Monocytes mediate shaving of B-cell-bound anti-CD20 antibodies

Abstract

Anti-CD20 monoclonal antibodies are promising for the treatment of B-cell malignancies such as chronic lymphocytic leukaemia and autoimmune diseases where auto-antibodies play an important role. Anti-CD20 such as rituximab (RTX) mediates B-cell depletion through mechanisms such as complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. However, in haematological malignancies, such effector mechanisms can be saturated and result in release of malignant B cells with reduced levels of CD20. It has been hypothesized that this is the result of monocyte-mediated shaving of the CD20/RTX complex from the B-cell surface. Here, we confirm, that in vitro co-culture of human monocytes and RTX-labelled syngeneic B cells results in reduced expression of CD20/RTX complex on the B cell surface. This shaving mechanism was the result of active protease activity because EDTA and PMSF were able to mediate partial inhibition. Also, a series of alternative anti-CD20 antibodies representing both type I and type II antibodies were tested for their ability to induce the shaving reaction. These results demonstrate that a monocyte-mediated shaving reaction can lead to complete loss of most anti-CD20 antibodies from the surface of B cells even from healthy donors and this is an important obstacle for antibody-mediated immune therapy. The findings demonstrate the necessity of developing novel antibodies that maintain high effector functions without enabling activation of the shaving reaction.

Figure 1

Human monocytes mediate shaving of B-cell bound rituximab (RTX): 1 × 10⁶ B cells were co-cultured with 1 × 10⁴ to 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with RTX antibody. Remaining RTX on the B-cell surface was analysed after 18 hr using flow cytometry. Data are presented as original histograms from the experiment (a–f) and as % shaving ± SD (g). Also, the effect of in-vitro-generated monocyte-derived dendritic cells was tested (g). Data presented are representative of at least three independent experiments with similar findings.

Figure 2

Monocyte-mediated shaving is dependent on the Fc part of rituximab (RTX): 1 × 10⁶ B cells were co-cultured with 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with RTX antibody or an Fc-truncated analogue. Remaining RTX on the B-cell surface was analysed after 18 hr using flow cytometry. Data are presented as % shaving, which indicates % loss of RTX. Data presented are representative of at least two independent experiments with similar findings.

Figure 3

Monocyte-mediated shaving is independent of endocytosis: 1 × 10⁶ B cells were co-cultured with 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with rituximab (RTX) antibody and 0·4 m hyperosmolar sucrosis. Remaining RTX on the B-cell surface was analysed after 18 hr using flow cytometry. Data are presented as % shaving, which indicates % loss of RTX. Data presented are representative of at least two independent experiments with similar findings.

Figure 4

Monocyte-mediated shaving is dependent on protease activity: 1 × 10⁶ B cells were co-cultured with 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with rituximab (RTX) antibody and 10 mm EDTA. Remaining RTX on the B-cell surface was analysed after 18 hr using flow cytometry. Data are presented as % shaving, which indicates % loss of RTX. Data presented are representative of at least three independent experiments with similar findings. *P < 0·05.

Figure 5

Monocyte-mediated shaving is dependent on protease activity: 1 × 10⁶ B cells were co-cultured with 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with rituximab antibody and 10 mm EDTA. Remaining RTX on the B-cell surface was analysed after 18 hr using flow cytometry. Data are presented as % shaving, which indicates % loss of RTX. Data presented are representative of at least two independent experiments with similar findings. *P < 0·05.

Figure 6

AT80 mouse anti-human CD20 antibody induces shaving at lower levels: 1 × 10⁶ B cells were co-cultured with 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with different mouse anti-human CD20 antibodies. Remaining antibody on the B-cell surface was analysed after 18 hr using flow cytometry with fluochrome-conjugated polyclonal rabbit anti-mouse antibody. Data are presented as % shaving, which indicates % loss of anti-CD20 antibody. Data presented are representative of at least three independent experiments with similar findings.

Figure 7

Human rituximab (RTX) analogues have similar shaving capacity: 1 × 10⁶ B cells were co-cultured with 1 × 10⁶ syngeneic CD14⁺ monocytes from healthy donors together with various human anti-CD20 antibodies. Remaining antibody on the B-cell surface was analysed using fluorochrome-conjugated anti-human Fc antibody SB2H2 after 18 hr using flow cytometry. Data are presented as % shaving, which indicates % loss of RTX. Data presented are representative of at least two independent experiments with similar findings.