scholarly journals Elution Profiles of Antibody-Drug Conjugates in Preparative Chromatography

2021 ◽  
Vol 333 ◽  
pp. 14001
Author(s):  
Takuro Tanaka ◽  
Koichiro Ikeda ◽  
Shuichi Yamamoto ◽  
Noriko Yoshimoto
Keyword(s):  
Thiol Group ◽  
Gradient Elution ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Preparative Chromatography ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Maleimide Group ◽  
Antibody Ratio ◽  
Antibody Drug

Monoclonal antibody drug conjugate (ADCs) have received much attention as pharmaceutical agents for treating serious diseases such as cancer. However, it is difficult to separate them on the basis of the drug to antibody ratio, DAR. Hydrophobic chromatography (HIC) is commonly used for the analysis of the drug to antibody ratio, DAR. The retention of ADCs on HIC can be controlled by the hydrophobic nature of ADCs, depending on the mobile phase conditions. They are sometimes performed at the restricted conditions where the solubility is too low. Ion exchange chromatography (IEC) using electrostatic interaction is an orthogonal method to HIC. IEC is widely used because of its higher capacity than HIC. We investigated the retention behavior of the protein conjugated with surrogate drugs on IEC. The surrogate drugs employed are 7-diethylamino-3-(4’-maleimidylhenyl) 4-methylcoumarin (CPM), N-(1-pyrenyl) maleimide (NPM). Bovine serum albumin (BSA) was used as a model protein. The molar ratio (CPM and NPM to protein) was set to 3. The maleimide group of CPM and NPM reacts with the thiol group of the proteins. On the linear gradient elution experiments, the elution salt concentrations of the conjugated and non-conjugated proteins were measured to obtain chromatographic parameter of the number of binding sites, B.

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

2021 ◽  
Author(s):  
Neelie Zacharias ◽  
Vladimir Podust ◽  
Kimberly Kajihara ◽  
Douglas leipold ◽  
Geoffrey Del Rosario ◽  
...  
Keyword(s):  
Cancer Therapeutics ◽  
Therapeutic Index ◽  
Tumor Xenograft ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Anti Cancer ◽  
Rapid Clearance ◽  
Antibody Ratio ◽  
Antibody Drug

Abstract Antibody-drug conjugates (ADCs) enable cell-specific delivery of small molecules and are validated anti-cancer therapeutics. One factor limiting ADC advancement and broader application is the drug-to-antibody ratio (DAR), which dictates the number of payloads that can be delivered per antibody. With few exceptions, efficacious ADCs with DAR > 4 are inaccessible due to aggregation or rapid clearance in vivo. Here, we report a versatile platform for the generation of homogeneous ADCs with DAR up to 18, combining Cys-engineered THIOMAB antibodies and XTEN polypeptides to give “TXCs”. We show that high-DAR TXCs are stable biochemically and in vivo. We demonstrate that two different cytotoxic TXCs directed toward a tumor xenograft and one TXC targeting Staphylococcus aureus have comparable pharmacokinetics, but significantly enhanced efficacy in vivo versus conventional low-DAR ADCs. Our data suggest that high-DAR TXCs may ultimately offer several advantages versus conventional ADCs, including increased therapeutic index and efficacious delivery of less potent payloads.

scholarly journals Development of a Single-Step Antibody–Drug Conjugate Purification Process with Membrane Chromatography

2021 ◽  
Vol 10 (3) ◽  
pp. 552
Author(s):  
Juan Carlos Cordova ◽  
Sheng Sun ◽  
Jeffrey Bos ◽  
Srinath Thirumalairajan ◽  
Sanjeevani Ghone ◽  
...  
Keyword(s):  
Downstream Processing ◽  
Single Step ◽  
High Yield ◽  
Antibody Drug Conjugate ◽  
Membrane Chromatography ◽  
Drug Conjugates ◽  
Host Cell Proteins ◽  
Viral Particles ◽  
Antibody Ratio ◽  
Antibody Drug

Membrane chromatography is routinely used to remove host cell proteins, viral particles, and aggregates during antibody downstream processing. The application of membrane chromatography to the field of antibody-drug conjugates (ADCs) has been applied in a limited capacity and in only specialized scenarios. Here, we utilized the characteristics of the membrane adsorbers, Sartobind® S and Phenyl, for aggregate and payload clearance while polishing the ADC in a single chromatographic run. The Sartobind® S membrane was used in the removal of excess payload, while the Sartobind® Phenyl was used to polish the ADC by clearance of unwanted drug-to-antibody ratio (DAR) species and aggregates. The Sartobind® S membrane reproducibly achieved log-fold clearance of free payload with a 10 membrane-volume wash. Application of the Sartobind® Phenyl decreased aggregates and higher DAR species while increasing DAR homogeneity. The Sartobind® S and Phenyl membranes were placed in tandem to simplify the process in a single chromatographic run. With the optimized binding, washing, and elution conditions, the tandem membrane approach was performed in a shorter timescale with minimum solvent consumption and high yield. The application of the tandem membrane chromatography system presents a novel and efficient purification scheme that can be realized during ADC manufacturing.

