Patent ID: 9499634
Filing Date: 2016-11-22
CPC Classification: C07K,C12P

Claim Text:
1. A method of producing an asymmetric antibody product comprising a first heavy chain polypeptide, a second heavy chain polypeptide, and at least one light chain polypeptide in stable mammalian cells, the method comprising: A. selecting a first heavy chain polypeptide sequence and a second heavy chain polypeptide sequence, each of the first and second heavy chain polypeptide sequences comprising a variant CH3 sequence, wherein the variant CH3 sequence of the first heavy chain polypeptide sequence is different from the variant CH3 sequence of the second heavy chain polypeptide sequence, wherein the variant CH3 sequence of the first heavy chain polypeptide comprises amino acid modifications at positions F405 and Y407, optionally in combination with an amino acid modification at position L351, and the variant CH3 sequence of the second heavy chain polypeptide comprises amino acid modifications at positions T366 and T394, optionally in combination with a modification at position K392, wherein the amino acid modification at position T366 is T366I, T366L, T366M or T366V; the amino acid modification at position T394 is T394W; the amino acid modification at position F405 is F405A, F405T, F405S or F405V; the amino acid modification at position Y407 is Y407V; the amino acid modification at position L351 is L351Y; and the amino acid modification at position K392 is K392L, K392M, K392V or K392F, and wherein the variant CH3 sequences of the first and second heavy chain polypeptides promote formation of a heterodimeric Fc region with a CH3 domain having a Tm greater than 75° C.; B. (i) transfecting at least two different mammalian cells with different ratios of a first DNA sequence encoding the first heavy chain polypeptide sequence, a second DNA sequence encoding the second heavy chain polypeptide sequence and at least one DNA sequence encoding a light chain polypeptide such that each of the at least two mammalian cells expresses the heavy chain polypeptides and the at least one light chain polypeptide in a different ratio, and (ii) selecting, as a pre-determined ratio, a ratio of the first DNA sequence, the second DNA sequence and the at least one DNA sequence encoding a light chain polypeptide that results in a mammalian cell line that produces greater than 75% asymmetric antibody product, less than 10% monomers, and undetectable amounts of homodimers of said first or second heavy chain polypeptide; C. transfecting at least one mammalian cell with the pre-determined ratio of the first DNA sequence, the second DNA sequence, and the at least one DNA sequence encoding a light chain polypeptide to generate stable mammalian cells; D. culturing said stable mammalian cells under conditions suitable for expression of the first and second heavy chain polypeptides and the light chain polypeptide to produce the asymmetric antibody product, whereby stable mammalian cells producing greater than 75% asymmetric antibody product, less than 10% monomers, and undetectable amounts of homodimers of said first or second heavy chain polypeptide are obtained at a frequency of greater than 1 in 500.