Recruitment of properdin by bi-specific nanobodies activates the alternative pathway of complement

TY - JOUR

T1 - Recruitment of properdin by bi-specific nanobodies activates the alternative pathway of complement

AU - Pedersen, Dennis V

AU - Rösner, Thies

AU - Hansen, Annette G

AU - Andersen, Kasper R

AU - Thiel, Steffen

AU - Andersen, Gregers R

AU - Valerius, Thomas

AU - Laursen, Nick S

N1 - Copyright © 2020 Elsevier Ltd. All rights reserved.

PY - 2020/8

Y1 - 2020/8

N2 - The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation. Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces.

AB - The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation. Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces.

KW - Alternative pathway

KW - Complement dependent cytotoxicity

KW - Complement system

KW - Epidermal growth factor receptor

KW - Nanobody

KW - Properdin

UR - http://www.scopus.com/inward/record.url?scp=85086848078&partnerID=8YFLogxK

U2 - 10.1016/j.molimm.2020.06.005

DO - 10.1016/j.molimm.2020.06.005

M3 - Journal article

C2 - 32599335

SN - 0161-5890

VL - 124

SP - 200

EP - 210

JO - Molecular Immunology

JF - Molecular Immunology

ER -