Combined modality immunotherapy and chemotherapy: a new perspective

Abstract

The results of recent clinical trials have demonstrated that cancer vaccines continue to struggle to achieve tangible clinical benefits as monotherapy. Tumor-induced abnormalities in the immune system hamper anti-tumor T cell responses limiting the effectiveness of cancer immunotherapy. Recently, evidence has been mounting to suggest that immunotherapy has the possibility of achieving better success when used in combination with conventional chemotherapy. In clinical trials, immune responses elicited by cancer vaccines appear to augment the effectiveness of subsequent conventional cancer therapies.

Introduction

The treatment of advanced stage cancer remains a difficult clinical problem. Despite advances in the development of new chemotherapeutic drugs and improvements in radiation therapy, conventional cancer therapy often falls short of the goal of controlling tumor progression. The development of tumor cell resistance to chemotherapy and radiation therapy as well as the toxicity of these modalities limit the success of treatment and necessitate the search for better treatment options. Targeting tyrosine kinases and other components of signal transduction pathways has shown promising results. However, the genetic heterogeneity of cancer and the multifactorial nature of drug resistance limit the efficacy of these strategies. Cancer immunotherapy is an attractive approach to cancer treatment. Therapeutic cancer vaccines offer advantages over other cancer therapy strategies. Due to the high specificity for cancer cells the toxicity of vaccine treatment is low. The phase of cell cycle of cancer cells should not hinder recognition and elimination of tumor by cytotoxic T cells (CTL). These vaccines would trigger immunologic memory, thus theoretically would be very responsive to re-vaccination and provide long-lasting effect. Very importantly, the tumors that develop drug resistance would still be a suitable target for immunotherapy. A number of tumor-associated antigens (TAA) like TRP-2, p53, HER-2, Sca-2, survivin are effective T cell targets and can functionally mediate drug resistance [31, 54]. However, clinical trials performed in recent years demonstrated a lack of clinical efficacy [43]. It appears that cancer immunotherapy is faced with a number of challenges. They include the ability of vaccination to generate potent immune responses given the presence of numerous immunosuppressive factors, the ability of cytotoxic T cells to penetrate tumor parenchyma and recognize tumor-associated antigen, the correct choice of antigen for immunization, etc. It has become apparent that therapeutic cancer vaccines given as a single agent will not likely produce substantial clinical benefits. An emerging strategy is one whereby multiple pathways of tumor cell survival and drug resistance are targeted by using immunotherapy in combination with chemotherapy or radiation therapy [15, 25].

Small-cell lung cancer

Lung cancer is the leading cause of cancer death for both men and women. More people die of lung cancer than colon, breast and prostate cancers combined. In 2007 alone, there were 213,380 new cases of lung cancer. About 15–20% of all lung cancers are small-cell lung cancer (SCLC). Diagnosis of extensive stage (ES) disease comprises approximately two-thirds of new SCLC cases, and the median survival of these patients is only 2–4 months if untreated, and survival increases to 6–8 months with chemotherapy. This disease is very responsive to first line chemotherapy with response rates of greater than 50% routinely observed. However, these responses almost invariably are short-lived and disease recurrence in ES patients occurs frequently. After relapse or failure to respond to chemotherapy, patients generally succumb to their disease within a few months [45]. Treatment of patients with relapsed SCLC is especially challenging if patients are platinum-resistant (i.e., disease progression occurs within 3 months of completion of a platinum containing regimen), where median survival ranges from 3.7 to 4.7 months.

Current treatments for SCLC include combination chemotherapeutic regimens of agents that alone have anti-cancer activities. Despite these therapies, response rates to second line chemotherapy in platinum-resistant patients are 3.7–6.4%. Treatment options extend life only by months and a mere 1–2% of patients with extensive stage SCLC survive 5 years after the diagnosis.

In SCLC, paclitaxel is primarily considered a second line therapy after the failure of platinum-based treatment regimens. Paclitaxel kills tumor cells by several different mechanisms. The drug is known to inhibit cell growth by promoting microtubule assembly which inhibits disassembly, arresting cells in G1 and G2/M phases of the cell cycle [2, 11, 53, 57]. Paclitaxel can also promote apoptosis of cancer cells; however, the exact mechanism of this activity has not been fully elucidated and is somewhat controversial and highly dependent on the target cell type and the concentration of the drug in a given assay. It has been reported that paclitaxel exerts an effect on both caspase-dependent and independent apoptosis pathways [18, 19, 39, 41, 56] and also regulates the expression of apoptosis-related proteins such as Bcl family members, Bad, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, namely death receptors 4 and 5 (DR4 and DR5, respectively) [6, 24, 37].

