A nanobody that can only bind its target when a VH1 domain is bound to a specific antigen on the same cell surface — AND-gate logic.
── INACTIVE (OFF state) ──────────────────────────────────────────
[Nanobody] ← CDRs blocked
+++
[Minibinder] ← bridges BOTH surfaces simultaneously
|||
[VH1] ← CH1-face exposed (CH1 domain deleted)
|
[Antigen] ··· membrane ··· [Target]
── ACTIVE (ON state, VL1 pairs with VH1) ────────────────────────
[Nanobody CDRs free] ────────────────────────── binds [Target]
↑
long linker (nanobody swings away)
|
[Minibinder]──short linker──[VH1]──[VL1/CL] ← VL1 occupies CH1-face
|
[Antigen]
- Take native Fab (VH + VL + CH1_heavy + CL)
- Delete heavy-chain CH1 — exposes VH1's CH1-contact surface
- Position Nanobody in the CH1 slot — CDR face (+++) points at VH1's exposed CH1-contact surface
- Design Minibinder — bridges VH1-CH1-face AND Nanobody CDRs simultaneously (bispecific)
- VH1 and VL1 engineered to be independent — don't spontaneously pair in solution
- Asymmetric linkers:
- Short (5-10 res): VH1 → Minibinder — MB stays at VH1 after displacement
- Long (30-50 res): Minibinder → Nanobody — CDRs can swing far away
In the OFF state, the Minibinder acts as a molecular staple holding VH1 and the Nanobody CDRs together in the geometry where CH1 used to sit.
When VH1 binds the antigen AND VL1 pairs with VH1:
- VL1's CL domain occupies VH1's CH1-contact surface (the same surface the Minibinder was using)
- The Minibinder is displaced from VH1's surface
- Short linker keeps the Minibinder physically near VH1 (it cannot escape)
- Long linker allows the Nanobody to swing completely away from the displaced Minibinder
- CDRs are free to bind the Target
| Previous designs | This design | |
|---|---|---|
| What minibinder bridges | Single surface (VL1 FR2 or nanobody CDRs alone) | Two surfaces simultaneously (VH1-CH1 face + nanobody CDRs) |
| Displacement trigger | VH1 surface concentration / CL steric kick | VL1 directly occupies VH1-CH1 face (same surface MB uses) |
| OFF-state lock | Thermodynamic (Kd windows) | Geometric — staple holds shape |
| Linker requirement | Symmetric | Asymmetric — the switch mechanism |
- VH1-CH1 contact residues (28): FR1(11-15), FR2(39-41), FR3(79-115)
- Nanobody CDR hotspots (27): CDR1(27-33), CDR2(52-57), CDR3(99-112)
- Gap between surfaces: 20 Å
- Minibinder size: 55 residues
- Input PDB:
structures/domains/vh1_nanobody_design_target.pdb
── INACTIVE ──────────────────────────────────────────────────────────
[Nanobody CDRs blocked by Minibinder]
|
~flexible linker~
|
[CL]──[VL1] ← CL/CH1 positioned away from nanobody
(free, unanchored)
[VH1]
(not yet bound to antigen)
[Antigen] ··· membrane ··· [Target]
── ACTIVE ────────────────────────────────────────────────────────────
[Nanobody]────────────────────────────╮
| binds [Target]
~flexible linker~
|
[CL] ← CH1/CL physically KICKS Minibinder off Nanobody CDRs
[VL1]──[VH1] ← VH1-VL1 pairing brings CL into kicking position
|
[Antigen] ··· membrane ··· [Target]
| Component | Role | How to obtain |
|---|---|---|
| VH1 | Binds antigen autonomously; anchors construct to membrane | Engineered from existing anti-antigen VH |
| VL1 | Pairs with VH1 to form scFv; triggers the kicker mechanism | From same antibody as VH1 |
| CL (CH1) | "Kicker" domain — physically displaces minibinder from nanobody CDRs upon VH1-VL1 pairing | Natural constant light chain, attached to VL1 C-terminus |
| Minibinder | Blocks nanobody CDR face in free state | Designed against nanobody paratope (anti-idiotypic design) |
| Flexible linker | Connects VL1-CL block to nanobody; must be long enough for kicking geometry | (G₄S)ₙ, optimised |
| Nanobody | Anti-target single-domain antibody | Selected from library or existing |
-
Free state: Minibinder occupies the nanobody's CDR face. Nanobody cannot bind target. VH1 and VL1 are not paired (or floating freely in solution).
