Antibody-Drug Conjugates Used in Breast Cancers

The prognosis of breast cancer has radically changed in recent years and continues to improve due to the broad application of effective therapies. New targeting strategies including targeted delivery of cytotoxic drugs via receptor-targeting agents have been developed. We summarize recent publications and developments of novel antibody-drug conjugates (ADCs) used to control breast cancer.


Introduction
Cancer is the second main cause of mortality worldwide [1]. Breast cancer is the most common cancer in women, and the most common cancer overall [2]. A subtype of breast cancer overexpresses HER2 receptors and is called HER2-positive (HER2+); HER2+ breast cancer accounts for 15-20% of all breast cancers and is associated with poor patient outcome and aggressive phenotype [3]. For many years, the therapies of the breast cancer were based on known biomarkers such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [4][5][6]. One of these treatments is trastuzumab; a humanized monoclonal antibody that targets HER2 receptor, leading to angiogenesis inhibition, diminished microvessel density, and better overall survival rates in patients with HER2positive breast cancer [7]. However, resistance to trastuzumab has been reported [8,9], in addition to several severe adverse effects such as cardiac toxicity [10]. In general, naked monoclonal antibodies, despite their importance in cancer research, have not offered the expected curative results, so the need for more potent agents was clear in order to completely treat cancer. Further studies using monoclonal antibodies as a treatment were made [11] introducing us to the amazing therapeutic properties of them, especially the selective delivery of cytotoxic agents to tumor cells, creating what is called antibody-drug conjugates (ADCs) as a solution to increase the therapeutic index of a cytotoxic chemical agent [11,12]. Although these HER2-targeting therapies have improved the overall survival rate, many more cases are still not affected by these treatments. A large population of them have breast cancer which does not overexpress HER2 receptors, those are clinically categorized as "HER2-negative." e word "negative" does not mean that the tumor does not express any HER2 receptors; it actually means that the amount of the receptors is not enough for the anti-HER2 antibodies to be used as a treatment [13]. If the tumor also does not express hormone receptors (HR), then it is called triple negative breast cancer (TNBC). In this review, we are going to describe ADCs generally and ADCs used in managing breast cancers specifically.

Antibody-Drug Conjugates (ADCs) Structure
In order to achieve the desired results, each ADC must contain three parts ( Figure 1) [19]: Monoclonal antibody: it binds the ADC to a specific tumor cell surface protein [19]. e antibody should bind tumor cells with high avidity and have little crossreactivity with healthy cells so that it does not affect them. All the antibodies developed or currently in clinical trials are immunoglobulin G (IgG); taking advantage of that, they contain multiple native sites for conjugation and can be modified for additional reactive sites [20,21]. Most of the ADCs are built on IgG1 scaffolds because the antibodydependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are much stronger in IgG1 and IgG3 than IgG2 and IgG4 [22,23].
Linker: it is a chemical spacer between the cytotoxic drug and the monoclonal body. It is usually stable in the circulation, but most of the linkers are easily displaced inside the cells. However, if the linker stays stable inside the cell, it requires degradation to release the drug. e linker must allow the release of the drug in its active form within or close enough to the target cells, because early release of drugs in the circulation can result in systemic toxicity and a lower therapeutic index [24,25].
ere are 2 types of linkers: cleavable and noncleavable, both of them are used in the ADCs developed or currently in clinical trials [26]. Cleavable linkers depend on the differences between conditions in the bloodstream and the cytoplasmic conditions within cancer cells (low pH, proteolytic cleavage, and high glutathione concentrations). Depending on their response to intracellular conditions, there are three types of cleavable linkers: hydrazone, disulfide, and peptide linkers [16,27]. On the other hand, noncleavable linkers release the drug after internalization in the target cell [16,27]; they rely on complete proteolytic degradation of the antibody to the amino acid level within the lysosome [27], that means they require appropriate internalization and degradation inside the cell to be active. e most common example of noncleavable linkers is the thioether linker.
Cytotoxic drug: cytotoxic compounds are divided into two main categories: microtubule inhibitors and DNAdamaging agents. ere are also other small molecules under investigation [27,28]. All the cytotoxic compounds used in the ADC structure must have higher toxic potency compared with standard chemotherapeutic agents, be able to kill cancer cells by induction of apoptosis, have a suitable functional group for linkage to an antibody, and be soluble in aqueous solutions to enable the reaction with antibodies [24,29,30].

Development of Antibody-Drug Conjugates
ere are three generations of ADCs: first, second, and third. Table 1shows the main differences between them.

Mechanism of Action of HER2-Directed
ADCs [34] 5.1. Classical Mode of Action. e monoclonal anti-HER2 binds to the HER2 expressed on the cells of the tumor and gets internalized by endocytosis. e proteases in the lysosomes cleavage the linker, releasing the payload and starting the cytotoxic effects.

Bystander Killing Effect.
is effect happens when the ADC is designed that the antibody releases the payload before internalization so that the surrounding cells get affected by its cytotoxic effects even if they do not express the receptor.
(3) Development status [43]: BAT8001 is in phase III clinical evaluation as a treatment of HER2-positivemetastatic breast cancer that is treated previously with trastuzumab..
(2) Evaluations showed that they were only moderately potent and less active than the parent drugs. (1) Huge improvements in mAbs technology were made, increasing selective binding to tumor cells and reducing crossreactivity with healthy cells. Payloads with smaller molecules were also discovered.
(1) Site-specific conjugation was developed, improving the therapeutic index, stability, and potency.
(4) Development status: phase I and phase II .
(3) Development status: phase I and phase II.

Conclusion
Breast cancer has become the most common cancer in the world; a lot of treatment methods and technologies were used in order to control it, but all of them did not achieve the required goal, until the invention of the antibody-drug conjugates. e concept of targeted delivery of anticancer drugs helped the oncologists to improve the tumor selectivity of anticancer drugs and to lower their systemic toxicity. Meaning that these drugs could be administered at higher doses, providing better therapeutic benefit to their patients. e tumor selectivity of antibodies offered a chance to achieve this goal by using them as guide for the drug towards the tumor. is seemingly simple concept had great attention from researchers at academic institutions and in the pharmaceutical industry. e current breed of ADCs uses antibodies that are humanized, not immunogenic, and linkers that are designed to be stable in circulation, but are cleaved upon delivery into a cell. e recent FDA approvals of new ADCs have generated tremendous excitement. ere are a lot of ADCs currently in clinical evaluation and almost every major pharmaceutical company has embraced this technology. ere is active research by medicinal chemists to develop new linkers and discover new potent effector molecules suitable for use in ADCs, while biologists have focused on identifying cell-surface targets suitable for antibody development.