Induced pluripotent stem cells as a novel cancer vaccine

Abstract

Introduction:

Although many current cancer therapies are effective, the mortality rate globally is unacceptably high. Cancer remains the second leading cause of death worldwide after heart disease and has caused nearly 10 million deaths in 2018. Additionally, current preventive therapies for cancer are underdeveloped, undermining the quality of life of high-risk individuals. Therefore, new treatment options for targeting cancer are urgently needed. In a recent study, researchers adopted an autologous iPSC-based vaccine to present a broad spectrum of tumor antigens to the immune system and succeeded in orchestrating a strong prophylactic immunity towards multiple types of cancer in mice.

Areas covered:

In this review, we provide an overview of how cancer develops, the role of immune surveillance in cancer progression, the current status and challenges of cancer immunotherapy as well as the genetic overlap between pluripotent stem cells and cancer cells. Finally, we discuss the rationale for an autologous iPSC-based vaccine and its applications in murine cancer models.

Expert opinion:

The autologous iPSC-based vaccine is a promising preventive and therapeutic strategy for fighting various types of cancers. Continuing efforts and clinical/translational follow-up studies may bring an autologous iPSC-based cancer vaccination approach from bench to bedside.

1. Introduction

1.1. What do we know about cancer

Cancer refers to an abnormal and uncontrollable cell growth that invades the surrounding or distant tissues and leads to eventual mortality if left untreated [1]. Over the past two decades, landmark discoveries in cancer research have stabilized or reduced the death rates of certain types of cancer, but the number of total cancer cases and associated fatality will still continue to climb in the foreseeable future [2]. In 2019, an estimated 1,762,450 new cancer cases will be diagnosed and 606,880 people will die from cancer in the United States alone [2,3]. The lack of effective cancer prevention strategies and therapies for late-stage cancer patients greatly impedes the goal of ending cancer.

Cancer cells arise from the accumulation of somatic genetic alterations (in some cases superposed on a background of germline cancer-predisposing mutation). Currently, there are two popular models to explain the tumor initiation and progression in cancer. The first is the clonal evolution model, which posits that cancer originates from the same set of cells that went through a series of genetic and epigenetic changes leading to the natural selection of the most robust cell clones [4]. The second model follows the cancer stem cell theory, which hypothesizes that cancer cells are composed of a majority of cells with no or little cancerous capacities but also a small stem cell subpopulation that is highly tumorigenic to maintain the tumor immortality [4]. Irrespective of the ongoing debates over the two models, cancer cells have indeed shown some stem cell-like characters as illustrated by the resemblance to oncofetal antigens expressed by cancer cells and stem cells as well as the unceasing replication ability and high migration potential seen in both types of cells [5–9]. Furthermore, like stem cells, immature cancer cells have the capacity to differentiate into other cell types during metastasis [8,10,11]. Therefore, specifically targeting cancer stemness has also become a focus of current cancer research. Since genomic instability alone may not be sufficient to trigger cancer or promote its progression, to dissect the crucial steps of cancer development, Weinberg and Hanahan described the hallmarks of cancer that contribute to cancer malignancies [12]. Briefly, cancer cells can acquire many unique features to support their continuous division, including the abilities to sustain their growth signals, escape the common cell death pathways, hijack the cell cycle machinery, induce angiogenesis, and evade the host’s defense mechanisms [12,13]. Hence, many basic and clinical studies have attempted to attack these critical cancer hallmarks hoping to slow or stop cancer progression [14–16]. Among the most promising approaches is the idea of re-stimulating the immune system to eliminate cancer cells that are known to express tumor-associated or tumor-specific antigens, which has made cancer immunotherapy a hot area of active research [17].

