Frank Fan M.D. Ph.D.

1 Summary

Cancer currently remains a leading cause of mortality worldwide, as the development of conventional therapeutic methods, such as surgery, chemotherapy and radiotherapy, gradually steps into a plateau stage. Immunotherapy, a comparatively non-invasive and effective approach, has been gaining more attention since 1960s. However, traditional cancer immunotherapy is now facing great challenges due to its poor precision and duration. Immuno-gene therapy approaches may become potentially more efficient, so that these issues can be overcome. This review focuses exclusively on gene-transfer-based strategies employed in active immunotherapy for cancer.

2 Introduction

To date, cancer remains a leading cause of mortality worldwide, as the development of conventional therapeutic methods, such as surgery, chemotherapy and radiotherapy, gradually steps into a plateau stage. Immunotherapy, a comparatively non-invasive and effective approach, has been gaining more attention. After it was demonstrated that the immune system could recognize and reject tumors in 1960s[1, 2]. During the past two decades, the rapid increase in knowledge surrounding the immune system and molecular biology techniques has led to a resurgence of interest in immunologic approaches against cancer[3, 4].

Immunity of healthy people can not only protect the body from infections, but also inhibit the development of tumor cells, in a processed termed "immunosurveillance". It is a concept that envisages the prevention of tumor development through the early destruction of abnormal cells by the host's immune system[4]. However, tumor cells usually have the ability to escape this sentinel function via several mechanisms, such as tolerance induction due to self-origin of tumour antigens, antigen loss, down-regulation of human lymphocyte antigen (HLA) expression, resistance to cytotoxic T lymphocytes (CTLs), tumor environmental immunosuppression, etc. Thus, a clear understanding of how to enhance tumor-specific immunity or increase immunogenicity of established tumors has beem intensively targeted by many immunologists.

An intact T cell response against tumor cells should have three important properties, following the "3R's" rule: right antigen, right adjuvant, and right immune response[5]. Regarding the goal of arming or strengthening anti-tumor immunity, there have been two main types of approaches: active and passive immunotherapy. Active immunotherapy involves instigating an immune response in a cancer patient to fight cancer cells, through the stimulation of host immunity by cytokines or vaccines. Vaccine strategies involve utilizing peptide, viral-vector, or dendritic cells (DCs)- based vaccines. In passive immunotherapy, immune molecules or cells are given to patients who do not produce them. For example, the host can be treated with tumor-specific antibodies or cytotoxic T cells via adoptive transfer[6]. Recently, adoptive transfer of genetically modified immune cells has become a potentially promising treatment method, which consists of components of both active and passive immunotherapy. The present review focuses exclusively on gene-transfer-based strategies employed in both active and passive immunotherapies for cancer.

3 Viral vectors used in immunotherapy against cancer

Despite the existence of conventional plasmids, various viral vectors are still predominately adapted for the transfer of specific genes into host cells. In comparison with the nonviral vectors, viral vectors have several advantages. They can efficiently infect host cells and mimic natural infection, thus providing appropriate inflammatory signals for subsequent inductions of immune response[7].Some viral vectors can even transfer target genes into the host's nucleus for stable expression. Therefore, viral vectors are an attractive means of transferring tumor-associated antigens (TAAs) and TAA-specific T cell receptors (TCR) to target cells. Poxvirus, adenovirus, and retrovirus are three main vehicles applied to the area of cancer gene therapy.

3.1 Poxviral vectors

Poxviruses make up a family of linear double-strained DNA viruses. During the past two decades, poxviruses, such as vaccinia, fowlpox and canarypox, have been widely used as vaccine vectors in cancer research, due to their ability to safely and effectively induce host immune responses[8]. For the vaccinia virus, new generations of attenuated strains, such as NYVAC and MVA, have been widely used because of low incidences of associated vaccination complications. Recombinant vaccinia viruses have been evaluated in numerous cancer immunotherapy clinical trials. They can safely transduce various TAAs in most cases, such as ones involving carcinoembryonic antigen (CEA)[ 9], prostate specific antigen (PSA)[ 10], MUC-1[11], 5T4[12], etc. However, vaccinia virus-infected cells can only express TAAs for approximately 2 days before cell death. In contrast, fowlpox can express transgenes for up to 3 weeks; consequently, a T cell response may be dramatically enhanced. Rosenberg et al[13]applied fowlpox to the delivering of melanoma associated antigen gp100 to patients with metastatic melanoma. All patients tolerated the recombinant viral vectorswell. Indeed, between 1989 and 2004, 82 poxvirus-based gene therapy clinical trials were carried out[14].

