Product Overview
The Human IgG1 (dsFab-KiH Pair Set) is a modular 6-vector mammalian expression platform engineered for high-yield, correct assembly of asymmetric bispecific antibodies (bsAbs). By pairing a Knobs-into-Holes (KiH) CH3 heterodimerization backbone with an inter-chain disulfide-engineered Fab (dsFab) on Arm 1, this system completely eliminates light-chain mispairing (heavy-light chain crosstalk).
- Universal Isotype Compatibility: Accommodates target variable domains of either Kappa (κ) or Lambda (λ) light chains across both antigen-binding arms without re-engineering constant vector backbones.
- Native Orientation: Produces full-length asymmetric bispecific IgGs without requiring domain crossovers (CrossMab) or common light chains.
- High-Throughput Screening Ready: Maintains native variable domain orientation to preserve epitope binding kinetics.
Expression Kit Setup
The kit consists of 6 optimized mammalian expression vectors allowing flexible setup for any κ or λ combination:
Engineering Architecture & Mechanism
Engineering Mechanism Breakdown
| Domain / Region | Arm 1 (Knob / Ag 1) | Arm 2 (Hole / Ag 2) | Role & Numbering Scheme |
|---|---|---|---|
| Heavy Chain VH | G44C | Wild-Type | Kabat: Forms artificial VH–VL inter-chain disulfide bridge in FR2 |
| Heavy Chain CH1 | C220A | Wild-Type | EU Index: Removes natural hinge cysteine, preventing mispairing with native L2 |
| Heavy Chain CH3 (S-S) | S354C | Y349C | EU Index: Inter-heavy chain disulfide bond locking H1–H2 heterodimer |
| Heavy Chain CH3 (Packing) | T366W | T366S / L368A / Y407V | EU Index: Steric complementary packing driving heterodimerization |
| Light Chain VL | κ: Q100C λ: 100C |
Wild-Type | Kabat: Pairs specifically with G44C on Arm 1 Heavy Chain |
| Light Chain CL | κ: ΔC214 λ: C215A |
Wild-Type | EU Index: Removes C-terminal cysteine so L1 cannot pair with H2 |
Plasmid Selection Guide
Select 4 plasmids (1 Heavy + 1 Light for Arm 1; 1 Heavy + 1 Light for Arm 2) depending on your target antibody isotypes:
| Arm 1 Isotype | Arm 2 Isotype | Arm 1 Heavy | Arm 1 Light | Arm 2 Heavy | Arm 2 Light |
|---|---|---|---|---|---|
| κ | κ | pHC-hIgG1-dsFab-Knob | pLC-hIgk-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgk-WT |
| κ | λ | pHC-hIgG1-dsFab-Knob | pLC-hIgk-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgλ-WT |
| λ | κ | pHC-hIgG1-dsFab-Knob | pLC-hIgλ-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgk-WT |
| λ | λ | pHC-hIgG1-dsFab-Knob | pLC-hIgλ-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgλ-WT |
Transfection & Ratio Optimization
For standard mammalian expression (ExpiCHO™ / Expi293™), start with a 1:1:1:1 equimolar ratio across all 4 selected plasmids.
Storage & Handling
- Plasmid DNA: Store at −20 °C.
- Purified Antibody: Store at 2–8 °C for short-term (<2 weeks) or −80 °C for long-term storage.
- Avoid repeated freeze–thaw cycles; use sterile handling practices.
