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). (C) Percentage of cell death in DuoHexaBody-CD37 pre-treated Oci-Ly8 cells (eFluor780-positive) upon co-culturing with healthy donor-derived fixed PBMC, monocytes, NK cells, B cells and T cells compared to Fc-crosslinker. Measurements from three individual donors (mean +/- SD) are shown, with each donor represented by a distinct colored symbol.

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

(A,B) Airyscan images depicting clustering (fluorescence/area) upon DuoHexaBody-CD37 (DHB-CD37) treatment or B12 isotype control in BJAB (A) and Oci-Ly8 (B) cells with or without Fc-crosslinking (a-Fc). Data shown from five independent experiments. Bar is 10 µm. Significance was calculated comparing DuoHexaBody-CD37-treated cells to isotype control antibody in the presence and absence of Fc-crosslinking (a-Fc) respectively, using Kruskal-Wallis test followed by Dunn’s multiple comparisons test (****p<0.0001, *p<0.05). (C) Percentage decrease (mean +/- SD) in cell surface binding by DuoHexaBody-CD37 or rituximab (anti-CD20) in indicated cell lines as measured by flow cytometry. Data is shown from at least three independent experiments. Significance was calculated comparing respective time points 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 pSYK(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 cell lines

(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 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-ΔY13) 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-ΔY13 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-IL4) 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 in HBL-1 cells (the only IL-6 responsive cell line in this study) 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).

Fc-crosslinking-mediated apoptotic cell death induced by DuoHexaBody-CD37 is not correlated with CD37 membrane expression

(A) Flow cytometric comparison of Annexin V stained, Annexin V + 7AAD stained and unstained cells in presence or absence of DuoHexaBody-CD37, with and without Fc-crosslinking on Oci-Ly8, Oci-Ly7 and HBL 1 cell lines. Data (mean +/- SD) show unstained, Annexin V-positive and Annexin V + 7AAD double positive populations from three independent experiments. (B) Flow cytometric quantification of CD37 expressed on membranes of DLBCL and Burkitt cell lines using two different CD37 antibodies (clones WR-17 and M-B371). Data (mean +/- SD) represent three independent experiments. MFI=mean fluorescence intensity.

Fc-crosslinking does not lead to increased CD37 internalization from the cell surface of DLBCL cell lines upon DuoHexaBody-CD37 treatment

Percentage decrease in cell surface binding by DuoHexaBody-CD37 with or without Fc-crosslinker in Oci-Ly8, BJAB, Oci-Ly7, U2932 and DAUDI cell lines as measured by flow cytometry. Data (mean +/- SD) is derived from four independent experiments.

Principal component analysis of (phospho)protein expression profiles after DuoHexaBody-CD37 treatment

Principal component analysis (PCA) of 484 proteins (102 phospho targets and 382 total proteins) in primary B cells (CD19+ purified) and two DLBCL cell lines (Oci-Ly7 and U2932) either untreated or treated with DuoHexaBody-CD37 in presence or absence of a-Fc using reverse phase protein array (RPPA). The first two components of the cluster analysis are shown.

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

(A-C) Phosphoflow analysis of (A) p-SYK(Y348), p-SHP1(Y564), p-AKT(S473); (B) p-BTK(Y223), p-PLCy2(Y759), p-PKCα/βII(T638/641) and (C) p38(T180/Y182), p-ERK-1/2(T202/Y204) on primary B cells (top row) or DLBCL cells (bottom row) either anti-IgM treated or treated with DuoHexaBody-CD37 and/or goat F(ab’)2 anti-human IgG (a-Fc). Dot plots depict fold change (mean +/- SD) of mean fluorescence intensity (MFI) of indicated treatment to untreated cells. Each dot represents individual donor (B cells) or experimental replicate (DLBCL). Significance of fold change to untreated control was calculated using Wilcoxon Signed Rank test (*p<0.05, **p<0.01, ****p<0.0001).

Knockout of SHP1 does not influence DuoHexaBody-CD37 mediated cytotoxicity

(A) Western blot showing SHP-1 levels in Oci-Ly7 and HBL1 cells for parental (NT), empty vector (EV) and SHP-1 gRNA1/gRNA4 transduced cells. Tubulin was used as loading control. (B) Percentage of dead (Annexin V + 7AAD double positive) populations in parental, transduction control (gRNA EV), and SHP1 knockout (gRNA SHP1-1 and gRNA SHP1-4) in Oci-Ly7 and HBL1 cells measured by flow cytometry. Data (mean +/- SD) shown from three independent experiments.