Does augmenting the complement system improve Rituximab induced cell lysis?


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Katy Plant1
1Cardiff University School of Medicine

Abstract

Background

Rituximab (RTX) is a therapeutic antibody used in the treatment of haematological malignancies. RTX induces complement dependant cytotoxicity (CDC). An individual’s phenotype of complement components, their “complotype”, is determined by genetic polymorphisms and governs their system functionality. We hypothesised that augmenting the complotype, by addition of purified complement components, may increase the effectiveness of RTX.

Method

Normal human serum was obtained from a healthy donor (NHS1) and from Sigma Aldrich (NHS2). The CDC elicited by NHS was assessed in Sheep Erythrocyte (ShE) haemolytic assays +/- addition of purified complement components. Studies were performed on Ramos human burkitt lymphoma cells lines. Ramos sublines, R1H (CD55/CD59 positive) and R2J (CD55/CD59 negative) were generated in house. To assess RTX induced CDC, cell viability assays were performed on Ramos cells +/- purified complement components.

Results

NHS1 induced greater CDC than NHS2. Addition of C2, C3, C5 components to NHS1 increased CDC. An estimated 2.5-fold increase of C5 concentration in NHS1 increased ShE lysis by 20.2%. An estimated 10-fold increase in the C2 and C3 concentrations in NHS1 increased ShE lysis by 39.7% and 21.8% respectively. Addition of the other C-Components did not augment CDC on ShEs.

R1H cells are resistant to RTX induced CDC. R2J cell viability post RTX sensitization was 23.8%. Addition of C2, C3 and C5 decreased cell viability to 9.7%, 6.5% and 11% respectively. The other C-Components did not augment RTX induced CDC.

Conclusion

Our data implicate C2, C3 and C5 as limiting factors of CDC and suggests addition of these to RTX therapy could improve efficacy. This is the first preliminary data exploring therapeutic antibodies and complement addition. Research into this hypothesis, the theoretical clinical potential and safety profile is warranted. The next step in developing knowledge in this area would be further R1H studies to prove utility in refractory cancer cells.