2. Immune surveillance and cancer immunotherapy

The notion of immune surveillance to prevent cancer incidence was first postulated in 1909 by Paul Ehrlich, who believed that the immune system could detect and clear transformed cells before they expand and cause disorders clinically [18,19]. In the mid-20th century, with extensive research on transplantation models, the importance of the immune system became widely recognized [20,21]. Based on these findings, Burnet and Thomas proposed that the immune system can destroy cancerous/precancerous cells by sensing the tumor-specific antigens (TSAs) expressed only by tumors cells or tumor-associated antigens (TAAs) that are also seen on some somatic cells [21–23]. Subsequently, the cancer immune surveillance model was developed after antigen-presenting cells (APCs) were shown to cross-present antigens to T cells and activate them [24,25]. However, questions were raised about this model because tumors can occur even after a measurable immune response is mounted, and immune-compromised nude mice have been found not to incur a higher rate of developing spontaneous tumors than wild type mice [21,26,27]. To resolve these issues, the concept of tumor immunoediting seeks to explain the phase changes of the immune system throughout tumor initiation and progression. This theory proposes that after most tumor cells were recognized and destroyed during the immune elimination phases, some remaining cells become dormant, resulting in a temporary equilibrium between the tumor and the immune system [26,28]. As susceptible tumor cells are eliminated by the immune system, the more immune-resistant/immune-suppressed and TSA-hypermutated cells survive and evolve under selection pressure, which explains the immune surveillance failure. As a result, this leads to the last phase, immune escape, and new tumor cell populations emerge to resume cancer progression.

To deal with these challenges, a main goal of cancer immunotherapy is to restore full tumor immunogenicity and ultimately allow the immune system to eliminate all cancer cells. In principle, cancer immunotherapy has the potential to be superior to traditional treatment methods, which are disadvantaged by the lack of specificity and by causing non-selective damage to cells in general. In addition, unlike conventional approaches, cancer immunotherapy can create a sustained anti-cancer immunity by exploiting memory T cells.

Cancer immunotherapy can be roughly divided into five categories: monoclonal antibodies, chimeric antigen receptor (CAR)-T cell therapies, immune checkpoint inhibitors, interleukins and cytokines, as well as anti-cancer vaccines. As a form of monoclonal antibody, trastuzumab is the only approved adjuvant therapy for treating human epidermal growth factor receptor 2 (HER2) positive breast cancer [29]. Trastuzumab eliminates HER2-expressing cancer cells through various mechanisms such as preventing HER2 cleavage and its dimerization, thus suppressing the HER2 downstream signaling pathways [30]. Moreover, it elicits anti-tumor immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) [31]. Besides, trastuzumab also promotes the endocytic degradation of HER2 receptor, cell cycle arrest, and apoptosis [29,32]. However, an intractable obstacle for monoclonal antibody therapies is the identification and selection of the appropriate tumor antigens to target, which requires intensive studies on the involvement of the targeted antigen in both tumor growth and its roles in normal tissues as well [33]. A well-studied example of CAR-T cell therapy uses the engineered CD19-specific CAR T cells to attack CD19-positive cells, which has demonstrated efficiency in treating acute lymphoblastic leukemia (ALL) [34,35]. However, CAR-T cell treatment also faces the hurdle of having limited targets. Immunotherapies like interleukin-2 (IL-2) and interferon-gamma can broadly increase the immune response, but the lack of specificity and undesirable side effects have restricted their clinical application [36,37].

As a rapidly growing field, the potential of anti-cancer vaccines is attracting more and more attention. Depending on treatment needs, anti-cancer vaccines can either serve as a prophylactic to delay cancer onset or a therapeutic agent to target pre-existing malignancies [38]. Currently, progress has been made in prophylactic anti-cancer vaccines, with the two most well-known ones targeting hepatitis B virus (HBV) and human papillomavirus (HPV), respectively. Universal immunization for HBV infection has greatly reduced the hepatocellular carcinoma (HCC) incidences in countries with high HBV prevalence [39,40]. The commercially available vaccination for HPV could potentially prevent cervical cancer onset by 90% globally [41,42]. Additionally, VGX-3100, an HPV DNA plasmid therapeutic vaccine has achieved tumor regression in 48.2% of the vaccinated women with grade 2/3 cervical intraepithelial dysplasia in a phase 2b clinical trial [43,44]. Anti-cancer vaccines can be designed to treat one type or multiple types of cancer [38]. They can also exist in various forms. Using antigen vaccine formulated from tumor-specific antigens as an example, direct administration of this vaccine into the tumor site stimulates a specific immune reaction to eliminate tumors. In addition to the antigen epitope-based immunization, anti-cancer vaccines can also be given in a whole-cell format. Pretreating tumor cells with irradiation or freeze/thaw cycles will deplete their replication ability before in vivo delivery to generate immunity. It is worth mentioning that both the unmodified whole tumor cells and the processed tumor lysates/oncolysates, as well as genetically transduced cancer cells, can all be utilized to vaccinate the host [38]. Dendritic cells also occupy an important position in the anti-cancer vaccine field. Loading dendritic cells with tumor cells/lysates or innate immunity stimulators (CpG) has generated many exciting results, both in animal models and clinical studies [38,45,46]. Other anti-cancer vaccines such as DNA vaccines, viral vector-based vaccines, and anti-idiotype antibody-based vaccines have induced varying levels of anti-tumor immunity in preclinical research and even early-stage clinical trials [47]. However, to date, much of the clinical success for anti-cancer vaccines has been found in preventing viral-associated cancers. By comparison, non-viral cancers, especially solid tumors, are more refractory to the currently available vaccines [48]. Some common challenges faced by anti-cancer vaccines include: the high level of immune tolerance due to the shared self-antigens presented by both tumor cells and normal cells; the lack of immunogenicity in many types of tumor cell-based antigens; and, as in the case of the CAR-T cell therapy, the therapy-induced autoimmune toxicity also significantly limits the generalization of autologously based anti-cancer vaccines.

