Articles tagged ”GlyCLICK”

GlyCLICK® and Middle-up LC-MS Enables Robust ADC Development

Scientists at the University of Geneva and CNRS present site-specific ADCs generated using the GlyCLICK technology and an analytical middle-up LC-HRMS workflow as a potential core module for ADC development.

 

Antibody-drug conjugates (ADCs) are efficient therapeutic agents that possess the cell-targeting properties of monoclonal antibodies combined with the potency of cytotoxic drugs. Early generation ADCs were predominantly obtained through non-selective conjugation methods by incorporation of a drug payload at randomly distributed sites. Such methods result in highly heterogenous subpopulations of varying antibody-drug ratio (DAR) leading to potential loss of efficacy and impaired pharmacokinetics. While alternative strategies exploring genetic engineering have emerged for conjugation at non-natural amino acids, challenges related to both production and analytical characterization persist.

 

Glycan-mediated bioconjugation using the GlyCLICK technology is an attractive option to overcome the challenges of conventional bioconjugation without the need for genetic engineering to produce custom ADCs. By utilizing a unique combination of enzymes, the conserved Fc-glycans are remodeled and site-specifically conjugated using click chemistry for ADCs carrying two payloads per antibody (DAR=2.0) having controlled drug stoichiometry and preserved immunoreactivity. In this paper, Duivelshof et al. developed a site-specific ADC by coupling trastuzumab to DM1 using the GlyCLICK technology and evaluated the quality of the conjugation process using complementary reversed phase (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS).

 

The trastuzumab antibody was site-specifically conjugated to DBCO-functionalized DM1 (DBCO-PEG4-Ahx-DM1) using the GlyCLICK technology. To reduce sample complexity, the antibodies were digested with FabRICATOR® (Ides) or FabALACTICA® (IgdE) and reduced for comparison of native and GlyCLICK conjugated trastuzumab at the subunit level. The complementary HILIC and RPLC workflow allowed the authors to observe the significant shift in retention between the lipophilic drug payloads on the ADC and the hydrophilic N-glycans on native trastuzumab. These results enabled the scientists to confirm site-specific conjugation at the Fc-glycans sites, while hyphenation to HRMS detection allowed accurate determination of a DAR of 2.0 for GlyCLICK conjugated trastuzumab, which was not possible at the intact ADC level.


“Most ADCs are produced with non-selective bioconjugation of drug payloads to lysine or cysteine residues creating a wide variety of drug-antibody ratios (DAR). In the frame of new ADC product development, we believe that having control over the DAR and drug load distribution (DLD) is of crucial importance, as is the ability to accurately monitor these two CQAs. Therefore, the combination of the GlyCLICK technology to create homogeneous site-specific ADCs with the middle-up LC/HRMS approach to rapidly determine both the DLD and DAR has a great potential for ADC development.”

 

Duivelshof et al., 2020. Glycan-mediated technology for obtaining homogenous site-specific conjugated antibody-drug conjugates: synthesis and analytical characterization by using complementary middle-up LC/HRMS analysis. Analytical Chemistry. doi: 10.1021/acs.analchem.0c00282

 

GlyCLICK PET-tracers in Quantitative Imaging to Predict Immunotherapy Response

February 26, 2020 | References |

Scientists at Minerva Imaging demonstrate the potential of site-specific immuno-PET tracers as early identifiers of immune response activation using in vivo imaging. 

 

The programmed cell death protein (PD-1) on immune cells and its corresponding tumor-associated ligand (PD-L1) have emerged as effective targets for immunocheckpoint therapy. To date, the selection of patients eligible for PD-L1 blockade therapy and response rate monitoring is guided by immunohistochemistry of randomly sampled biopsies. This method is not only invasive and prone to errors, but also poorly reflects the heterogeneity and potential metastasis of the tumor.

 

Immunoimaging using PET-tracers is an attractive option to overcome these challenges since it provides a more comprehensive portrayal of the tumor and its temporal dynamics in vivo. In this study, Christensen et al. developed a site-specifically labeled immuno-PET tracer using a GlyCLICK-conjugated anti-PD-L1 antibody. The PET-tracer was used for quantitative detection of PD-L1 expression in order to non-invasively monitor radiotherapy-induced changes and demonstrate the predictive value of such tracers prior to PD-L1 blockade immunotherapy.

 

The anti-PD-L1 antibody was site-specifically conjugated with DIBO-functionalized DFO chelators using the GlyCLICK technology. Chelated conjugates were then radiolabeled with 89Zr to generate PET-tracers carrying two radioisotopes per antibody (DOL=2). Comparing conjugation strategies, the authors found that the GlyCLICK-conjugated antibodies displayed higher immuno-reactivity, stability and affinity compared to random conjugates. In vivo PET imaging and ex vivo biodistribution showed clear PD-L1-specificity of the GlyCLICK-tracers that allowed for the detection of different PD-L1 expression levels among mouse models of human and murine cancer. Importantly, the authors were also able to monitor therapy-induced changes of syngenetic mouse models in a combination study using XRT and anti-PD-L1 therapy. The tumor-to-muscle ratio of GlyCLICK-tracers enabled the scientists to obtain results predictive of response to PD-L1 immunocheckpoint inhibition.

 

Christensen et al., 2019. Quantitative PET imaging of PD-L1 expression in xenograft and syngeneic tumour models using a site-specifically labelled PD-L1 antibody. Journal of Nuclear Medicine and Molecular Imaging. doi: 10,1007/s00259-019-04646-4

 

Learn more about how GlyCLICK works by watching the GlyCLICK Movie.

 

Read more about GlyCLICK on the Product page or download our GlyCLICK Poster

 

 

 

 

 

Improved antibody-PET tracers for in vivo imaging with GlyCLICK®

Radioactively labelled antibodies are excellent immuno-PET tracers for evaluating in vivo distribution and performance of therapuetic agents. Site-specific conjugation at the antibody Fc glycan site by enzymatic remodeling allows for a uniform label distribution of such PET-tracers, compared to conjugates generated with conventional random labelling strategies.

In an article by Kristensen et al. (2019), the authors evaluated the stability, immunoreactivity and in vivo biodistribution of the radioactively labelled mAb Trastuzumab (Herceptin). Using GlyCLICK, the antibody was enzymatically modified with GlycINATOR (EndoS2) and conjugated with a DIBO-DFO chelator prior to 89Zr radioactive labelling. Comparing the GlyCLICK technology with ß-galactosidase remodelled conjugates and two random labelling techniques, the authors obtained valuable data on the overall performance of the various PET-tracers.

Antibodies subjected to site-specific labelling showed significantly increased in vitro stability and immunoreactivity compared to randomly labeled Trastzumab. Furthermore, using in vivo immuno-PET imaging, these conjugates also displayed superior tumor-targeting properties based on the successful detection of HER2-positive tumors in mouse models. These results highlight the advantages of site-specific antibody conjugation.
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Reference:
Kristensen, L. et al., 2019. Site-specifically labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake for immuno-PET in a subcutaneous HER2-positive xenograft mouse model. Theranostics, 9(15). pp.4409-4420.