Aptamer-Based Strategies To Boost Immunotherapy in TNBC Part 1

Simple Summary:

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor outcomes and limited therapeutic options. It is characterized by a more pronounced immunogenicity compared with other breast cancer subtypes, suggesting immunotherapy is a viable strategy. Aptamers are short oligonucleotides that, similar to antibodies, recognize their protein target with high specificity and affinity. However, compared with antibodies, they present several advantages in terms of size, production, modification, and stability, which make them excellent candidates for the development of novel targeted anticancer therapies. Recently, to restore an immunoreactive and anticancer tumor microenvironment, effective aptamer-based strategies have been developed. Here, we discuss the most recent approaches aimed at using aptamers to enhance or restore the anticancer immune response in TNBC.

Triple-negative breast cancer is a subtype of breast cancer that is difficult to treat and prone to recurrence and metastasis and is closely related to immunity.

Studies have found that triple-negative breast cancer is significantly different from other types of breast cancer at the immunological level. Triple-negative breast cancer has a high degree of immune cell infiltration and a strong inflammatory response, which may lead to increased difficulty in treatment and increased recurrence rates. In addition, certain protein molecules on the surface of triple-negative breast cancer may induce antigen presentation by the immune system, which further stimulates the immune response.

Therefore, triple-negative breast cancer research with immunotherapy as the main approach is currently underway. One of the research directions is to use immune monitoring to monitor the efficacy of immunotherapy and the immune status of patients; the other research direction is to develop specific immune cells, vaccines, and immunomodulatory drugs for triple-negative breast cancer to promote the role of immune system. From this, we know that immunity is very important. Cistanche can significantly improve immunity. The polysaccharides in meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance the bactericidal effect of immune cells.

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Abstract:

The immune system (IS) may play a crucial role in preventing tumor development and progression, leading, over the last years, to the development of effective cancer immunotherapies. Nevertheless, immune evasion, the capability of tumors to circumvent destructive host immunity, remains one of the main obstacles to overcome for maximizing treatment success. In this context, promising strategies aimed at reshaping the tumor immune microenvironment and promoting antitumor immunity are rapidly emerging. Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with poor outcomes, is highly immunogenic, suggesting immunotherapy is a viable strategy. 

As evidence of this, already, two immunotherapies have recently become the standard of care for patients with PD-L1 expressing tumors, which, however, represent a low percentage of patients, making more active immunotherapeutic approaches necessary. Aptamers are short, highly structured, single-stranded oligonucleotides that bind to their protein targets at high affinity and specificity. They are used for therapeutic purposes in the same way as monoclonal antibodies; thus, various aptamer-based strategies are being actively explored to stimulate the IS’s response against cancer cells. This review aims to discuss the potential of the recently reported aptamer-based approaches to boost the IS to fight TNBC.

Keywords:

immune system; immunotherapy; aptamer; TNBC; active cancer targeting.

1. Introduction

Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype that accounts for approximately 10–20% of all diagnosed BCs. It generally affects younger women, is more likely to recur, and is associated with a poorer overall survival rate compared with other BC subtypes [1,2]. The term TNBC refers to a heterogeneous group of tumors having different histological, genomic, and immunological characteristics and response to therapy, which have in common the lack of estrogen receptor, progesterone receptor, and epidermal growth factor receptor 2 (HER2); thus, TNBC patients are not eligible for hormone or anti-HER2 therapy [2,3].

Based on gene expression profiling, Lehman et al. [4] initially classified TNBC into six distinct molecular subtypes, namely basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR). Subsequently, the IM and MSL subtypes were removed from the initial classification because it was recognized that their transcriptomic features were not derived from tumor cells but from tumor-infiltrating lymphocytes (TILs) and tumor-associated stromal cells, respectively, abundantly present in the TNBC tumor microenvironment (TME) [5]. 

Another classification for TNBC was suggested by Burstein et al. [6], who confirmed four subtypes reported by Lehman: LAR, M, and two BL. Basal subtypes described by Burstein, based on immune signaling, are divided into basal-like immune-suppressed, which exhibits downregulation of B cell, T cell, and natural killer (NK) cell immune-regulating pathways and cytokine pathways, and basal-like immune-activated, which is characterized by upregulation of genes controlling B cell, T cell, and NK cell functions. Burstein and colleagues demonstrated that immunosuppressed and immunoactivated tumors have the worst and best prognoses, respectively. Both classifications highlight the immune system (IS) signature as an important prognostic factor in TNBC and a potential target in the treatment of some TNBC subtypes.