3. hESCs and iPSCs are immunogenic and genetically overlapped with cancer cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are cells that are capable of differentiating into any type of adult cells [49,50]. Owing to their pluripotency and capacity for self-replication, they are widely utilized in the field of disease modeling [49,51]. For instance, cardiac iPSCs have been used to characterize cardiomyopathies and conduct drug screening [52,53]. Another potentially important application of pluripotent stem cells is to promote anti-cancer immunity in host organisms. Dating back to the early twentieth century, German scientist Schöne reported that immunization of mice with fetal tissues led to the rejection of implanted tumors [8,54]. Adult cells are less immunogenic than their parental-undifferentiated stem cells. Stem cell HLA mismatching will provoke a strong immune response that further leads to rejection of tumor engraftments [55]. This phenomenon is not limited to allogenic transplantation, as even autologous iPSCs can incite an immune response due to differences in histocompatibility antigens [56]. Although the immunogenicity of stem cells has given clinicians ‘headaches’ in the field of regenerative medicine, it also opens a new avenue for stimulating the immune response towards other invading diseases like cancer. In 2011, several studies demonstrated that both hESCs and iPSCs share many tumor antigens with common human cancers (e.g. mammary carcinoma, myeloid leukemia, glioblastoma multiforme, prostate cancer, and pancreatic cancer), but not with healthy tissues [57–59]. In 2014, seminal work illustrated that undifferentiated mouse iPSCs could elicit a potent immune response following transplantation, whereas differentiated iPSC-derivatives lost the immunogenicity and led to induction of tolerance [60]. While hESCs and iPSCs share many similarities, single-cell analysis has revealed that iPSCs are more heterogeneous and pluripotent than hESCs [61], suggesting that iPSCs may be a better surrogate to prime the anti-tumor immune response than hESCs, because they not only bypass the ethical concerns associated with hESCs but also provide the host with APCs that have a broader collection of neoantigens. Besides directly using iPSCs to elicit an anti-cancer response, many studies have already explored the regeneration and expansion of immune cells from iPSCs, which are initially reprogrammed from specific T cell clones. The re-differentiated dendritic cells or invariant natural killer cells can maintain the equivalent anti-tumor cytotoxicity as the original T cell clones [62–65]. However, due to the limitation of current differentiation methods, the iPSCs-derived T cells have also been reported to have acquired some unconventional T cell features which deviates from the conventional T cells but more resembles γδT cells or innate lymphoid cells [63,66–68]. Collectively, aforementioned studies point to the prospect for utilizing pluripotent stem cells as a new cell source in cancer immunotherapy.

3.1. Autologous iPSC-based vaccine as a universal anti-cancer whole-cell vaccine in vivo

In 2018, utilizing an autologous iPSC-based vaccine to replace ESCs, Kooreman and colleagues were able to induce effective immunity in mice against multiple cancer types (Figure 1) [69]. The team first showed that both human and mouse iPSCs share similar gene expression profiles with the ESCs and cancer cells, but not with the normal cells, providing a rationale for using iPSCs as an alternative vaccination to boost host immunity. Mice were vaccinated either with PBS (vehicle) or irradiated autologous murine iPSCs plus adjuvant CpG (iPSC+CpG) for 4 weeks, followed by subcutaneous inoculation of murine breast cancer cells or melanoma cells. The 4-week iPSC+CpG vaccination increased the secretion of antibodies that preferentially bound to iPSCs and cancer cells than fibroblast cells [69,70]. At the end of the breast cancer experiments, tumor regression was observed in 7 out of 10 mice primed with the iPSC+CpG vaccine, and 2 surviving mice from the iPSC+CpG group established sustained immunity against breast tumor engraftment and detectable iPSCs up to 1 year after the initial vaccination.

