Total Synthesis in Search of Potent Antibody-Drug Conjugate Payloads. From the Fundamentals to the Translational. Nicolaou Kyriacos C,Rigol Stephan Accounts of chemical research The emergence and evolution of antibody-drug conjugates (ADCs) as targeted cancer therapies in recent years is a living example of the "magic bullet" concept of Paul Ehrlich, introduced by him more than a century ago. Consisting of three components, the antibody serving as the delivery system, the payload drug that kills the cancer cell, and the chemical linker through which the payload is attached to the antibody, ADCs represent a currently hotly pursued paradigm of targeted cancer therapies. While the needed monoclonal antibody falls in the domains of biology and biochemistry, the potent payload and the linker belong to the realm of chemistry. Naturally occurring molecules and their derivatives endowed with high cytotoxic properties have proven to be useful payloads for the first approved ADCs (i.e., Mylotarg, Adcetris, Kadcyla, and Besponsa). The latest approaches and intensifying activities in this new paradigm of cancer therapy demands a variety of payloads with different mechanisms of action in order to address the medical needs for the various types of cancers, challenging synthetic organic chemists to enrich the library of potential payloads. Total synthesis of natural and designed molecules not only provides a powerful avenue to replicate rare naturally occurring compounds in the laboratory but also offers a unique opportunity to rationally design and synthesize analogues thereof for biological evaluation and optimization of ADC payloads. In this Account, we describe our efforts in this area highlighting a number of total synthesis endeavors through which we rendered scarce naturally occurring molecules readily available for biological evaluations and, most importantly, employed the developed synthetic strategies and methods to construct, otherwise unavailable or difficult to reach, designed analogues of these molecules. Specifically, we summarize the total syntheses of natural and designed molecules of the calicheamicin, uncialamycin, tubulysin, trioxacarcin, epothilone, shishijimicin, namenamicin, thailanstatin, and disorazole families of compounds and demonstrate how these studies led to the discovery of analogues of higher potencies, yet some of them possessing lower complexities than their parent compounds as potential ADC payloads. The highlighted examples showcase the continuing impact of total synthesis of natural products and their analogues on modern medicine, including the so-called biologics and should prove useful and inspirational in advancing both the fields of total synthesis and biomedical research and the drug discovery and development process. 10.1021/acs.accounts.8b00537
    Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Boshuizen Julia,Koopman Louise A,Krijgsman Oscar,Shahrabi Aida,van den Heuvel Elke Gresnigt-,Ligtenberg Maarten A,Vredevoogd David W,Kemper Kristel,Kuilman Thomas,Song Ji-Ying,Pencheva Nora,Mortensen Jens Thing,Foppen Marnix Geukes,Rozeman Elisa A,Blank Christian U,Janmaat Maarten L,Satijn David,Breij Esther C W,Peeper Daniel S,Parren Paul W H I Nature medicine Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy. 10.1038/nm.4472
    Enzyme-activatable polymer-drug conjugate augments tumour penetration and treatment efficacy. Zhou Quan,Shao Shiqun,Wang Jinqiang,Xu Changhuo,Xiang Jiajia,Piao Ying,Zhou Zhuxian,Yu Qingsong,Tang Jianbin,Liu Xiangrui,Gan Zhihua,Mo Ran,Gu Zhen,Shen Youqing Nature nanotechnology A tumour microenvironment imposes barriers to the passive diffusion of molecules, which renders tumour penetration an unresolved obstacle to an effective anticancer drug delivery. Here, we present a γ-glutamyl transpeptidase-responsive camptothecin-polymer conjugate that actively infiltrates throughout the tumour tissue through transcytosis. When the conjugate passes on the luminal endothelial cells of the tumour blood vessels or extravasates into the tumour interstitium, the overexpressed γ-glutamyl transpeptidase on the cell membrane cleaves the γ-glutamyl moieties of the conjugate to generate positively charged primary amines. The resulting cationic conjugate undergoes caveolae-mediated endocytosis and transcytosis, which enables transendothelial and transcellular transport and a relatively uniform distribution throughout the tumour. The conjugate showed a potent antitumour activity in mouse models that led to the eradication of small solid tumours (~100 mm) and regression of large established tumours with clinically relevant sizes (~500 mm), and significantly extended the survival of orthotopic pancreatic tumour-bearing mice compared to that with the first-line chemotherapeutic drug gemcitabine. 10.1038/s41565-019-0485-z
    A Novel HER3-Targeting Antibody-Drug Conjugate, U3-1402, Exhibits Potent Therapeutic Efficacy through the Delivery of Cytotoxic Payload by Efficient Internalization. Hashimoto Yuuri,Koyama Kumiko,Kamai Yasuki,Hirotani Kenji,Ogitani Yusuke,Zembutsu Akiko,Abe Manabu,Kaneda Yuki,Maeda Naoyuki,Shiose Yoshinobu,Iguchi Takuma,Ishizaka Tomomichi,Karibe Tsuyoshi,Hayakawa Ichiro,Morita Koji,Nakada Takashi,Nomura Taisei,Wakita Kenichi,Kagari Takashi,Abe Yuki,Murakami Masato,Ueno Suguru,Agatsuma Toshinori Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody-drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models. EXPERIMENTAL DESIGN: pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys. RESULTS:U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys. CONCLUSIONS:U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells. 10.1158/1078-0432.CCR-19-1745
    Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy. Kang Jeffrey C,Sun Wei,Khare Priyanka,Karimi Mostafa,Wang Xiaoli,Shen Yang,Ober Raimund J,Ward E Sally Nature biotechnology We improve the potency of antibody-drug conjugates (ADCs) containing the human epidermal growth factor receptor 2 (HER2)-specific antibody pertuzumab by substantially reducing their affinity for HER2 at acidic endosomal pH relative to near neutral pH. These engineered pertuzumab variants show increased lysosomal delivery and cytotoxicity towards tumor cells expressing intermediate HER2 levels. In HER2 xenograft tumor models in mice, the variants show higher therapeutic efficacy than the parent ADC and a clinically approved HER2-specific ADC. 10.1038/s41587-019-0073-7
    Enzyme-Triggered Transcytosis of Dendrimer-Drug Conjugate for Deep Penetration into Pancreatic Tumors. Wang Guowei,Zhou Zhuxian,Zhao Zhihao,Li Qunying,Wu Yulian,Yan Sheng,Shen Youqing,Huang Pintong ACS nano The dense fibrotic stroma in pancreatic ductal adenocarcinoma (PDA) resists drug diffusion into the tumor and leads to an unsatisfactory prognosis. To address this problem, we demonstrate a dendrimer-camptothecin (CPT) conjugate that actively penetrates deep into PDA tumors through γ-glutamyl transpeptidase (GGT)-triggered cell endocytosis and transcytosis. The dendrimer-drug conjugate was synthesized by covalent attachment of CPT to polyamidoamine (PAMAM) dendrimers through a reactive oxygen species (ROS)-sensitive linker followed with surface modification with glutathione. Once the conjugate was delivered to the PDA tumor periphery, the overexpressed GGT on the vascular endothelial cell or tumor cell triggers the γ-glutamyl transfer reactions of glutathione to produce primary amines. The positively charged conjugate was rapidly internalized caveolae-mediated endocytosis and followed by vesicle-mediated transcytosis, augmenting its deep penetration within the tumor parenchyma and releasing active CPT throughout the tumor after cleavage by intracellular ROS. The dendrimer-drug conjugate exhibited high antitumor activity in multiple mice tumor models, including patient-derived PDA xenograft and orthotopic PDA cell xenograft, compared to the standard first-line chemotherapeutic drug (gemcitabine) for advanced pancreatic cancer. This study demonstrates the high efficiency of an active tumor-penetrating dendrimer-drug conjugate transcytotic transport with ROS-responsive drug release for PDA therapy. 10.1021/acsnano.0c00974
    Antibody-Mediated Endocytosis of Polysialic Acid Enables Intracellular Delivery and Cytotoxicity of a Glycan-Directed Antibody-Drug Conjugate. Cox Emily C,Thornlow Dana N,Jones Michaela A,Fuller Jordan L,Merritt Judith H,Paszek Matthew J,Alabi Christopher A,DeLisa Matthew P Cancer research The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody-drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells . Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. SIGNIFICANCE: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx. 10.1158/0008-5472.CAN-18-3119
    Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry. Sonzini Silvia,Greco Maria Laura,Cailleau Thais,Adams Lauren,Masterson Luke,Vijayakrishnan Balakumar,Barry Conor,Howard Phillip,Ravn Peter,van der Walle Christopher F Bioconjugate chemistry Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification. 10.1021/acs.bioconjchem.9b00809
    Streamlined Characterization of an Antibody-Drug Conjugate by Two-Dimensional and Four-Dimensional Liquid Chromatography/Mass Spectrometry. Goyon Alexandre,Kim Michael,Dai Lu,Cornell Christopher,Jacobson Fred,Guillarme Davy,Stella Cinzia Analytical chemistry This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody-drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5-3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches. 10.1021/acs.analchem.9b02454
    Comprehensive Middle-Down Mass Spectrometry Characterization of an Antibody-Drug Conjugate by Combined Ion Activation Methods. Watts Eleanor,Williams Jon D,Miesbauer Laura J,Bruncko Milan,Brodbelt Jennifer S Analytical chemistry Antibody-drug conjugates (ADCs) are an increasingly prevalent drug class utilized as chemotherapeutic agents. The complexity of ADCs, including their large size, array of drug conjugation sites, and heterogeneous compositions containing from zero to several payloads, demands the use of advanced analytical characterization methods. Tandem mass spectrometry (MS/MS) strategies, including a variety of bottom-up, middle-down, and even top-down approaches, frequently applied for the analysis of antibodies are increasingly being adapted for antibody-drug conjugates. Middle-down tandem mass spectrometry, often focusing on the analysis of ∼25 kDa protein subunits, offers the potential for complete sequence confirmation as well as the identification of multiple conjugation states. While middle-down studies have been extensively developed for monoclonal antibodies, middle-down characterization of ADCs has been limited by the high complexity of the drug molecules. This study seeks to bridge the gap by utilizing a combination of 193 nm ultraviolet photodissociation (UVPD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). The compilation of these MS/MS methods leads to high sequence coverages of 60-80% for each subunit of the ADC. Moreover, the combined fragmentation patterns provide sufficient information to allow confirmation of both the sequence of the complementarity-determining regions and the payload conjugation sites. 10.1021/acs.analchem.0c01232
