Glycoengineering eCD4-Ig

ABSTRACTWe are developing eCD4-Ig, an antibody-like molecule constructed from CD4, a coreceptor-mimetic peptide, and an antibody Fc, as a long-acting injectable therapeutic for HIV. Our progress to-date includes having extended the plasma half-life of eCD4-Ig to the point that it now rivals or surpasses that of the leading broadly neutralizing antibodies (bNAbs) being developed as long-acting injectables. In a head-to-head comparison of pharmacokinetics (PK) in the human FcRn-transgenic mouse model, eCD4-Ig had significantly better PK than a VRC01 protein that had a half-life of 71 days in humans (Gaudinski et al., PLoS Med, 2018). With support from NIAID, we have developed a CHO cell line for manufacturing eCD4-Ig under cGMP conditions. However, in-depth analysis of the composition of the N-linked glycans (NLGs) at N297 of the Fc of the protein made by the cell line has highlighted a problem shared by the entire field: The large majority of the NLGs are forms detrimental to PK. This problem is particularly pronounced among the afucosylated forms that are active for antibody- dependent cell-mediated cytotoxicity (ADCC). Indeed, only a few percent of the NLGs are afucosylated forms that allow both ADCC and a long half-life. Therefore, we propose glycoengineering our cell line for manufacturing eCD4-Ig. To do so, we will develop a glycoengineering gene cassette, introduce it into the cell line, and derive a glycoengineered clone for manufacturing eCD4-Ig under cGMP conditions. Although our primary goal is to manufacture eCD4-Ig protein consisting almost entirely of a single long-lived glycoform that is active for ADCC, the same glycoengineering gene cassette can be used to manufacture other antibody therapeutics, including bNAbs, to similarly extend effector function and plasma half-life. This work will reduce the therapeutic concentration and increase the interval at which long-acting protein therapeutics for treating and preventing HIV infection can be dosed.PROJECT NARRATIVEThe HIV field is moving towards the use of long-acting injectable drugs. This project will extend the longevity of eCD4-Ig in the body by engineering which sugar molecules that are attached to it. The same approach can be used to extend the longevity of other therapeutics for treating and preventing HIV infection.