Figure 1.

Schema showing that mice receiving different vaccine treatments had distinct outcomes after tumor implant. (a) Autologous iPSC vaccine significantly reduced tumor growth in syngeneic murine models. (b) Adoptive transfer of splenocytes isolated from iPSC+CpG vaccine-vaccinated mice slowed down tumor growth in unvaccinated tumor-bearing mice. (c) Injection of iPSC vaccines to tumor resected mice could prevent future cancer recurrence. (Adapted from Kooreman N … Wu JC. Cell Stem Cell, 2018)[69].

To confirm whether the observed anti-cancer immunity is iPSC-specific and to exclude confounding factors such as FBS containing media associated with cross-reactivity, mice were also primed with iPSC-derived endothelial cells as a control, but no serum IgG binding was detected when they were co-incubated with breast cancer cell lines in vitro. Further analysis on immune cell profiling revealed that the iPSC+CpG vaccine induces strong cancer-immunity by selectively upregulating mature APCs and increasing the percentages of effector/memory helper T cells and cytotoxic T cells, as well as decreasing the percentage of regulatory T cells (Tregs).

To determine whether the anti-cancer response of the iPSC+CpG-vaccinated mice can be attributed to the common epitopes between cancer cells and iPSCs, the researchers conducted two-way immunity experiments by either adoptively transferring iPSC+CpG-primed splenocytes to orthotopic breast tumor-bearing mice, or transplanting the tumor-experienced lymphocytes (TELs) from breast cancer mice to iPSC-inoculated non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice. In both cases, adoptive transfer of splenocytes or TELs isolated from iPSC+CpG-vaccinated mice was found to prevent tumor or teratoma engraftment (Figure 1). In addition, they also examined the tumor-infiltrating lymphocytes (TILs) in mice inoculated with mesothelioma after 4 weeks of vaccination. The results again substantiated the augmentation of CD4/CD8-positive T cells and reduction in Tregs in mice immunized with iPSC+CpG. Intracellular interleukin staining and TCR sequencing further revealed that the combination vaccination induced the generation of specific and diverse T cell clones that were not seen in the vehicle control group. Moreover, IL-2, IL-4, and IL-5 expressing T cells or B cells were linked to tumor regression. Finally, the systematic cytokine level was low in iPSC+CpG-vaccinated mice, which implies a lower risk in developing autoimmune disease.

Kooreman et al. then proceeded with a more clinically relevant approach by assessing the strength of the iPSC-based vaccine as an adjuvant therapy. In this scenario, the melanoma-bearing mice first went through tumor resection with microscopic (R1) or macroscopic (R2) residual tumor tissue left at the tumor-draining lymph nodes, followed by injection with PBS, CpG, or iPSC+CpG vaccine at the resection site. Encouragingly, tumor load shrinkage was observed in CpG and iPSC+CpG groups, but only mice receiving iPSC+CpG were less likely to have local or distant tumor relapse after primary tumor resection (Figure 1).

4. Conclusion

In summary, Kooreman and colleagues describe a new anti-cancer vaccine that is derived from autologous iPSCs. This whole cell-based immunotherapy can function both as a prophylactic vaccine and an adjuvant therapy when combined with other treatments to retard cancer growth in multiple mouse strains.