3.2 Adenoviral vectors

Adenoviruses are double-stranded DNA viruses that have cloning capacity of up to 35 kb. There has been widespread interest in the use of adenoviruses for cancer treatment due to their efficient nuclear entry mechanism, their ability to engineer high level transient transgene expressions, and their low pathogenicity for humans.. Adenoviral vectors pass through three generations to prevent viral replication (first generation) and to reduce immunogenicity (second and third generations)[ 15]. Moreover, conditionally replicating adenoviruses (CRAds) are being developed as a promising new tool for cancer therapy. CRAds specifically replicate in cancer cells and thus lead to specific killing of cancer cells[16]. Within a solid tumor mass, the release of newly formed infectious particles from infected cancer cells allows additional cell layers to be infected and destroyed. There were 240 Ad-vectors gene therapy trials reported between 1989 and 2004[14].

3.3 Retroviral vectors

Compared with other viral vectors, the key advantage of retroviruses is that they have the ability to generate long-term transgene expression. Single-stranded RNA in retroviruses are reverse-transcribed into double-stranded DNA and are integrated into host genome. Lentiviral vectors (LVs) are a novel type of retroviruses used in this field.

The application of LVs for gene therapy evolved and popularized only ten years ago; they have received increased interest in recent years. The wild-type genome of lentiviruses was derived from immunodeficiency viruses, such as human immunodeficiency virus-1 (HIV-1). To enhance biosafety, essential genes for the packaging of HIV-1 have been split into three separate packaging plasmids to minimize their potential capacity of producing replication-competent lentiviral vectors[17]. The LV property of being able to be pseudotyped significantly broadened the tropism of the vector. Currently, vesicular stomatitis virus G glycoprotein (VSV-G) is widely used as an envelope glycoprotein for pseudotyped lentivirus vectors because the vectors are easily concentrated by ultra-centrifugation and can transduce a wide range of cell types in animals. Another important advantage of lentiviral vectors is their abilities to infect non-dividing cells, which distinguishes them from oncogenic retroviruses that only productively infect dividing cells. Recently, several investigators have successfully utilized LVs to deliver melanoma TAAs by transducting DCs in vitro[18, 19], while some others have attempted to transfer TAA-specific TCRs by LVs in order to exert their anti-tumor capacity. In addition, lentiviral vectors can be engineered to co-express more than one transgene, which may further enhance their potential application[20]. Yang et al[21]recently reported development of a very promising bicistronic lentiviral vector which harbour two TCR transgene of different specificity. The LV co-expressed TCRs directly against the melanoma associated antigens gp100 and MART-1 respectively and maintained intact anti-tumour function when transduced to naive CTL. They eventually gained robust melanoma tumor cell regression in vitro. Although very few clinical practices have applied LVs as a therapeutic approach until now, there is no doubt that LVs have strong potency and promising prospects.

4 Anti-tumor vaccines

Therapeutic cancer vaccines are potential strategies that are being developed with the intention of treating, or preventing the recurrence of, tumors. As a type of active specific immunotherapy, there are three main immuno-gene therapy approaches of anti-tumor vaccination: (1) Peptide-based vaccines: the peptide itself, DNA expression plasmids, or viral gene expression vectors are directly injected into the host, and TAAs can be presented to T cells via local DCs; (2) DCs-based vaccines: DCs can be genetically engineered to express specific TAAs by viral vector based gene transfer in vitro; they can then be injected into hosts to induce anti-tumor CTL responses. (3) Modified tumor cell vaccines: tumor cells transfected by viral vectors that encode immunostimulatory cytokines (such as GVAX?) can be injected to hosts and attract local DCs to acquire, process, and present TAAs in the context of MHC[3]. We will discuss the rationale and experiences of these widely used approaches respectively.

4.1 Peptide-based vaccines

The definition of peptide-based vaccines in this discussion is generalized, instead of peptide vaccines-specific. We consider all vaccines that directly deliver TAA peptide as peptide-based vaccines; these include the TAA peptide itself, directly delivering naked DNA or RNA encoding TAAs. and delivering DNA or RNA through recombinant viral vectors[4, 5].

The usage of peptides in therapeutic vaccines is based fundamentally on the observation that short peptide segments (8-10 amino acids) fit into a groove in the MHC molecule. Obviously, individual peptides used in the situation have to be presented by appropriately matched MHC molecules, which limit their application to HLA haplotype, unmatched patients. Numerous peptide vaccines have been used in several clinical trials. Appropriate epitope enhancement and adjuvants are very important because they can greatly enhance the efficacy of peptide vaccines in clinical trials. They can increase the affinity of a peptide for binding MHC molecules, triggering TCR signal transduction, and inducing CD4+and CD8+T cell responses[22].

