High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate

Abstract

CD19 and CD21 (CR2) are co-receptors found on B-cells and various B-cell lymphomas, including non-Hodgkin lymphoma. To evaluate their suitability as targets for therapy of such lymphomas using internalization-dependent antibody-drug conjugates [such as antibody-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) (MCC-DM1) conjugates, which require lysosomal degradation of the antibody moiety for efficacy], we examined uptake of antibodies to CD19 and CD21 in a panel of B-cell lines. Anti-CD21 antibodies were not sufficiently internalized even in the highest CD21-expressing Raji cells, resulting in lack of efficacy with anti-CD21-MCC-DM1 conjugates. Anti-CD19 antibody uptake was variable, and was unexpectedly negatively correlated with CD21 expression. Thus, high CD21-expressing Raji, ARH77 and primary B-cells only very slowly internalized anti-CD19 antibodies, while CD21-negative or low expressing cells, including Ramos and Daudi, rapidly internalized these antibodies in clathrin-coated vesicles followed by lysosomal delivery. Anti-CD19-MCC-DM1 caused greater cytotoxicity in the faster anti-CD19-internalizing cell lines, implying that the rate of lysosomal delivery and subsequent drug release is important. Furthermore, transfection of Ramos cells with CD21 impeded anti-CD19 uptake and decreased anti-CD19-MCC-DM1 efficacy, suggesting that CD21-negative tumours should respond better to such anti-CD19 conjugates. This may have possible clinical implications, as anti-CD21 immunohistochemistry revealed only approximately 30% of 54 diffuse large B-cell lymphoma patients lack CD21 expression.

Fig 1

Anti-CD21 antibodies are not significantly internalized, while anti-CD19 antibodies only internalize readily in CD21^lo or CD21⁻ cells. Various B-cell lines were incubated with anti-CD21 (HB135) for 20 h at 37°C in the presence of lysosomal protease inhibitors, and the total antibody distribution detected post fixation and permeabilization with Cy3-conjugated anti-mouse (left panels). Insets show surface binding of anti-CD21 following 1 h incubation on ice. Ramos (A) and DoHH2 (B) cells lack surface expression of CD21 and consequently failed to internalize any antibody, as expected. Anti-CD21 is not significantly internalized in the low CD21-expressing Namalwa (C) or Daudi (D) cells, or even in the higher expressing ARH77 (E) or Raji (F) cells, or in freshly isolated primary human B-cells (G). The same cell lines were incubated with anti-CD19 (B496) antibodies on ice for 1 h (insets in middle panels), or at 37°C for 3 h (middle panels) or 20 h (right panels) with detection as above. The CD21-negative cell lines Ramos (H,O) and DoHH2 (I,P) readily internalized anti-CD19 within 3 h, while the low CD21-expressing Namalwa (J,Q) and Daudi (K,R) cells internalized it less extensively, as judged by the faint plasma membrane staining remaining even after 20 h uptake. The high CD21-expressors, ARH77 and Raji did not detectably internalize anti-CD19 after 3 h (L,M), and after 20 h still had not internalized nearly as much as the CD21-negative cells did in 3 h (S,T). Primary human B-cells did not internalize anti-CD19 within 3 h (N), but did by 20 h (U). Virtually all the cells in each field readily internalized Alexa488-transferrin (with the exception of transferrin-receptor negative primary B-cells), indicating that any lack of antibody uptake was not due to loss of viability (not shown). Gamma levels were adjusted where appropriate. Scale bar = 20 μm.

Fig 2

Transfection of CD21 into Ramos cells impedes anti-CD19 uptake. Upper panels: freshly isolated human B-cells do not internalize Alexa555-labeled anti-CD19 within 3 h (A, red channel in C), although it does redistribute into patches on the cell surface (arrows) that co-localize with Alexa488-labeled anti-CD21 added post-uptake on ice (B, green channel in C). Scale bar = 20 μm. Lower panels: Ramos cells (left panels) stably expressing a high (clone 1, right panels) or medium (clone 3, middle panels) level of CD21 were incubated with Alexa555-labeled anti-CD19 (G–I and red channel in M–O) and Alexa647-transferrin (J–L and M–O, shown in green channel for better contrast) for 3 h at 37°C, then chilled and incubated with Alexa488-conjugated anti-CD21 antibodies (D–F) on ice prior to fixation and imaging. Anti-CD19 uptake is impeded by increased CD21 expression, while transferrin uptake is unaffected. Internalized anti-CD19 antibodies do not significantly co-localize with the recycling transferrin at this time-point, as seen by lack of yellow colour in the respective overlaid images (M–O). Gamma levels were adjusted where required for clarity. Scale bar = 20 μm.

