12 months of treatment, patients were divided into groups with either optimal or non-optimal outcome. Patients with increased cytokine levels tended to react optimally to the therapy more frequently than those others. TGF-ß3 was a notable exception; its levels were significantly higher in patients with non-optimal outcomes. Further analysis enabled us to define two combinations of cytokine cut-off levels - namely low TGF-ß3 and either high IL-8 or high MCP-1-each of which corresponded to therapy outcome better than either Sokal or EUTOS scores.

Kynurenine and uric acid levels in chronic myeloid leukemia patients.

Indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO) represent some of the key immune regulators. Their increased activity has been demonstrated in a number of human malignancies but not yet in chronic myeloid leukemia (CML). In the present study, the activity of these enzymes was tested in 29 CML patients and 28 healthy subjects by monitoring the kynurenine (KYN)/tryptophan ratio. Serum samples taken prior to the therapy displayed a highly significant difference in KYN levels between the patient and control groups. However, increased KYN levels were detected in only 13 (44.8%) of these CML patients. The KYN levels in pretreatment sera of the patients correlated with the tumor burden. There was also a strong correlation between KYN levels and uric acid levels (UA). This suggests but does not prove the possible involvement of UA in activating IDO family of enzymes. Whenever tested, the increased KYN levels normalized in the course of the therapy. Patients with normal KYN levels in their pretreatment sera and subsequently treated with interferon-α, showed a transitory increase in their KYN levels. The present data indicate that CML should be added to the malignancies with an increased activity of the IDO family of enzymes and suggest that IDO inhibitors may be used in the treatment of CML patients.

[Indoleamine 2,3-dioxygenase in oncology and psychiatry]. / Indolamin 2,3-dioxygenáza v onkologii a psychiatrii.

In the last years an attention has been paid to the indoleamine 2,3-dioxygenase (IDO), an enzyme catabolising L-tryptophan to kynurenine. Growing evidence has been accumulated that kynurenine and other metabolites of tryptophan play an important role in the pathogenesis of malignant tumours and some neurological and psychiatric disorders. The gradual recognition of mechanisms operative in their development may help to identify etiological factors involved and becomes prerequisite for the progress in their diagnostics and therapy. In oncology, great effort is directed to the development and testing of substances inhibiting IDO activity. It is expected that some of them will be utilized in the immunotherapy of cancer. In the field of psychiatric disorders, namely in schizophrenia and depression, the role of IDO is linked to immune dysregulation. In those diseases, IDO represents a potential mediator between immunological reactivity and alterations of the brain function. Changes in the IDO activity may also mediate interaction between the genetic predisposition and environmental factors.

Immunology of chronic myeloid leukemia: current concepts and future goals.

Although chronic myeloid leukemia is a rare malignancy, it has developed into a model system for the study of a variety of aspects of cancer biology and immunology. The introduction of tyrosine kinase inhibitors has resulted in a significant prolongation of the survival rates of chronic myeloid leukemia patients but has not resulted in a cure. There is a growing conviction that this aim can be achieved through immunotherapy. For this concept to be successful, a considerable increase in the present understanding of chronic myeloid leukemia immunology is required. The authors attempt to review and evaluate the current findings that demonstrate a number of immunological aberrations in patients prior to the start of any therapy and their normalization after achieving remission. They also discuss the recent clinical trials with experimental therapeutic vaccines and then present their own strategy on how to address the problem.

Administration of a plasmid that expresses SDF-1α affects the oncogenic potential of mouse bcr-abl-transformed cells.

Stromal-derived factor 1α (SDF­1α, also known as CXCL12) is a chemokine that exerts its effects through the G-protein coupled receptors, C-X-C chemokine receptor type 4 (CXCR4) and 7 (CXCR7). There is marked evidence that the SDF-1/CXCR4 axis is involved in the pathogenesis of leukemia and therapies that target this axis are under development. The present study aimed to increase the efficacy of a DNA-based bcr-abl vaccine by simultaneously immunizing mice with a plasmid carrying the whole SDF-1α gene. Bcr-abl­transformed 12B1 cells were used to challenge the mice. These cells have the oncogenic potential to induce both leukemia following intravenous inoculation and lymphoma-type solid tumors after subcutaneous inoculation. Administering an SDF­1 carrying plasmid together with the bcr-abl vaccine resulted in increased survival following a challenge with subcutaneously administered 12B1 cells, although the difference was not statistically significant. However, there was a difference when the animals that developed subcutaneous tumors were only taken into consideration. In doubly-treated mice, significantly more mice failed to develop solid tumors than mice that had only received the bcr-abl vaccine. By contrast, the occurrence of fatal leukemia was significantly higher in the mice that were treated with the SDF-1 plasmid, regardless of whether they were immunized with the bcr-abl-vaccine. No humoral or cellular immune responses against SDF­1 were detected in the treated mice, which suggested that the changes in oncogenic potential of 12B1 cells were due to the activity of SDF-1 itself.

