Calcein release assay as a method for monitoring serum complement activity during monoclonal antibody therapy in patients with B-cell malignancies

Stasiłojć, Grzegorz; Felberg, Anna; Urbán, A; Kowalska, Daria; Ma, Shuo; Blom, Anna M.; Lundin, Jeanette; Österborg, Anders; Okrój, Marcin

doi:10.1016/j.jim.2019.112675

Cited by 6 publications

(9 citation statements)

References 9 publications

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“…Importantly, such exhaustion did not overlap with the next infusion indicating that a four-week interval is enough for the restoration of the complement pool. These results are in agreement with another study, which analyzed the effect of ofatumumab, a stronger CDC-activating anti-CD20 antibody (60), applied in a 2-week interval (46).…”

Section: Discussionsupporting

confidence: 92%

“…Raji cell line is characterized as a moderately sensitive to rituximab compared to other B-cell lymphomas, thus enabling observation of either depressed or higher than average complement activity in CDC assays (43). Previously we demonstrated the utility of this model for the mirroring of the anti-CD20 antibody-driven complement consumption and found superior sensitivity of the assay when 10% instead of 50% serum was used (46). Importantly, 10% serum is a surrogate of the complement content in lymph or extravascular fluids, a natural microenvironment of lymphoma (13,57,58).…”

Section: Discussionmentioning

confidence: 99%

“…Previously we proposed a calcein release assay on Raji cells as a method for monitoring CDC potential of serum collected from patients treated with type I anti-CD20 antibodies (46). There are several advantages of this method over the routinely used CH 50 assay performed on sensitized sheep erythrocytes: i) usage of human tumor cells bearing both molecular target (CD20) for dedicated immunotherapeutics and human complement inhibitors (CD46, CD55, CD59) (43) fully compatible with human complement, ii) adequate sensitivity of target cells to complement-mediated lysis and iii) lower inter-assay variability compared to CH 50 assay (46). Using this approach, we measured the CDC potential of serum samples collected before and after each infusion of rituximab in 17 patients with various B cell malignancies.…”

Section: Rituximab (Type I) or Type II Anti-cd20 Immunotherapeutics?mentioning

confidence: 99%

“…Complement-dependent cytotoxicity (CDC) functional assay was performed as described in (46). Briefly, cells previously loaded with calcein-AM (Sigma) were pelleted onto V-shape microplate wells and overlaid with 50 ml of the indicated serum with or without addition of rituximab.…”

Section: Cdc Assaymentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Rituximab (Type I) or Type II Anti-cd20 Immunotherapeutics?mentioning

confidence: 99%

Section: Cdc Assaymentioning

confidence: 99%

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

et al. 2020

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“…Complement-dependent cytotoxicity (CDC) assay was performed in Raji cells as described in ( 8 ). Briefly, cells were loaded with 1mM calcein-AM (Sigma) for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein

et al. 2021

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The impairment of the alternative complement pathway contributes to rare kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). We recently described an aHUS patient carrying an exceptional gain-of-function (GoF) mutation (S250C) in the classical complement pathway component C2 leading to the formation of hyperactive classical convertases. We now report the identification of the same mutation and another C2 GoF mutation R249C in two other patients with a glomerulopathy of uncertain etiology. Both mutations stabilize the classical C3 convertases by a similar mechanism. The presence of R249C and S250C variants in serum increases complement-dependent cytotoxicity (CDC) in antibody-sensitized human cells and elevates deposition of C3 on ELISA plates coated with C-reactive protein (CRP), as well as on the surface of glomerular endothelial cells. Our data justify the inclusion of classical pathway genes in the genetic analysis of patients suspected of complement-driven renal disorders. Also, we point out CRP as a potential antibody-independent trigger capable of driving excessive complement activation in carriers of the GoF mutations in complement C2.

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A Cell-Based Assay to Measure the Activity of the Complement Convertases

Stasiłojć,

Kuźniewska

et al. 2024

Kidney International Reports

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Calcein release assay as a method for monitoring serum complement activity during monoclonal antibody therapy in patients with B-cell malignancies

Cited by 6 publications

References 9 publications

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

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

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein

A Cell-Based Assay to Measure the Activity of the Complement Convertases

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