Product Overview
The AFV-35 Human IgG1 CrossMab + KiH Bispecific Antibody Expression Vector Set is a modular mammalian expression platform designed for generating full-length asymmetric human IgG1 bispecific antibodies.
The platform combines Knobs-into-Holes (KiH) Fc engineering with CrossMab CH1–CL constant-domain swapping to promote heavy-chain heterodimerization and cognate heavy–light chain pairing.
Available Product Options
- AFV-35-KK: Conventional κ arm + CrossMab κ arm — 4 plasmids, 20 µg total.
- AFV-35-KL: Conventional κ arm + CrossMab human Cλ2 arm — 4 plasmids, 20 µg total.
- AFV-35-LK: Conventional human Cλ2 arm + CrossMab κ arm — 4 plasmids, 20 µg total.
- AFV-35-LL: Conventional human Cλ2 arm + CrossMab human Cλ2 arm — 4 plasmids, 20 µg total.
- AFV-35-6V: Complete six-vector platform supporting all four configurations — 6 plasmids, 30 µg total.
Key Advantages
- Flexible Kit Selection: Purchase only the four plasmids required for your selected antibody configuration or the complete six-vector platform.
- Heavy-Chain Heterodimerization: KiH engineering promotes preferential assembly of the two different heavy chains.
- Light-Chain Pairing: CrossMab CH1–CL domain swapping helps reduce noncognate heavy–light chain pairing.
- Human κ and Cλ2 Compatibility: Supports conventional and CrossMab configurations using the specified human light-chain constant domains.
- Modular Expression System: Each plasmid is supplied separately for cloning and mammalian expression.
Research Use Only. Antibody expression, assembly efficiency, and purity depend on the antibody sequences and experimental conditions.
Expression Kit Setup
The AFV-35 platform consists of six mammalian expression vector backbones. Customers may select one of four predefined four-plasmid configurations (KK, KL, LK, or LL), or purchase the complete six-vector set (AFV-35-6V).
Each four-plasmid kit includes one KiH-knob heavy-chain vector, one conventional wild-type light-chain vector, one CrossMab-hole heavy-chain vector, and one universal CrossMab light-chain vector.
All plasmids are supplied at 5.0 µg each. The four-plasmid kits contain 20 µg total DNA, and the complete six-vector set contains 30 µg total DNA.
AFV-35 Modular Expression Platform — Select KK, KL, LK, LL (4 plasmids) or Complete 6V (6 plasmids).
Engineering Architecture & Mechanism
AFV-35 Human IgG1 CrossMab CH1–CL Domain Crossover and Knobs-into-Holes Bispecific Antibody Pairing Mechanism.
Engineering Mechanism Breakdown
| Domain / Region | Arm 1 Modifications (Knob / Ag 1) | Arm 2 Modifications (Hole / Ag 2) | Role & Numbering Scheme |
|---|---|---|---|
| Heavy Chain CH1 | Wild-Type (C220) | Swapped to Cκ or Cλ | EU Index: Native hinge cysteine on Arm 1 forms native H–L bond |
| Heavy Chain CH3 Disulfide | S354C | Y349C | EU Index: Inter-heavy disulfide bond (CH3–CH3) locking H1–H2 |
| Heavy Chain CH3 Packing | T366W | T366S / L368A / Y407V | EU Index: Steric complementary packing driving heterodimerization |
| Light Chain Constant | Wild-Type (Cκ/Cλ) | Swapped to CH1 | Converts Arm 2 light chain to universal VL-CH1 backbone |
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-IgG1-KiH-Knob | pLC-hIgk-WT | pHC-hIgG1-CrossMab-Cκ-Hole | pLC-hIg-CrossMab |
| κ | λ | pHC-IgG1-KiH-Knob | pLC-hIgk-WT | pHC-hIgG1-CrossMab-Cλ-Hole | pLC-hIg-CrossMab |
| λ | κ | pHC-IgG1-KiH-Knob | pLC-hIgλ-WT | pHC-hIgG1-CrossMab-Cκ-Hole | pLC-hIg-CrossMab |
| λ | λ | pHC-IgG1-KiH-Knob | pLC-hIgλ-WT | pHC-hIgG1-CrossMab-Cλ-Hole | pLC-hIg-CrossMab |
Complete Six-Vector Option (AFV-35-6V): Includes all six AFV-35 plasmids, allowing researchers to configure KK, KL, LK, or LL antibody expression systems without purchasing additional AFV-35 backbone vectors. The complete set contains 5.0 µg of each plasmid (30 µg total).
Cloning-Site Reference: The four CrossMab/KiH plasmids are annotated with AgeI (798 bp) and XhoI (815 bp). The conventional pLC-hIgκ-WT and pLC-hIgλ2-WT vectors are annotated with AgeI (798 bp) and XhoI (1,157 bp) or XhoI (1,143 bp), respectively. Verify insert compatibility against the supplied vector sequences before cloning.
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.
