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. 2020 Nov 19:11:584509.
doi: 10.3389/fimmu.2020.584509. eCollection 2020.

Monitoring of the Complement System Status in Patients With B-Cell Malignancies Treated With Rituximab

Anna Felberg  1 , Michał Taszner  2 , Aleksandra Urban  1 , Alan Majeranowski  2 , Kinga Jaskuła  1 , Aleksandra Jurkiewicz  1 , Grzegorz Stasiłojć  1 , Anna M Blom  3 , Jan M Zaucha  2 , Marcin Okrój  1
Affiliations

Monitoring of the Complement System Status in Patients With B-Cell Malignancies Treated With Rituximab

Anna Felberg et al. Front Immunol. .

Abstract

Rituximab is a pioneering anti-CD20 monoclonal antibody that became the first-line drug used in immunotherapy of B-cell malignancies over the last twenty years. Rituximab activates the complement system in vitro, but there is an ongoing debate on the exact role of this effector mechanism in therapeutic effect. Results of both in vitro and in vivo studies are model-dependent and preclude clear clinical conclusions. Additional confounding factors like complement inhibition by tumor cells, loss of target antigen and complement depletion due to excessively applied immunotherapeutics, intrapersonal variability in the concentration of main complement components and differences in tumor burden all suggest that a personalized approach is the best strategy for optimization of rituximab dosage and therapeutic schedule. Herein we critically review the existing knowledge in support of such concept and present original data on markers of complement activation, complement consumption, and rituximab accumulation in plasma of patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphomas (NHL). The increase of markers such as C4d and terminal complement complex (TCC) suggest the strongest complement activation after the first administration of rituximab, but not indicative of clinical outcome in patients receiving rituximab in combination with chemotherapy. Both ELISA and complement-dependent cytotoxicity (CDC) functional assay showed that a substantial number of patients accumulate rituximab to the extent that consecutive infusions do not improve the cytotoxic capacity of their sera. Our data suggest that individual assessment of CDC activity and rituximab concentration in plasma may support clinicians' decisions on further drug infusions, or instead prescribing a therapy with anti-CD20 antibodies like obinutuzumab that more efficiently activate effector mechanisms other than complement.

Keywords: chronic lymphocytic leukemia; complement system; non-Hodgkin’s lymphoma; obinutuzumab (GA101); rituximab.

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Figures

Figure 1
Figure 1
CDC potential and rituximab concentration in serum samples collected from CLL patients. CDC potential was assessed in calcein release assay performed using Raji cells incubated with 10% patient’s serum. Dark bars represent CDC levels of patients’ sera non-supplemented with extra rituximab, grey bars represent CDC levels when sera were supplemented with 50 µg/ml of rituximab. Dotted line represents rituximab concentration (right Y axis). Each serum was tested in three independent experiments, error bars indicate standard deviation.
Figure 2
Figure 2
CDC potential and rituximab concentration in serum samples collected from NHL patients. CDC potential was assessed in calcein release assay performed using Raji cells incubated with 10% patient’s serum. Dark bars represent CDC level of patients’ sera non-supplemented with extra rituximab, grey bars represent CDC level when sera were supplemented with 50 µg/ml of rituximab. Dotted line represents rituximab concentration (right Y axis). Each serum was tested in three independent experiments, error bars indicate standard deviation.
Figure 3
Figure 3
CDC exerted in 50% normal human serum by rituximab at concentration range 10-100 µg/ml. CDC was examined on four CD20-positive cell lines: SU-DHL-4 (A), Ramos (B), Namalwa (C), Raji (D) and fresh culture of CLL cells (E). Supernatant collected from calcein-labelled cells lysed with 30% DMSO diluted in PBS served as the indicator of 100% (full) lysis. Readout obtained for heat-inactivated serum (Δ NHS) served as negative control, i.e. background lysis independent on complement activation. Cells were tested at quantities 1, 2, 5, and 10 × 105 cells/50 µl, Δ 100 group represents heat-inactivated normal human serum supplemented with 100 µg/ml of rituximab. Data were collected from three independent experiments, error bars indicate standard deviation.
Figure 4
Figure 4
Determination of levels of C4d and TCC, complement activation markers. Graphs show C4d concentration (A) and TCC concentration (B) in sera collected before (black bars) and after (grey bars) consecutive rituximab infusions in CLL patients. Data were collected from three independent measurements, error bars represent standard deviation.
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