Naked DNA vaccines can be packed into bacterial plasmids cheaply and conveniently. Following delivery, the host cell that has taken up the DNA synthesizes the encoded tumor antigen, which is then presented to T cells or recognized by B cells by DC. The major advantage of DNA vaccines is that they boost both CD4+and CD8+T cell responses, as well as humoral immune responses. DNA vaccines are being developed to induce immunity against oncogenic viruses. The most successful and well-known DNA vaccine is the human papillomavirus (HPV) vaccine. It has been commercially popularized and is being widely used in the prevention of cervical carcinoma[23]. As a more recent approach, the usage of RNA instead of DNA can avoid the integration problem[24].

Viral vectors can also be utilized to transfer DNA and RNA encoding tumor antigens into host cells. As discussed earlier, viral vectors are an attractive choice for antigen delivery because they can mimic a natural progress of infection and thus provide the necessary danger signals to activate antigen presenting cells (APCs). During the past 20 years, various viral vectors, such as modified vaccinia virus Ankara (MVA)[ 25], fowlpox[26], canarypox[27], and recombinant adenovirus[28], have been used in trials.

4.2 DC-based vaccines

DCs are the sentinels of the host immune system. After immature DCs encounter pathogens, they are very effective in antigen processing. Inflammatory mediators in the antigenic environment subsequently prompt DCs maturation. Mature DCs then travel to lymph nodes, where they activate antigen-specific T cells. Therefore, the demonstration of how DCs mature and activate T cell response in cancer patients suggests a rationale of using TAA-loaded DCs as anti-tumor vaccines[4]. To date, numerous DC-based vaccines have been developedm including DCs pulsed with tumor peptides directly, DCs loaded with mRNA or DNA encoding TAAs of interest, and DCs transfected by viral vectors containing the expressing cassettes of tumor antigens.

4.3 Modified tumor cell vaccines

Actually, the richest source of TAAs is the tumor itself. Since the 1990s[29], investigators have attempted to use irradiated autologous tumor cells transduced to express immuno-stimulatory molecules as anti-tumor vaccines in murine models. Among the stimulatory molecules studied, GM-CSF (an activator of APCs) appeared to have been the most potent, long-lasting and effective. The GVAX vaccine, containing whole tumor cells genetically modified by an adenovirus to secret GM-CSF, has been tested successfully in a number of clinical trials[30, 31]. This vaccine can be well-tolerated and shows effective anti-tumor function in various cancers such as melanoma, lung cancers, prostate, and renal cancers[32]. In addition, some genetically altered autologous or allogenetic tumor cell vaccines expressing IL-2, IL-4 and,B7.1 have also been tested in clinical trials[33, 34].

Although cancer vaccines have been widely developed and provide reasonable hope in numerous clinical trials, objective tumor responses in humans, to date, have been disappointing[35]. The human immune system is very complex; strategies dealing with one aspect of the immune system may be insufficient to gain sufficient anti-tumor effects. A recent trend of cancer vaccines using immunogenetic approaches is a shift towards more complex or combined approaches. Brave et al[36]recently reported a phase II clinical trial combining 10 cocktail peptide vaccines targeting different tumor antigens (IDM-2101) to treat metastatic, non-small cell lung cancer patients. They found this cocktail vaccine to have been well-tolerated, and suggested this as evidence of its efficacy. In the future, novel strategies of cancer vaccines may be better established based on these new concepts, involving the stimulation of both tumor-specific immunity and tumor-induced tolerance inhibition[37].

5 Adoptive cell transfer (ACT) therapy

ACT-based immunotherapy was first described in 1988 by Rosenberg et al[38]. It is a treatment that utilizes a cancer patient's autologous immune cells with anti-tumor activity, which is expanded in vitro and infused back into the patient[39]. Currently, T lymphocytes, including both CD4+and CD8+T cells, are the main choices of ACT.

5.1 Cell sources of ACT therapy

Conventional ACT therapy has usually used some tumor infiltration lymphocytes (TIL) in the early stages of the 1990s. Rosenberg et al initially described that the cultures derived from TILs were expanded in vitro with IL-2 and displayed marked anti-tumor effect in both murine models[40]and human cancers[41]. Initial clinical efforts with TIL were evaluated in a study using autologous TIL plus IL-2 in the treatment of 86 patients with metastatic melanoma[42]. They demonstrated that this approach was safe and illustrated the potential value of immune lymphocytes for treating patients with melanoma. The other important source of ACT is peripheral blood lymphocytes (PBL), which are easier to isolate from patients than TIL. In several phase I clinical trials, CD8+T cell clones targeting TAAs, such as MART-1, gp100, were derived from PBL and were expanded in vitro for treatment of cancer patients[43, 44]. Most clinical trials used to utilize cytotoxic T cells as regimen. Noticeably, a recent clinical case reported by Yee et al[45]in 2008 demonstrated that a CD4+T cell clone targeting NY-ESO-1 peptide were derived from a patient's PBL with metastatic melanoma and expanded in vitro to a 5 billion count. These anti-tumor specific CD4+T cells were infused back into the patient, who subsequently achieved a long-term complete remission. The NY-ESO-1 specific CD4+T cells were still detectable until 3 months post-infusion, in contrast to transferred CD8+T cells, which survive only briefly (<20 days) in vivo, in the absence of exogenous cytokines[46]. This finding suggests that CD4+T cells are more efficient than CD8+CTLs, and that it may be necessary to combine helper T cells with cytotoxic T cells as regimens in the future.

