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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.

SUBMITTER: Cai CX

PROVIDER: S-EPMC9120178 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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

Cai Cindy X CX Doria-Rose Nicole A NA Schneck Nicole A NA Ivleva Vera B VB Tippett Brad B Shadrick William R WR O'Connell Sarah S Cooper Jonathan W JW Schneiderman Zachary Z Zhang Baoshan B Gowetski Daniel B DB Blackstock Daniel D Demirji Jacob J Lin Bob C BC Gorman Jason J Liu Tracy T Li Yile Y McDermott Adrian B AB Kwong Peter D PD Carlton Kevin K Gall Jason G JG Lei Q Paula QP

Scientific reports 20220519 1

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, whi ...[more]

PMID: 35589938

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Project description:BackgroundThe experience from clinical trials indicates that anti-Aβ immunotherapy could be effective in early/pre-clinical stages of AD, whereas at the late stages promoting the clearing of Aβ alone may be insufficient to halt the disease progression. At the same time, pathological tau correlates much better with the degree of dementia than Aβ deposition. Therefore, targeting pathological tau may provide a more promising approach for the treatment of advanced stages of AD. Recent data demonstrates that the N-terminal region of tau spanning aa 2-18 termed "phosphatase activation domain" that is normally hidden in the native protein in 'paperclip'-like conformation, becomes exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and in aggregation of tau. Hence, we hypothesized that anti-Tau2-18 monoclonal antibodies (mAb) may recognize pathological, but not normal tau at very early stages of tauopathy and prevent or decrease the aggregation of this molecule.MethodsMouse mAbs were generated using standard hybridoma methodology. CDR grafting was used for humanization of mouse mAb. Humanized mAb (Armanezumab) was characterized and tested in vitro/ex vivo/in vivo using biochemical and immunological methods (HPLC, Biacore, ELISA, IHC, FRET, etc.). Stable DG44 cell line expressing Armanezumab was generated by clone selection with increased concentrations of methotrexate (MTX).ResultsA panel of mouse mAbs was generated, clone 1C9 was selected based on binding to pathological human tau with high affinity and humanized. Fine epitope mapping revealed conservation of the epitope of human tau recognized by the parent murine mAb and Armanezumab. Importantly, Armanezumab (i) bound to tau with high affinity as determined by Biacore; (ii) bound pathological tau in brains from AD, FTD and Pick's disease cases; (iii) inhibited seeding effect of aggregated tau from brain lysate of P301S Tg mice; (iv) inhibited cytotoxic effect of tau oligomers; (v) reduced total tau (HT7) and AT100, PHF1, AT8, AT180, p212, p214-positive tau species in brains of tau transgenic mice after intracranial injection. A stable CHO cell line producing >1.5 g/l humanized mAb, Armanezumab was generated.ConclusionThese findings suggest that Armanezumab could be therapeutic in clinical studies for treatment of AD.

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

Publications

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

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