Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Clinical pharmacology strategies to accelerate the development of polatuzumab vedotin and summary of key findings.

The favorable benefit-risk profile of polatuzumab vedotin, as demonstrated in a pivotal Phase Ib/II randomized study (GO29365; NCT02257567), coupled with the need for effective therapies in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), prompted the need to accelerate polatuzumab vedotin development. An integrated, fit-for-purpose clinical pharmacology package was designed to support regulatory approval. To address key clinical pharmacology questions without dedicated clinical pharmacology studies, we leveraged non-clinical and clinical data for polatuzumab vedotin, published clinical data for brentuximab vedotin, a similar antibody-drug conjugate, and physiologically based pharmacokinetic and population pharmacokinetic modeling approaches. We review strategies and model-informed outcomes that contributed to regulatory approval of polatuzumab vedotin plus bendamustine and rituximab in R/R DLBCL. These strategies made polatuzumab vedotin available to patients earlier than previously possible; depending on the strength of available data and the regulatory/competitive environment, they may also prove useful in accelerating the development of other agents.

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis.

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/ß-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Application of a Biocatalytic Strategy for the Preparation of Tiancimycin-Based Antibody-Drug Conjugates Revealing Key Insights into Structure-Activity Relationships.

Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Protocol for 3D screening of lung cancer spheroids using natural products.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for ∼84% of all lung cancer cases. NSCLC remains one of the leading causes of cancer-associated death, with a 5-year survival rate less than 25%. This type of cancer begins with healthy cells that change and start growing out of control, leading to the formation of lesions or tumors. Understanding the dynamics of how the tumor microenvironment promotes cancer initiation and progression that leads to cancer metastasis is crucial to help identify new molecular therapies. 3D primary cell tumor models have received renewed recognition due to their ability to better mimic the complexity of in vivo tumors and as a potential bridge between traditional 2D culture and in vivo studies. Vast improvements in 3D cell culture technologies make them much more cost effective and efficient largely because of the use of a cell-repellent surfaces and a novel angle plate adaptor technology. To exploit this technology, we accessed the Natural Products Library (NPL) at UF Scripps, which consists of crude extracts, partially purified fractions, and pure natural products (NPs). NPs generally are not very well represented in most drug discovery libraries and thus provide new insights to discover leads that could potentially emerge as novel molecular therapies. Herein we describe how we combined these technologies for 3D screening in 1536 well format using a panel of ten NSCLC cells lines (5 wild type and 5 mutant) against ∼1280 selected members of the NPL. After further evaluation, the selected active hits were prioritized to be screened against all 10 NSCLC cell lines as concentration response curves to determine the efficacy and selectivity of the compounds between wild type and mutant 3D cell models. Here, we demonstrate the methods needed for automated 3D screening using microbial NPs, exemplified by crude extracts, partially purified fractions, and pure NPs, that may lead to future use targeting human cancer.

Intramolecular C-C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis.

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the α/ß-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. This aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the α/ß-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

A homogeneous high-DAR antibody-drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Rational Approach to Identify RNA Targets of Natural Products Enables Identification of Nocathiacin as an Inhibitor of an Oncogenic RNA.

The discovery of biofunctional natural products (NPs) has relied on the phenotypic screening of extracts and subsequent laborious work to dereplicate active NPs and define cellular targets. Herein, NPs present as crude extracts, partially purified fractions, and pure compounds were screened directly against molecular target libraries of RNA structural motifs in a library-versus-library fashion. We identified 21 hits with affinity for RNA, including one pure NP, nocathiacin I (NOC-I). The resultant data set of NOC-I-RNA fold interactions was mapped to the human transcriptome to define potential bioactive interactions. Interestingly, one of NOC-I's most preferred RNA folds is present in the nuclease processing site in the oncogenic, noncoding microRNA-18a, which NOC-I binds with low micromolar affinity. This affinity for the RNA translates into the selective inhibition of its nuclease processing in vitro and in prostate cancer cells, in which NOC-I also triggers apoptosis. In principle, adaptation of this combination of experimental and predictive approaches to dereplicate NPs from the other hits (extracts and partially purified fractions) could fundamentally transform the current paradigm and accelerate the discovery of NPs that bind RNA and their simultaneous correlation to biological targets.

Functional Characterization of Cytochrome P450 Hydroxylase YpmL in Yangpumicin A Biosynthesis and Its Application for Anthraquinone-Fused Enediyne Structural Diversification.

Comparative analyses of four anthraquinone-fused enediyne biosynthetic gene clusters (BGCs) identified YpmL as a cytochrome P450 enzyme unique to the yangpumicin (YPM) BGC. In vitro characterization of YpmL established it as a hydroxylase, catalyzing C-6 hydroxylation in YPM A biosynthesis. In vivo application of YpmL enabled engineered production of four new tiancimycin analogues (14-17). Evaluation of their cytotoxicity against selected human cancer cell lines shed new insights into the enediyne structure-activity relationship.

