CPC C07K 14/5434 (2013.01) [A61K 38/208 (2013.01); C07K 14/54 (2013.01); C07K 14/59 (2013.01); A61K 38/00 (2013.01); C07K 2319/30 (2013.01)] | 20 Claims |
1. A method of producing a heterodimeric Fc-fused protein, the method comprising:
(a) introducing one or more than one polynucleotide encoding a heterodimeric Fc-fused protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12 into a cell,
wherein the p40 and p35 subunits of IL-12 are linked separately to the first Fc region and the second Fc region, or to the second Fc region and the first Fc region, respectively,
wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc regions, and
wherein CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations selected from the group consisting of:
(i) glutamic acid (E), arginine (R), methionine (M), aspartic acid (D), or histidine (H) substitution of the amino acid residue at position 370 in the CH3 domain of the first Fc region;
(ii) glutamic acid (E) substitution of the amino acid residue at position 360 in the CH3 domain of the first Fc region;
(iii) tryptophan (W) substitution of the amino acid residue at position 409 in the CH3 domain of the first Fc region;
(iv) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I), glycine (G), or methionine (M) substitution of the amino acid residue at position 357 in the CH3 domain of the second Fc region, and threonine (T) or tryptophan (W) substitution of the amino acid residue at position 364 in the CH3 domain of the second Fc region; and
(v) threonine (T) substitution of the amino acid residue at position 405 in the CH3 domain of the second Fc region, and valine (V) substitution of the amino acid residue at position 399 in the CH3 domain of the second Fc region,
and wherein mutation positions are numbered according to the EU index, and
(b) culturing the cell for a sufficient period of time to express the heterodimeric Fc-fused protein.
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