Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

Abstract

Abbreviations:A1AT, alpha-1-antitrypsin; AATD, alpha-1-antitrypsin deficiency; AUC, area under curve; B3gnt2, UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2; Cas9, CRISPR-associated protein 9; C1INH, C1 esterase inhibitor; CHO, Chinese hamster ovary; CRISPR, clustered regularly interspaced short palindromic repeats; FACS, fluorescence-activated cell sorting; FITC, Fluorescein isothiocyanate; Fut8, alpha-(1,6)-fucosyltransferase; Glul, glutamate-ammonia ligase; HAE, hereditary angioedema; HM, high-mannose; indel, insertion or deletion; IVC, integral of viable cells; KO, knock-out; mAb, monoclonal antibody; Mgat4A, mannosyl (alpha-1,3-)-glycoprotein beta-1,4-Nacetylglucosaminyltransferase isozyme A; Mgat4B, mannosyl (alpha-1,3-)glycoprotein beta-1,4-N-acetylglucosaminyltransferase isozyme B; Mgat5, mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase; MSX, methionine sulfoximine; sgRNA, single guide RNA; SNA, sambucus nigra agglutinin; Sppl3, signal peptide peptidase like 3; St3gal3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; St3gal4, ST3 beta-galactoside alpha-2,3-sialyltransferase 4; St3gal6, ST3 beta-galactoside alpha-2,3-sialyltransferase 6; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; VCD, viable cell density; WT, wild type Abstract Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency.Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

“…The addition of a signal peptide or fusion protein is a common technique used to increase recombinant protein expression, and has been used to improve α 1 ‐AT yields from E. coli , yeast, and plants . In the first study to overexpress α 1 ‐AT using cultured mammalian cells, modification to the 5′ untranslated region and endogenous signal peptide improved expression up to fivefold, however, more recent studies using the wild type endogenous signal peptide achieved superior yields . There are therefore a variety of protein and DNA modifications that can be made to α 1 ‐AT to improve the yield, which is largely dependent on the recombinant expression system used.…”

“…Functional expression of wild type recombinant α 1 ‐AT in CHO and HEK293 cells has been reported, although without pharmacokinetic information . Amann et al engineered CHO cells, combining CRISPR/Cas9‐mediated disruption of 10 rodent glycosylation‐related genes and overexpression of human St6 β galactoside α‐2,6‐sialyltransferase 1 (ST6GAL1), to produce recombinant α 1 ‐AT with N‐linked glycan structures more closely resembling those in plasma‐derived α 1 ‐AT than in standard CHO‐derived α 1 ‐AT …”

Awakening dormant glycosyltransferases in CHO cells with CRISPRa

“…For recombinant therapeutic protein production, CHO cells are the most widely used expression host. Despite the ubiquity of CHO cells, the resulting recombinant proteins are not perfect matches to their native human form, in part due to posttranslational modifications, such as differences in glycosylation (Amann et al, ). For example, the bisecting N‐acetylglucosamine attached by Mgat3 (β‐1,4‐mannosyl‐glycoprotein 4‐β‐N‐acetylglucosaminyltransferase) and the alpha 2,6 sialic acid attached by St6gal1 (β‐galactoside α‐2,6‐sialyltransferase 1) are common on many human glycoproteins (Amann et al, ; Popp et al, ), but missing from proteins expressed in CHO cells.…”

“…Despite the ubiquity of CHO cells, the resulting recombinant proteins are not perfect matches to their native human form, in part due to posttranslational modifications, such as differences in glycosylation (Amann et al, ). For example, the bisecting N‐acetylglucosamine attached by Mgat3 (β‐1,4‐mannosyl‐glycoprotein 4‐β‐N‐acetylglucosaminyltransferase) and the alpha 2,6 sialic acid attached by St6gal1 (β‐galactoside α‐2,6‐sialyltransferase 1) are common on many human glycoproteins (Amann et al, ; Popp et al, ), but missing from proteins expressed in CHO cells. While the lack of these epitopes has not led to immunogenic responses (Butler & Spearman, ), these human epitopes may impact drug activity (Chiang et al, ; Reusch & Tejada, ; Tian et al, ).…”

“…are common on many human glycoproteins (Amann et al, 2018;Popp et al, 2018), but missing from proteins expressed in CHO cells. While the lack of these epitopes has not led to immunogenic responses (Butler & Spearman, 2014), these human epitopes may impact drug activity (Chiang et al, 2016;Reusch & Tejada, 2015;Tian et al, 2019).…”