Trastuzumab does not bind rat or mouse ErbB2/neu: implications for selection of non-clinical safety models for trastuzumab-based therapeutics.

PURPOSE: Assessment of non-clinical safety signals relies on understanding species selectivity of antibodies. This is particularly important with antibody-drug conjugates, where it is key to determine target-dependent versus target-independent toxicity. Although it appears to be widely accepted that trastuzumab does not bind mouse or rat HER2/ErbB2/neu, numerous investigators continue to use mouse models to investigate safety signals of trastuzumab and trastuzumab emtansine (T-DM1). We, therefore, conducted a broad array of both binding and biologic studies to demonstrate selectivity of trastuzumab for human HER2 versus mouse/rat neu. METHODS: Binding of anti-neu and anti-HER2 antibodies was assessed by ELISA, FACS, IHC, Scatchard, and immunoblot methods in human, rat, and mouse cell lines. In human hepatocytes, T-DM1 uptake and catabolism were measured by LC-MS/MS; cell viability changes were determined using CellTiter-Glo. RESULTS: Our data demonstrate, using different binding methods, lack of trastuzumab binding to rat or mouse neu. Structural studies show important amino acid differences in the trastuzumab-HER2 binding interface between mouse/rat and human HER2 ECD. Substitution of these rodent amino acid residues into human HER2 abolish binding of trastuzumab. Cell viability changes, uptake, and catabolism of T-DM1 versus a DM1 non-targeted control ADC were comparable, indicating target-independent effects of the DM1-containing ADCs. Moreover, trastuzumab binding to human or mouse hepatocytes was not detected. CONCLUSIONS: These data, in total, demonstrate that trastuzumab, and by extension T-DM1, do not bind rat or mouse neu, underscoring the importance of species selection for safety studies investigating trastuzumab or trastuzumab-based therapeutics.

Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase.

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

Discovery of ammosesters by mining the Streptomyces uncialis DCA2648 genome revealing new insight into ammosamide biosynthesis.

The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure-activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity.

Preclinical Characterization of the Distribution, Catabolism, and Elimination of a Polatuzumab Vedotin-Piiq (POLIVY®) Antibody-Drug Conjugate in Sprague Dawley Rats.

Polatuzumab vedotin (or POLIVY®), an antibody-drug conjugate (ADC) composed of a polatuzumab monoclonal antibody conjugated to monomethyl auristatin E (MMAE) via a cleavable dipeptide linker, has been approved by the United States Food and Drug Administration (FDA) for the treatment of diffuse large B-cell lymphoma (DLBCL). To support the clinical development of polatuzumab vedotin, we characterized the distribution, catabolism/metabolism, and elimination properties of polatuzumab vedotin and its unconjugated MMAE payload in Sprague Dawley rats. Several radiolabeled probes were developed to track the fate of different components of the ADC, with 125I and 111In used to label the antibody component and 3H to label the MMAE payload of the ADC. Following a single intravenous administration of the radiolabeled probes into normal or bile-duct cannulated rats, blood, various tissues, and excreta samples were collected over 7-14 days post-dose and analyzed for radioactivity and to characterize the metabolites/catabolites. The plasma radioactivity of polatuzumab vedotin showed a biphasic elimination profile similar to that of unconjugated polatuzumab but different from unconjugated radiolabeled MMAE, which had a fast clearance. The vast majority of the radiolabeled MMAE in plasma remained associated with antibodies, with a minor fraction as free MMAE and MMAE-containing catabolites. Similar to unconjugated mAb, polatuzumab vedotin showed a nonspecific distribution to multiple highly perfused organs, including the lungs, heart, liver, spleen, and kidneys, where the ADC underwent catabolism to release MMAE and other MMAE-containing catabolites. Both polatuzumab vedotin and unconjugated MMAE were mainly eliminated through the biliary fecal route (>90%) and a small fraction (<10%) was eliminated through renal excretion in the form of catabolites/metabolites, among which, MMAE was identified as the major species, along with several other minor species. These studies provided significant insight into ADC's absorption, distribution, metabolism, and elimination (ADME) properties, which supports the clinical development of POLIVY.

Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): Mechanistic insights into MMAE ADC peripheral neuropathy.

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.

Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production.

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates, utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019 mg/l), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here, we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1 mg/l through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications.

Challenges and Opportunities to Develop Enediyne Natural Products as Payloads for Antibody-Drug Conjugates.

Calicheamicin, the payload of the antibody-drug-conjugates (ADCs) gemtuzumab ozogamicin (Mylotarg®) and inotuzumab ozogamicin (Besponsa®), belongs to the class of enediyne natural products. Since the isolation and structural determination of the neocarzinostatin chromophore in 1985, the enediynes have attracted considerable attention for their value as DNA damaging agents in cancer chemotherapy. Due to their non-discriminatory cytotoxicity towards both cancer and healthy cells, the clinical utilization of enediyne natural products relies on conjugation to an appropriate delivery system, such as an antibody. Here we review the current landscape of enediynes as payloads of first-generation and next-generation ADCs.

