Afuco™ Anti-Human ICAM1 ADCC Recombinant Antibody (BI-505), ADCC Enhanced (CAT#: AFC-361CL)

Figure 1 Observed BI-505 serum concentration versus time curves (mean ± SD) in dose groups from 0.9 mg/kg and higher.

A. Pharmacokinetics after first dose. B. Pharmacokinetics after multiple doses. LLOQ indicates lower limit of quantification; and arrow indicate BI-505 infusions.

Hansson, M., Gimsing, P., Badros, A., Niskanen, T. M., Nahi, H., Offner, F., ... & Sundberg, A. (2015). A phase I dose-escalation study of antibody BI-505 in relapsed/refractory multiple myeloma. Clinical Cancer Research, 21(12), 2730-2736.

Figure 2 BI-505 Has Significant In Vivo Antitumor Activity against CD20-Expressing Tumors Compared with Rituximab

(A–H) Mean tumor volumes (A, C, E, and G) and survival (B, D, F, and H) of mice xenografted with CD20-expressing ARH-77 (A–D) or Daudi (E–H) cells and treated with BI-505 (bright red line = 20 mg/kg BI-505; maroon line = 2 mg/kg BI-505; and orange line = 0.2 mg/kg BI-505), rituximab (20 mg/kg, blue line), or isotype control (20 mg/kg, black line) antibodies in prophylactic (A, B, E, and F) or established (C, D, G, and H) tumor models. There were eight to ten animals per treatment/dose group. Tumor cells were injected day 0, and antibody treatment started as indicated in the graphs (black arrow). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars show ± SD. (I and J) FACS analysis of BI-505 and rituximab epitopes on the surface of ARH-77 (I) and Daudi (J) tumor cells. Antibodies were used at binding saturating concentrations.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.

Figure 3 BI-505 Dose-Dependent Antitumor Activity Correlates with ICAM-1 Receptor Occupancy on Tumor Cell Surfaces

(A and B) Mean tumor volumes (A) and mean survival (B) of mice treated with different doses of BI-505 in the ARH-77 tumor model. Error bars show ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. (C–E) BI-505 concentration-dependent in vivo antitumor activity (C), in vitro antitumor (tumor PCD) activity (D), and receptor occupancy of tumor cell-expressed ICAM-1 (E). (F) A combined plot of (C)–(E). There were eight to ten animals per treatment group.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.

Figure 4 The BI-505 Epitope Is Highly Expressed on the Surface of Primary MM Plasma Cells.

FACS analysis of BI-505 epitope expression on the cell surface of patients' MM cells (red bars) versus normal B cells (yellow bars). Numbers on top of bars indicate fold increase of the BI-505 epitope on surface of MM cells compared to normal B cells.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.

Figure 5 BI-505 Has Broad and ICAM-1- Dependent Anti-MM Activity In Vivo.

(A) Tumor volume (mean ± SD) of NCI-H929 (ICAM-1+), EJM (ICAM-1+), RPMI-8226 (ICAM-1+), and OPM-2 (ICAM-1
) MM models after treatment with 2 mg/kg BI-505 (filled circles) or control (open circles) antibody. (B) Relative tumor volumes following treatment with 2 mg/kg BI-505 (filled bars) or control (open bars) antibody in NCI-H929, EJM, RPMI-8226, and OPM-2 MM models. Graph shows tumor volumes (mean ± SD) relative to the mean tumor volume of control IgG-treated animals. There were eight animals per treatment group.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.

Figure 6 BI-505 Confers Enhanced Survival Compared to Currently Used Treatments in Disseminated Experimental Models of Advanced MM.

(A) Animal survival in advanced disseminated RPMI-8226 myeloma model following treatment with control antibody, lenalidomide, bortezomib, dexamethasone (DXH), melphalan, or BI-505. ***p < 0.001. (B) Human immunoglobulin G (hIgG) (mean ± SD) in SCID-hu mice after myeloma cell inoculation and drug treatment. Graph shows pooled data from two independent experiments, each with MM cells obtained from two different patient donors (n = 4). The percentage of hIgG levels compared to start of treatment (arrow) was monitored. ***p < 0.001. (C) Myeloma tumor burden in implanted bones harvested from drug-treated mice. Pictures show representative images of tumor burden as assessed by immunohistochemistry following staining for human CD138-expressing cells. Arrows indicate human CD138-positive myeloma cell regions. Scale bar = 50 mm. (D) X-radiographic quantification of bone mineral density. Radiographs of implanted human bones receiving drug or control treatment were harvested from mice at end of experimentation (10 weeks postmyeloma cell injection and following 6 weeks of drug treatment). Upper panel shows representative radiographs of bones from healthy mice, control IgG-treated mice, BI-505-treated mice, or bortezomib-treated mice (left to right). Lower panel shows mean ± SD bone mineral density of mice receiving treatment as indicated. *p < 0.05. (E and F) Representative images of trap staining (purple stain) for detection of osteoclasts (E) or hematoxylin and eosin staining for detection of infiltrated nucleated cells (F) performed on healthy and MM cell-injected bones harvested from SCIDhu mice treated as indicated at end of experimentation. Scale bar = 100 mm.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.

