N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.
The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.
Antibody drug conjugates
(ADCs) can undergo in vivo biotransformation
(e.g., payload metabolism, deconjugation) leading to reduced or complete
loss of activity. The location/site of conjugation of payload-linker
can have an effect on ADC stability and hence needs to be carefully
optimized. Affinity capture LC–MS of intact ADCs or ADC subfragments
has been extensively used to evaluate ADC biotransformation. However,
the current methods have certain limitations such as the requirement
of specific capture reagents, limited mass resolution of low mass
change metabolites, low sensitivity, and use of capillary or nanoflow
LC–MS. To address these challenges, we developed a generic
affinity capture LC–MS assay that can be utilized to evaluate
the biotransformation of any site-specific ADC independent of antibody
type and site of conjugation (Fab and Fc) in preclinical studies.
The method involves a combination of some or all of these steps: (1)
“mono capture” or “dual capture” of ADCs
from serum with streptavidin magnetic beads coated with a generic
biotinylated antihuman capture reagent, (2) “on-bead”
digestion with IdeS and/or PNGase F, and (3) reduction of interchain
disulfide bonds to generate ∼25 kDa ADC subfragments, which
are finally analyzed by LC–HRMS on a TOF mass spectrometer.
The advantages of this method are that it can be performed using commercially
available generic reagents and requires sample preparation time of
less than 7 h. Furthermore, by reducing the size of intact ADC (∼150
kDa) to subfragments (∼25 kDa), the identification of conjugated
payload and its metabolites can be achieved with excellent sensitivity
and resolution (hydrolysis and other small mass change metabolites).
This method was successfully applied to evaluate the in vitro and
in vivo biotransformation of ADCs conjugated at different sites (LC,
HC-Fab, and HC-Fc) with various classes of payload-linkers.
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