Over the last decade multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein, gp120, have been described. Surprisingly many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of HIV envelope (Env) proteins compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan dependent epitopes has been challenging. Here we report the development of a stable suspension adapted CHO cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the CRISPR/Cas9 gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan dependent bN-mAbs directed to the V1/V2 domain (e.g. PG9) and the V3 stem (e.g. PGT128 and 10–1074) while preserving the structure of the important glycan independent epitopes (e.g. VRC01). The ability of the MGAT1-CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway, without impairing the doubling time or ability to grow at high cell densities, suggest that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.
Proteolysis associated with recombinant protein expression in Chinese HamsterOvary (CHO) cells has hindered the development of biologics including HIV vaccines.When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1 s (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s −/− MGAT1 − CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs).The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation.Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases. K E Y W O R D S
The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10–100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.
Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid–containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1− CHO cell line exhibit improved binding to prototypic glycan-dependent bN-mAbs directed to the V1/V2 domain (e.g., PG9) and the V3 stem (e.g., PGT128 and 10–1074) while preserving the structure of the important glycan-independent epitopes (e.g., VRC01). The ability of the MGAT1− CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway without impairing the doubling time or ability to grow at high cell densities suggests that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.
2 The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging.3 The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is 4 typically 10-100 fold lower than other glycoproteins of pharmaceutical interest.5 Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing 6 glycans compared to virus associated Envs that possess mainly high-mannose 7 carbohydrates. This difference alters the net charge and biophysical properties of Envs 8 and impacts their antigenic structure. Here we employ a novel gene-edited CHO cell 9 line (MGAT1 -CHO) to address the problems of low expression, high sialic acid content, 10 and poor antigenic structure. We demonstrate that stable cell lines expressing high 11 levels of gp120, potentially suitable for biopharmaceutical production can be created 12 using the MGAT1 -CHO cell line. We also show that the efficiency of this process can be 13 greatly improved with robotic selection. Finally, we describe a MGAT1 -CHO cell line 14 expressing A244-rgp120 that exhibits improved binding of three major families of bN-15 mAbs compared to Envs produced in normal CHO cells. The new strategy described 16 has the potential to eliminate the bottleneck in HIV vaccine development that has limited 17 the field for more than 25 years. 18 3 19 1 Introduction 20The development of a safe, effective, and affordable HIV vaccine is a global 21 public health priority. After more than 30 years of HIV research, a vaccine with these 22properties has yet to be described. To date, the only clinical study to show that 23 vaccination can prevent HIV infection is the 16,000-person RV144 trial carried out in 24 Thailand between 2003 and 2009 (1). This study involved immunization with a 25 recombinant canarypox virus vector to induce cellular immunity (2-4) and a bivalent 26 recombinant gp120 vaccine designed to elicit protective antibody responses (5-7).27 Although statistically significant, the protective efficacy of this vaccination regimen was 28 low (31.2%, P=0.04). Several correlates of protection studies suggested that the 29 protection observed was primarily due to antibodies to rgp120 (8-10). Thus, there is 30 considerable interest in finding ways to improve the level of protection that can be 31 achieved with rgp120 vaccine regimens. Improving an existing vaccine such as RV144, 32 with an established record of safety, would be faster and more cost-effective than 33 developing a new vaccine concept from scratch. A roadmap to improve the rgp120 34 vaccine used in the RV144 trial has been provided by the recent studies of broadly 35 neutralizing monoclonal antibodies (bN-mAbs) to gp120 as well as studies of the 36 carbohydrate content of virion associated Env proteins. Beginning in 2009, studies of 37 bN-mAbs isolated from HIV infected subjects revealed that many recognized unusual 38 glycan dependent epitopes requiring high-mannose glycans that are early intermediates 39 in the N-linked glycosylation pathway (11)(12)(13)(14)(15)(16)(17)(18)(...
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