Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

Abstract

Antibody-dependent cellular phagocytosis (ADCP) by monocytes and macrophages contributes significantly to the efficacy of many therapeutic monoclonal antibodies (mAbs), including anti-CD20 rituximab (RTX) targeting CD20⁺ B-cell non-Hodgkin lymphomas (NHL). However, ADCP is constrained by various immune checkpoints, notably the anti-phagocytic CD47 molecule, necessitating strategies to overcome this resistance. We have previously shown that the IgG2 isotype of RTX induces CD20-mediated apoptosis in B-cell lymphoma cells and, when combined with RTX-IgG1 or RTX-IgG3 mAbs, can significantly enhance Fc receptor-mediated phagocytosis. Here, we report that the apoptotic effect of RTX-IgG2 on lymphoma cells contributes to changes in the tumor cell's CD47 profile by reducing its overall expression and altering its surface distribution. Furthermore, when RTX-IgG2 is combined with other lymphoma-targeting mAbs, such as anti-CD59 or anti-PD-L1, it significantly enhances the ADCP of lymphoma cells compared to single mAb treatment. In summary, RTX-IgG2 acts as a potent phagocytic enhancer by promoting Fc-receptor mediated phagocytosis through apoptosis and reduction of CD47 in CD20⁺ malignant B-cells. RTX-IgG2 represents a valuable therapeutic component in enhancing the effectiveness of different mAbs targeting B-cell NHL.

Keywords: CD47; anti-CD20 antibody; apoptosis; cancer immunotherapy; monocyte; phagocytosis; rituximab.

Figure 1

RTX-IgG2 enhances phagocytosis of RTX-IgG1- or RTX-IgG3-treated CD20⁺ B-cell lymphoma cells. Flow cytometry analysis of ADCP of Granta-519 B-cell lymphoma cells, treated with anti-CD20 RTX isotypes or human isotype control Abs (1.5 µg/ml), by MonoMac-6 cells at an E:T ratio of 1:1. (A) Percentage phagocytosis of Granta-519 cells induced by single RTX isotypes, or dual combinations of RTX-IgG2 with RTX-IgG1 or RTX-IgG3. Untreated cells (UT), and human Ab isotypes: hIgG1, hIgG2, hIgG3, were used as controls. For dual treatments, Granta-519 cells were pre-opsonized with 1.5 μg/mL of RTX-IgG2 for 30 min followed by 1.5 μg/mL of RTX-IgG1 or RTX-IgG3. Results are shown as mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (****p<0.0001, **p<0.01). (B) Representative bivariate plots showing phagocytosis of RTX-treated CFSE-labeled Granta-519 target cells by CTFR-labeled MonoMac-6 effector cells. Phagocytosis was quantified as the percentage of double positive CFSE⁺ CTFR⁺ MonoMac-6 cells (square gate). Increased phagocytosis was observed when RTX-IgG2 was combined with RTX-IgG1 or RTX-IgG3.

Figure 2

Analysis of cell death in CD20⁺ B-cell lymphoma cells treated with STR or RTX isotypes. Granta-519 cells treated with STR (6 hours) or RTX isotypes (30 min) were analyzed for apoptosis or necrosis compared to untreated cells (UT). Dimethyl sulfoxide (DMSO) was used as vehicle control of STR treatment. (A) Percentage apoptosis in UT or treated Granta-519 cells. (B) Representative bivariate plots of Granta-519 cells, showing apoptosis and necrosis, in UT and after treatment with RTX isotypes or STR. Apoptotic cells were identified as Annexin V⁺ DC-Violet⁻ cells, while double positive (Annexin V⁺ DC-Violet⁺) cells were identified as necrotic cells with compromised cell membrane. (C) Percentage necrosis in UT or treated Granta-519 cells. Results in (A, C) show mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (****p<0.0001; ns, not significant).

Figure 3

Apoptosis induced by STR in CD20⁺ B-cell lymphoma cells enhances ADCP. Flow cytometry analysis of phagocytosis of Granta-519 cells, untreated (UT) or incubated with STR for 6 hours before addition of RTX isotypes or isotype controls (1.5 μg/mL), by MonoMac-6 cells (E:T ratio = 1:1). (A) Percentage phagocytosis of UT Granta-519 cells, treated with STR, RTX-IgG1 or RTX-IgG3 or combinations thereof. Results shows mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (****p<0.0001). (B) Representative bivariate plots showing phagocytosis of CFSE-labeled Granta-519 target cells, treated with RTX isotypes alone or in combination with STR. Phagocytosis was quantified as the percentage of double positive CFSE⁺ CTFR⁺ MonoMac-6 cells (square gate). Increased phagocytosis was observed when RTX-IgG1 or RTX-IgG3 is combined with STR.

