title: Highly reduced binding to high and low affinity mouse Fc gamma receptors by L234A/L235A and N297A Fc mutations engineered into mouse IgG2a.
abstract: The effects of the Fc silencing mutations such as leucine (L) to alanine (A) substitution at the position 234 and 235 (LALA) and the alanine (A) to asparagine (N) substitution at position 297 (N297A) are well investigated for human IgG. However, the effects of the same two silencing Fc mutations in a mouse IgG backbone are not yet well investigated in respect to binding to mouse Fc gamma receptors (FcγRs), complement and subsequent effector functions. By using a mouse IgG2a tool antibody directed against mouse OX40L, we demonstrate a strongly reduced binding of the two Fc mutants to high and low affinity recombinant and cell expressed mouse FcγRs, when compared to the mouse IgG2a with the wild type (wt) backbone. Reduced FcγR binding by the two investigated Fc mutants could further be confirmed on primary mouse macrophages expressing their native FcγRs. In addition, we reveal that the LALA and N297A mutations in the mIgG2a also slightly reduced binding to C1q of human origin. Thus, here we provide experimental evidence that the two investigated Fc mutations in the mouse IgG backbone lead to similar "silencing" properties as previously demonstrated for the human IgG and thus represent a useful method to alter effector functions in tool antibodies to be used in mouse models.
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: E
authors.initials: E
authors.lastname: Arduin
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Arora
authors.affiliation: Novartis Pharma AG, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: P R
authors.initials: PR
authors.lastname: Bamert
authors.affiliation: Novartis Pharma AG, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Kuiper
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Popp
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Geisse
authors.affiliation: Novartis Pharma AG, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: R
authors.initials: R
authors.lastname: Grau
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Calzascia
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland.
authors.forename: G
authors.initials: G
authors.lastname: Zenke
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland. Electronic address: jiri.kovarik@novartis.com.
authors.forename: J
authors.initials: J
authors.lastname: Kovarik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015922:Complement C1q / Q000378:metabolism; D005434:Flow Cytometry; D005818:Genetic Engineering; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D053278:OX40 Ligand / Q000378:metabolism; D011485:Protein Binding; D017452:Receptors, IgG / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D007074:Immunoglobulin G; D050505:Mutant Proteins; D053278:OX40 Ligand; D017452:Receptors, IgG; D011994:Recombinant Proteins; D015922:Complement C1q
keywords: Complement; Fc silencing mutations; Mouse FcγRs binding; Mouse IgG2a