Overcoming challenges associated with the bioanalysis of cysteine-conjugated metabolites in the presence of antibody–drug conjugates

Bioanalysis ◽  
2021 ◽  
Vol 13 (18) ◽  
pp. 1427-1439
Author(s):  
Andrew P Mayer ◽  
Hermes Licea-Perez ◽  
Sharon Boram ◽  
Kristen E Pannullo ◽  
Jonathan Kehler ◽  
...  
Keyword(s):  
Extraction Process ◽  
Critical Importance ◽  
Sample Processing ◽  
Antibody Drug Conjugate ◽  
Bioanalytical Method ◽  
Drug Conjugates ◽  
Spe Method ◽  
Conjugated Metabolites ◽  
Antibody Drug

Aim: Investigations have shown that for the antibody–drug conjugate (ADC) belantamab mafodotin, concentrations of the cysteine-conjugated metabolite, Cys-mcMMAF, were overestimated in the presence of the ADC during sample processing when utilizing a historical SPE method. Results: A new assay was developed utilizing an acidic protein precipitation to remove the ADC early in the extraction process, thus eliminating the risk of overestimating Cys-mcMMAF in the presence of belantamab mafodotin. In vitro experiments demonstrated a linear relationship between the concentration of belantamab mafodotin and the release of Cys-mcMMAF. Extensive stability assessments were performed to cover storage of study samples. Conclusion: This work emphasized the critical importance of understanding the performance of a bioanalytical method for free toxic payload in the presence of the ADC.

scholarly journals ATRT-15. LY6D – A CANDIDATE FOR NANOPARTICLE-BASED TARGETED THERAPIES OF ATRT

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii278-iii278
Author(s):  
Lea Hagemeier ◽  
Marthe Sönksen ◽  
Natalia Moreno ◽  
Romy Ettlinger ◽  
Hana Bunzen ◽  
...  
Keyword(s):  
Targeted Therapies ◽  
Surface Proteins ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Interesting Candidate ◽  
Atypical Teratoid Rhabdoid Tumors ◽  
Rhabdoid Tumors ◽  
Antibody Drug

Abstract Atypical Teratoid Rhabdoid Tumors (ATRT) are aggressive brain malignancies of the infant. Despite intensive multimodal therapy, the overall prognosis remains poor, making investigations on targeted therapies crucial. Arsenic trioxide (ATO) is known to inhibit cell growth of ATRT in vitro and in vivo but its efficacy in solid tumors is limited by its adverse effects. We aimed to characterize whether a nanoparticle-based drug delivery could overcome these limitations. Therefore metal-organic frameworks containing ATO (MOF-ATO) were constructed. To improve drug specificity further, we searched for unique proteins on the surface of ATRT, in order to create antibody-drug-conjugates out of MOF-ATO and an ATRT-specific ligand. ATRT are marked by a biallelic loss of SMARCB1, which results in an activation of the repressive histone methyltransferase EZH2. After chemical inhibition of EZH2 with GSK126, a mass spectrometric based screening for differentially expressed surface proteins was performed. Treatment with ATO, as well as MOF-ATO and GSK126 each reduces the cell viability of ATRT cell lines. It results in a cell cycle arrest and an induction in apoptosis, being analysed via MTT test and flow cytometry. GSK126 treatment causes a significant upregulation of several cell surface proteins, upon them the Lymphocyte antigen 6 family member D (LY6D). Being rarely expressed on other human cells, this protein is an interesting candidate. An antibody-drug-conjugate consisting of MOF-ATO and LY6D-ligands could be a promising approach for future targeted therapies of ATRT.

scholarly journals A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1029
Author(s):  
Ricarda M. Hoffmann ◽  
Silvia Crescioli ◽  
Silvia Mele ◽  
Eirini Sachouli ◽  
Anthony Cheung ◽  
...  
Keyword(s):  
Tumor Regression ◽  
Chondroitin Sulphate ◽  
Alkylating Agents ◽  
Residual Tumor ◽  
Similar Efficacy ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Antibody Drug

