Afuco™ Anti-Human SLAMF7 ADCC Recombinant Antibody (PDL241), ADCC Enhanced (CAT#: AFC-548CL)

Figure 1 Binding of PDL241 to leukocytes.

(A) PBMC subsets were labeled for immunofluorescence analysis on the basis of co-staining: CD3⁺ CD4⁺ and CD3 ⁺ CD8⁺ (T cells); CD3-CD56⁺ (NK cells) and CD3 ⁺ CD56⁺ (NKT cells); CD19⁺ (B cells) and CD14⁺ (monocytes); CD27⁺CD38⁺ CD138⁺ (plasmablasts and plasma cells). Lineage negative cells were first gated and then separated into HLA-DR ⁺ CD11c ⁺ (mDC), HLA-DR ⁺ CD123⁺(pDC) and HLA-DR-CD123⁺ (basophils). Data representative of 1 of 10 donors. (B)  Immunofluorescence analysis of co-staining of PDL241 (green) with anti-CD3 (T cells), anti-CD20 (B cells) or VS38c Ab (plasma cells) in red on OCT embedded frozen tissues from normal human tonsil. Nuclei were counter-stained with DAPI (blue). Double stained cells appeared yellow. Ab, antibody; DAPI, 4′,6-diamidino-2-phenylindole; NK, natural killer; OCT, optimal cutting temperature; PBMC, peripheral blood mononuclear cells.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 2 PDL241 inhibits Ig production from PBMC by specifically depleting plasmablasts and plasma cells.

(A) Time course of inhibition of IgM production from PBMC. Representative of n = 2 experiments. (B) Inhibition of IgM by PDL241 is dependent on mAb concentration and intact mAb, as F(ab')2 fragments of PDL241 showed no activity. The dose dependent inhibition of IgM by PDL241 was demonstrated in>10 donors; F(ab')2 experiment was a representative experiment of two. (C) Depletion of NK cells but not monocytes from PBMC reduces the inhibitory activity of PDL241. Representative experiment of n = 4; P = 0.01 for NK cell depleted compared to PBMC. (D) PDL241 treatment results in specific reduction in plasmablast counts in PBMC cultures. In contrast to rituximab (open bars), PDL241 treatment (closed bars) of PBMC specifically depleted plasma cells but not B cells from PBMC cultures. Counts were expressed as% of cIgG1-treated cultures. Data represent the mean and SD of n = 6 experiments from distinct donors. P ≤0.001 for B cell depletion by rituxan compared to PDL241 at 1 and 10 μg/ml. (E) CD319 was highly expressed on plasmablasts in PBMC cultures, (left panel, black histogram). Dot plots showing CD27 ⁺ CD38⁺ plasmablasts following treatment with cIgG1 control (middle panel) or PDL241 (right panel). Ig, immunoglobulin; mAB, monoclonal antibody; NK, natural killer; PBMC, peripheral blood mononuclear cells; SD, standard deviation.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 3 PDL241 treatment of RA synovial fibroblast-PBMC co-cultures leads to reduction on plasmablasts with no effect on B cells.

(A) B cells differentiated into CD19⁺CD27̶ʰⁱ CD38̶ʰⁱ CD319⁺ plasmablasts after co-culture with RA-synovial fibroblasts. PBMC from normal donors were cultured with RA synovial fibroblasts. At day 0 and day 7, cells in the culture were analyzed by flow cytometry. After co-culture for seven days, differentiated plasmablast cells (CD19⁺CD27hiCD38hi) (gate 1) could be detected and were found to be CD319 positive (solid line) as compared to isotype control staining (dotted line); in contrast, memory (CD19⁺CD27⁺) (gate 2) and naïve B cells (CD19⁺CD27⁻) (gate 3) did not express CD319. (B) Removal of CD319⁺ plasmablast cells by PDL241 in RA synovial fibroblast-PBMC co-cultures. Addition of PDL241 to cultures specifically depleted CD19⁺CD27̶ʰⁱ CD38̶ʰⁱ  plasmablasts, as compared to rituximab, which depleted only B cells. A FcR-binding deficient mutant of PDL241 (241-G2M3) had no effect on plasmablast cell numbers. The number in the gated population was calculated based on 10,000 events recoded. A representative experiment of n = 4 is shown. PBMC, peripheral blood mononuclear cells; RA, rheumatoic arthritis.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 4 PDL241 binds human leukocytes and inhibits human Ig production in huSCID mice.

