Antibody Variants and Cell Killing Assays

Example 10

Several 4D5 liability fixed affinity variants were selected for further characterization. Y55E.H91A.N54E.D98T and Y55E.H91A.N54E.D98T.Y102V variants were formatted into a 1Fab-IgG TDB antibody having a natural (short) linker (hinge; DKTHT, SEQ ID NO: 50) and compared to H91A-1Fab-IgG TDB in a dose response assay to quantify killing of SKBR3 (FIG. 33A) or MCF7 (FIG. 33B) target cells. Each well was seeded with 1.5×10⁴ SKBR3 or MCF7 target cells and co-cultured with PMBC-derived CD8⁺ effector cells at a 1:3 effector:target ratio. Results are summarized in Table 12, below.

The binding of 4D5 Y55E.H91A.N54E.D98T-1Fab-IgG TDB and 4D5 Y55E.H91A.N54E.D98T.Y102V antibodies to SKBR3 cells (FIG. 34A) and MCF7 cells (FIG. 34B) was also characterized by flow cytometry. In general, 4D5 Y55E.H91A.N54E.D98T.Y102V-1Fab-IgG TDB were similar to 4D5 H91A-1Fab-IgG TDB in terms of cytotoxicity and binding to SKBR3 cells and MCF7 cells.