Are Ovarian Cancer Stem Cells the Target for Innovative Immunotherapy?

IMMUNE SYSTEM AND IMMUNOTHERAPEUTIC STRATEGIES RELEVANT TO CSCs/OCSCs

Immune surveillance is the barrier against tumor initiation. CSCs can use immune evasion to grow, differentiate, spread, and generate primary lesion and metastatic lesion. Nowadays, many immune-suppressive molecules have been identified including programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD1-L1), transforming growth factor β (TGF-β), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), B- and T-lymphocyte attenuator, and CD200.^11,63 There is a hypothesis called “immunoediting,” which indicates that the immune system plays a dual contradictory role in cancer not only suppressing tumor growth but also promoting tumor outgrowth with the ability of host-protection and tumor promotion.^64,65 The innate mechanism underlying cancer and immune system needs more researches, and the role of CSCs in the immunoediting hypothesis has not been revealed yet. Because of the resistance to conventional therapeutic strategies of CSCs and the ability to recapitulate original tumor to be the source of recurrent tumor, the immunotherapy may be a processing way and has been partly proved in the clinic. There are many immunotherapeutic strategies targeting CSCs in various types of cancers, such as NK cell, cancer therapeutic vaccine, monoclonal antibody (mAb) immunotherapy, and blockade of immune checkpoints.

NK cells

NK cells can target and eliminate CSCs in an major histocompatibility complex (MHC) unrestricted fashion. CSCs can upregulate NK cell ligands (MICA and MICB) after treatment and conceal those ligands for immune evasion. NK cell immunotherapy targeting CSCs has been researched in many cancers, such as colorectal, glioblastoma, melanoma, and breast cancer.⁶⁶ A study reported that NK cells activated by interleukin-2 (IL-2) and IL-15 are effective for eliminating CD44⁺CD24⁻ human breast CSCs with the upregulation of NKG2D (the receptor on NK cell) ligands ULBP1, ULBP2, and MICA on these CSCs.⁶⁷

Cancer therapeutic vaccine

Cancer therapeutic vaccine requires the participation of innate and adaptive immunity of a patient by first stimulating the host by a tumor-specific antigen to activate large amounts of tumor antigen-specific cytotoxic T lymphocytes (CTLs) to remove tumor.⁶⁸ Cancer vaccine is superior to other therapy because the immunological memory will prevent cancer recurrence for a period. It has implication in the field of OCSCs therapy now. For example, Wu et al reported that the SKOV3 CD117⁺CD44⁺ CSC vaccine depressed ovarian cancer growth in xenograft mice. This base-CD177/CD44 vaccine can reduce the CD117⁺CD44⁺ CSC and ALDH1-positive cell populations in the immunized mice with enhanced serum interferon-γ (IFN-γ), decreased TGF-β levels, and increased cytotoxic activity of NK cells.⁶⁹ CD117, CD44, and ALDH are specific surface markers of many types of cancers which can be targeted in ovarian cancer therapy.

Monoclonal antibody

Monoclonal antibody (mAb) immunotherapy uses antigen–antibody response to exploit the immunocompetence of the host to remove the targeted cells, which resulted in mAb is widely detected for decades. The mechanism underlying it is to activate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, inhibit receptor-mediated signaling, prime antigen-presenting cells, effector and memory T cells.⁷⁰ Many preclinical and clinical research studies have been exploring this therapy targeting CSCs, considering ovarian cancer is an aggressive malignancy, and mAbs targeting OCSCs are under intense scrutiny. For example, a study has examined the reactivity of anti-CD133 Mab CC188 to CD133⁺ ovarian cancer cells which are believed to be OCSCs by using immunofluorescence staining methods and tissue microarray technique, and the results showed that Mab CC188-based imaging and therapeutic reagents may provide a promising method to detect ovarian tumors in an earlier stage and also in treatment.⁷¹ Besides mAbs, antibody constructs that are designed flexibly can induce a more effective outcome, for instance, Catumaxomab, a trifunctional antibody construct consisting of two half antibodies, each with one light and one heavy chain that originate from parental mouse IgG2a and rat IgG2b isotypes which can bind to antigens CD133 and EpCAM. In a Phase II/III clinical study of patients suffering malignant ascites with advanced ovarian, gastric, pancreatic cancer and other origins, the results showed that CD133⁺/EpCAM+ CSCs are vanished in the catumaxomab samples.⁷²

Blockade of immune checkpoints

Immune checkpoints are cell surface molecules that are crucial for maintaining self-tolerance and regulating physiological immune responses by mediating co-inhibitory signaling pathways, such as CTLA-4, PD-1, and PD-L1 the antibodies of which can be blockers of immunosuppression.⁷³ These regulatory pathways result in a suppressive tumor microenvironment and tumor cell niche which is entrenched especially in CSCs that protect cancer cells.⁷⁴ Nowadays, clinical trials of antibodies of immune checkpoints in ovarian cancer have been partly completed and partly ongoing, for example, ipilimumab (anti-CTLA-4 antibody), nivolumab (anti-PD-1 antibody), avelumab (anti-PD-L1 antibody), which are clearly effective but still limited especially with some adverse effects, and those are reviewed by Mittica et al.⁷⁵ Recently, MYC, an oncogene code for a transcription factor which regulates the expression of CD47 (innate immune regulator and discussed above because of its role in cancer evading immune surveillance in the CSC tumorigenesis) and PD-L1 (adaptive immune checkpoint) are inactivated to enhance the antitumor immune response, and it will be an important implication in immunotherapy.⁷⁶ Until now, immune checkpoints in CSCs/OCSCs carcinogenesis are still interesting to be studied.

