Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

Henrik Pedersen; Rasmus Kjeldsen Jensen; Annette Gudmann Hansen; Steen Vang Petersen; Steffen Thiel; Nick Stub Laursen; Gregers Rom Andersen

doi:10.3389/fimmu.2022.872536

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

Henrik Pedersen, Rasmus Kjeldsen Jensen, Annette Gudmann Hansen, Steen Vang Petersen, Steffen Thiel, Nick Stub Laursen, Gregers Rom Andersen^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

3 Citations (Scopus)

Abstract

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

Original language	English
Article number	872536
Journal	Frontiers in Immunology
Volume	13
Number of pages	13
ISSN	1664-3224
DOIs	https://doi.org/10.3389/fimmu.2022.872536
Publication status	Published - Jul 2022

Keywords

Animals
Complement Activation
Complement C3
Complement C3 Convertase, Alternative Pathway
Complement C3-C5 Convertases/metabolism
Complement C3b/metabolism
Female
Fibrinogen/metabolism
Humans
Rats
Sheep

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@article{b322b3749d624cdfb8cd33ce28cdd2b3,

title = "Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity",

abstract = "The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.",

keywords = "Animals, Complement Activation, Complement C3, Complement C3 Convertase, Alternative Pathway, Complement C3-C5 Convertases/metabolism, Complement C3b/metabolism, Female, Fibrinogen/metabolism, Humans, Rats, Sheep",

author = "Henrik Pedersen and Jensen, {Rasmus Kjeldsen} and Hansen, {Annette Gudmann} and Petersen, {Steen Vang} and Steffen Thiel and Laursen, {Nick Stub} and Andersen, {Gregers Rom}",

note = "Copyright {\textcopyright} 2022 Pedersen, Jensen, Hansen, Petersen, Thiel, Laursen and Andersen.",

year = "2022",

month = jul,

doi = "10.3389/fimmu.2022.872536",

language = "English",

volume = "13",

journal = "Frontiers in Immunology",

issn = "1664-3224",

publisher = "Frontiers Media SA",

}

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity. / Pedersen, Henrik; Jensen, Rasmus Kjeldsen; Hansen, Annette Gudmann et al.
In: Frontiers in Immunology, Vol. 13, 872536, 07.2022.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

AU - Pedersen, Henrik

AU - Jensen, Rasmus Kjeldsen

AU - Hansen, Annette Gudmann

AU - Petersen, Steen Vang

AU - Thiel, Steffen

AU - Laursen, Nick Stub

AU - Andersen, Gregers Rom

PY - 2022/7

Y1 - 2022/7

N2 - The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

AB - The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

KW - Animals

KW - Complement Activation

KW - Complement C3

KW - Complement C3 Convertase, Alternative Pathway

KW - Complement C3-C5 Convertases/metabolism

KW - Complement C3b/metabolism

KW - Female

KW - Fibrinogen/metabolism

KW - Humans

KW - Rats

KW - Sheep

U2 - 10.3389/fimmu.2022.872536

DO - 10.3389/fimmu.2022.872536

M3 - Journal article

C2 - 35935935

SN - 1664-3224

VL - 13

JO - Frontiers in Immunology

JF - Frontiers in Immunology

M1 - 872536

ER -

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

Abstract

Keywords

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