The limited options of molecular therapies and the highly heterogeneous nature of these tumors make TNBC the most challenging BC to treat. To date, the standard of care for both early and advanced TNBC remains chemotherapy, generally using combined regimens of taxane, anthracycline, cyclophosphamide, cisplatin, and fluorouracil [7,8]. Unfortunately, chemotherapy efficacy is limited by the high toxicity toward normal cells [9]. Moreover, after a brief initial response to treatment, TNBC tends to reappear in a more aggressive and chemoresistant form, showing a significantly high pathological remission rate [10]. Therefore, it is increasingly necessary to identify specific molecular signatures that can be targeted to establish new effective treatments for TNBC. Years of intense research into TNBC led to the identification of the first molecular therapies.

The first clinically validated biomarker for TNBC therapy was the BRCA mutational status. Up to 19% of TNBC patients carry mutations in genes encoding for BRCA1 and BRCA2, proteins involved in DNA double-strand break repair, resulting in sensitivity to poly-ADP-ribose polymerase (PARP) inhibitors [2,11]. In 2018, the Food and Drug Administration (FDA) approved two PARP inhibitors, Olaparib [12,13] and Talazoparib [14], as monotherapy for the treatment of patients with BRCA-mutated tumors. Currently, the efficacy of PARP inhibitors is also being investigated in BRCA wild-type TNBC [15].

TNBC is more immunogenic than other BC subtypes due to its high propensity to generate neoantigens that can be recognized as “nonself” by the adaptive IS [16] and the presence of immune cell infiltrates, which are responsible for developing antitumor immune responses [17,18]. In addition, approximately 20% of TNBC expresses anti-programmed cell death-ligand 1 (PD-L1) [19], a surface protein that binds programmed cell death protein 1 (PD-1) receptors on TILs, turning off their activity. In this way, the PD-1/PD-L1 axis induces an immunosuppressive TME responsible for tumor immune evasion. All these features are fundamental prerequisites for benefiting from immunotherapy. Indeed, in 2019, the FDA approved the first immunotherapy with anti-PDL1 Atezolizumab monoclonal antibody (mAb) to treat PD-L1-positive unresectable, locally advanced, and metastatic TNBC in combination with nab-paclitaxel [20,21]. 

In the following 2 years, Pembrolizumab, an anti-PD-1 mAb, was also approved by FDA in combination with standard chemotherapy: first, in 2020, for patients with locally recurrent inoperable or metastatic TNBC and later, in 2021, for newly diagnosed and operable early-stage TNBC by adding the antibody to both neoadjuvant and adjuvant chemotherapy [22]. However, the mentioned therapies are approved only for TNBC expressing high levels of PD-L1, making it necessary to find new strategies to expand the applications of immunotherapy for TNBC.

More recently, the FDA approved the first antibody-drug conjugate (ADC) as a second-line treatment for patients with unresectable locally advanced or metastatic TNBC who have received two or more prior systemic therapies, at least one of them for metastatic disease. 

The name of this ADC is Sacituzumab govitecan, which is composed of a humanized trophoblast cell surface antigen-2 antibody coupled via a linker to the SN-38 payload, the active metabolite of the topoisomerase 1 inhibitor irinotecan [23].

Oligonucleotide aptamers are highly selective compounds emerging as alternative or complement to mAbs for active cancer targeting, and the application of aptamers as innovative therapeutic agents in different human cancers, including TNBC, is increasing rapidly [24]. In this review, we will first discuss the features of aptamers against cancer-related biomarkers as innovative therapeutics. Then, we will focus on the recently emerged aptamer-based strategies that can prevent immune evasion and stimulate anticancer immune responses, discussing their potential to manage TNBC in the next future.

2. Aptamers for Targeted Cancer Therapy

Aptamers are synthetic, short, single-stranded DNA or RNA oligonucleotides that fold into unique three-dimensional (3D) structures interacting at high affinity and specificity with targets of different natures. Aptamers were first discovered in 1990 when two papers [25,26] described the in vitro SELEX (systematic evolution of ligands by exponential enrichment) technology for aptamer’s generation and introduced the term aptamer, which derives from the Latin word “Aptus” (to fit) and the ancient Greek word “meros” (part), thus meaning “fitting parts” (Figure 1).