Tumor protective activity of CD4+ but not of CD8+ T cells in DNA-vaccinated mice challenged with bcr-abl-transformed cells.

In the recent past, it has repeatedly been reported that CD4 cells play an important role in the immunology of chronic myeloid leukaemia. It was therefore of interest to test their activity in an animal model using bcr-abl-transformed cells. BALB/c mice were four times immunized with a DNA vaccine carrying the bcr-abl fusion gene. Two weeks after the last vaccine dose, the animals were challenged with syngeneic bcr-abl-transformed 12B1 cells which form solid tumors after subcutaneous administration. At the time of challenge, animals were treated with antibodies against the CD8+ T cells or CD4+ T cells. The efficacy of the depletion was monitored and found highly effective. All nonimmunized animals developed tumors. All animals untreated with the antibodies as well as those in which CD8+ T cells had been depleted, were fully protected against the challenge. On the other hand, almost all mice treated with anti-CD4+ antibody developed tumors. These results strongly suggested that the CD4+ T cells acted as effectors in the present system.

Properties of bcr-abl-transformed mouse 12B1 cells secreting interleukin-2 and granulocyte-macrophage colony stimulating factor (GM-CSF): II. Adverse effects of GM-CSF.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is considered to be the most effective immunostimulating factor for the construction of gene-engineered anti-cancer vaccines. In some tumour cells, this type of genetic modification has resulted in the loss of the oncogenic potential. This was not the case with bcr-abl-transformed mouse 12B1 cells. A cell line, designated 12B1/GM-CSF/cl-5 producing more than 100 ng/106 cells/24 h, displayed higher pathogenicity than the parental, non-transduced cells. Although the tumours induced by the parental 12B1 cells and 12B1/GM-CSF/cl-5 cells appeared nearly at the same time and then grew at an approximately equal rate, the latter mice were in a much poorer clinical condition. In these animals the growth of the tumours was associated with gradually increasing blood levels of GM-CSF. In both groups of animals splenomegaly was observed; it was much more pronounced in the case of 12B1/GM-CSF/cl-5-inoculated animals. While in the case of animals inoculated with the parental cells the splenomegaly was probably mainly due to infiltration with tumour cells, in the animals inoculated with the GM-CSF-secreting cells splenomegaly and derangement of parenchymal organs, such as lungs, liver and kidneys, were more complex, including congestion and infiltration with hemopoietic cells, predominantly immature cells of myeloid lineage. The most conspicuous of these changes was the hyperaemia of the lungs. No such alterations were seen in animals inoculated with the parental cells. On the other hand, the contents of T regulatory cells were comparable in both groups and they increased in parallel at the end of the observation period. When GM-CSF neutralizing antibody was administered to animals inoculated with the 12B1/GM-CSF/cl-5 cells, the pathological changes observed within the organs were suppressed, this proving that the overproduced GM-CSF and not any other substance, played the key role in their induction.

Properties of bcr-abl-transformed mouse 12B1 cells secreting interleukin-2 and granulocyte-macrophage colony-stimulating factor: I. Derivation, genetic stability, oncogenicity and immunogenicity.

The highly oncogenic bcr-abl-transformed mouse (Balb/c) 12B1 cells were transfected with plasmids carrying genes for either mouse interleukin-2 (IL­2) or the mouse granulocyte-macrophage colony-stimulating factor (GM­CSF) and the gene for blasticidine resistance. From the transduced cells several clones widely differing in the production of either cytokine were isolated. For further experiments, clones with the highest secretion of the cytokines were selected. When administered subcutaneously to mice, the IL-2-secreting cell line was approximately hundred times less pathogenic than the parental cells. A portion of animals developed small, spontaneously regressing tumours and most of them became resistant to challenge with the parental cells. Cell populations from either solid tumours or from organs infiltrated by the tumour cells predominantly consisted of cells which did not produce IL-2 and had lost resistance to blasticidine. This indicated that the IL-2 secreting cells were genetically unstable in the course of their propagation in vivo. On the other hand, the GM­CSF­secreting cells were more pathogenic than the parental cells, induced extensive organ damage and remained genetically stable in the course of their growth in vivo. The pathogenicity of different GM­CSF secreting clones directly depended on the magnitude of production of this cytokine. When used in the form of inactivated vaccines, the GM-CSF-secreting cells were more immunogenic than the IL-2-secreting cells. In comparative experiments, similar results were obtained with GM­CSF- and IL-2-secreting cells derived from B210 cells, another bcr-abl transformed cell line.

Unexpected properties of endostatin-producing mouse BCR-ABL-transformed cells.

