NeuroMab™ Anti-ICAM-1 BBB Shuttle Antibody(NRZP-1022-ZP2809)

Figure 1 Binding of seven unique scFv clones to Ramos cells (solid bars) and Jurkat cells (open bars). Control scFv (ctrl) did not bind to any cells.

Figure 2 Apoptosis induction by anti-Ramos scFv.

Ramos B lymphoma cells were sequentially incubated with anti-Ramos scFv, anti-His mAb, and anti-mouse polyclonal Ab on ice (with intermittent washing to remove excess unbound antibody), and were incubated in a humidified atmosphere of 5 % CO2 at 37 C for 24 hours. Cells were then harvested and subjected to combined staining with AnnexinTM V-AF488 (AV) and propidium iodide (PI).

Figure 3 Apoptosis induction by anti-Ramos scFv.

The seven unique scFv clones were incubated with Ramos B lymphoma cells at 37 C for 24 hours at various concentrations and the effect on apoptosis induction studied. Three scFv; Bl, B11, and C11, show titratable activity towards both cell lines, whereas apoptosis inducing capability

Figure 4 Specificities of isolated antibodies include HLA-DR/DP, IgM, and ICAM-1.

50-600 x 10^6 Raji B lymphoma cells were lysed with the non-ionic detergent Triton XlOOTM at 0.5 % v/v and immunoprecipitated with 100 g of the fully human IgG1 format of B1 (lane 1) and B11 (lane 2) antibody, followed by crosslinking with Protein A SepharoseTM. Ramos B lymphoma cell lysates, from 50 x 106 cells, were used for the precipitation of 20 ,g C11 (lane 3).

Figure 5 Specificities of isolated antibodies include HLA-DR/DP, IgM, and ICAM-1.

B1 IgG, B11 IgG, and Cll IgG binding to B lymphoma cells is specifically blocked by pre-incubation with anti-HLA-DR/DP, anti-ICAM-1 or anti-IgM antibodies, respectively.

Figure 8 Binding of B1, B11, C11 IgG1 to tumor cell lines of different origin. The antigen distribution of antigens targeted by B1, B11 and C11 antibodies on different cancer cell lines was studied by flow cytometry.

Figure 9 Induction of apoptosis in B11 IgG1 cells in cancer cells.

Bar graph shows the mean percentage of apoptotic PC-3 cells following incubation with serially diluted B11 IgGI or 20 g/m1 B1 IgGi.

Figure 10 B1 IgG1 and B1 IgG4 induced direct cytotoxicity against Raji B Lymphoma cells without cross-linking agent.

Raji cells were incubated with B1 IgGI, B1 IgG4, Rituximab IgGI, or control CT-17 IgGI at 20, 6.7 or 2.2 jig/m1 for 24 hours. Cells were harvested and viability was determined as the percentage of Annexin V and Propidium Iodide double negative cells.