Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1

Abstract

(2016) Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1, mAbs, 8:4, 761-774, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTWe previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.

PotentIn VivoNK Cell-Mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-Bispecific and Hexavalent Broadly Neutralizing Fusion Protein

“…While LSEVh-LS-F plasma levels averaged about 100 g/ml for the first 2 days after administration, levels subsequently declined more rapidly than those of bNAb such as VRC01 and 3BNC117. As previously described, several factors may contribute to the shorter half-life of LSEVh-LS-F than of native bNAb, including the reduced ability of the CCH1-CK interaction to generate extremely stable heterodimers and the increased susceptibility to proteolytic degradation associated with the use of polypeptide linkers to link mD1.22 and m36.4 to the Fc scaffold (12). We are investigating new approaches to enhance the half-life of LSEVh-LS-F.…”

“…Nevertheless, its potent capacity to neutralize HIV-1 strains resistant to other bNAb and to eliminate HIV-1infected cells should counterbalance its shorter half-life, and this supports the potential inclusion of short, intense courses of LSEVh-LS-F as a component of therapeutic regimens that combine immunostimulants, such as ALT-803, and latency-reactivating agents to reduce the HIV-1 reservoir and contribute to the final goal of HIV-1 eradication. Expression and purification of VRC01, 4Dm2m, LSEVh-LS, and LSEVh-LS-F. 4Dm2m and LSEVh-LS were produced by transfecting 293-F cells with either the 4Dm2m or LSEVh-LS vector constructed by PCR cloning or the VRC01 plasmid provided by John Mascola (Vaccine Research Center [VRC], NIH, Bethesda, MD) and processing the cell culture supernatant as described previously (10,12). Defucosylated LSEVh-LS (LSEVh-LS-F) was produced using CHO cells with their GDP-fucose transporter (GFT) genes inactivated using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system as described previously (19).…”

“…Plasma concentrations of VRC01 and LSEVh-LS-F were determined by enzyme-linked immunosorbent assay (ELISA) with plates coated with HIV-1 Env gp140 89.6. Standard curves were generated using VRC01 or LSEVh-LS-F standards as described previously (12).…”

“…22 was designed to mirror the CD4 structure, mutations in gp120 which reduce mD1.22 binding should be paralleled by decreased CD4 binding, which would diminish HIV-1 replicative capacity and thereby inhibit the emergence of mD1.22 escape mutations. We generated a structural variant of 4Dm2m, LSEVh-LS, with a significantly increased in vivo half-life due to its improved structural stability and increased binding to the FcRn (12). We further augmented the capacity of LSEVh-LS to mobilize ADCC activity by defucosylating its Fc domain to increase its affinity for Fc␥RIIIa and thereby amplify its ability to recruit effector cells (13).…”

Establishment of a Novel Humanized Mouse Model To Investigate In Vivo Activation and Depletion of Patient-Derived HIV Latent Reservoirs

“…SPHIR mice were intravenously injected with 0.5 mg of either 10-1074 or 10-1074-FcR null on day 0. Plasma samples were collected weekly and plasma concentrations of 10-1074 and 10-1074-FcR null were determined by ELISA with plates coated with HIV Env gp140 89.6 , and standard curves were generated using 10-1074 or 10-1074-FcR null standards as described previously (54).…”