Plasmid Name
pMCIgG2aAgly(N297Q)
Mutation Set
N297Q
Selection Marker
Ampicillin
Promoter
CMV
Fc Engineering Overview
The mouse IgG2a (N297Q) Fc variant is a primary aglycosylated mouse Fc vector engineered to eliminate the conserved N297 biantennary glycan in mouse IgG2a. Disables murine FcγR (I, IIb, III, IV) and complement C1q binding via structural collapse of the internal CH2 domain interface rather than primary lower-hinge sequence alteration.
Functional Profile
| Property | Effect |
|---|---|
| Murine FcγRI/IIb/III/IV binding | Abolished / Undetectable ↓↓↓↓ |
| ADCC (Mouse Effector Cells) | Completely Abolished ↓↓↓↓ |
| ADCP (Macrophage Phagocytosis) | Completely Abolished ↓↓↓↓ |
| C1q binding (Murine Complement) | Severely Reduced / Minimal ↓↓↓ |
| CDC | Severely Reduced / Minimal ↓↓↓ |
| FcRn binding & half-life | Normal / WT Baseline |
| Glycosylation & Thermal Stability | Aglycosylated (N297 glycan absent); reduced CH2 thermal stability (Tm ≈ 60°C) |
Mechanism of Action
The N297Q substitution operates through a glycosylation-dependent conformational collapse within the murine CH2 domain:
- Glycan Elimination: Mutating Asparagine to Glutamine at position 297 removes the consensus Asn-X-Ser/Thr motif, preventing N-linked carbohydrate addition during post-translational modification.
- Conformational Collapse: The conserved N297 glycan acts as a molecular spacer keeping the two CH2 domains in an open, receptive orientation. In its absence, the CH2 domains collapse inward, occluding the binding sites for murine FcγRs and C1q head groups.
Phenotypic Effects
- Complete loss of binding to all murine Fcγ receptors (FcγRI, FcγRIIb, FcγRIII, FcγRIV).
- Elimination of NK cell-mediated ADCC, neutrophil activation, and macrophage ADCP in mouse models.
- Severe attenuation of classical complement activation (CDC).
- High structural homogeneity (completely free of glycoform microheterogeneity).
- Preserves endosomal FcRn recycling dynamics and systemic pharmacokinetic half-life in rodents.
- Displays reduced CH2 domain thermal melting temperature (Tm reduced by ~10–12°C).
Applications
- Preclinical in vivo syngeneic mouse models requiring zero Fc-mediated effector background.
- Murine surrogate antibodies targeting immune cell receptors (e.g., anti-mPD-1, anti-mPD-L1, anti-mTIGIT) where Fc activation induces toxic cell depletion.
- Receptor-blocking antibodies and ligand traps in rodent disease models.
- Mechanistic studies evaluating the contribution of Fc glycosylation vs. peptide sequence silencing.
- Agonist surrogate antibodies requiring pure target cross-linking without FcγR co-engagement.
Plasmid Map & Feature Annotation
Insert Structure: VH – CH1 – hinge(WT) – CH2(N297Q) – CH3(WT)
- VH: Variable heavy domain
- CH1: Constant heavy 1
- Hinge: Mouse IgG2a hinge
- CH2 (N297Q): Murine CH2 domain containing the aglycosylating N297Q mutation
- CH3 (WT): Native mouse IgG2a CH3 domain
Product Note: The mIgG2a-N297Q Fc variant serves as the standard aglycosylated mouse control for preclinical target validation. While N297Q offers complete effector silencing in rodent models, researchers should note that aglycosylation lowers the thermal stability (Tm) of the CH2 domain. For in vivo preclinical studies requiring maximum biophysical stability, sequence-silenced variants such as mouse IgG2a (LALA-PG) are recommended.
Fusion BioLabs EffectorNull Family Comparison Matrix
| Variant | Mechanism | Silencing Strength | Notes |
|---|---|---|---|
| human IgG1 (WT) | Wild-Type baseline | Baseline | Human WT Control. Native FcγR and C1q binding; fully active baseline control. |
| mouse IgG2a (WT) | Wild-Type baseline | Baseline | Murine WT Control. Native mouse FcγR and complement binding; active control for syngeneic models. |
| human IgG1 (EN / Armour) E233P / L234V / L235A / ΔG236 + A327G / A330S / P331S |
Lower hinge & CH2 chimeric sequence substitution | ★★★★★ | Ultra-Silent Chimeric Null. Cross-subclass IgG2/IgG4 sequence replacement; abolishes FcγR & CDC engagement. |
| human IgG1 (LALA-PG) L234A / L235A / P329G |
Lower hinge & CH2 proline-sandwich null | ★★★★★ | Clinical Gold Standard. Complete, double-silent suppression of all human FcγRs and C1q/CDC. |
| mouse IgG2a (LALA-PG) L234A / L235A / P329G |
Murine lower hinge & CH2 proline-sandwich | ★★★★★ | In Vivo Murine Gold Standard. Complete silencing of murine FcγRs and complement in syngeneic mouse models. |
| human IgG1 (LALA) L234A / L235A |
Lower hinge double alanine substitution | ★★★☆☆ | Classic Benchmark Control. Widely used legacy variant; retains minor residual FcγRI (CD64) binding. |
| human IgG1 (ΔG236) ΔG236 |
Lower hinge single residue deletion | ★★★☆☆ | Selective FcγR-Null. Dislocates FcγR binding site while preserving native N-glycan structure and C1q loops. |
| human IgG1 (N297Q) N297Q |
CH2 domain N-glycan site elimination | ★★★★☆ | Human Aglycosylated Control. Broad effector silencing via glycan removal; lowers thermal stability (Tm). |
| mouse IgG2a (N297Q) N297Q |
Murine CH2 domain N-glycan site elimination | ★★★★☆ | This Product. Murine Aglycosylated Control. Aglycosylated mouse control for in vivo preclinical rodent studies. |
Storage & Handling
- Plasmid: Store at −20°C.
- Purified Antibody: Store at 2–8°C for short-term storage; −80°C for long-term storage.
- Avoid repeated freeze–thaw cycles.
- Use sterile technique for all handling.
- Suitable for transient or stable mammalian expression (CHO, HEK293).
References
- Tao MH, Morrison SL. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol. 1989;143(8):2595-2601.
- Nimmerjahn F, Ravetch JV. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science. 2005;310(5753):1510-1512.
- Baudino L, Shinohara Y, Nimmerjahn F, Furukawa J, Nakata M, Martínez-Soria E, Petry F, Ravetch JV, Nishimura S, Izui S. Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions. J Immunol. 2008;181(9):6664-6669.