    Native size-exclusion chromatography-mass spectrometry: suitability for antibody-drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels. Jones Jay,Pack Laura,Hunter Joshua H,Valliere-Douglass John F mAbs Native size-exclusion chromatography-mass spectrometry (nSEC-MS) is an analytical methodology that is appropriate for accurately quantitating the drug-to-antibody ratio (DAR) on a wide variety of interchain cysteine-linked antibody-drug conjugates (ADCs), irrespective of chemotype. In the current preclinical environment, novel ADCs conjugated with unique drug-linkers need to progress toward the clinic as quickly as possible. Platform analytical approaches can reduce time-to-clinic because key process development and optimization activities can be decoupled from the development of bespoke, molecule-specific analytical methods. In this work, we assessed the potential of nSEC-MS as a platformable, quantitative DAR method. The nSEC-MS method was evaluated according to performance characteristics and parameters described in the ICH guideline Validation of Analytical Procedures: Text and Methodology Q2(R1). In order to comprehensively assess the accuracy and bias of nSEC-MS DAR quantitation, ADCs were generated using three different drug-linker chemotypes with DARs ranging from 2 to 8. These molecules were tested by hydrophobic interaction chromatography (HIC) and nSEC-MS, and DARs obtained from both methods were compared to assess the degree to which nSEC-MS quantitation aligned with the HIC release assay. Our results indicated that there is no bias introduced by nSEC-MS quantitation of DAR and that SEC-MS data can be bridged to HIC data without the need for a correction factor or offset. nSEC-MS was also found to be suitable for unbiased DAR quantitation in the other ADC chemotypes that were evaluated. Based on the totality of our work, we conclude that, used as intended, nSEC-MS is well suited for quantitating DAR on a variety of interchain cysteine-linked ADCs in an accurate, unbiased manner. 10.1080/19420862.2019.1682895
    A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis. MacGregor Paula,Gonzalez-Munoz Andrea L,Jobe Fatoumatta,Taylor Martin C,Rust Steven,Sandercock Alan M,Macleod Olivia J S,Van Bocxlaer Katrien,Francisco Amanda F,D'Hooge Francois,Tiberghien Arnaud,Barry Conor S,Howard Philip,Higgins Matthew K,Vaughan Tristan J,Minter Ralph,Carrington Mark PLoS neglected tropical diseases Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics. 10.1371/journal.pntd.0007373
    CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity. Tsui C Kimberly,Barfield Robyn M,Fischer Curt R,Morgens David W,Li Amy,Smith Benjamin A H,Gray Melissa Anne,Bertozzi Carolyn R,Rabuka David,Bassik Michael C Nature chemical biology Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets. 10.1038/s41589-019-0342-2
    Controlled drug release from polyelectrolyte-drug conjugate nanoparticles. Catarata Ruginn,Azim Nilab,Bhattacharya Santanu,Zhai Lei Journal of materials chemistry. B Encapsulating drugs in functional nanoparticles provides controlled and targeted release of drugs. In this study, a general approach for encapsulating hydrophobic drugs in polyelectrolyte nanoparticles was developed for a controlled drug release. Gemcitabine (GEM), an anticancer drug for pancreatic ductal adenocarcinoma (PDAC), was used as a model drug to produce poly(acrylic acid) (PAA)-GEM conjugate nanoparticles to achieve a controlled release of GEM in cells. The PAA-GEM conjugate nanoparticles were fabricated by coupling GEM onto PAA through the formation of amide bonds. The hydrophobic interactions of GEM molecules induced the formation of the nanoparticles with the GEM core and PAA shell. Fabrication conditions such as the PAA/GEM ratio and pH were optimized to achieve high structure stability and drug loading efficiency. The size and surface charge of the nanoparticles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurement. The optimized PAA-GEM nanoparticles had a size around 12 nm, 30 nm and 60 nm in dry state, water, and phosphate buffered saline (PBS), respectively. The encapsulation efficiency was 29.29 ± 1.7%, and the loading capacity was 9.44 ± 0.46%. Less than 7% GEM was released from the PAA-GEM nanoparticles after 96 hour incubation in phosphate buffered saline. The cytotoxic efficacy of the PAA-GEM nanoparticles in cancer cells was investigated through viability studies of PANC-1, a human pancreatic cancer cell line. It was found that the PAA-GEM nanoparticles had more than a 48 hour delay of releasing GEM and had the same cytotoxic efficacy in PANC-1 cells as free GEM. The uptake of the PAA-GEM nanoparticles by PANC-1 cells was investigated using PAA-GEM labeled by rhodamine G6. Fluorescence and bright field overlay images indicated that the PAA-GEM nanoparticles were taken up by PANC-1 cells within 2 hours. It is believed that the PAA-GEM nanoparticles were decomposed in PANC-1 cells and GEM was released from the nanoparticles. 10.1039/d0tb00012d
