ABSTRACT We 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.