Figure 4

Decreased CD47 expression in RTX-IgG2-treated CD20⁺ B-cell lymphoma cells. CD47 expression was evaluated in Granta-519 cells after incubation with STR (6 hours) or treatment with RTX isotypes (1.5 μg/mL) (30 min). (A) Representative histograms of CD47 expression in Granta-519 cells after treatment with RTX-IgG1, RTX-IgG2, RTX-IgG3, or STR. Grey dashed line indicates the level of CD47 on untreated cells (UT). (B) Bar graph representation of fold change (left Y-axis) and percentage difference (right Y-axis) in CD47 expression on Granta-519 cells induced by STR or RTX isotypes, normalized to CD47 expression on UT. To obtain the fold change values, mean fluorescence intensity (MFI) of treated samples was first adjusted by subtracting MFI of isotype controls and then normalized to the MFI of UT samples. The percentage difference was calculated by the following formula: ((MFI_{treated sample} – MFI_UT)*100)/MFI_UT). Results show mean fold change ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (****p<0.0001). (C) Confocal microscopy images of CD47 expression in untreated, RTX-IgG1-, RTX-IgG2-, and STR-treated Granta-519 cells. Cells were counterstained with Hoescht 33342 nucleus stain. Disruptions in CD47 cell surface pattern are indicated by white arrows. Scale bar: 10 μm.

Figure 5

RTX-IgG2 enhances phagocytosis of CD20⁺ B-cell lymphoma cells induced by anti-CD59 or anti-PD-L1 mAb. Representative histograms of (A) CD59 and (B) PD-L1 expression on Granta-519 cells. (C, D) Percentage phagocytosis of Granta-519 cells by MonoMac-6 cells (E:T ratio = 1:1), induced by c) mouse anti-human CD59 mAb (αCD59, 2.5 μg/mL) and d) human anti-PD-L1 mAb (αPD-L1, 1.5 μg/mL) alone, or in combination with a pre-treatment with RTX-IgG2. Data are presented as mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (****p<0.0001, *p<0.05; ns, not significant).

Figure 6

Blocking of CD47 on CD20⁺ B-cell lymphoma cells enhances FcR-mediated ADCP. Percentage phagocytosis of Granta-519 cells treated with RTX-IgG1 or RTX-IgG3, and in combination with blocking of CD47 by mouse anti-human CD47 mAb with a functional Fc domain (αCD47-fuFc, 1 μg/mL) or humanized anti-CD47 mAb with a silenced Fc domain (αCD47-siFc, 10 μg/mL), by MonoMac-6 cells (E:T ratio = 2:1). Data are presented as mean ± SEM of three independent experiments, each with three biological replicates. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (****p<0.0001, **p<0.01, *p <0.05; ns, not significant).

Figure 7

Effects of RTX-IgG2 on Raji B-cell lymphoma cells. (A) Representative histograms of CD20, CD47, and CD59 expression on Raji and Granta-519 B-cell lymphoma cells. (B) Percentage apoptosis in untreated (UT) and STR- or RTX-IgG2-treated Raji cells. (C) Fold change in CD47 expression on Raji cells induced by STR or RTX-IgG2, normalized to CD47 expression on untreated cells (UT). (D) Percentage phagocytosis of Raji cells by MonoMac-6 cells (E:T ratio = 1:1), induced by single RTX isotypes, or dual combinations of RTX-IgG2 with RTX-IgG1 or RTX-IgG3. Untreated cells (UT), and human Ab isotypes (hIgG1, hIgG2, hIgG3) were used as controls. For dual treatments, Raji cells were pre-opsonized with 2.5 μg/mL of RTX-IgG2 for 30 min followed by 1.5 μg/mL of RTX-IgG1 or RTX-IgG3. (E) Percentage phagocytosis of Raji cells by MonoMac-6 cells (E:T ratio = 1:1), induced by mouse anti-human CD59 mAb (αCD59, 2.5 μg/mL) alone, or in combination with a pre-treatment with RTX-IgG2. Results in (B-E) are shown as mean ± SEM of three biological replicates from one representative experiment out of two. Statistical analysis by one-way ANOVA with Tukey-Kramer post-hoc test (**p<0.01, *p<0.05; ns, not significant).