DuoHexaBody-CD37-induces direct cytotoxicity in DLBCL-derived tumor cell lines

(A) Percentage decrease (mean +/- SD) in viability upon DuoHexaBody-CD37 treatment in presence or absence of goat F(ab’)2 anti-human IgG (a-Fc) in indicated cell lines after 48h. Data is shown from at least three independent experiments. Significance was calculated compared to untreated control using Kruskal-Wallis test followed by Dunn’ multiple comparison test correction (**p<0.01) (B) Percentage increase in cell death (PI-positive) upon co-culturing healthy donor-derived fixed PBMCs with DuoHexaBody-CD37 pre-treated HBL-1 and U2932 cells compared to untreated cells for indicated time points. Duplicates from each individual donor (n=4) are indicated in separate colors. Significance was calculated compared to 4h treated cells using Kruskal-Wallis test followed by Dunn’ multiple comparison test correction (*p<0.05, **p<0.01, ***p<0.001).

DuoHexaBody-CD37 induces CD37 clustering without modulating CD37 cell surface expression

(A,B) Airyscan images depicting clustering (fluorescence/area) upon DuoHexaBody-CD37 (DHB) treatment or B12 isotype control in BJAB (A) and Oci-Ly8 (B) cells. Data shown from three independent experiments. Bar is 10 μm. Significance was calculated comparing DuoHexaBody-CD37 treated cells to isotype control antibody using unpaired T-test (****p<0.0001). (C) Percentage decrease (mean +/- SD) in cell surface binding by DuoHexaBody-CD37 or rituximab in indicated cell lines as measured by flow cytometry. Data is shown from at least three independent experiments. Significance was calculated comparing respective time point using unpaired T-test (*p<0.05, **p<0.01, ***p<0.001).

DuoHexaBody-CD37 treatment results in activation of different downstream signaling pathways

(A) Heatmap depicting log-normalized signal intensities of 26 phosphoproteins with two-fold increase in signal upon DuoHexaBody-CD37 treatment in presence or absence of goat F(ab’)2 anti-human IgG (a-Fc) in primary B cells, U2932 and Oci-Ly7. (B) Oncogenic hallmark signatures obtained from the MSigDB enriched using 26 phosphoproteins with two-fold increase in signal upon DuoHexaBody-CD37 treatment in presence or absence of goat F(ab’)2 anti-human IgG (a-Fc) in primary B cells, U2932 and Oci-Ly7. (C) Phosphoflow analysis of p-SYK(Y348), p-SHP1(Y564), p-AKT(S473) on primary B cells (top row) or DLBCL cells (bottom row) either untreated or treated with anti-BCR (F(ab’)2 anti-IgM), DuoHexaBody-CD37 and/or goat F(ab’)2 anti-human IgG (a-Fc). Dot plots depict quantification (mean +/- SD) of mean fluorescence intensity (MFI). Each dot represents individual donor (B cells) or experimental replicate (DLBCL). Significance was calculated compared to untreated control using Kruskal-Wallis test followed by Dunn’ multiple comparison test correction (**p<0.01, ***p<0.001, ****p<0.0001).

DuoHexaBody-CD37 treatment results in differential activation of BCR and RAS/MAPK downstream signaling proteins in primary B cells and DLBCL

(A, B) Phosphoflow analysis of (A) p-BTK(Y223), p-PLCy2(Y759), p-PKCα/βII(T638/641) and (B) p38(T180/Y182), p-ERK-1/2(T202/Y204) on primary B cells (top row) or DLBCL cells (bottom row) either untreated or treated with anti-BCR (F(ab’)2 anti-IgM), DuoHexaBody-CD37 and/or goat F(ab’)2 anti-human IgG (a-Fc). Dot plots depict quantification (mean +/- SD) of mean fluorescence intensity (MFI). Each dot represents an individual donor (primary B cells) or experimental replicate (DLBCL tumor cell lines). Significance was calculated compared to untreated control using Kruskal-Wallis test followed by Dunn’ multiple comparison test correction (*p<0.05, **p<0.01, ****p<0.0001).

CD37 N-terminus is involved in DuoHexaBody-CD37-mediated cytotoxic signaling

(A) Histogram showing CD37 cell surface expression determined by flow cytometry analysis of NALM6 cell lines expressing wild type CD37 (CD37-WT), or mutations of Tyr13 to phenylalanine (CD37-Y13F), or deletion of Tyr13 (CD37-DY13) in the cytosolic regions of the CD37 molecule. (B) p-AKT(S473) and (C) p-SHP1(Y564) on NALM6 cell lines transfected with CD37-WT, CD37-Y13F or CD37-DY13 treated with DuoHexaBody-CD37 (DHB) and goat F(ab’)2 anti-human IgG (a-Fc) or crosslinker alone (a-Fc). Dot plots depict p-AKT and p-SHP1 levels in treated (DHB+a-Fc or a-Fc) vs untreated cells in CD37-GFP-positive (transfected) cells using phosphoflow analysis, corrected for background signal from GFP-negative cells. Significance was calculated using Student t-test (*p<0.05). Data shown from three independent experiments.

DuoHexaBody-CD37 treatment decreases cytokine-mediated pro-survival signaling in DLBCL cell lines

(A) Percentages of p-STAT6-positive cells (mean and SD) upon recombinant human IL-4 (rh-IL-4) stimulation in DLBCL cells pre-treated with DuoHexaBody-CD37 in presence or absence of goat F(ab’)2 anti-human IgG (a-Fc). Data is shown from at least three independent experiments. Percentages of p-STAT3-positive cells (mean +/- SD) upon (B) recombinant human IL-6 (rh-IL-6) stimulation or (C) recombinant human IL-21 (rh-IL-21) stimulation in DLBCL cells pre-treated with DuoHexaBody-CD37 in presence or absence of a-Fc. Data is shown from at least three independent experiments. (A-C) Significance was calculated compared to untreated control using Kruskal-Wallis test followed by Dunn’ multiple comparison test correction (**p<0.01).