Common modifications to Immunoglobulin Fc sites to Silence Immune Effector Functions

Common modifications to Immunoglobulin Fc sites to Silence Immune Effector Functions

2nd Nov 2023


  • Introduction
  • IgG1 vs IgG4
  • What is the LALA mutation in antibodies?
  • What is the LALAPG mutation in antibodies
  • What is the N297A mutation in antibodies
  • ichorbio Products
  • Introduction

    Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Below we describe some common modifications of immunoglobulins and their effects on the immune response.

    IgG1 vs IgG4

    IgG1 mAbs bind to target antigens through the variable (Fab) region and can participate in other effector functions through the constant (Fc) region. Many immune cells express receptors that specifically bind to the Fc region of IgGs (FcγRs). FcγRs recognize IgG-coated targets such as opsonized pathogens or immune complexes; cross-linking leads to internalization and activation of downstream signaling cascades. Two effector cell activities elicited by FcγR activation include antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). The IgG1 Fc region also contains binding sites for the complement component C1q which can initiate the complement cascade leading to complement-dependent cytotoxicity (CDC). IgG Fc can also bind to the neonatal Fc receptor, FcRn, that regulates antibody recycling - impacting the half life of IgG.

    For many years, scientists have investigated the particular residues involved in binding to these various ligands, with the aim of modifying the natural properties of antibodies either to enhance effects deemed to be positive (e.g. to improve killing of tumour cells, or to extend half-life) or to reduce effects deemed to be negative (e.g. to avoid over stimulating the immune response).

    Many therapeutic IgG4 antibodies have been developed as it was once believed that this isotype was devoid of effector function. However, that is not the case, as was shown by a disastrous trial of the IgG4 CD28 antibody TGN1412 which resulted in a cytokine storm in 6 healthy human volunteers, with one losing toes and fingers and all facing potential health complications in the future.

    What is the LALA Mutation in antibodies?

    One of the most widely used IgG1 variants is L234A/L235A (LALA). These substitutions reduce binding to the IgG Fc receptors FcγRI, FcγRII and FcγRIII as well as to complement component C1q. Such antibodies are useful where binding and activation of Fc receptors is undesirable, for example when the product is being used as an antagonist of a cytokine or similar. Numerous therapeutic antibodies using the LALA mutations have entered clinical trials (e.g. bimagrumab, cemiplimab, galcanezumab, progolimab, risankizumab, spesolimab, teplizumab). However, it is now known that LALA variants still have substantial binding to Fc receptors and so numerous others have been developed in the quest to completely eliminate binding to the Fcγ receptors

    What is the LALAPG / LALA-PG Mutation in Antibodies?

    LALAPG includes three point mutations: L234A, L235A, and P329G. LALAPG variants inhibit binding to FcγRs and C1q while FcRn binding and Fc stability remain unaffected. Several antibodies with this set of mutations have been taken into clinical trials (e.g. cergutuzumab, cibisatamab, faricimab, RG7386).

    What is the N297A Mutation in Antibodies?

    N297A is a mutation whereby alanine (A) is substituted to asparagine (N) at position 297 in mouse and human Immunoglobulins.

    The Fc region is glycosylated and the carbohydrate is easily eliminated by mutation of N297, resulting in a reduction in binding to Fc receptors and C1q. At least three therapeutic antibodies using the N297A mutation entered clinical trials (e.g. atezolizumab, clazakizumab, otelixizumab). However, removal of carbohydrate is a drastic change which significantly destabilises the Fc structure, leading to potential problems with manufacturability, stability and pharmacokinetics.

    ichorbio Products

    ichorbio has a range of Fc silenced antibodies that can be found here.