Malignant tissues produce divergent antibody glycosylation of relevance for cancer gene therapy effectiveness

Abstract

Gene therapy approaches now allow for the production of therapeutic antibodies by healthy or cancerous human tissues directly in vivo, and, with an increasing number of gene delivery methods available, the cell type for expression can be chosen. Yet, little is known about the biophysical changes introduced by expressing antibodies from producer cells or tissues targeted by gene therapy approaches, nor about the consequences for the type of glycosylation. The effects of different glycosylation on therapeutic antibodies have been well studied by controlling their glycan compositions in non-human mammalian production cells, i.e., Chinese hamster ovary cells. Therefore, we investigated the glycosylation state of clinically approved antibodies secreted from cancer tissues frequently targeted by in vivo gene therapy, using native mass spectrometry and glycoproteomics. We found that antibody sialylation and fucosylation depended on the producer tissue and the antibody isotype, allowing us to identify optimal producer cell types according to the desired mode of action of the antibody. Furthermore, we discovered that high amounts (>20%) of non-glycosylated antibodies were produced in cells sensitive to the action of the produced antibodies. Different glycosylation in different producer cells can translate into an altered potency of in-vivo produced antibodies, depending on the desired mode of action, and can affect their serum half-lives. These results increase our knowledge about antibodies produced from cells targeted by gene therapy, enabling development of improved cancer gene therapy vectors that can include in vivo glycoengineering of expressed antibodies to optimize their efficacies, depending on the desired mode of action.

The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody

“…1A). This expression format allows for stochiometric expression of antibody chains, which, following signal sequence cleavage, cleavage by furin, and subsequent posttranslational modifications, indeed results in antibodies identical in protein sequence to their recombinantly produced counterparts (21)(22)(23).…”

“…1D) and functional tests (see Transduced Tumor Cells Secrete Functional Therapeutic Antibodies). For an extensive characterization of the glycosylation status of antibodies produced by malignant tissues, including TZB produced from BT474 cells and the potential impact on antibody effector functions, we refer to an earlier study of our group (21).…”

iMATCH: an integrated modular assembly system for therapeutic combination high-capacity adenovirus gene therapy

“…Chains were expressed using a F2A 68 , 69 sequence with an optimized furin site (RKRR) 70 for expression from a single open reading frame, with the orientation HC-F2A-LC as described previously. 71 …”

Approaches to Heterogeneity in Native Mass Spectrometry

“…Much discussion in the literature to date, ,,, especially early in the history of this field, has focused on approaches that aim to reduce complexity and heterogeneity at the sample preparation stage with additional or refined purification steps and chromatographic separations or through gas-phase fragmentation/dissociation (such as in tandem MS, ,,− native top-down MS, − and other methods ,,,,,− ) of the heterogeneous subunits. However, advancement in structural biology relies fundamentally upon accurate understanding of biomolecular structure and function in physiologically relevant states, and examples illustrating the importance of heterogeneous features, such as different proteo- and glycoforms, stoichiometries and identities of bound ligands and other cofactors, and multiple coexisting stoichiometries or conformations, in both functional and disease-associated systems abound. ,,− …”