Fig 3

Quantitation of CD19 and CD21 surface levels and anti-CD19 uptake by flow cytometry confirms the immunofluorescence results. (A) B-cell lines were incubated on ice with 2 μg/ml mouse anti-CD21 (HB135) or mouse anti-CD19 (B496), followed by rat anti-mouse-phycoerythrin and analyzed by flow cytometry to determine surface expression. Results are the average mean fluorescence intensity (MFI) of triplicates ± standard deviation from a representative of three independent experiments (average of five independent experiments shown for the more variable ARH77 cells). Shown in increasing order of CD21 expression are: (1) SuDHL-4, (2) Ramos, (3) DoHH2, (4) Namalwa, (5) Daudi, (6) Ramos-CD21 clone 3, (7) ARH77, (8) Raji, (9) Ramos-CD21 clone 1. (10) Freshly isolated human B-cells have lower fluorescence values for both antigens than expected due to their small size, but their relative ratio of CD21 to CD19 is similar to that of ARH77 and Raji cells. Ramos-CD21 clone 1 expresses CD21 even more highly than Raji, while Ramos-CD21 clone 3 is intermediate between that of ARH77 and Daudi. (B) The rate of internalization of Alexa488-anti-CD19 in Ramos (▪), Ramos-CD21 clone 1 (□), Ramos-CD21 clone 3 (△) and CD21^hi ARH77 (▴) cells was determined by pre-binding to cells then incubating at 37°C (without washing) for the indicated times, washing and fixing either with or without surface fluorescence quenching with anti-Alexa488. Results are the average and standard deviation of two duplicate experiments each normalized to their respective initial surface binding levels after subtraction of background signals.

Fig 4

Anti-CD19 is internalized by dynamin-dependent, clathrin-mediated endocytosis and is delivered to lysosomes. (A) Ramos cells were pre-incubated for 30 min at 37°C with the following reagents: dimethyl sulphoxide (DMSO) (1); 1 μmol/l chlorpromazine (Cpmzn) (2), a clathrin-mediated endocytosis inhibitor; 80 μmol/l dynamin inhibitor dynasore, preincubated for 5 min only (3); 2 mmol/l methyl-β-cyclodextrin (MbC) (4) or 5 μg/ml filipin (5), both inhibitors of caveolar and lipid raft endocytosis. Alexa488-anti-CD19 (black bars) or Alexa488-transferrin (grey bars) were then added in the continuous presence of inhibitors for 30 min and surface quenched as in Fig 3B. Results were plotted as a percentage of uptake compared with the DMSO control and represent the average and standard deviation of three independent triplicate experiments. (B–D) Alexa488-anti-CD19 (green channel in B and D) was co-internalized with Alexa647-transferrin (shown in the red channel in C and D) in Ramos cells for 5 min, surface quenched with anti-Alexa488, fixed and imaged. (E–G) Alexa488-anti-CD19 (green channel in E and G) was chased for 3 h in Ramos cells in the presence of lysosomal protease inhibitors prior to fixation and staining with Alexa555-anti-LAMP1 (red channel in F and G). Yellow colour in the merged images in panels D and G indicates colocalization. Gamma levels were adjusted where necessary to better illustrate marker overlap. Arrows indicate examples of co-localized staining. Scale bar is 20 μm in the main panels and 6·7 μm in the 3×-magnified insets of the boxed region indicated in D.

Fig 5

Anti-CD19-MCC-DM1 is less efficacious in high CD21 expressing cells. (A) CD21^hi Raji were incubated for 3 d with anti-CD21-MCC-DM1 (○), negative control Trastuzumab-MCC-DM1 (▴), free L-DM1 dimer (▪) or naked anti-CD21 antibodies (•) and assessed for viability by measuring ATP levels. CD21^hi ARH77 (B), CD21^lo Daudi (C), and CD21⁻ DoHH2 cells (D) were incubated for 3 d with anti-CD19-MCC-DM1 (♦), negative control Trastuzumab-MCC-DM1 (▴), free L-DM1 dimer (▪) or naked anti-CD19 antibodies (×) and assessed for viability by measuring ATP levels. (E) Ramos (solid symbols and lines) and Ramos-CD21 clone 1 (open symbols and dashed lines) were treated with anti-CD19-MCC-DM1 (♦,⋄) or free DM1 (▪,□) as in B-D. Inset bar graph shows percentage killing of Ramos (♦) and Ramos-CD21 clone 1 (⋄) at the highest anti-CD19-MCC-DM1 concentration used (3·33 μg/ml). (F) Ramos (solid symbols and lines) and Ramos-CD21 clone 1 (open symbols and dashed lines) were treated with control Trastuzumab-MCC-DM1 (▴,△), free DM1 (▪,□) or unconjugated anti-CD19 (×) as in B. Data are shown in all panels as a percentage viability of untreated control cells (mean and standard deviation of three independent duplicate experiments) versus ADC concentration in μg/ml on the lower x-axes or free DM1 concentration in M on the upper x-axes. * denotes data points statistically different (P < 0·01) from the control untreated cells using the analysis of variance (anova) test. +, data points significantly different between Ramos and Ramos-CD21 clone 1 cells by anova analysis (P < 0·01). The CD21^hi Raji and Ramos-clone 1 cells showed greater resistance (compared with their respective free L-DM1 sensitivities) than the CD21⁻ Ramos, DoHH2 and CD21^lo Daudi cells.