Figure 7 BI-505 Confers Fc-FcgR-Dependent Antitumor Activity through Macrophages.

(A) Mean tumor volume of SCID mice bearing established ARH-77 tumors and treated with isotype control antibody or BI-505 IgG1, BI-505 IgG4, or BI-505 IgG1 N297Q (Fc-variant) antibodies. *p < 0.05, **p < 0.01. (B) BiaCore analysis of BI-505 Fc-variant antibodies binding to mouse FcgRIV. (C) BiaCore analysis of BI-505 Fc-variant antibodies binding to human FcgRIIIa. (D) Immunohistochemical quantitation of F4/80+ macrophages (top panel) or NK cells (lower panel) in tumor tissue of animals bearing established ARH-77 tumors treated with control antibody or BI-505. Graphs show mean F4/80+ and NK-cellpositive tumor areas, respectively. Bar = 40 mm. *p < 0.05, ***p < 0.001. (E) Tumor growth in macrophage or NK-celldepleted SCID mice bearing established RPMI- 8226 myeloma tumors treated with BI-505 or control antibody. ***p < 0.001. (F) Animal survival following BI-505 or control antibody treatment in a disseminated NK-celldeficient NOD/Shi-scid/IL-2Rg/mouse model comprising i.v. grafted U266 myeloma cells. ***p < 0.001. (G) Tumor growth in BI-505 or control antibodytreated NK-cell-deficient NOD/Shi-scid/IL-2Rg/mice transplanted with RPMI-8226 myeloma cells. ***p < 0.001. (H) Macrophage ADCP of RPMI-8226 and OPM-2 myeloma cells. n = 4, ***p < 0.001. (I) Macrophage-mediated ADCP of primary multiple myeloma cells. n = 2, ***p < 0.001. (J) Macrophage ADCP of ICAM-1+ EJM myeloma cells. n = 2, ***p < 0.001.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.

Figure 8 BI-505 Does Not Induce Apoptosis, ADCC, CDC, T Cell Proliferation, or Cytokine Release in Resting or Stimulated Normal Cells Expressing ICAM-1.

(A) Apoptosis in peripheral blood B cells. Graph shows percent live (nonapoptotic) B cells following no treatment or treatment with isotype control IgG, anti-HLADR (positive control IgG), or BI-505 (0, 1.5, 6, or 24 mg/ml). Values were normalized to untreated cells, where percent living cells was set to 100. (B) ADCC of peripheral blood B cells. Graph shows specific lysis of target peripheral blood B cells following treatment with BI-505 or anti-HLA-DR IgG1 (positive control). Values were normalized to treatment with isotype control IgG, where specific lysis was set to 0%. (C) CDC of peripheral blood B cells and Daudi Burkitt's lymphoma cells. Cells were incubated with BI-505, rituximab, or isotype control IgG and analyzed for CDC. (D) Antibody-induced PBMC cytokine release. PBMC cytokine release was measured by ELISA of cell culture supernatants for IL-1b, IL-2, IL-6, IL-8, IFN-g, and TNF-a following incubation of cells in plates coated with hypercrosslinked (air-dried ''A'' or wet-coated ''W'') BI-505, isotype control, or positive-control Okt-3 antibody. (E) Antibody-induced cytokine release in lipopolysaccharide (LPS)-primed PBMCs. PBMCs were incubated with titrated LPS, and concentrations yielding submaximal cellular release of IL-1b (100 pg/ml), IL-6 (10 pg/ml), IL-8 (10 pg/ml), and TNF-a (10 pg/ml) were determined (arrows, left panel) and used in subsequent experiments assessing antibody (BI-505 or control IgG) effects on cytokine release from LPS-primed PBMCs (right panel). Treatment with 100 ng/ml LPS served as positive control for robust cytokine release. (F) Antibody-induced T cell proliferation. CFSE-labeled T cells were incubated with BI-505, isotype control IgG, or anti-CD3 (okt-3) IgG hyperimmobilized to cell culture plates by air-drying ''A'' or wet-coating ''W.'' Cells were cultured for 6 days, and T cell proliferation was monitored by flow cytometry as decreased CFSE signals. (G) Antibody-induced endothelial cell apoptosis. HUVEC or HMVEC endothelial cells were incubated with paclitaxel (positive control), BI-505, or isotype control IgG in the presence or absence of crosslinking mAb. Apoptosis was measured by flow cytometry following staining of cells with annexin V-AF488. **p < 0.01, ***p < 0.001. Error bars show ± SD.

Veitonmäki, N., Hansson, M., Zhan, F., Sundberg, A., Löfstedt, T., Ljungars, A., ... & Danielsson, L. (2013). A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer cell, 23(4), 502-515.