    Heterogeneous Drug Efficacy of an Antibody-Drug Conjugate Visualized Using Simultaneous Imaging of Its Delivery and Intracellular Damage in Living Tumor Tissues. Gonda Kohsuke,Negishi Hiroshi,Takano-Kasuya Mayumi,Kitamura Narufumi,Furusawa Naoko,Nakano Yasushi,Hamada Yoh,Tokunaga Masayuki,Higuchi Hideo,Tada Hiroshi,Ishida Takanori Translational oncology Anticancer drug efficacy varies because the delivery of drugs within tumors and tumor responses are heterogeneous; however, these features are often more homogenous in vitro. This difference makes it difficult to accurately determine drug efficacy. Therefore, it is important to use living tumor tissues in preclinical trials to observe the heterogeneity in drug distribution and cell characteristics in tumors. In the present study, to accurately evaluate the efficacy of an antibody-drug conjugate (ADC) containing a microtubule inhibitor, we established a cell line that expresses a fusion of end-binding protein 1 and enhanced green fluorescent protein that serves as a microtubule plus-end-tracking protein allowing the visualization of microtubule dynamics. This cell line was xenografted into mice to create a model of living tumor tissue. The tumor cells possessed a greater number of microtubules with plus-ends, a greater number of meandering microtubules, and a slower rate of microtubule polymerization than the in vitro cells. In tumor tissues treated with fluorescent dye-labeled ADCs, heterogeneity was observed in the delivery of the drug to tumor cells, and microtubule dynamics were inhibited in a concentration-dependent manner. Moreover, a difference in drug sensitivity was observed between in vitro cells and tumor cells; compared with in vitro cells, tumor cells were more sensitive to changes in the concentration of the ADC. This study is the first to simultaneously evaluate the delivery and intracellular efficacy of ADCs in living tumor tissue. Accurate evaluation of the efficacy of ADCs is important for the development of effective anticancer drugs. 10.1016/j.tranon.2020.100764
    Kinetic reaction modeling for antibody-drug conjugate process development. Andris Sebastian,Seidel Jonathan,Hubbuch Jürgen Journal of biotechnology By combining the specificity of monoclonal antibodies (mAbs) and the efficacy of cytotoxic drugs in one molecule, antibody-drug conjugates (ADCs) form a promising class of anti-cancer therapeutics. This is emphasized by around 65 molecules in clinical trials and four marketed products. The conjugation reaction of mAbs with small-molecule drugs is a central step during production of ADCs. A detailed kinetic model for the conjugation reaction grants enhanced process understanding and can be profitably applied to process optimization. One example is the identification of the optimal amount of drug excess, which should be minimized due to its high toxicity and high cost. In this work, we set up six different kinetic model structures for the conjugation of a cysteine-engineered mAb with a maleimide-functionalized surrogate drug. All models consisted of a set of differential equations. The models were fit to an experimental data set, and the best model was selected based on cross-validation. The selected model was successfully validated with an external validation dataset (R² of prediction: 0.978). Based on the modeling results, process understanding was improved. The model shows that the binding of the second drug to the mAb is influenced by the attachment of the first drug molecule. Additionally, an increase in reaction rate was observed for the addition of different salts to the reaction. In a next step, the model was applied to an in silico screening and optimization, which illustrates its potential for making ADC process development more efficient. Finally, the combination of the kinetic model with a PAT tool for reaction monitoring was demonstrated. In summary, the proposed modeling approach provides a powerful tool for the investigation of ADC conjugation reactions and establishes a valuable in silico decision support for process development. 10.1016/j.jbiotec.2019.09.013
    Evaluation of Quantitative Relationship Between Target Expression and Antibody-Drug Conjugate Exposure Inside Cancer Cells. Sharma Sharad,Li Zhe,Bussing David,Shah Dhaval K Drug metabolism and disposition: the biological fate of chemicals Antibody-drug conjugates (ADCs) employ overexpressed cell surface antigens to deliver cytotoxic payloads inside cancer cells. However, the relationship between target expression and ADC efficacy remains ambiguous. In this manuscript, we have addressed a part of this ambiguity by quantitatively investigating the effect of antigen expression levels on ADC exposure within cancer cells. Trastuzumab-valine-citrulline-monomethyl auristatin E was used as a model ADC, and four different cell lines with diverse levels of human epidermal growth factor receptor 2 (HER2) expression were used as model cells. The pharmacokinetics (PK) of total trastuzumab, released monomethyl auristatin E (MMAE), and total MMAE were measured inside the cells and in the cell culture media following incubation with two different concentrations of ADC. In addition, target expression levels, target internalization rate, and cathepsin B and MDR1 protein concentrations were determined for each cell line. All the PK data were mathematically characterized using a cell-level systems PK model for ADC. It was found that