Axially-substituted silicon phthalocyanine payloads for antibody-drug conjugates

Synlett ◽  
2021 ◽  
Author(s):  
Kazuki Takahashi ◽  
Akira Sugiyama ◽  
Kei Ohkubo ◽  
Toshifumi Tatsumi ◽  
Tatsuhiko Kodama ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Antibody Drug Conjugate ◽  
Antibody Drug Conjugates ◽  
Drug Conjugate ◽  
Drug Conjugates ◽  
Antibody Drug ◽  
Silicon Phthalocyanine

IR700, a silicon phthalocyanine (SiPc) photosensitizer, is an antibody-drug conjugate payload used clinically. It is, however, the sole SiPc payload to date, possibly due to the difficulty of its synthesis, resulting from its asymmetric phathalocyanine skeleton. Here we report a new axially-substituted SiPc payload with easier synthesis. Trastuzumab conjugated with the SiPc showed light- and antigen-dependent cytotoxicity in HER2-overexpressed cancer cell lines.

scholarly journals Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation

2019 ◽  
Author(s):  
Susanna K. Elledge ◽  
Hai L. Tran ◽  
Alec H. Christian ◽  
Veronica Steri ◽  
Byron Hann ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Xenograft Model ◽  
Antibody Engineering ◽  
Antibody Drug Conjugate ◽  
Site Specific ◽  
Drug Conjugates ◽  
Mouse Xenograft Model ◽  
Random Modification ◽  
The Stability ◽  
Antibody Drug

AbstractChemical modification of antibodies is one of the most important bioconjugations utilized by biologists and biotechnology. To date, the field has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently we have developed oxaziridine chemistry for highly selective and efficient sulfimide modification of methionine called redox-activated chemical tagging (ReACT). Here, we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and generally good reaction efficiencies with the panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than ten-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, likely because of their reduced access to water. There was also a ten-fold variation in stability depending on the nature of the oxaziridine, which we determined was inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs and antibody drug conjugate potencies were comparable to those reported for cysteine-maleimide modifications of trastuzumab. We also found our antibody drug conjugates to be potent in a breast cancer mouse xenograft model. These studies provide a roadmap for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation.

scholarly journals Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry

2021 ◽  
Author(s):  
Syed Usama ◽  
Sierra Marker ◽  
Donald Caldwell ◽  
Nimit Patel ◽  
Yang Feng ◽  
...  
Keyword(s):  
Near Infrared ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Lysosomal Accumulation ◽  
Cellular Signal ◽  
In Vivo Analysis ◽  
Cellular Permeability ◽  
Therapeutic Platform ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitively assess cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits dramatically in-creased cellular signal due to improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ~50X brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or non-hindered disulfides – an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

The role of enfortumab vedotin and sacituzumab govitecan in treatment of advanced bladder cancer

2021 ◽  
Author(s):  
Kirollos S Hanna ◽  
Samantha Larson ◽  
Jenny Nguyen ◽  
Jenna Boudreau ◽  
Jennifer Bulin ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Second Line ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Advanced Bladder Cancer ◽  
Checkpoint Inhibitor Therapy ◽  
Antibody Drug

Abstract Disclaimer In an effort to expedite the publication of articles pandemic, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. Purpose The treatment landscape of advanced bladder cancer continues to evolve with novel therapeutics approved in recent years and many in the pipeline. Here we review the role of the novel agents enfortumab vedotin and sacituzumab govitecan in treatment of advanced disease. Summary Patients with advanced bladder cancer often first receive platinum-based therapy, while immune checkpoint inhibitors offer a maintenance option following cytotoxic chemotherapy or a second-line option. Despite various first- and second-line options, patients with significant comorbidities and treatment-related adverse events will experience disease progression requiring alternative treatment. Enfortumab vedotin and sacituzumab govitecan are novel antibody-drug conjugates approved in patients with advanced bladder cancer following platinum-based and immune checkpoint inhibitor therapy. Following platinum-based therapy and immunotherapy in patients with advanced bladder cancer, enfortumab vedotin, targeting Nectin-4, improves overall survival while sacituzumab govitecan, targeting Trop-2, is associated with a 27% response rate. With these new approaches to disease management, however, it remains critical to understand safety, efficacy, and operational considerations to optimize outcomes. Conclusion When selecting an antibody-drug conjugate to treat patients with bladder cancer, it is important to note the adverse event profile of each agent to optimize outcomes and safety for patients.

Targeting CD30 in Hodgkin Lymphoma: Antibody-Drug Conjugates Make a Difference

2012 ◽  
pp. 162-166 ◽  
Author(s):  
Catherine S. M. Diefenbach ◽  
John P. Leonard
Keyword(s):  
Hodgkin Lymphoma ◽  
Large Cell ◽  
Brentuximab Vedotin ◽  
Antibody Drug Conjugate ◽  
Lymphoid Malignancies ◽  
Drug Conjugates ◽  
First Line Therapy ◽  
Resistant Disease ◽  
Relapsed Disease ◽  
Antibody Drug

Overview: CD30 expression is characteristic of the malignant Reed-Sternberg cell in Hodgkin lymphoma (HL) and several other lymphoid malignancies, such as anaplastic large-cell lymphoma (ALCL). Although unconjugated anti-CD30 antibodies have had minimal therapeutic activity in patients with HL as single agents, the CD30-directed antibody-drug conjugate (ADC) brentuximab vedotin has demonstrated activity that has resulted in its recent regulatory approval for the treatment of patients with relapsed HL and ALCL. Approximately 75% of patients with recurrent HL achieve objective responses, with the principal toxicity being peripheral neuropathy. Ongoing studies are evaluating treatment with this agent as part of first-line therapy, for patients with relapsed disease, and for patients with resistant disease and limited other options. Brentuximab vedotin demonstrates the therapeutic value of antibody-drug conjugation and serves as a model of how a novel, targeted approach can be employed to potentially further improve outcomes in settings where curative chemotherapeutic regimens are already available.

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