Product Overview
Product Overview
The AFV-34 Human IgG1 dsFab + KiH Bispecific Antibody Expression Vector Set is a modular mammalian expression platform designed for the production of full-length asymmetric human IgG1 bispecific antibodies.
The platform combines Knobs-into-Holes (KiH) Fc engineering with disulfide-engineered Fab (dsFab) technology. KiH mutations promote heavy-chain heterodimerization, while an engineered VH–VL disulfide interface on Arm 1 helps favor cognate heavy–light chain pairing.
Available Product Options
- AFV-34-KK: Engineered κ dsFab arm + conventional κ Fab arm — 4 plasmids.
- AFV-34-KL: Engineered κ dsFab arm + conventional human λ2 Fab arm — 4 plasmids.
- AFV-34-LK: Engineered human λ2 dsFab arm + conventional κ Fab arm — 4 plasmids.
- AFV-34-LL: Engineered human λ2 dsFab arm + conventional human λ2 Fab arm — 4 plasmids.
- AFV-34-6V: Complete six-vector platform supporting all four configurations.
Key Advantages
- Flexible Kit Selection: Purchase the four plasmids required for your selected antibody configuration or the complete six-vector platform.
- Engineered Heavy-Chain Pairing: KiH mutations promote preferential heavy-chain heterodimerization.
- Disulfide-Engineered Fab Pairing: Engineered VH/VL cysteine residues support selective Fab-chain pairing on Arm 1.
- Precloned Reference Inserts: Avastin and Lc06 variable-region inserts provide reference constructs and reusable cloning backbones.
- Human κ and Cλ2 Compatibility: Supports κ and human λ2 light-chain constant-region options.
Research Use Only. Expression yield, antibody assembly, and purity depend on the antibody sequences and experimental conditions.
Expression Kit Setup
The AFV-34 platform consists of six mammalian expression vectors. Customers may select one of four predefined configurations (KK, KL, LK, or LL), each containing four plasmids, or purchase the complete six-vector kit.
Each plasmid is supplied at 5.0 µg. Four-plasmid configurations contain 20 µg total DNA, while the complete AFV-34-6V kit contains 30 µg total DNA.
All six plasmids contain precloned Avastin or Lc06 reference variable-region inserts. These inserts can be replaced with customer-specific antibody VH or VL sequences using the designated cloning sites. The engineered dsFab arm requires the appropriate variable-region cysteine mutations.
AFV-34 Modular Expression Platform — Select KK, KL, LK, LL (4 plasmids) or Complete 6V (6 plasmids).
Engineering Architecture & Mechanism
AFV-34 dsFab-KiH Architecture — Engineered VH–VL disulfide pairing on Arm 1 and Knobs-into-Holes Fc heterodimerization.
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:
| SKU | DNA supplied | Arm 1 dsFab Heavy | Arm 1 dsFab Light | Arm 2 Fab Heavy | Arm 2 Fab Light |
|---|---|---|---|---|---|
| AFV-34-KK | 20 µg (4 × 5.0 µg) | pHC-hIgG1-dsFab-Knob | pLC-hIgk-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgk-WT |
| AFV-34-KL | 20 µg (4 × 5.0 µg) | pHC-hIgG1-dsFab-Knob | pLC-hIgk-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgλ2-WT |
| AFV-34-LK | 20 µg (4 × 5.0 µg) | pHC-hIgG1-dsFab-Knob | pLC-hIgλ2-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgk-WT |
| AFV-34-LL | 20 µg (4 × 5.0 µg) | pHC-hIgG1-dsFab-Knob | pLC-hIgλ2-dsFab | pHC-hIgG1-KiH-Hole | pLC-hIgλ2-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.
