Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p -Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2 + cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

IntroductionDespite advances in clinical therapy, metastasis is still the leading cause of death in breast cancer patients (1). A clearer understanding of molecular mechanisms that drive metastasis will help to develop more effective therapies (2). Our present study focused on metabolism as an essential driver of tumor growth and metastasis, potentially common to all breast cancer types. Normal cells primarily use mitochondrial oxidative phosphorylation (OXPHOS) for energy production, whereas cancer cells depend on aerobic glycolysis (the Warburg effect) to generate energy and glycolytic intermediates for enhanced growth (3, 4). Tumor cells also generate high levels of reduced forms of NAD + , NADH, and NADPH as important cofactors and redox components (4, 5). These altered metabolic activities can be linked to mitochondrial dysfunction that inhibits OXPHOS, increases ROS, promotes uncontrolled growth, and causes DNA damage that further supports a metastatic phenotype (6, 7). Mitochondrial dysfunctions can be caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial proteins (6,8) that are essential for the respiratory chain/OXPHOS system. Due to the lack of protective histones and limited DNA repair (8), mtDNA mutations occur at high rates and were found in tumors including breast cancer (6,(9)(10)(11)(12)(13)(14), which suggests that defects in OXPHOS might contribute to tumorigenesis.By combining the nuclear genome of a recipient cell with the mitochondrial genome of a donor cell using cybrid technology, mitochondria from the triple-negative aggressive breast cancer cell lines MDA-MB-435 (15) and MDA-MB-231 facilitated tumor progression and metastasis in nonmetastatic tumor cells (7, 10). The donor cell lines harbor mtDNA mutations in tRNAs, in the

SUMMARY Bacterial biofilms in the colon alter the host tissue microenvironment. A role for biofilms in colon cancer metabolism has been suggested but to date has not been evaluated. Using metabolomics, we investigated the metabolic influence that microbial biofilms have on colon tissues and the related occurrence of cancer. Patient-matched colon cancers and histologically normal tissues, with or without biofilms, were examined. We show the upregulation of polyamine metabolites in tissues from cancer hosts with significant enhancement of N1, N12-diacetylspermine in both biofilm positive cancer and normal tissues. Antibiotic treatment, which cleared biofilms, decreased N1, N12-diacetylspermine levels to those seen in biofilm negative tissues, indicating that host cancer and bacterial biofilm structures contribute to the polyamine metabolite pool. These results show that colonic mucosal biofilms alter the cancer metabolome, to produce a regulator of cellular proliferation and colon cancer growth potentially affecting cancer development and progression.

B-cell chronic lymphocytic leukemia (B-CLL IntroductionThe tumor suppressor TP53 plays an important role in the control of key genes involved in the regulation of DNA repair, cell cycle, and apoptosis. 1,2 p53 is activated in response to DNA damage or other forms of stress, protecting cells from malignant transformation. This is the reason why p53 is frequently inactivated in human cancer. p53 is a short-lived protein, and its cellular level is controlled by the rate at which it is degraded. Although several U3 ubiquitin ligases have been implicated in p53 ubiquitylation and degradation, MDM2 appears to function as a master regulator of p53. 3,4 MDM2 not only facilitates p53 degradation, but it also binds p53 and inhibits its transcriptional activity. Therefore, inhibitors of p53-MDM2 binding are expected to stabilize and activate p53. Recently, the first potent and selective small-molecule antagonists of MDM2, the nutlins, have been shown to activate the p53 pathway in cancer cells with wild-type p53 in vitro and in vivo. 5 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5 ϩ B lymphocytes. 6 TP53 is mutated in only 5% to 10% of B-CLL cases at diagnosis, but in nearly 30% in chemotherapy-resistant tumors. TP53 mutation is associated with poor clinical outcome, shorter survival, and lack of response to therapy with purine nucleoside analogs or alkylating agents. [7][8][9][10][11] In fact, alterations in the TP53 gene are among the worst prognostic indicators for B-CLL. [12][13][14] Most of the chemotherapeutic drugs currently used induce cell cycle arrest or apoptosis through activation of p53, and p53 inactivation leads to chemoresistance. 1,2 Chemotherapeutic drugs, including purine analogs, topoisomerase inhibitors, and alkylating agents, have been shown to effectively increase p53 levels in B-CLL. 15,16 Thus, p53 activation is considered among the critical molecular events in chemotherapy-induced apoptosis in B-CLL cells. Although TP53 is mutated in only 5% to 10% of patients, the p53 pathway could be altered at a higher frequency, thus effectively attenuating p53 function. One of the mechanisms involved in p53 stabilization in response to DNA damage is its phosphorylation by ataxia telangiectasia mutated (ATM) protein. 1,2 Interestingly, ATM is inactivated in 10% to 20% of B-CLL cases, thus providing an alternative way to disable p53 function. [17][18][19][20] Tumors with alterations upstream of p53 would not respond adequately to genotoxic chemotherapeutics that act through the p53 pathway (eg, alkylating agents such as chlorambucil and cyclophosphamide; purine nucleosides such as fludarabine and cladribine; or topoisomerase inhibitors such as doxorubicin and mitoxantrone). Therefore, new therapies that overcome these For personal use only. on May 11, 2018. by guest www.bloodjournal.org From defects by acting directly on p53 stability may benefit these patients. Nutlins activate p53 by releasing it from MDM2-mediated negative control and thus compensate for d...