Single-domain antibodies (VHHs, also known as nanobodies) derived from camelid heavy-chain-only immunoglobulins offer a compelling combination of small size (~15 kDa), high stability, deep tissue penetration, and access to epitopes that are sterically inaccessible to conventional IgG antibodies. These properties make VHHs ideal building blocks for bispecific and multispecific therapeutic formats, inhaled or intranasal delivery, enzyme inhibitors, and imaging agents.
ICDMO's VHH discovery platform is built around a pre-humanised phage display library constructed from 65 naïve llamas, with a diversity of 1×10¹⁰ unique clones. By replacing llama framework residues with human germline sequences at the library construction stage — rather than after lead selection — we eliminate the 3–4 month humanisation step that is unavoidable with conventional llama immunisation workflows. The result is a fully characterised, humanised lead antibody in approximately 4 months from project initiation.
Lead optimisation and multi-specific format assembly are guided by Computer-Aided Antibody Design (CAAD), which applies structural modeling, binding affinity prediction, and immunogenicity analysis to rank candidates and design VHH-Fc, bispecific (BsAb), and trispecific (TriAb) constructs with optimal geometry and cooperativity.
Platform Highlights
65
Naïve Llamas
PBMC repertoire sourced from 65 non-immunised llamas to maximise CDR diversity
1×10¹⁰
Library Size
Largest pre-humanised VHH phage display library available for therapeutic discovery
>15
Case Studies
Validated across oncology, virology, immunology and GPCR targets
~4
Months to Lead
Skip immunisation and humanisation — deliver characterised leads in ~4 months
Time Savings vs. Traditional Workflow
Workflow Step
Traditional Technology
ICDMO Pre-humanised VHH Library
Immunisation
~2–3 months
✓ N/A — not required
VHH library construction
~1 month
✓ N/A — library ready
Panning & screening
~1 month
~1 month
Lead selection
~1 month
~1 month
Lead characterisation
~2 months
~2 months
Lead humanisation
~3–4 months
✓ N/A — pre-humanised
Total Time
~10–12 months
~4 months!
Save 6 to 8 months with ICDMO's pre-humanised VHH platform!
Discovery Workflow
The ICDMO VHH discovery workflow integrates pre-humanised library construction, multi-strategy phage panning, format engineering, and computer-aided lead optimisation into an end-to-end service:
65 Llama PBMC Preparation
PBMCs isolated from 65 naïve llamas; IgG heavy-chain-only B cells enriched
→
VHH cDNA Amplification
RT-PCR amplification of VHH-encoding cDNA from total B-cell RNA
→
Pre-humanised VHH Library (1×10¹⁰)
Llama FRs replaced with human germline FRs; CDR1/2/3 shuffled via overlapping PCR
VHH Phage Panning (3 rounds)
P: Recombinant Protein Panning
Three rounds of panning against purified recombinant antigen protein. Optimal for well-characterised, soluble extracellular domains.
C: Cell-Based Panning
Panning directly on antigen-expressing cells. Enables discovery of VHHs that recognise native conformational epitopes under physiological membrane context.
B: Biotinylated Antigen Panning
Panning with streptavidin-bead-captured biotinylated antigen. Provides controlled antigen orientation and improved sensitivity for low-density epitopes.
Panning strategy combinations (PPP, CCC, BBB, BBC, BCC, PCC…) selected based on antigen type and target epitope requirements.
Format Engineering
VHH · VHH-Fc · Bispecific (BsAb) · Trispecific (TriAb) — format selected based on MOA and clinical indication
→
Computer-Aided Antibody Design (CAAD)
Lead selection and ranking by predicted affinity, stability, immunogenicity and manufacturability; multi-specific structural assembly
Panning Strategy Selection Guide
P
Recombinant Protein Panning (PPP)
Three rounds of panning against purified recombinant antigen protein. Optimal for well-characterised, soluble extracellular domains.
C
Cell-Based Panning (CCC)
Panning directly on antigen-expressing cells. Enables discovery of VHHs that recognise native conformational epitopes under physiological membrane context.
B
Biotinylated Antigen Panning (BBB)
Panning with streptavidin-bead-captured biotinylated antigen. Provides controlled antigen orientation and improved sensitivity for low-density epitopes.
Case Studies
The following validated studies demonstrate the breadth of ICDMO's VHH discovery platform across antiviral therapy, immuno-oncology T-cell engagement, and epitope-selective membrane protein targeting.
Case Study 1
World's First Trispecific Nanobody Targeting SARS-CoV-2 and Its Variants
A trispecific VHH-Fc antibody was engineered to simultaneously engage three distinct epitopes on the SARS-CoV-2 spike protein, including receptor-binding domains targeted by multiple circulating variants. By covering multiple non-overlapping epitopes, the trispecific design maintains broad-spectrum neutralisation against wildtype, Delta, Omicron, and other variant strains through a combination of direct receptor-binding blockade and Fc-mediated immune effector recruitment.