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

scholarly journals A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Tobias Weber ◽  
Athanasios Mavratzas ◽  
Stefan Kiesgen ◽  
Stephanie Haase ◽  
Benedikt Bötticher ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Cancer Therapeutics ◽  
Size Exclusion ◽  
Single Chain ◽  
Antibody Drug Conjugate ◽  
Disulfide Linkage ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significantin vitrocytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies.

scholarly journals Synthesis and Enhanced Cellular Uptake In Vitro of Anti-HER2 Multifunctional Gold Nanoparticles

Cancers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 870 ◽  
Author(s):  
Cruz ◽  
Kayser
Keyword(s):  
Gold Nanoparticles ◽  
Cellular Uptake ◽  
Therapeutic Index ◽  
Active Targeting ◽  
Cell Penetrating Peptide ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Cell Penetrating ◽  
Antibody Drug

Nanoparticle carriers offer the possibility of enhanced delivery of therapeutic payloads in tumor tissues due to tumor-selective accumulation through the enhanced permeability and retention effect (EPR). Gold nanoparticles (AuNP), in particular, possess highly appealing features for development as nanomedicines, such as biocompatibility, tunable optical properties and a remarkable ease of surface functionalization. Taking advantage of the latter, several strategies have been designed to increase treatment specificity of gold nanocarriers by attaching monoclonal antibodies on the surface, as a way to promote selective interactions with the targeted cells—an approach referred to as active-targeting. Here, we describe the synthesis of spherical gold nanoparticles surface-functionalized with an anti-HER2 antibody-drug conjugate (ADC) as an active targeting agent that carries a cytotoxic payload. In addition, we enhanced the intracellular delivery properties of the carrier by attaching a cell penetrating peptide to the active-targeted nanoparticles. We demonstrate that the antibody retains high receptor-affinity after the structural modifications performed for drug-conjugation and nanoparticle attachment. Furthermore, we show that antibody attachment increases cellular uptake in HER2 amplified cell lines selectively, and incorporation of the cell penetrating peptide leads to a further increase in cellular internalization. Nanoparticle-bound antibody-drug conjugates retain high antimitotic potency, which could contribute to a higher therapeutic index in high EPR tumors.

scholarly journals Targeting RON receptor tyrosine kinase for treatment of advanced solid cancers: antibody–drug conjugates as lead drug candidates for clinical trials

2020 ◽  
Vol 12 ◽  
pp. 175883592092006
Author(s):  
Hang-Ping Yao ◽  
Sreedhar Reddy Suthe ◽  
Xiang-Min Tong ◽  
Ming-Hai Wang
Keyword(s):  
Clinical Trials ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Targeted Therapeutics ◽  
Antibody Drug Conjugates ◽  
Drug Candidates ◽  
Drug Conjugates ◽  
Ron Receptor ◽  
Antibody Drug

The recepteur d’origine nantais (RON) receptor tyrosine kinase, belonging to the mesenchymal-to-epithelial transition proto-oncogene family, has been implicated in the pathogenesis of cancers derived from the colon, lung, breast, and pancreas. These findings lay the foundation for targeting RON for cancer treatment. However, development of RON-targeted therapeutics has not gained sufficient attention for the last decade. Although therapeutic monoclonal antibodies (TMABs) targeting RON have been validated in preclinical studies, results from clinical trials have met with limited success. This outcome diminishes pharmaceutical enthusiasm for further development of RON-targeted therapeutics. Recently, antibody–drug conjugates (ADCs) targeting RON have drawn special attention owing to their increased therapeutic activity. The rationale for developing anti-RON ADCs is based on the observation that cancer cells are not sufficiently addicted to RON signaling for survival. Thus, TMAB-mediated inhibition of RON signaling is ineffective for clinical application. In contrast, anti-RON ADCs combine a target-specific antibody with potent cytotoxins for cancer cell killing. This approach not only overcomes the shortcomings in TMAB-targeted therapies but also holds the promise for advancing anti-RON ADCs into clinical trials. In this review, we discuss the latest advancements in the development of anti-RON ADCs for targeted cancer therapy including drug conjugation profile, pharmacokinetic properties, cytotoxic effect in vitro, efficacy in tumor models, and toxicological activities in primates.

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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