SKBR-3, MDA-MB-453, MCF-7, and MDA-MB-468 cells had ∼800,000, ∼250,000, ∼50,000, and ∼10,000 HER2 receptors per cell, respectively. A strong linear relationship ( > 0.9) was observed between HER2 receptor count and released MMAE exposure inside the cancer cells. There was an inverse relationship found between HER2 expression level and internalization rate, and cathepsin B and multidrug resistance protein 1 (MDR1) expression level varied slightly among the cell lines. The PK model was able to simultaneously capture all the PK profiles reasonably well while estimating only two parameters. Our results demonstrate a strong quantitative relationship between antigen expression level and intracellular exposure of ADCs in cancer cells. SIGNIFICANCE STATEMENT: In this manuscript, we have demonstrated a strong linear relationship between target expression level and antibody-drug conjugate (ADC) exposure inside cancer cells. We have also shown that this relationship can be accurately captured using the cell-level systems pharmacokinetics model developed for ADCs. Our results indirectly suggest that the lack of relationship between target expression and efficacy of ADC may stem from differences in the pharmacodynamic properties of cancer cells. 10.1124/dmd.119.089276
    Assessing localized conformational stability of antibody-drug conjugate by protein conformation assay. Fu Cexiong,Zhang Zhaorui,Zhou Shiyue,Pritts Wayne A,Zhang Qunying Journal of pharmaceutical and biomedical analysis Antibody-drug conjugates (ADCs) are a class of attractive therapeutic agents to fight cancer with conjugation of potent chemical agents on target-selective antibodies. The conceptually elegant approach has encountered mounting practical challenges in combining the mAb and potent drug while maintaining the conformational and physiochemical stability of the bioconjugates. The attachment of hydrophobic drug-linker with antibody could potentially alter the antibody conformational scaffold, locally or globally. Here we propose to use a protein conformation assay (PCA) to measure the higher-order structure of antibodies upon drug-linker conjugation. The PCA analysis provides insights into the formation of partially unfolded ADCs, which may correlate with protein stability and aggregation propensity. To further elucidate the cause of the unfolding events, in-depth peptide mapping combined with the PCA conformational footprints were performed on a commercial ADC trastuzumab emtansine in this study. The locally altered conformational hot-spots observed in PCA matched with conjugation sites with high occupancy rate identified in peptide mapping. In summary, by combining PCA and in-depth peptide mapping, a snapshot of ADC structural conformation and stability profile could be obtained and provide a swift and convenient measurement of the 'fitness' of ADC to facilitate payload selection, conjugation process development and early predictive developability assessment. 10.1016/j.jpba.2019.113020
    Quantification of an Antibody-Conjugated Drug in Fat Plasma by an Affinity Capture LC-MS/MS Method for a Novel Prenyl Transferase-Mediated Site-Specific Antibody-Drug Conjugate. Lee Byeong Ill,Park Min-Ho,Byeon Jin-Ju,Shin Seok-Ho,Choi Jangmi,Park Yuri,Park Yun-Hee,Chae Jeiwook,Shin Young G Molecules (Basel, Switzerland) The novel prenyl transferase-mediated, site-specific, antibody-drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration), with the equation y = ax + bx + c, was used to fit calibration curves over the concentration range of 19.17~958.67 ng/mL for acDrug. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions (short-term: 1 day, long-term: 10 months, Freeze/Thaw (F/T): 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats. 10.3390/molecules25071515
    UPLC-based assay to assess the hydrophobicity of Antibody-Drug Conjugate (ADC) payloads. Pysz Ilona,Jackson Paul J M,Barlow David J,Rahman Khondaker Miraz,Thurston David E Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Antibody-Drug Conjugates (ADCs) consist of antibodies attached to cytotoxic small molecules or biological agents (i.e., payloads) through chemical linkers which may be cleavable or non-cleavable. The development of new ADCs is challenging, particularly the process of attaching the linker-payload construct to the antibody (i.e., the conjugation process). One of the major problems associated with conjugation is high hydrophobicity of the payload which can lead to low yields of the ADC through aggregation and/or lower than desired Drug-Antibody Ratios (DARs). We report here a UPLC-based assay that can be used to study the physicochemical properties of ADC payloads at an early stage of development, and to provide information on whether the hydrophilic-hydrophobic balance is suitable for conjugation or further physicochemical optimization is required. The assay is relatively simple to establish and should be of use to those working in the ADC area. 10.1016/j.jchromb.2020.122075
