No other higher MW adducts were observed in ADCs 5 and 6. linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs. Keywords: antibodyCdrug conjugate, ADC, homogeneous, bifunctional, linker, site-specific, conjugation, maleimide, dibromomaleimide, MMAF, auristatin, interchain, disulfide, DAR, hinge, cysteine, trastuzumab, Her2, CD98 Graphical Abstract INTRODUCTION AntibodyCdrug conjugates (ADCs) are a promising new class of targeted therapeutic agents for treatment of cancer.1,2 Most ADCs are synthesized by conjugating a cytotoxic compound or payload to a tumor specific monoclonal antibody. The payloads are conjugated using amino or sulfhydryl specific linkers that react selectively with lysines or cysteines on the antibody surface. A typical antibody contains over 50 lysines and eight interchain cysteines as potential conjugation sites. The optimal DAR (drugs/antibody ratio) for most ADCs, however, ranges from two to four drugs/antibody.3 RGS4 ADCs with suboptimal DARs are prone to aggregation, poor solubility, and instability, which often lead to increased toxicity and/or inadequate efficacy in vivo. 4 The discrepancy between the number of potential conjugation sites and the desired DAR, combined with the use of linkers that lack site-specificity, results in heterogeneous ADCs that vary in both DAR and the conjugation sites.5 Consequently, most of the ADCs in clinical development for cancer indications contain dozens or more of chemically distinct ADC Tucidinostat (Chidamide) molecules, each with unique pharmacological properties.6,7 Conjugation through antibody cysteines minimizes ADC heterogeneity relative to lysine conjugation because there are fewer potential conjugation sites.8 The process typically involves partial reduction of four antibody interchain disulfide bonds to generate up to eight reactive cysteine thiol groups, followed by conjugation of payloads containing thiol-specific maleimide linkers.9 The resulting ADCs are composed of dozens of chemically distinct molecules with DARs ranging from zero to eight payloads per antibody. The maleimide linkers typically used for cysteine conjugation result in thio-succinimide linkages between the payload and the antibody known to undergo side reactions such as elimination or thiol exchange, resulting in premature release of the payloads from the ADCs.10 New site-specific conjugation methods have emerged in order to reduce ADC heterogeneity and other undesirable properties associated with conventional methods. Most are recombinant methods focused on modification of the antibody with unique functional groups to enable site-specific conjugation with orthogonally modified linkers.11 For example, cysteine mutations have been introduced into different antibodies to provide free thiol groups for conjugation with payloads containing conventional thiol-specific maleimide linkers.12,13 The process affords homogeneous ADCs containing approximately two drugs/antibody, but additional antibody reduction/oxidation steps are required to obtain mutants suitable for conjugation. Later studies revealed that subtle differences in the ADC microenvironments significantly affect linker stability, which correlates with improved efficacy.14 The combined results indicate that ADC activity is highly dependent Tucidinostat (Chidamide) upon the conjugation sites and suggest that optimal conjugation sites are likely to be different for each antibody. Recombinant methods using nonnatural amino acids to enable site-specific conjugation have also been reported. For example, stop codon suppression methodology was used to produce antibodies containing phenyl ketone side chains for site-specific conjugation to hydroxylamine linkers.15 The Tucidinostat (Chidamide) approach was later combined with cell-free antibody expression technology to introduce unique functional groups into over one hundred different conjugation sites in trastuzumab, an anti-Her2 antibody approved for treatment of breast cancer.16 The results were consistent with previous findings in that ADC activity was highly dependent on the conjugation site. Antibody expression and conjugation efficiency were also site-dependent, suggesting that site optimization is required for each ADC. Alternative semisynthetic methods for site-specific conjugation have been described in which recognition sequences were engineered into different locations on antibodies for subsequent enzymatic modification in order to create unique Tucidinostat (Chidamide) functional groups for site-specific conjugation. For example, a microbial transglutaminase recognition sequence was introduced into 90 different positions on an anti-EGFR antibody.17 Twelve.