5.2 TCR gene transfer

One of the notable deficits of traditional ACT is the rapid disappearance of anti-tumor specific T cell clones from peripheral circulation after transfer. Dudley et al[47]reported that transferred cells rapidly declined to undetectable levels at 2 weeks after infusion. Moreover, tumor-reactive T cell clones can only be achieved in partial patients, which make up approximately 50% of patients with melanoma. A durable alternative method for the production of tumor-reactive cells relies on genetic engineering to transfer tumor antigen specificity to non-specific patients' T cell. By using viral vectors, it is now possible to transfer the genes of the alpha and beta chains of antigen-specific TCR into T cells, thus “arming” almost all harvested T cells with a strong weapon.

Bulk T cell populations are currently used in most cases of TCR gene transfer. Polyclonal activators, such as the anti-CD3 antibody, alone or in combination with anti-CD28 antibody, were usually used to trigger the proliferation of both CD4+and CD8+T cells. However, CD4+CD25+regulatory T cells (Treg) are usually triggered simultaneously, which is highly undesirable, since Treg can suppress the anti-tumor capacity of TCR gene modified T cells[48]. Therefore, depleting the CD4+CD25+regulatory T cell population of the host before TCR gene transfer may efficiently prevent the triggering of Treg proliferation.

Retroviral vectors are main tools used in the field of TCR gene transfer. In 2002, retroviral gene transfer was initially used to transduce PBL, derived from patients with melanoma, with the cassette encoding the alpha and betachains of a MART-1 peptide specific TCR (HLA-A*0201 restriction)[ 49]. The engineered T cells persisted, and the two patients with the highest levels of circulating anti-melanoma T cells showed objective regression of metastaic lesions and remained in remission 18 months after treatment. Although TCR gene transfer protocols using retroviral vectors can obtain efficient infection of T cells, it does not lead to efficient or appropriate TCR expression on the surface of infected T cells[48]. Alpha and betachains of the introduced TCR could usually "mispair" with beta and alphachains of the endogenous TCR. A number of strategies have recently been employed to overcome this issue. Hybrid TCRs have been exploited to incorporate human variable regions with murine constant regions, since the murine TCRs express more efficiently on human T cell surfaces[50]. These hybrid TCRs display superior cell surface expression and biological activity[50].

Generally speaking, TCR gene transfer opened a new window for cancer immunotherapy. It provides the possibility of employing anti-tumor specific T cell therapy to a much wider patient population. However, few TCR can serve a large patient population, since the genotype of the human HLA allele is varied, and TCR gene transfer is HLA-restriction defined. In the near future, it will be necessary to perform more trials with TCRs of different specificities, which will provide valuable information about the potential benefits and the risks of this approach.

6 Biosafety concerns of immuno-gene therapy

Biosafety is the biggest concerns of future clinical application of retroviral vector based gene therapy. Despite several retroviral gene therapy trials without serious adverse effects, the theoretical risk of insertional oncogenesis was notoriously realized in the X-linked SCID trial, in which two of the nine children cured developed leukemia within three years after treatment with a retroviral vector distinct from the lentiviral class; the leukemia was subsequently linked to vector-associated insertional oncogenesis. Accordingly, the risks of insertional oncogenesis and clonal outgrowth of modified cells have become a pressing question in the field of gene therapy[51].

Lentivirus gene therapy vectors may be safer than murine oncoretroviral-based (MLV) vectors recently used in the successful treatment of disease in three gene therapy trials to date, including the X-linked SCID trial mentioned above. Contrary to the MLV used in these trials, lentiviruses are not associated with oncogenesis[51, 52], and therefore may have a safety advantage over oncoretroviral gene therapy vectors. This is one of the significant technological advances of HIV-derived lentiviral gene therapy vectors.

7 Conclusion

Molecular biology has passed though an era of information explosion in the past two decades. Most medical research has been much more dependent on molecular biology techniques than before. In another words, state-of-the-art genetic engineering techniques have offered many more potent tools for investigators who are attempting to manipulate the immune response, so that they can modify or assemble receptors, proteins, and transductors of various cells according to their needs. Traditional cancer immunotherapy is now facing great challenges due to their poor precision and duration. Molecular biology techniques derived from gene-transfer-based strategies may be more valuable and efficient, so that these issues may be overcome. Of course, the development of immuno-gene therapy approaches for cancer are still in the early stages, and further intensive research and clinical trials are still needed for the optimization and evaluation of their benefits and risks.

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