We investigated whether a genetic modification of BCR-ABL-transformed mouse cells that resulted in endostatin (ES) production altered their oncogenic potential. Mouse B210 cells, which express p210bcr-abl fusion protein and induce leukemia-like disease and extremely rarely solid tumors after intravenous (i.v.) administration, were used. The cells were transfected with a plasmid carrying genes for mouse ES and resistance to blasticidine. Transduced cells were isolated in media supplemented with blasticidine. Production of ES was determined by Western blotting. For further tests, two clones were selected, and their pathogenicity after i.v. inoculation was tested. Compared with the parental B210 cells, the capability of both gene-modified cell clones to induce lethal leukemia was reduced. However, mice that did not succumb to leukemia subsequently developed solid tumors. They were composed of poorly differentiated cells with irregular nuclei and roughly granular chromatin and were well vascularized. FISH revealed the presence of the BCR-ABL fusion gene both in tumors and spleens. Immunohistological investigation of the tumors demonstrated the production of ES in vivo and the cell lines derived from the tumors produced detectable amounts of ES, this demonstrating that the formation of solid tumors was not associated with the loss or silencing of the ES gene.

Immunotherapy of chronic myeloid leukemia: present state and future prospects.

In spite of the considerable successes that have been achieved in the treatment of chronic myeloid leukemia (CML), cure for the disease can only be obtained by the present means in a rather small minority of patients. During the past decade, considerable progress has been made in the understanding of the immunology of CML, which has raised hopes that this disease may be curable by supplementing the current targeted chemotherapy with immunotherapeutic approaches. More than ten small-scale clinical trials have been carried out with experimental vaccines predominantly based on the p210bcr-abl fusion protein. Their results suggested beneficial effects in some patients. Recent data obtained in human patients as well as in animal models indicate that the p210bcr-abl protein does not carry the immunodominant epitope(s). These observations, combined with the recognition of an ever increasing number of other immunogenic proteins in CML cells, strongly support the concept that gene-modified, cell-based vaccines containing the full spectrum of tumor antigens might be the most effective immunotherapeutic approach. Recently created mathematical models have provided important leads for the timing of the combination of targeted drug therapy with vaccine administration. A strategy of how targeted drug therapy might be combined with vaccination is outlined.

Changes of immunological profiles in patients with chronic myeloid leukemia in the course of treatment.

In the previous paper of ours we compared, prior to start any treatment, a number of immunological parameters in 24 chronic myeloid leukemia patients with the same number of healthy subjects matched by age and sex. We found significant differences in the levels of immunoglobulins, the C4 component of complement, the C-reactive protein, interleukin 6, the composition of lymphocyte population and the production of some cytokines by stimulated CD3+ cells. Eleven of these patients were followed longitudinally. After treatment with hydroxyurea, interferon alpha, imatinib mesylate and dasatinib, or various combinations thereof, hematological remission was achieved in all patients and complete cytogenetic remission in nine of them. There was a nearly general tendency towards normalization of the abnormalities observed in the patients at their enrollment.

Bcr-Abl fusion sequences do not induce immune responses in mice when administered in mouse polyomavirus based virus-like particles.

Mouse polyomavirus-like particles (MPyV-VLPs) carrying inside a fragment of the Bcr-Abl hybrid protein containing the epitope of chronic myeloid leukemia fusion region were prepared. A sequence encoding 171 amino acids covering Bcr-Abl breakpoint was fused to the C-terminal part of VP3 minor protein connecting it to the VP1 capsomeres. Chimeric particles, the Bcr-Abl VLPs, were tested for their ability to induce Bcr-Abl specific immune response in mice after their intranasal (i.n.) or intraperitoneal (i.p.) administration without any other adjuvants. Bcr-Abl VLPs induced strong anti-VP1 immune response in both i.n. and i.p. immunized mice. As expected, neither IgG nor IgM anti-Bcr-Abl specific antibodies were detected in the sera of immunized animals. Surprisingly, no specific CTL (cytotoxic T-lymphocyte) activity was proved using two different methods (in vitro cytotoxicity assay with CFSE-labeled target cells and highly sensitive cytotoxicity assay using MHC class I Bcr-Abl specific pentamers). In addition, no proliferative response of T-cells of i.n. immunized mice after in vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells was observed. Taken together, Bcr-Abl breakpoint epitopes appeared to be weak immunogens and even MPyV-VLPs did not provide sufficient adjuvant ability to support induction of immune responses specific to Bcr-Abl fusion zone epitope.

DNA vaccination against bcr-abl-positive cells in mice.

A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

Effects of endostatin production on oncogenicity and metastatic activity of HPV16-transformed mouse cells: role of interleukin 1alpha.