    Advances in Antibody-Drug Conjugate Design: Current Clinical Landscape and Future Innovations. Gauzy-Lazo Laurence,Sassoon Ingrid,Brun Marie-Priscille SLAS discovery : advancing life sciences R & D The targeted delivery of potent cytotoxic molecules into cancer cells is considered a promising anticancer strategy. The design of clinically effective antibody-drug conjugates (ADCs), in which biologically active drugs are coupled through chemical linkers to monoclonal antibodies, has presented challenges for pharmaceutical researchers. After 30 years of intensive research and development activities, only seven ADCs have been approved for clinical use; two have received fast-track designation and two breakthrough therapy designation from the Food and Drug Administration. There is continued interest in the field, as documented by the growing number of candidates in clinical development. This review aims to summarize the most recent innovations that have been applied to the design of ADCs undergoing early- and late-stage clinical trials. Discovery and rational optimization of new payloads, chemical linkers, and antibody formats have improved the therapeutic index of next-generation ADCs, ultimately resulting in improved clinical benefit for the patients. 10.1177/2472555220912955
    Streamlined Expressed Protein Ligation: Site-Specific Antibody-Drug Conjugate. Frutos Silvia Methods in molecular biology (Clifton, N.J.) Protein semisynthesis is a powerful tool for studying proteins and has contributed to a better understanding of protein structure and function and also driven innovations in protein science. Expressed protein ligation (EPL) is a widely used method to generate chemically modified proteins. However, EPL has some limitations, particularly relevant to modify challenging proteins such as antibodies. The method termed streamlined expressed protein ligation (SEPL) overcomes some of the problems of EPL, and other methods of protein semisynthesis, to generate challenging modified proteins such as antibody-drug conjugates (ADCs). ADCs targeting highly cytotoxic molecules to cancer cells, offer an attractive strategy to selectively eliminate tumor cells with improved therapeutic index than the antibodies or cytotoxic molecules themselves. Despite the potential of ADCs, the development of such complex molecules is challenging. We provide here protocols to prepare site-specifically modified ADCs by streamlined expressed protein ligation (SEPL), which does not require the incorporation of unnatural modifications into the antibody. Therefore, fully native antibodies, with only the desired cytotoxic molecules attached, can be generated. 10.1007/978-1-0716-0434-2_9
    High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries. Yamazoe Sayumi,Hogan Jason M,West Sean M,Deng Xiaodi A,Kotapati Srikanth,Shao Xiang,Holder Patrick,Lamba Vandana,Huber Mary,Qiang Cong,Gangwar Sanjeev,Rao Chetana,Dollinger Gavin,Rajpal Arvind,Strop Pavel Bioconjugate chemistry Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs. 10.1021/acs.bioconjchem.0c00146
    Dual-mechanistic antibody-drug conjugate site-specific selenocysteine/cysteine conjugation. Nilchan Napon,Li Xiuling,Pedzisa Lee,Nanna Alex R,Roush William R,Rader Christoph Antibody therapeutics Background:While all clinically translated antibody-drug conjugates (ADCs) contain a single-drug payload, most systemic cancer chemotherapies involve use of a combination of drugs. These regimens improve treatment outcomes and slow development of drug resistance. We here report the generation of an ADC with a dual-drug payload that combines two distinct mechanisms of action. Methods:Virtual DNA crosslinking agent PNU-159682 and tubulin polymerization inhibitor monomethyl auristatin F (MMAF) were conjugated to a HER2-targeting antibody site-specific conjugation at engineered selenocysteine and cysteine residues (thio-selenomab). Results:The dual-drug ADC showed selective and potent cytotoxicity against HER2-expressing cell lines and exhibited dual mechanisms of action consistent with the attached drugs. While PNU-159682 caused S-phase cell cycle arrest due to its DNA-damaging activity, MMAF simultaneously inhibited tubulin polymerization and caused G2/M-phase cell cycle arrest. Conclusion:The thio-selenomab platform enables the assembly of dual-drug ADCs with two distinct mechanisms of action. 10.1093/abt/tbz009
    A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations. Zmolek Wesley,Bañas Stefanie,Barfield Robyn M,Rabuka David,Drake Penelope M Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step. 10.1016/j.jchromb.2016.05.055
    Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity. Christianson Helena C,Menard Julien A,Chandran Vineesh Indira,Bourseau-Guilmain Erika,Shevela Dmitry,Lidfeldt Jon,Månsson Ann-Sofie,Pastorekova Silvia,Messinger Johannes,Belting Mattias Oncotarget Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments. 10.18632/oncotarget.16921
    Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions. Huang Betty C B,Kim Yun Cheol,Bañas Stefanie,Barfield Robyn M,Drake Penelope M,Rupniewski Igor,Haskins William E,Rabuka David mAbs The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and - increasingly - the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches. 10.1080/19420862.2018.1512327
    Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads. Puthenveetil Sujiet,Loganzo Frank,He Haiyin,Dirico Ken,Green Michael,Teske Jesse,Musto Sylvia,Clark Tracey,Rago Brian,Koehn Frank,Veneziale Robert,Falahaptisheh Hadi,Han Xiaogang,Barletta Frank,Lucas Judy,Subramanyam Chakrapani,O'Donnell Christopher J,Tumey L Nathan,Sapra Puja,Gerber Hans Peter,Ma Dangshe,Graziani Edmund I Bioconjugate chemistry There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs. 10.1021/acs.bioconjchem.6b00291
