Anti his alexa fluor 647 antibody

Example 19

This example demonstrates the CLDN18.2-CD16-TandAbbinding activity to native positive cells or artificial overexpressed cells by flow cytometry.

Method: Tandab 307-CD16A and Tandab 358-CD16A as well as AFM13 were incubated with 150,000 cells in 50 μl 2% FBS/PBS at 4° C. for 30 mins followed by washing with 2% FBS/PBS; then incubated with anti-his Alexa Fluor© 647 antibody (Biolegend #652513) at 4° C. for 30 mins. The samples were then measured by BD Accuri™ C6 Flow Cytometer.

Brief Summary: The Tandab 307-CD16A and Tandab 358-CD16A showed good binding affinity with native CLDN18.2 expressing HUPT4 cells, while showing less activity against CLDN18.2 negative ARK2 cells (FIG. 26A-FIG. 26E). However, anti-his-AF647 (Biolegend #652513) has high background/non-specific binding. AFM13 antibody (bispecific to CD16A and CD30) was used as positive control for binding to CD16A in Jurkat-Lucia-NFAT-CD16A cells. The results in this flow assay showed that the Tandab 307-CD16A and Tandab 358-CD16A successfully bind to both CLDN18.2 and CD16A (FIG. 26A-FIG. 26E).

Example 18

This example demonstrates the CLDN18.2-CD16-TandAb (Tandem diabody) binding activity to native positive cells or artificial overexpressed cells by flow cytometry.

Method: A TandAb comprising antigen-binding moieties that each binds CD16A or CLDN18.2 was constructed using an antigen binding protein of 307 or 358 antibody. The structure of TandAb described herein is: CD16A VH-(G2S)3-CLDN18.2AbVL-(G2S)3-CLDN18.2AbVH-(G2S)3-CD16A VL-His Tag (FIG. 23A-FIG. 23C). The TandAb 307-CD16A and TandAb 358-CD16A as well as AFM13 were incubated with 150,000 cells in 50 μl 2% FBS/PBS at 4° C. for 30 mins followed by washing with 2% FBS/PBS; then incubated with anti-his Alexa Fluor© 647 antibody (Biolegend #652513) at 4° C. for 30 mins. The samples were then measured by BD Accuri™ C6 Flow Cytometer.

Brief Summary: The TandAb 307-CD16A and TandAb 358-CD16A showed good binding affinity by flow assay with native CLDN18.2 expressing HUPT4 cells, while showing less activity against negative control ARK2 cells. However, anti-his-AF647 (Biolegend #652513) has very high background/non-specific binding. AFM13 antibody (bispecific to CD16A and CD30) was used as positive control for binding to CD16A in Jurkat-Lucia-NFAT-CD16A cells. The results in this flow assay demonstrated that the TandAbs of 307-CD16A and 358-CD16A successfully bind to both CLDN18.2 and CD16A (FIG. 25A-FIG. 25E).

Example 17

This example demonstrates the CLDN18.2-CD3-bispecific T cell engager (BiTE) binding activity to native positive cells or artificially overexpressed cells by flow cytometry.

Method: The #307-CD3E-BiTE (2 batches) and 358-CD3E-BiTE were incubated with 150,000 cells in 50 μl 2% FBS/PBS at 4° C. for 30 mins followed by washing with 2% FBS/PBS; then incubated with anti-his Alexa Fluor© 647 antibody (Biolegend #652513) at 4° C. for 30 mins. The samples were then measured by BD Accuri™ C6 Flow Cytometer.

Brief Summary: CLDN18.2 BiTEs [#307-CD3E-BiTE (2 batches) and 358-CD3E-BiTE] showed good binding affinity by flow assay with native CLDN18.2 expressing HUPT4 cells, and artificial CLDN18.2 overexpressing HEK293T cells while showing no activity against negative control ARK2 cells (FIG. 24A-FIG. 24E). However, anti-his-AF647 (Biolegend #652513) has very high background/non-specific binding with HEK293T parental cells.