Personalized Cancer Vaccines: Clinical Landscape, Challenges, and Opportunities.

Tremendous innovation is underway among a rapidly expanding repertoire of promising personalized immune-based treatments. Therapeutic cancer vaccines (TCVs) are attractive systemic immunotherapies that activate and expand antigen-specific CD8+ and CD4+ T cells to enhance anti-tumor immunity. Our review highlights key issues impacting TCVs in clinical practice and reports on progress in development. We review the mechanism of action, immune-monitoring, dosing strategies, combinations, obstacles, and regulation of cancer vaccines. Most trials of personalized TCVs are ongoing and represent diverse platforms with predominantly early investigations of mRNA, DNA, or peptide-based targeting strategies against neoantigens in solid tumors, with many in combination immunotherapies. Multiple delivery systems, routes of administration, and dosing strategies are used. Intravenous or intramuscular administration is common, including delivery by lipid nanoparticles. Absorption and biodistribution impact antigen uptake, expression, and presentation, affecting the strength, speed, and duration of immune response. The emerging trials illustrate the complexity of developing this class of innovative immunotherapies. Methodical testing of the multiple potential factors influencing immune responses, as well as refined quantitative methodologies to facilitate optimal dosing strategies, could help resolve uncertainty of therapeutic approaches. To increase the likelihood of success in bringing these medicines to patients, several unique development challenges must be overcome.

Anti-Lymphocyte Antigen 6 Complex, Locus E-Seco-Cyclopropabenzindol-4-One-Dimer Antibody-Drug Conjugate That Forms Adduct with α1-Microglobulin Demonstrates Slower Systemic Antibody Clearance and Reduced Tumor Distribution in Animals.

Anti-Ly6E-seco-cyclopropabenzindol-4-one dimer antibody-drug conjugate (ADC) has been reported to form an adduct with α1-microglobulin (A1M) in animal plasma, but with unknown impact on ADC PK and tissue distribution. In this study, we compared the PK and tissue distribution of anti-Ly6E ADC with unconjugated anti-Ly6E mAb in rodents and monkeys. For PK studies, animals received an intravenous administration of anti-Ly6E ADC or unconjugated anti-Ly6E mAb. Plasma samples were analyzed for total antibody (Tab) levels and A1M adduct formation. PK parameters were generated from dose-normalized plasma concentrations. Tissue distribution was determined in tumor-bearing mice after a single intravenous dosing of radiolabeled ADC or mAb. Tissue radioactivity levels were analyzed using a gamma counter. The impact of A1M adduct formation on target cell binding was assessed in an in vitro cell binding assay. The results show that ADC Tab clearance was slower than that of mAb in mice and rats but faster than mAb in monkeys. Correspondingly, the formation of A1M adduct appeared to be faster and higher in mice, followed by rats, and slowest in monkeys. Although ADC tended to show an overall lower distribution to normal tissues, it had a strikingly reduced distribution to tumors compared with mAb, likely due to A1M adduct formation interfering with target binding, as demonstrated by the in vitro cell binding assay. Together, these data 1) demonstrate that anti-Ly6E ADC that forms A1M adduct had slower systemic clearance with strikingly reduced tumor distribution and 2) highlight the importance of selecting an appropriate linker-drug for successful ADC development. SIGNIFICANCE STATEMENT: Anti-lymphocyte antigen 6 complex, locus E, ADC with seco-cyclopropabenzindol-4-one-dimer payload formed adduct with A1M, which led to a decrease in systemic clearance but also attenuated tumor distribution. These findings demonstrate the importance of selecting an appropriate linker-drug for ADC development and also highlight the value of a mechanistic understanding of ADC biotransformation, which could provide insight into ADC molecule design, optimization, and selection.

Characterization of TnmH as an O-Methyltransferase Revealing Insights into Tiancimycin Biosynthesis and Enabling a Biocatalytic Strategy To Prepare Antibody-Tiancimycin Conjugates.

The enediynes are among the most cytotoxic molecules known, and their use as anticancer drugs has been successfully demonstrated by targeted delivery. Clinical advancement of the anthraquinone-fused enediynes has been hindered by their low titers and lack of functional groups to enable the preparation of antibody-drug conjugates (ADCs). Here we report biochemical and structural characterization of TnmH from the tiancimycin (TNM) biosynthetic pathway, revealing that (i) TnmH catalyzes regiospecific methylation at the C-7 hydroxyl group, (ii) TnmH exhibits broad substrate promiscuity toward hydroxyanthraquinones and S-alkylated SAM analogues and catalyzes efficient installation of reactive alkyl handles, (iii) the X-ray crystal structure of TnmH provides the molecular basis to account for its broad substrate promiscuity, and (iv) TnmH as a biocatalyst enables the development of novel conjugation strategies to prepare antibody-TNM conjugates. These findings should greatly facilitate the construction and evaluation of antibody-TNM conjugates as next-generation ADCs for targeted chemotherapy.