**1. Introduction**

Natural science has witnessed many breakthroughs during the past decades. The new biological insights gained, the improved biochemical protocols and analytical tools developed, and the constant advances in chemical reaction technologies have paved the way for exciting and multidisciplinary research fields such as the field of antibody-drug conjugates (ADCs). ADCs are modern drug-delivery molecules that combine the selective targeting capabilities of monoclonal antibodies (mab) with the potent cytotoxicity displayed by toxic organic compounds [1,2]. The interest toward ADCs and the investments in ADC research have increased exponentially in recent years as a result of the U.S. Food and Drug Administration (FDA) approval of brentuximab vedotin (Adcetris®) in 2011 (for relapsed cases of Hodgkin's lymphoma and anaplastic large cell lymphoma) [3], trastuzumab emtansine (Kadcyla®) in 2013 (for human epidermal growth factor receptor 2 -positive metastatic breast cancer) [4], gemtuzumab ozogamicin (Mylotarg®) in 2017 (for acute myeloid leukemia) [5], and inotuzumab ozogamicin (Besponsa®) in 2017 (for acute lymphoblastic leukemia) [6].

The development of modern ADCs requires systematic research on antibodies, bioconjugation technologies, as well as information on the properties of the payload molecules and the characteristics

of the end products. Recently, hydrophilic derivatization of payload molecules was reported to have beneficial effects on the overall properties of the ADCs, for example, on the therapeutic index and the pharmacokinetics [7,8]. In line with the current research trends, we developed an alternative strategy for increasing the hydrophilicity of the cytotoxic agents based on the incorporation of carbohydrates and we constructed a limited set of monomethyl auristatin E (MMAE)-carbohydrate hybrids [9].

MMAE (see the structure displayed in Figure 1) is an antineoplastic and antimitotic drug that appears as the cytotoxic agen<sup>t</sup> in at least sixteen ADCs which have progressed to clinical trials [10,11]. Among these is the ADC brentuximab vedotin, which is used in the treatment of relapsed cases of Hodgkin's lymphoma and anaplastic large cell lymphoma [3]. On a more general level, MMAE and other auristatins have become important cytotoxic agents for ADC development since they tolerate covalent structural modifications without substantial loss of cytotoxic activity. While this is beneficial, the hydrophobic nature of MMAE and especially current MMAE-linker conjugates is sub-optimal for the development of ADCs with high drug-to-antibody ratios. This is because the attachment of multiple drug-linker moieties of this kind may lead to devastating effects on the biocompatibility and pharmaceutical efficacy of the end products. These problems are reflected in the design of current ADCs where focus is placed on low drug-to-antibody ratios, typically in the range of 2–4. In addition, multi-drug resistant cancer cells tend to overexpress efflux pump proteins capable of removing hydrophobic cytotoxic agents from the intracellular environment, thus further diminishing their potential [12]. The incorporation of hydrophilic moieties in the cytotoxic agents or the payload molecules has been identified as a valid strategy for overcoming these challenges and circumventing issues related to unwanted aggregation and clearance of ADCs [13]. To date, hydrophilic linkers based on polyethylene glycol (PEG) [7], the sulfonate group [8], and carbohydrates [9] have been reported. We have previously focused on the inclusion of hydrophilic carbohydrates in the linker species and the cytotoxic agen<sup>t</sup> due to their biocompatibility, pre-existing degradation routes, further derivatization possibilities, and, just as important, low cost.

**Figure 1.** Chemical structures of the cytotoxic agents studied: from the left; monomethyl auristatin E (MMAE), 1 (β-d-glucuronyl-monomethylauristatin E, MMAU), and 2 (MMAE-glycolinker-substrate).

A thorough investigation of the change in the hydrophobic character was not included in the previous studies, even though it is important in understanding the nature of the modified molecules and the effects of the chosen strategy. Therefore, we continue our studies in this work by determining the relative hydrophobicities of MMAE and representatives of our own modified auristatins, namely, β-d-glucuronyl-monomethylauristatin E (MMAU, compound 1 in Figure 1) and an MMAE-glycolinker-substrate (compound 2 in Figure 1), by micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate and sodium cholate as surfactants. Furthermore, cytotoxic

assays reveal an obvious connection between the hydrophobic properties of the warhead molecules and their corresponding cytotoxicity. Due to the mechanism by which ADCs function (internalization followed by the release of the cytotoxic agent), it was important to analyze the cytotoxic profiles of eventual end products in addition to those of the free warhead molecules. As a result, trastuzumab-auristatin derivatives, which can be used to treat HER2-positive breast cancer patients, were constructed and their cytotoxicities were screened.

### **2. Materials and Methods**
