Tyrosine O-sulfation proteoforms affect HIV-1 monoclonal antibody potency

Abstract

CAP256V2LS, a broadly neutralizing monoclonal antibody (bNAb), is being pursued as a promising drug for HIV-1 prevention. The total level of tyrosine-O-sulfation, a post-translational modification, was known to play a key role for antibody biological activity. More importantly, here wedescribe for the first time the significance of the tyrosine-O-sulfation proteoforms. We developed a hydrophobic interaction chromatography (HIC) method to separate and quantify different sulfation proteoforms, which led to the direct functionality assessment of tyrosine-sulfated species. The fully sulfated (4-SO₃) proteoform demonstrated the highest in vitro relative antigen binding potency and neutralization efficiency against a panel of HIV-1 viruses. Interestingly, highly variable levels of 4-SO₃ were produced by different clonal CHO cell lines, which helped the bNAb process development towards production of a highly potent CAP256V2LS clinical product with high 4-SO₃ proteoform. This study presents powerful insight for any biotherapeutic protein development where sulfation may play an important role in product efficacy.

Figure 1

HIC-UV chromatograms and RPLC-mass spectrometry analysis distinguishing CAP256V2LS sulfation proteoforms. Traces on the left are CAP256V2LS analyzed by HIC-UV. (y-axis is the UV absorbance, normalized to the highest peak intensity in each chromatogram). On the right, deconvoluted Reversed phase coupled with intact mass analysis from each peak fraction corresponding to the left elution after deglycosylation. (y-axis is the mass intensity, normalized to the highest peak in each mass spectrum.) (a) Unfractionated CAP256V2LS showed five peaks on HIC-UV chromatograms, with the peak of 4-SO₃ and 3-SO₃ species detected in RPLC-MS. (b) 4-SO₃ eluted first at 13.7 min with mass confirmed at 150,526.5 Da. (c) 3-SO₃ eluted at 15.6 min with mass of 150,447.3 Da. (d) 2-SO₃ eluted at 17.2 min with mass observed at 150,366.9 Da. (e) 1-SO₃ eluted at 18.7 min with mass detected at 150,287.2 Da. (f) 0-SO₃ eluted last at 20.1 min with mass measured at 150,206.4 Da.

Figure 2

Increased levels of CAP256V2LS tyrosine sulfation correspond to increased antigen binding. (a) Octet antigen-binding curve for fractions of CAP256V2LS generated from CHO clone 206. (b) Relative antigen binding potency (%RP) for each level of tyrosine sulfation proteoform of CAP256V2LS. Octet antigen-binding curve for fractions of (c) CHO clone 386 and (d) CAP256V2LS expressed in HEK-293 cells. %RPs were normalized against the highest 4-SO₃ proteoform from the same parent material. Binding curve data points shown as average with error bars are from two independently prepared experiments.

Figure 3

Neutralization potency of CAP256V2LS versus tyrosine sulfation occupancy. Sulfation fractions from CHO clone 206 were assayed in one experiment for neutralization activity against 4 Env-pseudoviruses, (A) IC₈₀ and (B) relative neutralization potency of each virus, calculated as the IC₈₀ of the 4-SO₃ proteoform divided by the IC₈₀ of the tested fraction.

Figure 4

Abundance of CAP256V2LS sulfation proteoforms varied with different CHO cell clones. Sulfation proteoform quantitation of CAP256V2LS expressed from 18 CHO clonal cell lines cultured at 250-ml scale was performed once for each sample. Asterisk marks the three top clones selected for subsequent 3-l scale studies.

Figure 5

Lot-to-lot consistency of CAP256V2LS sulfation profile. Five independently produced lots of CAP256V2LS at the 50-l scale included one reference lot, one toxicology lot and three consistency-run lots. (a) % Relative potency values are shown for binding to HIV-1 antigen accordingly to procedures detailed in materials and methods. Reported value %RP for all lots is compared to reference lot. (b) Octet antigen-binding curves.