. Viability of colonies was then assessed using the WST-1 reagent as described above, and normalized relative to untreated controls.

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1 Cell viability analysis in the absence of disaggregation To assess cell viability in the absence of disaggregation, quintuplicate samples of cells at 5 x 1 5 /ml were treated with mab (1 µg/ml) for 24 hours in 96-well plates at 37ºC and WST-1 reagent (Roche), which works in a similar manner to XTT (i.e. reduction by viable cells to give a color change), was added gently (1:5 final dilution). Cells were further incubated for 2 or 1 hours at 37ºC and absorbance was detected as described above. Colony growth assay 16 cells per well were seeded in a 1:1 mixture of normal and conditioned media in round-bottom 96-well plates (Corning). MAbs (.1 µg/ml) were added and cells were incubated for 14 days at 37ºC, 5% CO 2. Viability of colonies was then assessed using the WST-1 reagent as described above, and normalized relative to untreated controls. Anti-CD mab characterization assays: CD raft redistribution, CDC and calcium flux To establish the type I or II status of anti-cd mabs, redistribution of CD into lipid rafts was assessed using a flow cytometry-based Triton-X insolubility assay 17. For CDC assays, after coating cells with mab, % normal human serum was added as a source of complement for 3 minutes at 37ºC, and cell lysis was analyzed using PI staining detected by flow cytometry. To assess calcium flux, cells were treated with anti-cd mab (1 µg/ml) and a secondary hyper-cross-linking mab ( µg/ml) and the induction of cytosolic calcium flux was determined by Indo-1-AM labeling as previously described 27. Transmission Electron Microscopy (TEM) processing protocol Samples were fixed overnight in a mixture of 2% formaldehyde and 2.5% glutaraldehyde in.1 M sodium cacodylate buffer in (ph 7.4). Cell pellets were then washed in.1 M sodium cacodylate buffer and post-fixed with reduced osmium in.1 M sodium cacodylate buffer for 1 hour. Pellets were then treated with 1% tannic acid in.1m cacodylate buffer for 1 hour, washed with water and stained with 1% uranyl acetate overnight at 4 C. Dehydration was performed in a graded series of ethanol followed by propylene oxide. Specimens were then infiltrated with TAAB LV resin and polymerized at 6 C for at least 24 hours. 5 7 nm sections were examined with FEI Tecnai 12 Biotwin electron microscope at 8kV acceleration voltage.

2 Table S1. B-NHL patient data Patient ID Age/Sex Disease Stage Previous therapy % cell death above control GA11 (NG) Rituximab LY564 39M MCL IV No prior therapy LY425 72M MCL IVA CVP, FC, PACEBOM LY43 61M MCL IVA No prior therapy.9.3 LY34 53M DLBCL (previous FL) n/a Chlorambucil, CHOP LY268 6M DLBCL n/a No prior therapy LY489 68F DLBCL IIA CHOP + RT n/a: not available; MCL: mantle cell lymphoma, DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; CVP: cyclophosphamide, vincristine, prednisolone; FC: fludarabine, cyclophosphamide; PACEBOM: prednisolone, doxorubicin, cyclophosphamide, etoposide, bleomycin, vincristine and methotrexate; CHOP: cyclophosphamide, doxorubicin, vincristine, prednisolone; RT: radiation therapy

3 A Figure S1 Relative cell growth (%) Raji Granta 519 control mab Daudi SU-DHL4 * GA11 Rituximab B Relative colony growth (%) Raji Daudi * control mab GA11 Rituximab

4 Figure S1. GA11 induces longer term loss of cell viability and inhibition of colony growth. A) Analysis of longer-term cell growth inhibition induced by anti-cd mabs. Cells were cultured with mabs (1 µg/ml) for 72 hours and cell viability was assessed by the colorimetric XTT assay as described in the materials and methods. Absorbance was detected and normalized relative to untreated cells. GA11 induced superior loss of cell viability compared to rituximab at 72 hours in all cell lines tested (Raji p<.1, Daudi and SU-DHL4 p<.1, Granta p<.3). B) The impact of anti-cd mabs on colony growth. 16 Raji or Daudi cells were seeded in 96-well plates and treated with.1 µg/ml mab. After 14 days colony growth was quantified by incubation with WST-1 reagent (gently added in a 1:5 final dilution) for 1 hours at 37ºC. Absorbance was detected and normalized relative to untreated cells. Mean + SEM of quintuplicates, representative of 2 independent experiments are shown. Compared to rituximab, GA11 induced greater inhibition of colony growth in Raji and Daudi cells (P<.1).

