Antigen-binding molecules capable of binding cd3 and cd137 but not simultaneously
An antigen-binding molecule, CD137 technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, drug combination, etc., can solve the problem of unknown antibody and so on
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[0872] Examples thereof may include a production method comprising the steps of:
[0873] (i) preparing a library of antigen-binding molecules that each bind CD3 or CD137 and have at least one amino acid altered in the antibody variable region, wherein the altered variable regions differ from each other in at least one amino acid;
[0874] (ii) selecting an antigen-binding molecule from the prepared library, the antigen-binding molecule comprising a variable region that has binding activity to CD3 and CD137 but does not simultaneously bind to CD3 and CD137;
[0875] (iii) culturing host cells comprising the nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and the nucleic acid encoding the variable region of the antigen-binding molecule that binds to the third antigen, to express the nucleic acid comprising the variable region capable of binding to CD3 and CD137, but not an antibody variable region that binds both CD3 and CD137 and...
Embodiment 1
[0900] [Example 1] Screening of affinity maturation variants derived from parental double Fab H183L072 for improved in vitro cytotoxicity against tumor cells
[0901] 1.1 Sequences of affinity matured variants
[0902] To increase the binding affinity of the parental double Fab H183L072 (heavy chain: SEQ ID NO: 1; light chain: SEQ ID NO: 57), 1,000 Multiple double Fab variants. Antibody Expi293 (Invitrogen) was expressed and purified by protein A purification followed by gel filtration if required. The sequences of 15 representative variants with multiple mutations are listed in Tables 1.1 and 1.2, and were used in Example 1.2.2 at 25°C and / or 37°C using the Biacore T200 instrument (GE Healthcare) described below. Binding kinetics were assessed at °C. Table 1.3 lists the fold change in affinity for human CD137 and human CD3 by single mutations in the variable regions.
[0903] [Table 1.1a]
[0904] SEQ ID NO of human CD3 and CD137 antigens used for affinity measurement
...
Embodiment 2
[0939] [Example 2] Evaluation of in vitro cytotoxicity of affinity matured variants derived from parental double Fab H183L072 on tumor cells
[0940] 2.1. In vitro assessment of CD3 agonistic activity of affinity matured variants
[0941] To evaluate CD3 agonistic activity due to affinity maturation, the NFAT-luc2 Jurkat luciferase assay was performed. Briefly, 4 × 10 of human GPC3 will be expressed on the cell membrane 3 SK-pca60 cells (reference example 13) of each cell / well were used as target cells, and in the presence of 0.02, 0.2 and 2 nM trispecific antibodies with 2.0 × 10 4 NFAT-luc2 Jurkat cells (E:T ratio of 5)) were co-cultured for 24 hours. variants are divided into Figure 1.1 Board 1 in the picture above and Figure 1.1 Plate 2 in the lower panel. After 24 hours, luciferase activity was detected using the Bio-Glo Luciferase Assay System (Promega, G7940) according to the manufacturer's instructions. Luminescence (unit) was detected using GloMax (registered tra...
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