Polypeptide variants with altered effector function
A variant, functional technology, applied in the field of polypeptide variants with altered effector function
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Embodiment 1
[0431] Phage display of human IgG1-Fc for selection of single-point mutants that optimize pH-dependent binding to human FcRn
[0432] For phage display, we used a "hingeless region" variant of human IgG1 Fc, in which the disulfide bond in the hinge region has been removed (C226 is deleted, and C229 is replaced with Ser), and the C-terminus is fused to the phage The C-terminal region of the M13 gIII protein (g3p) in the bacteriophage construct (pW0437). The protein sequence (SEQ ID. NO. 38) is shown in Figure 7. The huFcRn that we use as a phage selection target has been biotinylated and captured in a pH 6.0 buffer with a neutravidin-coated immunosorbent (Maxisorp). TM ), wash thoroughly with a pH 6.0 buffer to remove the lower affinity variants, and use a pH 7.4 buffer to elute the higher affinity pH-sensitive variants. After the third round of selection, N434W was the dominant clone (44 of the 48 sequenced clones were consistent with this variant), and N434Y (3 / 48) and N434F (1 / 4...
Embodiment 2
[0439] Identification of soluble Fc variant proteins
[0440] Soluble Fc variants with these mutations have been expressed, purified, and tested for their affinity for human, cynomolgus, rat, and mouse FcRn using the BIAcore binding assay. The Fc variants were also analyzed by size exclusion chromatography to determine their aggregation tendency.
[0441] The 34B8 E. coli cells were transformed with the variant pW0437 phagemid, grown in a phosphate-free medium at 30°C for 24 hours to induce the expression of the Fc gene, and the cells were collected to express the variant Fc fragment. The cell pellet was frozen overnight, and then dissolved in 10 mM Tris, 1 mM EDTA by osmotic shock. The lysate was separated by centrifugation, and then applied to the Protein A column. The column was washed with PBS, the soluble Fc was eluted by Protein A Citrate Elution Buffer (0.1M citrate, pH 3.0), and then neutralized with Tris of pH 7.5. Concentrate soluble Fc in Amicon Centriprep.
[0442] FcR...
Embodiment 3
[0445] Identification of humanized anti-CD20 IgG1 variants with FcRn mutations
[0446] The effects of mutations identified by human Fc phage display were also tested in the context of the complete antibody 2H7.v138. 2H7.v138 is a humanized anti-CD20 antibody, in which Fc was modified by the following mutations in order to enhance ADCC and CDC activities: S298A, K326A, E333A, K334A. The mutation at position N434 was introduced into this environment, and IgG was prepared by transient transfection of 293 cells as described previously (Table 2). In each case, the purified IgG variants showed low levels of protein aggregation by size exclusion chromatography as described above.
[0447] 2H7 variant
FcRn mutation
138
-
364
N434A
477
N434W
478
N434F
479
N434Y
[0448] Using biotinylated FcRn, the pH-dependent binding capacity of the human IgG1 variant of 2H7.v138 to human FcRn was determined in ELISA. Coat...
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