The production of recombinant therapeutic glycoproteins is an active area of research and drug development. Typically, improvements in therapeutic glycoprotein efficacy have focused on engineering additional N-glycosylation sites into the primary amino acid sequence or attempting to control a particular glycoform profile on a protein through process improvements. Recently, a number of alternative expression systems have appeared that are challenging the dominance of mammalian cell culture. Our laboratory has focused on the re-engineering of the secretory pathway in the yeast Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans. We have demonstrated that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. In this chapter we provide detailed protocols for the analysis of glycosylation on intact glycoproteins by MALDI-TOF and site specific N-glycan occupancy on digested glycoprotein using ESI-MS.
The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fcγ receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.
We describe KPMW101, which was created by chemical conjugation of a CD38-specific binder to clinical grade intravenous immunoglobulin (IvIg) pooled from healthy donors. Kleo's MATETM technology enables efficient site-directed chemical conjugation to 'off-the-shelf' IvIg and allows the development of antitumor agents with rapidly introduced target specificity. Our platform allows for chemical engineering of existing IvIg in a cost-efficient manner. This technology relies on synthetic compounds that consists of antibody binder with react-and-release mechanism.
Methods
Design of synthetic chemical reagents included antibody binding group capable of covalent bond formation with specific lysine, CD38 binding moiety proven to work in our clinical candidate KP1237, and tunable non-cleavable linker. Conjugation efficiency to polyclonal IvIg was evaluated using LC-MS analysis of IdeZ-digests. The binding of CD38, CD16a, and FcRn were determined by ELISA and BLI.For in vitro ADCC assays, PBMCs provided NK effector function. Daudi (CD38+) B lymphoblast cells were treated with KPMW101 or IvIg, PBMCs were introduced and incubated for 18h, and target cellular death was measured. For an in vivo IP macrophage lavage model of ADCP, SCID mice were implanted IP with CFSE-labeled Daudi cells. Mice were injected with IvIg or KPMW101 (0.21, 0.625, 1.875 mg/kg) SQ, and tumor cell counts were measured by flow cytometry. The pharmacokinetic profile of in vivo KPMW101 was determined from blood and analyzed utilizing a human Ig isotyping array.
Results
Synthetic chemical reagents with multiple linker types have been conjugated to IvIg and evaluated in biochemical assays. KPMW101 showed the highest conjugation efficiency. Binding affinity of KPMW101 to CD38 was 27nM. ELISA results show KPMW101 binds to CD16a and FcRn, indicating that conjugation does not interfere with FcR binding.In vitro ADCC results demonstrate that KPMW101 elicited CD38+ target cell killing with an EC50 of 0.91–2.09nM.In vivo studies showed that KPMW101 resulted in a 49.9–63.5% reduction of tumor cells. Pharmacokinetic profile showed stability of KPMW101 throughout the 144-hour study, whereby IgG1, IgG2, IgG3, and IgG4 isotypes were detectable.
Conclusions
KPMW101 is created by chemical conjugation of CD38-specific binder to IvIg using our proprietary MATETM technology, maintaining native binding to FcRs via the Fc domain. This ensures the stability of the molecule and retains immune-mediated mechanisms of action. KPMW101 induces IvIg to adopt Fc effector mechanisms like ADCC and ADCP. Our in vitro data and in vivo studies confirm KPMW101 ability to kill tumor cells, making IvIg into an active antitumor therapeutic agent.
Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia–produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.
