Highly reduced binding to high and low affinity mouse Fc gamma receptors by L234A/L235A and N297A Fc mutations engineered into mouse IgG2a

Arduin, Elodie; Arora, Seguinde; Bamert, Paulo Roky; Kuiper, Torsten; Popp, Simone; Geisse, Sabine; Grau, Roger; Calzascia, Thomas; Zenke, Gerhard; Kovařík, Jiří

doi:10.1016/j.molimm.2014.09.017

Cited by 62 publications

(63 citation statements)

References 23 publications

(31 reference statements)

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“…In human IgG1 antibodies, the LALA-PG variant is equivalent to the NG variant at eliminating binding to Fc-␥ receptors. Our data further demonstrate the superiority of using the LALA-PG variant over the LALA variant to silence effector function, as previous work has shown that the LALA variant in murine IgG2a retains attenuated binding to the murine Fc-␥ receptors RIII and RIV (26). In contrast to previous studies (26), we demonstrate residual binding of the murine IgG2a-DANG to Fc-␥RI.…”

Section: Figure 5 Murine Fc-␥ Binding Is Significantly Reduced By Lasupporting

confidence: 74%

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

Lo¹,

Kim

Tong³

et al. 2017

Journal of Biological Chemistry

221

201

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Edited by Peter CresswellThe antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-␥ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-␥-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

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Section: Figure 5 Murine Fc-␥ Binding Is Significantly Reduced By Lasupporting

confidence: 74%

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

Lo¹,

Kim

Tong³

et al. 2017

Journal of Biological Chemistry

221

201

View full text Add to dashboard Cite

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“…faecalis virulence, with a gls24 mutant significantly attenuated in establishing infection in a mouse peritonitis model. [13,21,22,34]. For further exploration of the down-regulation of gls24 in our transcriptome analysis, we performed Western blot analysis to assess the level of Gls24 expression in an RNase J2 deletion mutant by probing with a Gls24 specific monoclonal antibody we generated using techniques previously described [35,36].…”

Section: Resultsmentioning

confidence: 99%

“…Disruption of the ebp operon has previously been shown to significantly reduce E . faecalis biofilm formation and the ability to form vegetation in a rat endocarditis model [22]. Gls24 is an important E .…”

Section: Discussionmentioning

confidence: 99%

“…Monoclonal antibody mAb 9 against EbpA was developed in house using recombinant EbpA protein as immunogen [22] following standard hybridoma generation methods previously described [20]. Immunogold labeling was performed using mAb 69 against EbpC [20], mAb 9 against EbpA, or mAb70 against Ace[23] for primary binding, followed by gold bead-conjugated donkey anti-mouse IgG, using methods previously described [24].…”

Section: Methodsmentioning

confidence: 99%

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Functional studies of E. faecalis RNase J2 and its role in virulence and fitness

et al. 2017

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Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.

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“…ChMBC7 has a mouse IgG2a/κ isotype but contains 2 amino acid substitutions in the heavy chain (leucine at positions 234 and 235 to alanine; L234A/ L235A) to reduce effector functions. The presence of L234A/L235A in human IgG1 and mouse IgG2a render resulting antibodies to be Fc-silent (81,82). ChMBC7 and TM-β1 both bind to CD122 with similar affinity by binding titration flow cytometry on CTLL-2 cells, with a half maximal effective concentration (EC 50 ) of about 0.2 μg/ml.…”

Section: Methodsmentioning

confidence: 99%

CD122 blockade restores immunological tolerance in autoimmune type 1 diabetes via multiple mechanisms

Yuan¹,

Dong²,

Tsurushita³

et al. 2018

JCI Insight

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Highly reduced binding to high and low affinity mouse Fc gamma receptors by L234A/L235A and N297A Fc mutations engineered into mouse IgG2a

Cited by 62 publications

References 23 publications

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

Functional studies of E. faecalis RNase J2 and its role in virulence and fitness

CD122 blockade restores immunological tolerance in autoimmune type 1 diabetes via multiple mechanisms

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