Studies have suggested that tumor cells develop various mechanisms to avoid immune-mediated cytolysis. Twenty-two years ago Doyle et al. showed that SCLC cells can have decreased expression of MHC class I and β2 microglobulin (β2M) at both the mRNA and protein level, which could dramatically limit the expression of TAA on the surface of the tumor cells [12]. More recent studies show that there is a decrease in Fas expression on SCLC [47, 52] and that some lung cancer cells produce a soluble decoy receptor, DcR3, that mimics Fas and binds Fas L [42]. DcR3 could act to bind FasL on CTL and obstruct the interaction between the CTL ligand and the tumor cell receptor, allowing the tumor to escape T cell-mediated killing. Another mechanism of immune evasion involves the silencing or loss of DR5 and/or caspase 8 expression in SCLC [17, 47]. It has also recently been shown that there is an upregulation of Bcl-2 in SCLC and that this increased expression potentiates chemotherapy resistance in SCLC cell lines [44], possibly by blocking TRAIL-induced apoptosis [49]. Each of these described mechanisms relate directly to pathways utilized by CTLs to recognize and induce apoptosis of tumor cells.

p53 as a target for SCLC immunotherapy

The ideal TAA would not only be expressed in a significant proportion of cancer patients, but survival of tumor cells would require the continued presence of the TAA. With this approach the emergence of antigen-loss variants would not be possible. The tumor suppressor gene, p53, has many features of an ideal TAA. It plays an essential role as a regulator of cell growth and differentiation, however p53 is mutated in approximately 90% of SCLC [7]. The p53 protein is pivotal in the maintenance of genetic integrity damage, and alterations in the p53 pathway, including mutations in the p53 gene, greatly increase the probability of tumor formation. Wild-type p53 protein has a brief half-life and is therefore present in very low levels in normal cells, whereas mutant p53 has a significantly prolonged half-life and is present in much greater quantities in tumor cells. This differential level of expression of a protein between normal and malignant cells could provide a basis for immunotherapy. Survival of tumor cells with mutant p53 largely depends on the the presence of mutant p53. Pre-clinical studies using mouse models and an ex vivo human culture model [1] have demonstrated that the induction of an anti-p53 CTL response selectively kills tumor cells and spares normal cells. Our laboratory has previously observed that immunizations with activated DCs transduced with full-length murine wild-type p53 (Ad-p53 DC) protected mice against challenge with MethA sarcoma cell bearing p53 gene mutations [34]. Immunization with Ad-p53 provided complete tumor protection in 85% of mice challenged with tumor cells expression human mutant p53 [20]. Significantly, we were able to demonstrate that immunization with Ad-p53 DCs generated CTL responses against tumor cells with human wild-type p53. To test the hypothesis that wild-type p53 protein can be used for the generation of an immune response against tumor cells over-expressing p53 we obtained T cells from nine HLA-A2 positive cancer patients. Stimulation of T cells with Ad-p53 DCs resulted in generation of CTLs specific for p53-derived peptide, demonstrating the induction of a specific anti-tumor immune response [35].

With these encouraging results, our group conducted a clinical trial to test the safety and efficacy of a DC vaccine using p53 as a tumor antigen in SCLC patients. The rationale for using p53 as a tumor antigen is that the p53 mutation in SCLC produces a non-functional but stable p53 protein that is markedly over expressed. We sought to exploit this differential expression between tumor cells and normal cells. Adverse events associated with the vaccine administration were mild and infrequent and the vaccine was well tolerated. The majority of patients (57.1%) were observed to have p53-specific CTL responses and two patients demonstrated a clinical response to vaccination [3]. A high rate (61.9%) of objective clinical responses to chemotherapy, particularly paclitaxel, that immediately followed vaccination was observed and correlated positively with p53 responders. Since the typical response rate to second line therapy in SCLC is less than 20%, this was a remarkable observation [3]. The median overall survival of all the patients was 11.9 months from the time of the first vaccination with eleven out of the 29 patients surviving a year after administration of the first vaccine giving a 38.1% survival rate [3].

We also evaluated the correlation between the development of p53 specific T cell responses after immunization and the clinical response to subsequent chemotherapy. Chemotherapy in most of the patients under evaluation started 3–4 weeks after the end of vaccinations. An impressive 75% of the patients who had a positive immune response to the vaccinations developed a complete or partial response to second line chemotherapy as compared to 30% of patients who had no detectable immunologic response. The patients with a positive immunologic response to the vaccine also showed overall improvement in survival as compared to the non-responders [3]. This study suggests a synergistic effect of immune and chemotherapy. It also shows that after treatment with chemotherapy, patients lose the immune response induced by prior immunotherapy. However the period of time between the vaccination and chemotherapy was sufficient for CTLs to produce anti-tumor effects. It appears that timing is essential for the synergistic effect of combining chemotherapy and immunotherapy to be effective.

