The Second Extracellular Loop of CCR5 Contains the Dominant Epitopes for Highly Potent Anti-Human Immunodeficiency Virus Monoclonal Antibodies

Abstract

Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2.

Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains

“…Twenty thousand CHO-CCR5, CHO-CCR5-dN8, or CHO-CCR5-K171A cells per well were seeded into 96-well tissue culture plates and incubated overnight at 37°C as described earlier (40). Thereafter, the cell culture medium was aspirated, 50 l new medium containing serially diluted bispecific or control antibodies was added, and plates were incubated for 2 h at 4°C.…”

“…A cellbased ELISA was applied to assess specific binding of recombinant antibody derivatives to the N-terminal epitope (RoAb13 antibody) and the second extracellular loop (ECL-2) (MAb3952 antibody) within CCR5. For these experiments, two different recombinant cell lines were used: CHO cells expressing human CCR5 with a K171A mutation in the ECL-2 domain and CHO cells with a truncation of the N-terminal 8 residues (40). The truncation of the N-terminal 8 residues abolished binding of the RoAb13 antibody to the CHO/hCCR5dN8 cell line, whereas the MAb3952 antibody bound to the ECL-2 epitope (Fig.…”

“…Current therapeutics under development, such as the anti-CD4 antibody ibalizumab (formerly known as TNX-355) (10,12,14,22) and the anti-CCR5 antibodies PRO 140 and HGS004 (14,23), have shown efficacy against viral infections in vitro and in clinical trials (11,15,16,24,25). We recently described two CCR5 antibodies with high antiviral activity in vitro (16,18,40): RoAb13, which binds to the NTD of CCR5, and MAb3952, which recognizes extracellular domain 2 (ECL-2) of CCR5 (16).…”

Epitope Switching as a Novel Escape Mechanism of HIV to CCR5 Monoclonal Antibodies

“…1 and Table 1). We also tested the susceptibility of Bal_3952res and CC1/85_3952res to another ECL-2-binding antibody (2D7) and a NTD-binding antibody (RoAb13) (29). The MAb3952-resistant viruses are cross-resistant to the CCR5 MAb 2D7, while both NDC virus strains remain sensitive to 2D7 ( Fig.…”

“…We recently discovered two highly potent mouse anti-CCR5 MAbs: RoAb13, whose epitope lies in the NTD of CCR5 and RoAb14, which binds to extracellular loop 2 (ECL-2) (13,29). RoAb14 was chosen for further development due to its better antiviral potency and its broad activity against HIV strains from all clades, resulting in the deimmunized human antibody MAb3952.…”