Two mouse HPV16-transformed cell lines, viz. MK16 cells, which induce metastasizing tumors, and TC-1 cells, which induce non-metastasizing tumors were transduced with the gene for mouse endostatin. Two clones constitutively expressing endostatin were isolated from each of them. They were denoted ME3 and ME9, and TE2 and TE5, respectively. When inoculated into mice, ME3 cells were non-oncogenic. Nearly all mice inoculated with ME9 cells developed tumors, but considerably later than did the parental MK16 cells and metastasis formation was strongly reduced in these animals. On the other hand, TE2 and TE5 cells displayed oncogenic potential similar to that of the parental cells. To provide more information on these different effects of endostatin production, cell lysates of all six lines studied were tested for the content of 25 factors known to be involved in angiogenesis. The parental MK16 cells differed from the parental TC-1 cells and also from all endostatin producing sublines by a markedly higher production of interleukin 1alpha (IL-1alpha) and, to a lesser extent, by a higher production of several other factors tested. Additional experiments indicated that the suppression of the production of IL-1alpha by the parental MK16 caused by endostatin was due to an autocrine mechanism.

Combined chemo- and immunotherapy of tumors induced in mice by bcr-abl-transformed cells.

For our experiments we selected two oncogenic, bcr-abl-transformed mouse cell lines, viz. B210 and 12B1. Both cell types are capable of inducing leukemia-like disease in syngeneic BALB/c mice after intravenous inoculation. 12B1 cells can moreover form solid tumors after subcutaneous injection. Since immunotherapy would expectedly be most effective in animals in which the tumor mass had been reduced by other therapeutic means, we attempted to develop a combined therapeutic system for suppressing tumor growth. In the present study, mice inoculated with the aggressive 12B1 cells were treated with imatinib mesylate (IM), mouse interferon alpha (IFNalpha) and cyclophosphamide (Cy) in combination with genetically modified tumor cells engineered to produce various cytokines. These cell vaccines had been derived from B210 cells. Therapy with IM or IFNalpha alone or cell immunotherapy alone resulted in partial suppression of tumor growth. Of the different therapeutic regimens tested, a combination of repeated doses of IM, IFNalpha and cell vaccines with one relatively high dose of Cy (200 mg/kg) was the most effective, resulting in tumor-free survival of a large portion of mice. The spleens, livers and bone marrows of the successfully treated animals were tested for the presence of bcr-abl-positive cells by means of RT-PCR technique. Results were negative, this suggesting that the animals had been cleared of residual disease.

Efficacy of reovirus therapy combined with cyclophosphamide and gene-modified cell vaccines on tumors induced in mice by HPV16-transformed cells.

Oncolytic virotherapy is a novel approach to cancer treatment. In the present study we tested the ability of reovirus type 3, strain Dearing, to suppress the growth of tumors induced in mice by HPV16-transformed TC-1 cells. In vitro, these cells are highly susceptible to the virus. In repeated in vivo tests the intratumoral inoculation of the virus resulted in only a minor slow-down of tumor growth, never in a complete cure. The effect of the treatment was not enhanced by the simultaneous administration of non-oncogenic, genetically modified TC-1 cells expressing either IL-2, IL-12 or GM-CSF, and, in fact, the oncolytic effect of the virus was even less expressed in some instances. When cyclophosphamide was used in combination with the viral treatment, a synergistic effect resulting in tumor suppression was observed. In most instances the tumor regression was transitory, however, and was followed by tumor progression. The outcome of these experiments was dependent on the timing of the two treatments.

Human hair follicle and interfollicular keratinocyte reactivity to mouse HPV16-transformed cells: an in vitro study.

The role of stem cells in cancer formation and spreading has been established. As with normal tissue, the cancer stem cells need a special microenvironment to support their growth. This microenvironment may be represented by the tumor stroma. One of the possible ways of tumor stromal formation is the epithelial-mesenchymal transition of tumor epithelium. Following this mechanism, stromal cells must share the basic genetic alterations with the tumor cells. In an attempt to create a system capable of testing some aspects of the mesenchymal cell-keratinocyte interactions, we studied the effects of the fibroblastoid mouse TC-1 cells that were prepared by the introduction of human papillomavirus type 16 (HPV16) genes E6 and E7 to lung epithelial cells on the phenotype of normal human interfollicular and hair follicle keratinocytes. From this point of view, they may resemble stromal cells formed by the epithelial-mesenchymal transition of cells from HPV-induced squamous cell carcinoma. In contrast to 3T3 murine embryonic fibroblasts which were used as control cells, TC-1 cells influenced not only the size of the keratinocytes and the shape of their colonies, but also induced the expression of keratins 8 and 19 and vimentin. In conclusion, TC-1 cells exhibited a marked biological activity by influencing the behavior of the normal human follicular and intefollicular keratinocytes. This observation is compatible with the hypothesis that stromal cells play an important role in tumor progression and spreading.