    An Alternative Focus for Route Design for the Synthesis of Antibody-Drug Conjugate Payloads. Tiberghien Arnaud C,Howard Philip W,Goundry William R F,McCormick Marc,Parker Jeremy S The Journal of organic chemistry An analysis of Antibody-Drug Conjugate Payload manufacturing has revealed that the majority of the cost is associated with the use of high-containment facilities for the latter stages of the synthesis. To make a significant reduction in the Cost of Goods (CoGs), a new approach to route design has been introduced which focuses on minimizing the number of steps that require high containment. This approach has been exemplified in a new synthesis of tesirine, including the first application of a ring-closing copper(I)/TEMPO aerobic oxidation to the pyrrolobenzodiazepine ring system, affording a 60% reduction in CoGs. 10.1021/acs.joc.8b02876
    A RAGE-Targeted Antibody-Drug Conjugate: Surface Plasmon Resonance as a Platform for Accelerating Effective ADC Design and Development. Healey Gareth D,Frostell Asa,Fagge Tim,Gonzalez Deyarina,Conlan R Steven Antibodies (Basel, Switzerland) Antibodies, antibody-like molecules, and therapeutics incorporating antibodies as a targeting moiety, such as antibody-drug conjugates, offer significant potential for the development of highly efficacious drugs against a wide range of disorders. Despite some success, truly harnessing the superior targeting properties of these molecules requires a platform from which to effectively identify the best candidates for drug development. To streamline the development of antibody-drug conjugates targeting gynecological cancers within our laboratory, we incorporated surface plasmon resonance analysis (Biacore™ T200) into our development toolkit. Antibodies, selected based on positive ELISA screens as suitable for development as antibody-drug conjugates, were evaluated using surface plasmon resonance to determine a wide range of characteristics including specificity, kinetics/affinity, the effect of linker binding, the impact of the drug to antibody ratio, and the effect of endosomal pH on antibody-antigen binding. Analysis revealed important kinetics data and information regarding the effect of conjugation and endosomal pH on our antibody candidates that correlated with cell toxicity and antibody internalization data. As well as explaining observations from cell-based assays regarding antibody-drug conjugate efficacies, these data also provide important information regarding intelligent antibody selection and antibody-drug conjugate design. This study demonstrates the application of surface plasmon resonance technology as a platform, where detailed information can be obtained, supporting the requirements for rapid and high-throughput screening that will enable enhanced antibody-drug conjugate development. 10.3390/antib8010007
    A Switchable Site-Specific Antibody Conjugate. Lyu Zhigang,Kang Lei,Buuh Zakey Yusuf,Jiang Dawei,McGuth Jeffrey C,Du Juanjuan,Wissler Haley L,Cai Weibo,Wang Rongsheng E ACS chemical biology Genetic incorporation of unnatural amino acids (UAAs) provides a unique approach to the synthesis of site-specific antibody conjugates that are homogeneous and better defined constructs than random conjugates. Yet, the yield varies for every antibody, and the process is costly and time-consuming. We have developed a switchable αGCN4-Fab conjugate that incorporates UAA p-acetylphenylalanine. The GCN4 peptide is used as a switch, and antibodies fused by GCN4 can direct the αGCN4-Fab conjugate to target different cancer cells for diagnosis, imaging, or therapeutic treatment. More importantly, this switchable conjugate demonstrated an impressive potential for pretargeted imaging in vivo. This approach illustrates the utility of an orthogonal switch as a general strategy to endow versatility to a single antibody conjugate, which should facilitate the application of UAA-based site-specific conjugates for a host of biomedical uses in the future. 10.1021/acschembio.8b00107
    Computational Approaches in Antibody-drug Conjugate Optimization for Targeted Cancer Therapy. Melo Rita,Lemos Agostinho,Preto Antonio J,Almeida Jose G,Correia Joao D G,Sensoy Ozge,Moreira Irina S Current topics in medicinal chemistry Cancer has become one of the main leading causes of morbidity and mortality worldwide. One of the critical drawbacks of current cancer therapeutics has been the lack of the target-selectivity, as these drugs should have an effect exclusively on cancer cells while not perturbing healthy ones. In addition, their mechanism of action should be sufficiently fast to avoid the invasion of neighbouring healthy tissues by cancer cells. The use of conventional chemotherapeutic agents and other traditional therapies, such as surgery and radiotherapy, leads to off-target interactions with serious side effects. In this respect, recently developed target-selective Antibody-Drug Conjugates (ADCs) are more effective than traditional therapies, presumably due to their modular structures that combine many chemical properties simultaneously. In particular, ADCs are made up of three different units: a highly selective Monoclonal antibody (Mab) which is developed against a tumour-associated antigen, the payload (cytotoxic agent), and the linker. The latter should be stable in circulation while allowing the release of the cytotoxic agent in target cells. The modular nature of these drugs provides a platform to manipulate and improve selectivity and