Research Areas
- Innate Immunity
- Toll-Like Receptors
- RIG-I-Like Receptors
- Nod-Like Receptors
- C-type Lectins
- Inflammasomes
- Danger Signals
- Inhibitors
- Blues™ Reporter Cells
- Lentivirus Production
- Cytokine Signaling
- Immunoglobulin A
- RNA Interference
- Gene Therapy
- Cancer Research
- Cell Culture & Transfection
- Expression Vectors
Literature
IgG-Fc Engineering For Therapeutic Use
Recombinant fusion proteins consisting of the extracellular domain of immunoregulatory proteins and the constant (Fc) domain of immunoglobulin G (IgG) represent a growing class of human therapeutics.
The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region.
The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
In ADCC, the Fc region of an antibody binds to Fc receptors (FcγRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells.
In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. IgG isoforms exert different levels of effector functions increasing in the order of IgG4 < IgG2 < IgG1 ≤ IgG3. Human IgG1 displays high ADCC and CDC, and is the most suitable for therapeutic use against pathogens and cancer cells.
Under certain circumstances, for example when depletion of the target cell is undesirable, abrogating effector functions is required. On the contrary, in the case of antibodies intended for oncology use, increasing effector functions may improve their therapeutic activity[1].
Modifying effector functions can be achieved by engineering the Fc regions to either improve or reduce their binding to FcγRs or the complement factors.
The binding of IgG to the activating (FcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcγRs and complement C1q binding, and have unique sequences in IgG2 and IgG4. Substitution into human IgG1 of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduced ADCC and CDC[2,3].
Numerous mutations have been made in the CH2 domain of IgG and their effect on ADCC and CDC tested in vitro[3-6]. In particular, a mutation to alanine at E333 was reported to increase both ADCC and CDC[4,5].
Increasing the serum persistence of a therapeutic antibody is another way to improve its efficacy, allowing higher circulating levels, less frequent administration and reduced doses. This can be achieved by enhancing the binding of the Fc region to neonatal FcR (FcRn). FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation. Several mutations located at the interface between the CH2 and CH3 domains have been shown to increase the half-life of IgG1[7,8].
InvivoGen provides many of the engineered Fc regions mentioned in this short review. They are available in pFUSE-Fc, a plasmid specifically designed for the production of high levels of Fc fusion proteins in mammalian cells.
Engineered Fc properties and applications
| Engineered Fc | IgG Isotype | Mutations | Properties | Potential Benefits | Applications |
|---|---|---|---|---|---|
| hIgG1e1 | human IgG1 | T250Q/M428L | Increased plasma half-life | Improved localization to target; increased efficacy; reduced dose or frequency of administration | Vaccination; therapeutic use |
| hIgG1e2 | human IgG1 | M252Y/S254T/T256E + H433K/N434F | Increased plasma half-life | Improved localization to target; increased efficacy; reduced dose or frequency of administration | Vaccination; therapeutic us |
| hIgG1e3 | human IgG1 | E233P/L234V/L235A/ΔG236 + A327G/A330S/P331S | Reduced ADCC and CDC | Reduced adverse events | Therapeutic use without cell depletion |
| hIgG1e4 | human IgG1 | E333A | Increased ADCC and CDC | Increased efficacy | Therapeutic use with cell depletion |
| hIgG2e1 | human IgG2 | K322A | Reduced CDC | Reduced adverse events | Vaccination; therapeutic use |
| mIgG2Aae1 | mouse IgG2a | L235E + E318A/K320A/K322A | Reduced ADCC and CDC | Reduced adverse events | Therapeutic use without cell |
1. Carter PJ., 2006. Potent antibody therapeutics by design. Nature Reviews Immunology. Advance online publication.
2. Armour KL. et al., 1999. Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol. 29(8):2613-24.
3. Shields RL. et al., 2001. High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R. J Biol Chem. 276(9):6591-604.
4. Idusogie EE. et al., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol. 166(4):2571-5.
5. Idusogie EE. et al., 2000. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol. 164(8):4178-84.
6. Steurer W. et al., 1995. Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J Immunol. 155(3):1165-74.
7. Hinton PR. et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6.
8. Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8.