NSG mice were transfused with human PBMC. (A) Double staining of CD319 (green fluorescence) and human leukocyte cell surface markers as indicated (red fluorescence) in the spleen. Cells expressing both markers were co-stained and appear yellow. 600X magnification. (B) Mice were sorted into groups on day 10 post-transfusion, and dosed twice weekly with PDL241 or cIgG1. IgM levels in the serum were measured at day 32 and day 47. In this experiment, PDL241 treatment resulted in significant reduction of human IgM compared to cIgG1 (day 32 P = 0.003 and day 47 P = 0.004 two tailed t-test). Significant reduction in IgM was observed in 6 of 11 separate experiments. huSCID, human-severe combined immunodeficiency; Ig, immunoglobulin; PBMC, peripheral blood mononuclear cells.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 5 PDL241 binds to plasma cells in rhesus monkey.

(A) Immunofluorescence co-staining of PDL241 and anti-CD20 or VS38c in tonsils from normal rhesus monkey. PDL241 staining was green, CD20 and VS38c stained red and nuclei were counter-stained with DAPI (blue). Cells with double staining (PDL241 and VS38c) appeared yellow. (B) IHC staining of PDL241 to draining lymph nodes from normal (N) or collagen immunized (CIA) rhesus monkey. (C). PDL241 but not Fc mutant 241-G2M3 inhibited IgM production from rhesus monkey PBMC stimulated with the TLR-9 agonist ODN2006. CIA, collagen-induced arthritis; DAPI, 4′,6-diamidino-2-phenylindole; IgM, immunoglobulin M; IHC, immunohistochemistry; PBMC, peripheral blood mononuclear cells.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 6 PDL241 had no effect on clinical parameters in the rhesus monkey CIA model.

The analysis of all animals showed no effect of PDL241 on (A) bodyweight loss relative to day 0; (B) average clinical score; and (C) serum CRP levels. PDL241 treatment had no effect on (D) disease free survival or (E) overall survival. CIA, collagen-induced arthritis; CRP, C-reactive protein.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 7 PDL241 treatment reduced the severity of joint-related disease parameters in a rhesus CIA model.

Analyses are provided for 'all animals' and animals with 'early CRP onset' as described in the Methods section. Collagen type II-specific IgM (A) and IgG (B) were measured by ELISA. (C) The small joint swelling score is a representation of the number of joints affected (joints with soft tissues swelling) and the severity of swelling for each individual joint. Urinary excretion of the collagen crosslinks (D) hydroxylysylpyrridinoline (HP) and (E) lysylpyrridinoline (LP) was determined, with the levels normalized to creatinine levels (nmol levels per mmol creatinine) to compensate for a possible dilution by spilled drinking water. Pl = Placebo, 30 = 30 mg/kg and 100 = 100 mg/kg. * P ≤0.05; ** P ≤0.01. CIA, collagen-induced arthritis; CRP, C-reactive protein; Ig, immunoglobulin.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.

Figure 8 PDL241 treatment reduced the severity of joint-related histopathological parameters in the rhesus CIA model.

The histopathology score was based on a grading system. Analyses were performed for (A) 'all animal' or (B) the 'early CRP onset' subgroup. CIA, collagen-induced arthritis; CRP, C-reactive protein.

Woo, J., Vierboom, M. P., Kwon, H., Chao, D., Ye, S., Li, J., ... & Breedveld, E. (2013). PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis. Arthritis research & therapy, 15(6), R207.