🔬
Trispecific Design
Targets 3 independent spike epitopes
Multiple mechanisms of action (Fc fusion)
Sequence-optimised for manufacturability
⚡
Efficacy
Potent intranasal therapy — validated in vivo
Maintains neutralisation across key variants
~100-fold viral titer reduction in huACE2 mice
🏭
Manufacturability
Excellent thermostability maintained
Research-grade expression cell line established
Kd (trispecific) at 1×10⁻¹¹ M level
Cross-Variant Binding & Blocking Activity
SARS-CoV-2 Variant
Binding Activity
ACE2 Blocking
Wildtype
+++
+++
Other variants
+++/++
+++/++
Delta
++
++
Omicron
+++/++
+++/++
~1×10⁻¹¹ M
Kd — Trispecific VHH-Fc
~1×10⁻¹⁰ M
Kd — Single VHH domain
Methodology: (1) Generate library from 65 naïve llamas · (2) Triple protein panning against SARS-CoV-2 S1 · (3) S/ACE2 competition ELISA · (4) Pairwise competition ELISA to identify synergistic VHH pairs · (5) CAAD-guided trispecific VHH-Fc structural assembly · (6) In vivo functional validation in huACE2 mice
Case Study 2
Anti-CD3 VHH Development for T Cell Engager (TCE) Bispecific Antibody
VHHs binding human CD3-γ and CD3-ε subunits were identified by cell-based panning against Jurkat T cells, with counter-depletion on 293T cells to eliminate non-specific binders. Selected leads demonstrated selective binding to CD3-expressing Jurkat cells and competitive displacement of reference anti-CD3 antibodies (12F6 and the CD3 arm of REGN1979/Odronextamab), confirming epitope overlap with clinically validated binding sites.
Panning Methodology (CCC)
1
Depletion with parental 293T cells to remove non-specific and Fc-binding clones
2
Three-round cell panning on Jurkat cells (CD3+) under progressively stringent wash conditions
3
Phage-infected colony selection and monoclonal ELISA screening
4
Phage elution and sequence analysis of positive clones
5
Transfer enriched phage pool to next panning round for further selection pressure
Key Results
Selective cell binding
Lead VHHs bind Jurkat (CD3+) cells but show no binding to parental 293T (CD3−) cells, confirming CD3 specificity.
Competitive epitope mapping
Multiple leads show competitive displacement with reference anti-CD3 antibodies 12F6 and the REGN1979 CD3 arm, confirming overlap with clinically validated epitopes.
Cross-species activity
Binding to both human and cynomolgus macaque CD3-δ and CD3-ε subunits confirmed, supporting IND-enabling toxicology studies.
TCE functional validation
BCMA × CD3 bispecific constructs demonstrate dose-dependent T cell activation (CD69+) and tumour cell killing (7AAD+ cells) in PBMC/H929 co-culture assays.
Case Study 3
Epitope-Selective VHH Discovery Against a Membrane Protein Target (Antigen-A)
This programme required isolation of VHHs that bind specifically to a defined extracellular domain (ECD) epitope present on the full-length membrane protein but absent in an ECD-deletion construct. A hybrid panning strategy (BB/C) combining biotinylated antigen panning with epitope-directed counter-panning was designed to achieve this selectivity.
Panning Strategy (BBC)
Rounds 1 & 2 (BB)
Biotinylated antigen panning using streptavidin-bead capture
Selects initial pool of antigen-binding clones with high stringency
Round 3 (C — Counter/Panning)
Counter-panning with 293T-ECD-Deletion cells to deplete non-epitope binders
Positive panning on 293T-ECD-Full Length cells to enrich epitope-specific clones
FACS Binding Validation
Lead VHH specificity was confirmed by FACS binding assays across three cell lines:
✅
293T-ECD-Full Length
High binding (dose-dependent, EC₅₀ ~0.1 nM)
❌
293T-ECD-Deletion
No significant binding — epitope absent
❌
293T (parental)
No binding — confirms antigen specificity
FACS binding assay demonstrates that the lead VHH candidate binds exclusively to cells expressing the full-length antigen with the target epitope intact, confirming both specificity and correct epitope selectivity.
ICDMO's pre-humanised VHH discovery platform is the fastest route from target to characterised, clinical-ready nanobody lead. With a 1×10¹⁰-clone library, flexible multi-strategy panning, and integrated CAAD-guided lead optimisation and multi-specific format assembly, we deliver validated VHH candidates in approximately 4 months.
Contact our antibody discovery team to discuss your target, panning strategy, and desired therapeutic format.