Potential mechanisms of combined effect of immunotherapy and chemotherapy

In addition to our observations, the synergistic activity of combinations of immunotherapy and chemotherapy is supported by recent clinical and pre-clinical studies [9]. Cancer patients, who developed immunity to CYP1B1 after treatment with a cytochrome P450 1B1 vaccine had an unexpectedly high response to subsequent salvage therapy [16]. Arlen et al. immunized hormone refractory prostate cancer patients with recombinant viral vectors containing the PSA and B7.1 genes. Docetaxel was given at the time of progression. The investigators observed that docetaxel could be administered safely with immunotherapy without inhibiting vaccine specific T cell responses. Patients who received the vaccine followed by docetaxel had a longer time to progression than patients who received docetaxel alone [5]. In a review of three prostate cancer vaccine trials, researchers from National Cancer Institute (NCI) offer evidence that patients who receive cancer vaccines respond better to subsequent chemotherapy than those who do not [46]. Similar results were obtained in patients with glioblastoma. Wheeler et al. treated patients with an autologous tumor-pulsed DC vaccine [55] Chemotherapy given after vaccine appeared to be synergistic. A retrospective analysis demonstrated an apparent survival advantage for patients treated with the vaccine followed by chemotherapy as compared to patients treated with chemotherapy alone. All of these reports support the concept that utilization of immunotherapy to sensitize tumor cells can substantially enhance the effect of chemotherapy.

In mice, it was demonstrated that chemotherapy drugs (cyclophosphamide, doxorubicin, and paclitaxel) can enhance the anti-tumor effect of granulocyte/macrophage-colony stimulating factor-secreting cell-based vaccines in HER-2/neu mice [26]. It was also reported that doxorubicin is directly responsible for inducing an immunologic response to tumors in mice. Caspase inhibitors suppressed the doxorubicin-induced immunologic eradication of the tumor cells, although the cells still died as a result of treatment [8]. This indicates that caspases are likely involved in the regulation of the immunogenicity of the tumor. Another study has demonstrated that a combination of chemotherapy and cancer vaccines can efficiently break self-tolerance and induce anti-tumor immunity in a tolerogenic murine tumor model. Using a Her-2/neu expressing murine tumor model, Her-2/neu- or α-GalCer-loaded DC-based vaccines were most effective when given in combination with gemcitabine chemotherapy [22]. Indoleamine 2,3-dioxygenase (IDO), a trytophan degrading enzyme is highly expressed in many cancers and myeloid cells recruited to tumors. IDO activity suppresses T cell proliferation and differentiation and is thought to be a fundamental immune escape mechanism for tumor cells [14, 30] Immunotherapy with IDO inhibitors when administered in combination with chemotherapy to tumor-bearing mice was synergistic in producing tumor regressions [29].

The mechanisms responsible for this observed improvement in immunotherapy with chemotherapy remain mostly unknown and need to be explored. There are two general categories of mechanisms that could possibly be responsible: systemic factors and local tumor microenvironment factors.

Examples of possible systemic effects include:

• Elimination of cells with immunosuppressive activity such as myeloid-derived suppressor cells, and regulatory T cells.

• Non-specific activation of antigen-presenting cells (taxol, for example, can bind TLR).

• Improved cross-presentation of tumor antigens.

• Lymphopenia induction and resultant homeostatic T cell proliferation.

Examples of possible local effects within the tumor include:

• Disruption of tumor stroma that results in improved penetration of CTLs into the tumor site.

• Decreased local suppressive activity of tumor cells via mechanisms involving for example, program death ligands (PDL), indoleamine 2,3-dioxygenase (IDO), or immunosuppressive cytokines.

• Increased permeability of tumor cells to CTL-derived granzymes.

• Increased expression of tumor associated antigens by tumor cells (for instance p53) that could make them more available for targeting by CTLs.

• Up-regulation of Fas (and other death receptors) on tumor cells, or FasL on CTLs.

• Synergistic effect on caspase 3 activation between chemotherapeutics, granzymes, and Fas.