the toxicity of these molecules independently from each other. This in turn leads to generation of second- and third-generation ADCs, which have been more effective than the previous ones in terms of either selectivity or toxicity or both. Development of ADCs with improved efficacy requires knowledge at the atomic level regarding the structure and dynamics of the molecule. As such, we reviewed all the most recent computational methods used to attain all-atom description of the structure, energetics and dynamics of these systems. In particular, this includes homology modelling, molecular docking and refinement, atomistic and coarse-grained molecular dynamics simulations, principal component and cross-correlation analysis. The full characterization of the structure-activity relationship devoted to ADCs is critical for antibody-drug conjugate research and development. 10.2174/1568026618666180731165222
    Antibody conjugated nanoparticles as a novel form of antibody drug conjugate chemotherapy. Johnston Michael C,Scott Christopher J Drug discovery today. Technologies Antibody conjugated nanoparticles (ACNPs) represent a novel strategy for the development of therapies exploiting antibodies to augment the delivery of chemotherapy payloads. Following in the footsteps of the success of antibody drug conjugates (ADCs), ACNPs are only now reaching clinical evaluation. In this review we discuss the success of ADCs and explore the opportunities ACNPs offer, such as broad chemotherapy payload selection, high drug to antibody ratios and the ability to finely tailor drug release in comparison to ADCs. The ability of ACNPs to elicit increased avidity due to multivalent effects and the potential to use these modular platforms in immunotherapeutic approaches is also explored. Through addressing challenges that still remain in bringing these complex formulations to the clinic, ACNPs hold obvious potential for the treatment of a wide range of cancers and other diseases where selective targeting of drug agents is essential. 10.1016/j.ddtec.2018.10.003
    In-Depth Characterization of a Pro-Antibody-Drug Conjugate by LC-MS. Liu Boning,Guo Huaizu,Zhang Junjie,Xue Jingya,Yang Yun,Qin Ting,Xu Jin,Guo Qingcheng,Zhang Dapeng,Qian Weizhu,Li Bohua,Hou Sheng,Dai Jianxin,Guo Yajun,Wang Hao Molecular pharmaceutics Pro-antibody-drug conjugate (PDC) is a hybrid structural format of immunoconjugate, where the structural complexity of pro-antibody and intrinsic heterogeneity of ADCs impose a prominent analytical challenge to the in-depth characterization of PDCs. In the present study, we successfully prepared and characterized PanP-DM1 as a model of PDCs, which is an anti-EGFR pro-antibody following conjugation with DM1 at lysine residues. The drug-to-antibody ratio (DAR) of PanP-DM1 was determined by LC-MS after deglycosylation, and verified by UV/vis spectroscopy. Following reduction or IdeS digestion, the pro-antibody fragments linked with DM1 were investigated by middle-down mass spectrometry. Furthermore, more than 20 modified lysine conjugation sites were determined by peptide mapping after trypsin digestion. Additionally, more than ten glycoforms of PanP-DM1 were also identified and quantified. In summary, critical quality attributes (CQAs) of PDCs including DAR, drug load distribution, and conjugation sites were fully characterized, which would contribute to the development of other PDCs for cancer treatment. 10.1021/acs.molpharmaceut.6b00280
    Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release. Szot Christopher,Saha Saurabh,Zhang Xiaoyan M,Zhu Zhongyu,Hilton Mary Beth,Morris Karen,Seaman Steven,Dunleavey James M,Hsu Kuo-Sheng,Yu Guo-Jun,Morris Holly,Swing Deborah A,Haines Diana C,Wang Yanping,Hwang Jennifer,Feng Yang,Welsch Dean,DeCrescenzo Gary,Chaudhary Amit,Zudaire Enrique,Dimitrov Dimiter S,St Croix Brad The Journal of clinical investigation Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types. 10.1172/JCI120481
    Checkpoint Inhibitors Boost Power of Antibody-Drug Conjugate. Cancer discovery The antibody-drug conjugate T-DM1 spurs immune cells to infiltrate HER2-positive breast tumors in patients. Treating mice that carry breast tumors with T-DM1 improves survival, but the tumors suppress the infiltrating lymphocytes. Combining T-DM1 with checkpoint inhibitors that block CTLA-4 and PD-1 overcomes this effect, eliminating breast tumors from 95% of the mice. 10.1158/2159-8290.CD-NB2015-172
    An immunosuppressive antibody-drug conjugate. Wang Rongsheng E,Liu Tao,Wang Ying,Cao Yu,Du Jintang,Luo Xiaozhou,Deshmukh Vishal,Kim Chan Hyuk,Lawson Brian R,Tremblay Matthew S,Young Travis S,Kazane Stephanie A,Wang Feng,Schultz Peter G Journal of the American Chemical Society We have developed a novel antibody-drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib-antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC50 and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology. 10.1021/jacs.5b00620
    Antibody-Drug Conjugate Kills Neuroblastoma Cells. Cancer discovery Researchers have developed an antibody-drug conjugate with the potential to treat neuroblastoma. The agent targets ALK, which is found on neuroblastoma cells but not healthy cells, delivering a drug that causes DNA cross-links. Researchers have found that the antibody-drug conjugate kills ALK-expressing cells and increases survival in mouse models of neuroblastoma. 10.1158/2159-8290.CD-NB2019-052