Figure 1. Schematic representation of aptamer binding to its target and key steps of the SELEX method. (a) The aptamer adopts a 3D structure to bind to its target; (b) The SELEX starts with random libraries of ssDNA or RNA sequences and comprises reiterated rounds of binding to the target, partitioning of the target bound sequences from unbound and amplification of the bound sequences. Finally, the enriched library is analyzed by cloning and sequencing or, in the most recent approaches, high-affinity ligands are identified by next-generation sequencing (NGS) and bioinformatics. Created with BioRender.com (accessed on 2 March 2023).

The in vitro selection starts with the generation of a combinatorial library of oligonucleotides, each containing a central random sequence, approximately 20–100 nucleotides in length, flanked by two fixed regions necessary for primers annealing during amplification and in vitro transcription (to select RNA aptamers). The library is incubated with a target molecule, and a partitioning step is performed to separate oligonucleotides bound to the target from unbound nonspecific sequences. Targets for SELEX can include peptides, proteins, metabolites, carbohydrates, small organic molecules, other structured RNAs, and even whole cells or organisms, and different selection schemes that have been described so far adapted to the nature of the target [27–29]. Once eluted from the target, the sequences are directly amplified by PCR in the case of DNA-SELEX or first reverse-transcribed and then amplified in the case of RNA-SELEX. The amplified sequences are used to generate a new oligonucleotide library, which is subjected to a further round of selection. Repeating multiple rounds of incubation, partition, and amplification, this process leads to the generation of sequences with high affinity and specificity for the target (Figure 1).

Aptamers can be considered the nucleic acid version of protein antibodies because they share with them a high binding affinity and specificity for the target (Kd values, 10−8–10−12 M) and, analogous to antibodies, have a variety of applications as recognition elements, including therapeutics, biosensors, and diagnostics [30].

Aptamers discriminate among highly similar members of the same protein family, protein isoforms, and also proteins that differ in a single amino acid [27]. For example, by a contrast SELEX screening with agarose beads and magnetic beads coupled with wild-type and mutant proteins, respectively, RNA aptamers have been generated able to bind to p53R175, one of the hot spots of p53 mutation, discriminating the mutant protein from wild-type p53 [31].

Aptamers may function as anticancer therapeutics by different modalities (Figure 2).

Figure 2. Aptamer-based anticancer therapy. (a) Antagonistic therapy: aptamers bind to cancer cell surface targets, inhibiting protumoral pathways. (b) Targeted drug delivery: aptamers conjugated to drug-loaded nanoparticles or linked to drugs bind to cell surface targets and internalize into cancer cells, resulting in selective intracellular drug delivery. (c) Gene therapy: aptamers decorating small interfering RNA (siRNA)-loaded nanoparticles or conjugated directly to siRNA, bind to cell surface targets and internalize into cancer cells, resulting in selective gene silencing. (d) Immunotherapy: aptamers stimulate immune cells against cancer cells (see text for details). Created with BioRender. com (accessed on 2 March 2023).

As it happens for an antibody, the action of the aptamer as a cancer therapeutic relies on its ability to bind to a cancer-related protein target and interfere with its correct functioning, thus ultimately inhibiting tumor development and progression [27,32]. Alternatively, similar to mAbs linked with payloads in ADC [33], aptamers against unique cancer biomarkers can be applied as anticancer tools by exploiting them as targeting agents to transport therapeutic molecules specifically to tumor sites [28,34]. In this regard, the capability of a subset of cell-targeting aptamers to actively internalize into target cells via receptor-mediated endocytosis or micropinocytosis [35,36] has fueled an area of intense research aimed at developing smart aptamer-based modalities for targeted TNBC treatment by conjugation aptamers with chemotherapeutics [37], therapeutic RNAs [38], small inhibitors [39] or nanosystems loaded with drugs [40,41], which need to enter into the cell to exert their anticancer function. 

Furthermore, recent approaches are also attempting to confer to the aptamers the effector functions, typical of the antibody, of activation of the complement system [42]. Moreover, radiolabeling methods applied to antibodies can be easily used for conjugating aptamers with radionuclides, and the resulting conjugates have great potential in innovative theranostic applications [43]. Even if aptamers can be compared with antibodies for their mode of action, there is no doubt that they have several advantages to successfully replace or complement traditional antibodies for active cancer targeting (Table 1) [44,45]. 

First, they have smaller sizes (5–15 kDa) and more flexible structures compared to classical antibodies that allow them to penetrate solid tumors more easily and bind to small and hidden targets otherwise inaccessible to antibodies [46]. Importantly, the production of identified aptamers by chemical synthesis allows for to avoidance of batch-to-batch variability and overcomes expensive and labor-intensive steps that are required for antibody production.

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