There is published data supporting at least some of these mechanisms. Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape by suppressing T cell responses. The major functional characteristics of these cells are increased arginase activity and production of reactive oxygen species (ROS). In a clinical trial involving patients with renal carcinoma, we found that treatment with all-trans retinoic acid (ATRA) dramatically reduced the number of MDSC, improved the dendritic cell (DC)/MDSC cell ratio, DC function, and antigen-specific T cell responses [28]. Further studies using combination therapy with vaccines and ATRA in mouse tumor models showed that a decrease in the number of MDSC could dramatically improve anti-tumor immune responses and enhance the effect of vaccination [23]. Furthermore, we recently found that ATRA neutralizes the high ROS production in MDSC by specific up-regulation of glutathione synthase and accumulation of glutathione [33]. Cytotoxic chemotherapy may also improve immunotherapy in clinical trials by reducing the negative influence of MDSC. The treatment of tumor-bearing mice with gemcitabine has been shown to substantially reduce the numbers of MDSC and improve anti-tumor immune responses [22, 50]. Similar results were obtained in pre-clinical studies of amino-biphosphonates, which were able to reduce MDSC expansion both in bone marrow and peripheral blood. By reducing MDSC, amino-biphosphonates overcame tumor-induced immune suppression and improved the generation and maintenance of anti-tumor immune responses induced by immunization against p185/HER-2 [27].

Regulatory T cells (Tregs) are important negative regulators of immune responses in cancer [21]. Elimination of Tregs in combination with vaccination may dramatically improve immune responses even to poorly immunogenic tumors [32]. It is well established that chemotherapy can dramatically reduced the presence of these cells [40]. In our vaccine study involving patients with SCLC we did not detect the presence of Tregs in patients after completion of first line platinum-based chemotherapy [3]. This could partially explain the relatively high frequency of immune responses to subsequent vaccination with the DC-p53 vaccine. Therefore elimination of regulatory T cells may be an additional mechanism of the potential synergistic effect of immunotherapy and chemotherapy.

One possible effect of chemotherapy could be in creating a more targetable tumor cell by up-regulating TAA expression and/or tumor-associated death receptors Tumor cell death induced by radiation or chemotherapy promotes immune responses by enhancing cross-presentation of tumor antigens in a toll-like receptor 4 (TLR4)-dependent manner. This is a consequence of tumor cells dying after exposure to chemotherapy releasing the TLR4 ligand HMGB1 [4]. Recent studies have demonstrated that chemotherapy with anthracyclins may generate immunogeneic epitopes in tumor cells via involvement of calreticulin translocation [38]. If chemotherapy is combined with DC administration, this can help to develop potent anti-tumor immune responses. The chemotherapeutic drug Paclitaxel augments the efficacy of immune priming by binding to toll-like receptor on the surface of immature dendritic cells, facilitating maturation and maximizing the extent of dendritic cell activation [51, 58]. We have seen that a combination of γ-irradiation and DC administration produces a potent anti-tumor response in mice bearing MethA sarcoma and C3 tumors [36].

Chemotherapy can improve the penetration of CTLs into the tumor parenchyma [10]. It is well known that paclitaxel induces cytokine production patterns typical of the T helper type I phenotype, thereby promoting effective CTL responses. 5′-Aza-2′-deoxycytidine can re-induce the expression of MHC-I molecules on tumor cells in vitro thereby restoring melanoma- and renal cell carcinoma-specific CTL activity [13].

Chemotherapy may also improve immune-mediated destruction of tumors by facilitating the induction of an immune response against tumor stromal elements. Recent pre-clinical studies have suggested that targeting tumor stroma as immunotherapeutic strategy may be very effective [59, 60].

CTLs kill target cells via release of perforin and granzymes, primarily granzyme B and/or induce apoptosis of target cells via Fas–FasL interaction. Paclitaxel is known to up-regulate the expression of Fas on the surface of tumor cells, resulting in an increase in the Fas–FasL interaction [48]. The hypothesis that paclitaxel may increase the sensitivity of tumor cells to CTLs via increased permeability of tumor cell membranes to granzymes and/or via increased expression of Fas on the surface of tumor cells bears further investigation. It is also of interest to evaluate if chemotherapy has any effect on the apoptosis-inducing phenotype of the immune cells that mediate the response, specifically CTL.

Conclusions

Recent evidence clearly suggests that immunotherapy can be effectively combined with chemotherapy in the treatment of cancer. Most importantly, this combination may provide substantial clinical benefits for patients with advanced disease. The precise mechanisms of the synergistic effect of these modalities have not yet been completely defined. However, it is likely that multiple mechanisms are involved. Intensive studies that are currently being conducted by a number of groups will undoubtedly help to clarify the nature of this phenomenon. It appears that the clinical success of combination therapy will depend on careful selection of different treatment modalities, the dosage of drugs, and importantly, the timing of combination therapies.