5 A % raft redistribution CDC (PI +ve %) B Figure S2 control mab Rituximab GA11(G) GA11 (NG) Rituximab GA11 (NG) mab ( µg/ml) C 1 4 No mab GA11 (NG) Rituximab SU-DHL-4 FL1-H FL1-H FL1-H Calcium flux Time: Time (512. sec.) Time: Time (512. sec.) Time: Time (512. sec.) Daudi FL1-H FL1-H FL1-H Time: Time (512. sec.) Time: Time (512. sec.) Time: Time (512. sec.) Time (seconds)

6 Figure S2. GA11 shows characteristics of a type II anti-cd mab (A) 1 µg/ml of FITC-labeled anti-cd mab were added to SU-DHL4 cells for 3 minutes on ice, and the proportion of mab that had redistributed to the Triton X- 1-insoluble fraction was assessed by flow cytometry. GA11 (NG), the nonglycoengineered derivative of GA11, failed to redistribute CD into Triton X- 1 insoluble lipid rafts in contrast to rituximab (B) SU-DHL4 cells, which are highly sensitive to complement-dependent cytotoxicity (CDC) due to their very high expression of CD and lack of complement defense molecules, were treated with different concentrations of anti-cd mabs for 15 minutes at room temperature. % (vol/vol) normal human serum was then added as a source of complement, and cells were incubated for 3 minutes at 37ºC. Cell lysis was determined by PI staining, analysed by flow cytometry. Both GA11 and GA11 (NG), failed to induce CDC, in contrast to rituximab which was highly effective in CDC. (C) SU-DHL4 and Daudi cells were labelled with Indo-1-AM and treated with 1 µg/ml anti-cd mab for 15 minutes at room temperature and assessed by flow cytometry to set background fluorescence. A secondary hyper-crosslinking mab (Mouse anti-human IgG, µg/ml) was subsequently added (as indicated by arrows) and calcium flux was then detected as an increase in Indo-1 fluorescence, by flow cytometry. GA11-NG evoked less Calcium flux compared to rituximab upon cross-linking.

7 Figure S3 Cell death (% PI +ve) No complement % human serum complement ** * No treatment Rituximab GA11 Ofatumumab Figure S3. GA11 induces superior cell death in the presence of serum complement. Comparison of mab-induced cytotoxicity in the presence or absence of serum complement. Raji cells, which express more typical levels of CD and complement defense molecules, were coated with mab (1 g/ml) and % (vol/vol) normal human serum was added as a source of complement. Cell death was analysed after 4 hours by PI staining and flow cytometry. Mean + SEM of 3 independent experiments are shown. GA11 induced superior cell death compared to rituximab and ofatumumab in the presence of serum complement. * p=.18, ** p<.1

8 Figure S4 1 No inhibitor Lat B 1 Relative cell growth (%) 8 6 Control mab GA11 Rituximab Figure S4. GA11-induced cell growth inhibition is dependent on actin polymerization. Raji cells were pre-treated or not with the inhibitor of actin polymerization latrunculin B (LatB, 1µM) in 96-well plates and subsequently treated with mab (1 µg/ml) for 24 hours at 37ºC and WST-1 reagent (Roche) was added gently (1:5 final dilution). Cells were further incubated for 2 hours at 37ºC and absorbance was detected as described above. Mean + SEM of quintuplicate samples representative of 2 independent experiments are shown. GA11 induced greater growth inhibition compared to rituximab (32.5% versus 14.5%, P<.1), and mab-induced growth inhibition was completely attenuated by Lat B.

9 Figure S h 2h Figure S5. Relocalization of actin towards cell-cell junctions during homotypic adhesion induced by GA11. Time lapse microscopy of Raji cells expressing GFP-labelled actin following treatment with GA11 (1 µg/ml) at timepoint. Fluorescence images were captured every 5 minutes, with a sample of images captured at different timepoints shown. (Scale bar= µm).

10 Figure S6 Hypodiploid cells (%) Control mab GA11 Ritux STSP Time (hours) Control mab GA11 Rituximab STSP Figure S6. GA11-induced cell death is independent of nuclear fragmentation. Raji cells were treated with 1 µg/ml mab or 2 µm Staurosporine (STSP) as a positive control for apoptosis. Cells were harvested at different timepoints, resuspended in hypotonic propidium iodide solution and incubated overnight at 4ºC. Cell cycle profile was analysed using flow cytometry. Nuclear fragmentation was detected as the hypo-diploid (sub G1) fraction. Mean + SEM of 2 independent experiments are shown (Top) with representative flow cytometry histograms of samples harvested 24-hours post treatment (bottom). No significant nuclear fragmentation was observed with GA11 for up to 72 hours after treatment in contrast to staurosporine-induced apoptosis.

11 Figure S No inhibitor CMA Relative cell growth (%) Control mab GA11 Rituximab Figure S7. GA11-induced cell growth inhibition is dependent on vacuolar ATPase. Raji cells were pre-treated or not with the vacuolar ATPase inhibitor concanamycin A (CMA, 1 nm) in 96-well plates and subsequently treated with mab (1 µg/ml) for 24 hours at 37ºC and WST-1 reagent (Roche) was added gently (1:5 final dilution). Cells were further incubated for 2 hours at 37ºC and absorbance was detected as described above. Mean + SEM of quintuplicate samples representative of 2 independent experiments are shown. GA11 induced greater growth inhibition compared to rituximab (P<.1), and mab-induced growth inhibition was completely attenuated by CMA.

12 Figure S8 Control mab GA11 Figure S8. GA11 induces relocalization of lysosomes towards points of cellular adhesion. Fluorescence microscopy of LAMP-1 staining (green), used as a lysosomal marker, 4 hours after treatment with mabs. DNA was counter-stained with DAPI (blue). GA11 induces the relocalization of LAMP-1 positive lysosomes towards points of cellular adhesion. Scale bar = 15 µm