All those immunotherapeutic strategies have been shown to be effective in ovarian cancer to some extent, while there is a long way to go for an apparent cure in clinical practice. Nowadays, adoptive cellular immunotherapy has shed light on both hematologic and solid cancers. CAR-T cells have been a relatively successful method to mediate tumor rejection, especially in B-cell malignancies targeting CD19.⁷⁷ In the present study, the basic conception of CAR, the current progression, and the possibility to employ this method in OCSCs are reviewed.

THE PROMISING IMMUNOTHERAPY CARs TARGETING OCSCs

CARs are recombinant receptors for specific antigen, which can reprogram the specificity and function of T lymphocytes or other immune cells such as NK cells.^8,78 The basic approach of CAR is to redirect tumor targeted T cells, which is called CAR-T cells (CAR-T lymphocyte), bypassing major histocompatibility complex inducing immune reactions and leading to cytotoxicity. As an adoptive transfer immunotherapy, CAR-T has three primary components including the extracellular domain, transmembrane, and intracellular domain. The primary feature of the extracellular domain is the single-chain variable fragment (scFv) region which is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody specific for the antigen. The hinge/spacer and transmembrane domains are usually CD8α or CD28, which links the extramembrane domain and intramembrane and may contribute to the interaction with antigen and recruitment of signals resulting in immune activation of CAR.⁷⁹ Transmembrane domains can influence the immunogenicity depending on its length or flexibility.⁸⁰ The intracellular domain is to lead to T-cell activation.⁸¹ It comprises CD3ζ chain and is often incorporated with costimulatory molecules that include CD27, CD28, CD134 (OX-40), and CD137 (4-1BB). These costimulatory molecules aid the signals that influence the proliferation and the persistence of the T cells.⁸² CAR-T cells are separated by generation as the field has progressed. The first generation of CAR-T cells include only CD3ζ as an intracellular signaling domain. The second generation modifies the surface of the CAR and includes an additional costimulatory molecule like CD28. The third generation is on the basement of the second to add multiple costimulatory molecules on CAR such as OX-40/4-1BB (CD134/CD137). The fourth generation is significantly different in its function of releasing cytokines (IL-12) and is also known as T-cell redirected universal cytokine killing (TRUCK) (Figure 1).⁸³

(To view a larger version of Figure 1, click here.)

The primary standard protocol of CAR-T design implemented from bench to bedside is of several steps. First, T cells are extracted from the patient. Second, the T cells that can recognize the specific antigen of the cancer cells are engineered in vitro and called CAR-T cells. Then, the CAR-T cells are cultured millions and billions fold. Next, these CAR-T cells are injected back into the patient. Finally, with the antitumor ability of the T cells, the patient is expected to be healed (Figure 2). In addition, many advanced reconstruction strategies have solved the branch problems of CAR-T cells. For example, the traditional autologous adoptive transfer strategy is not appropriate for patients with a distempered immune system to isolate enough T cells in both quantity and quality; hence, recently allogeneic adoptive cell transfer strategy is developing. The CAR-T cells with two genes (TRAC and B2M) disrupted can make allogeneic T cells (these T cells come from healthy donors) adapt in acceptors. Mutation in T-cell receptor α constant (TRAC) leads to loss of αβ TCR on T-cell surface to avoid graft-versus-host-disease. Destruction of B2M can interfere with the expression of HLA-Is.⁸⁴ Such gene reconstruction method enlarges the source of T cells for engineering. A study showed that disruption of endogenous PD1 pathway enhances the efficacy of gene disrupted allogeneic CAR-T cells by CRISPR/cas9 system, that is, triple simultaneous ablation of TCR, B2M, and PD1 of the engineered CAR-T cells improves antitumor ability.⁸⁵ Using CRISPR/Cas9 system to direct a CD19-specific CAR to TRAC locus results in uniform CAR expression in human peripheral blood T cells and enhances T-cell potency in a mouse model of acute lymphoblastic leukemia which has avoided random CAR transduction compared with conventional retrovirus or lentivirus transfection methods.⁸⁶ Nowadays, the success of CAR-T cells in hematological malignancies is inspiring, however is less in solid cancers, which is mainly due to the heterogeneity of a solid tumor and the complex protection of tumor microenvironment that can reduce T-cell trafficking or killing kinetics, loss of CAR expression, or exhaustion of CAR-T cells.⁸⁷

(To view a larger version of Figure 2, click here.)