-
Anchoring: VH1 binds the antigen on the cell surface.
-
Pairing: VH1 recruits VL1; VH1-VL1 pair into a functional scFv. The CL domain comes along with VL1 (it is covalently attached at VL1's C-terminus).
-
Kicking: The CL domain, now in its new position relative to the nanobody (set by the VH1-VL1 pairing geometry), sterically clashes with the minibinder and physically displaces it from the nanobody's CDR loops.
-
Activation: Nanobody CDRs are now free. The flexible linker allows the nanobody to extend and bind the target.
| Property | Competitive displacement (old) | CH1-kicker (new) |
|---|---|---|
| Mechanism | Thermodynamic (VH1 surface conc. vs intramolecular minibinder) | Structural/mechanical (steric kick) |
| OFF-state fidelity | Good — intramolecular minibinder is always present | Good — minibinder blocks CDRs |
| ON-state trigger | Needs µM VH1 surface concentration | Needs VH1-VL1 pairing (binary event) |
| Precision needed | High — tight Kd windows, exact linker lengths | Low — CH1 kick is a physical event |
| Minibinders needed | 2 | 1 |
| Robustness | Sensitive to antigen expression level | Robust — switch is structural |
The CL domain must be positioned such that, after VH1-VL1 pairing, it overlaps with the minibinder's footprint on the nanobody CDRs. This is determined by:
- The Fab geometry (VH1-VL1-CL complex structure)
- The flexible linker length between CL and the nanobody
- The size and position of the minibinder on the nanobody CDR face
Use the structural analysis in binary_antibodies/kicker.py to assess geometry.
SP – [VL1] – [CL] – (G4S)n – [Minibinder] – (G4S)3 – [Nanobody VHH]
SP: signal peptideVL1: variable light domain (pairs with VH1 to trigger kicking)CL: constant light domain (the "kicker" — repositions upon VL1-VH1 pairing)(G4S)n: flexible linker (length sets kicking geometry)Minibinder: blocks nanobody CDRs in free state(G4S)3: short linkerNanobody VHH: anti-target single-domain antibody
Chain A (separate): VH1 (binds antigen; expressed as separate polypeptide)
| Component | Molecule | PDB |
|---|---|---|
| Antigen | EGFR | Cetuximab Fab 1YY9 (VH template) |
| Target | HER2 | — |
| VH1 + VL1 | Anti-HER2 scFv | Trastuzumab Fab 1N8Z |
| CL | Trastuzumab Cκ | 1N8Z chain B (C-terminal constant domain) |
| Nanobody | 2Rs15d anti-HER2 VHH | 5MY6 |
| Minibinder target | Nanobody CDR face (CDR1, CDR2, CDR3) | structures/domains/her2_nanobody_VHH.pdb |
binary_antibodies/
├── README.md
├── requirements.txt
├── binary_antibodies/
│ ├── polymer.py # linker physics
│ ├── design.py # ConditionalConstruct, SplitScFvConstruct
│ ├── kicker.py # CH1-kicker geometry and displacement model ← NEW
│ ├── split_scfv.py # competitive displacement model (reference)
│ ├── minibinder.py # minibinder design guidance
│ ├── sequences.py # linker sequences
│ └── structures.py # PDB download/analysis
├── scripts/
│ ├── setup_example.py
│ ├── interface_analysis.py # VH-VL interface (reference)
│ ├── nanobody_cdr_analysis.py # Nanobody CDR face analysis ← NEW
│ └── gpu_setup/
│ ├── run_minibinder_design_rfd3.sh # rfd3 pipeline (against nanobody CDRs)
│ └── ...
└── structures/
├── domains/
│ ├── her2_nanobody_VHH.pdb ← TARGET for minibinder design
│ ├── trastuzumab_VH.pdb
│ └── trastuzumab_VL.pdb
└── interface/