Variant fc domains and uses thereof
A domain and variant technology, applied in the field of variant Fc domain monomers, can solve problems such as decreased patient compliance and increased cost of health systems
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Embodiment 1
[0448] Example 1. General procedure for the synthesis of azido-Fc
[0449] Prepare PEG4-azido NHS ester solution (0.050M) in DMF / PBS: dissolve 16.75 mg PEG4-azido NHS ester in 0.100 mL of DMF at 0 °C and buffer by adding PBS 1x at 0 °C The solution was diluted to 0.837mL. Other PEG4-azido Fcs with various DAR values were prepared using this solution by adjusting the equivalents of this PEG4-azido NHS ester PBS solution.
[0450] Pretreatment h-IgG1 Fc (107.2 mg in 8.800 mL of pH 7.4 PBS, MW ~ 57891 Da, 1.852 μmol): Fc solution was transferred to four centrifugal concentrators (30,000 MWCO, 15 mL) and diluted with PBS x1 buffer to 15 mL and concentrated to a volume of -1.5 mL. The residue was diluted 1:10 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of four times, then diluted to 8.80 mL.
[0451] Preparation of PEG4-azido Fc: 0.050 M PEG4-azido NHS ester in PBS buffer (0.593 mL, 29.6 μmol, 16 equiv) was added to the above h-IgG1 Fc so...
Embodiment 2
[0452] Example 2. Synthesis of Conjugate 1 (including C220S / M252Y / S254T / T256E mutated Fc domains)
[0453] Prepare click reagent solution: 0.0050M CuSO in PBS buffer 4 : 10.0mg CuSO 4 Dissolve in 12.53mL PBS, then take 5.00mL of this CuSO 4 solution and added 43.1 mg BTTAA (CAS# 1334179-85-9) and 247.5 mg sodium ascorbate, resulting in a click reagent solution (0.0050M CuSO4, 0.020M BTTAA and 0.25M sodium ascorbate).
[0454] To the azido-functionalized Fc solution (Example 1; 65.5 mg, 10.0 mL, 1.13 μmol, azido DAR~5.9, SEQ ID NO: 10) in a 15 mL centrifuge tube was added an alkyne-derived small molecule virus inhibitor agent (22.7 mg per Fc azide, 15.2 μmol, 3.0 equiv). After gentle stirring to dissolve all solids, the mixture was treated with click reagent solution (1.80 mL). The resulting mixture was rotated at ambient temperature for 12 hours. The mixture was purified by affinity chromatography on a protein A column followed by size exclusion chromatography. Maldi TOF...
Embodiment 3
[0455] Example 3. Synthesis of Conjugate 2 (including C220S mutated Fc domain)
[0456] This conjugate was prepared by PEG4-azido-Fc (SEQ ID NO: 21, prepared as in Example 1) and a small molecule virus inhibitor in a similar manner to that of Conjugate 1 (Example 2) . Maldi TOF analysis of the purified final product gave an average mass of 62,927 Da (DAR=4.2).
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