5. Expert opinion

Anti-cancer vaccines are not a new concept in immunotherapy field [71,72]; however, thus far Imlygic (Talimogene Laherparepvec) is the only FDA and European Medicines Agency (EMA) approved anti-cancer therapeutic vaccine that is being used in clinics and actively manufactured in market for treating advanced melanoma [73,74]. The tumor-attacking immunity of T cells after vaccination is significantly limited by the few antigens presented by conventional vaccines. Autologous iPSC-based anti-cancer vaccines differ from current cancer immunotherapies by shifting the focus from stimulating a known antigen-specific immune response to broadly activating diverse immune profiles from unknown antigens. In addition to being potentially free of the lengthy process needed for cancer neo-antigen identification and CAR-T cell generation that is associated with current cellular immunotherapy approaches, autologous iPSC-based vaccines can also circumvent conventional barriers and present both known and unknown antigens to the immune system [69,75,76]. In the future, early immunization of high-risk groups with iPSC-based vaccines may prime their immune systems with various types of tumor antigens, which will create a repertoire of memory cells capable of orchestrating tumor-specific immune responses when encountering cancer cells. Furthermore, for patients who have undergone surgical removal of their primary tumors, iPSC-based vaccines may be used as a powerful adjuvant therapy to strengthen immune surveillance and help prevent cancer relapse (Figure 2). Importantly, iPSCs are a new form of individualized cell-based medicine that can induce a more patient-specific anti-cancer response compared to currently available immunotherapies. Notably, a common challenge that impedes the clinical application of many cancer immunotherapies is toxicity, including the cytokine release syndrome induced by CAR-T cell therapy in patients, and autoimmunity induced by PD(L)-1 checkpoint inhibition [77]. Given that immune surveillance may promote tumor elimination at the expense of self-tissue targeting, cancer patients have been found to develop hypophysitis, adrenal insufficiency, and thyroiditis [78–81]. By comparison, mice receiving the iPSC-based vaccine produced only very low levels of systematic cytokines and had no signs of autoimmunity [69]. The autologous iPSCs can be easily generated by a single transfection of four Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) into somatic cells, and the 4-in-1 CoMiP reprogramming method used in this paper allows for the production of iPSC clones within 2 weeks [82]. More recently, researchers even proposed a simple design, low-cost device that can produce large quantities of highly pure iPSCs for clinical use in about 20 days [83]. A common concern of whole tumor cell-based vaccine is the tumorigenicity of these cells. In the case of iPSCs, they can induce the formation of teratoma if live cells were injected [84,85]. As shown by Kooreman et al., iPSCs were irradiated at a lethal dose prior to tumor injection to prevent cell replication in vivo [69]. Given these advances and anticipated future progress, it may be routinely feasible to generate personalized iPSC-based vaccines at an attenable cost in the future.

Figure 2.

Schematic flowchart demonstrating the timeline of using an iPSC vaccine to immunize people against different types of cancer. Briefly, (1) patient’s blood is drawn in hospital, which will be used to (2) isolate peripheral blood mononuclear cells (PBMCs) and (3) reprogrammed into induced pluripotent stem cells (iPSCs) with the introduction of four Yamanaka factors (Oct4, Klf4, Sox2, and c-Myc). (4) The resulting patient-derived iPSCs are sorted by anti-SSEA-1 (CD15) microbeads to ensure that only cells that are expressing SSEA-1, a key pluripotency cell marker are isolated. Then (5) cells are irradiated at lethal dose to deplete their replication ability but maintain the diverse tumor markers on their surface. (6) Combined with other immune adjuvants for example, CpG oligodeoxynucleotides (CpG-ODN), which stimulates toll-like receptor 9 on antigen-presenting cells, such as dendritic cells, B cells and macrophages, the iPSC vaccine can be administered to cancer patients or high-risk individuals to orchestrate a strong anti-cancer immune response towards multiple types of cancers.

One of the compelling questions that remained to be answered is whether it is possible to use allogenic iPSCs in cancer immunotherapy in addition to autologous iPSCs. A previous study using a semi-allogenic whole cell-based vaccine has worked successfully in a murine glioblastoma model [86]. On top of that, a newly published paper applying CRISPR technology to deplete major histocompatibility complex (MHC) class I and II genes while overexpressing CD47 could mask the allogenic iPSCs in the host immune system [87]. Thus, it is reasonable to hypothesize that an allogenic source of iPSCs could be applied to form the vaccine. Then a careful comparison of different sources of iPSC (both allogenic and autologous) may provide more information about the most ideal form of iPSC-vaccine towards cancer.

Although more in-depth characterizations are required, encouraging results on autologous iPSC-based vaccines have opened a new paradigm for research into personalized cancer prevention and treatment. Combined with other anti-cancer therapies, this new approach has the potential to be a significant breakthrough towards the eventual elimination of cancer.

Article highlights.

• Proposed models in the field explaining the initiation and propagation of cancer.

• The importance of immune surveillance in cancer development and a summary of major cancer immunotherapies.

• Immunogenicity of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) and their genetic similarities with cancer cells.

• Utilization of autologous iPSC-based vaccines to target murine tumors.

This box summarizes key points contained in the article.