How to Produce Nanobodies - Generation and Design

Nanobodies, single-domain antibody fragments of approximately 15 kDa, combine the binding specificity of conventional IgGs with exceptional tissue penetration and stability.

The classical in vivo workflow to generate these camelid-derived binders encompasses antigen immunization, lymphocyte harvesting, cDNA synthesis, phage‐display library construction, biopanning, and downstream cloning and expression.

In the following sections, we detail each experimental phase of this established pipeline.

(Note: Ziab’s in silico platform streamlines and, in many cases, replaces these in vivo steps, dramatically reducing timelines, eliminating animal use, and offering rapid, sequence‐to‐function binder design.)

Building the Nanobody Library

The initial step requires obtaining a diverse repertoire of camelid heavy‐chain antibodies (HCAbs), whose variable domains (V_HH) constitute nanobodies.

Llamas and alpacas are the preferred species due to well‐characterized HCAb genetics and manageable husbandry. A representative immunization regimen is as follows:

• Prime (Week 0): Subcutaneous injection of 100 µg purified antigen (protein, peptide, or cell lysate) emulsified in alum or Freund’s adjuvant.
• Boosts (Weeks 2, 4, 6): Administer 50–100 µg antigen in adjuvant at biweekly intervals to sustain affinity maturation.
• Harvest (Week 8): Five days post-final boost, collect 50–100 mL peripheral blood.

This schedule ensures expansion of antigen-specific B cells expressing high-affinity V_HH fragments. Following collection, isolate peripheral blood lymphocytes (PBLs) by density gradient centrifugation.

These PBLs are then processed for total RNA extraction, setting the stage for downstream cDNA synthesis and library construction.

From Lymphocyte to cDNA

The transition from lymphocyte to clonable V_HH sequences involves three core steps: total RNA extraction, reverse transcription, and targeted PCR amplification of the V_HH domain.

1. Total RNA Extraction: Harvested PBLs are lysed and subjected to either phenol–chloroform extraction (e.g., TRIzol) or silica‐column purification. Verify RNA integrity (A_260/280 ≈ 1.8–2.0) and quantify to obtain 5–10 µg of high‐quality total RNA.
2. Reverse Transcription: Synthesize first‐strand cDNA using a high‐fidelity reverse transcriptase (e.g., SuperScript IV) with a mixture of oligo(dT) and random hexamer primers. This generates a comprehensive cDNA library encompassing all immunoglobulin transcripts.
3. Nested PCR Amplification of V_HH
   • First‐Round PCR: Use primers targeting conserved camelid heavy‐chain constant regions to amplify all HCAb transcripts.
   • Nested PCR: Employ internal primers specific to V_HH framework sequences, engineered with SfiI and NotI restriction sites. This yields a discrete ~400 bp amplicon corresponding to the isolated V_HH domains.

Following nested amplification, the resulting V_HH pool—typically comprising 10⁷–10⁸ unique fragments—provides the substrate for downstream cloning into display vectors.

Cloning Into a Phage Display Vector

Digest your ~400 bp V_HH PCR products with SfiI/NotI and ligate into a phagemid backbone (e.g., pMESy4 or pHEN6) that fuses each V_HH to the pIII coat protein.

Transform E. coli TG1 or XL1-Blue, select on ampicillin, then infect with M13KO7 helper phage to rescue virions.

After overnight outgrowth and PEG/NaCl precipitation, you’ll have a diverse library of ~10⁷–10⁸ phage particles—each displaying a unique nanobody—ready for antigen panning.

Phage Panning: Fishing for the Best Binders

Antigen Immobilization & Blocking

Coat ELISA plates or magnetic beads with 2–10 µg/mL antigen in PBS for 1–2 hours (4 °C or RT), then block with 2–5% BSA in PBS to minimize nonspecific binding.

Library Incubation & Washing

Add ~10¹² pfu of your phage library and incubate 1–2 hours at RT with gentle agitation. Wash away weak binders—start with 5–10 washes in PBS + 0.1% Tween-20 in Round 1, then increase stringency (e.g. 0.5% Tween or higher salt) in later rounds to enrich for high-affinity clones.

Elution and Amplification

Elute specifically bound phage by either:

• Acidic elution: 0.1 M glycine-HCl, pH 2.2 (then neutralize)
• Competitive elution: excess soluble antigen

Infect fresh E. coli with the eluted phage, rescue with helper phage, and repeat the panning cycle. By Round 4, you should have a phage population dominated by tight, specific binders.

Screening Individual Clones

1. Colony Picking: Plate final-round bacteria on selective agar and pick 48–96 colonies.
2. Phage Induction & Mini-ELISA: Grow overnight, induce phage production, then assay each supernatant against antigen-coated and control (e.g., BSA) wells using anti-M13–HRP detection.
3. Hit Selection & Sequencing: Identify clones with strong target signal and negligible control binding. Sequence their V_HH inserts, focusing on unique CDR3 loops and intact frameworks.
4. Subcloning for Expression: Clone top candidates into a soluble expression vector (e.g., pET22b with pelB leader and C-terminal His₆ tag) for milligram-scale nanobody production in E. coli.

Engineering and Affinity Maturation

Initial nanobody clones frequently exhibit dissociation constants (K_D) in the high-nanomolar to low-micromolar range. To achieve single-digit nanomolar affinities, iterative library diversification and selection are employed:

• Error-Prone PCR Libraries: Apply error-prone PCR across the full V_HH sequence (targeting ~1–2 nucleotide changes per kilobase). Recloned libraries undergo additional panning rounds under increased stringency (e.g., higher detergent concentrations or reduced antigen density), enriching for variants with slower off-rates (k_off).
• CDR-Focused Mutagenesis: When a parental CDR3 exhibits strong binding, construct targeted libraries randomizing select residues in CDR1 or CDR2. Combine these CDR variants with the parental CDR3 framework, then re-select to isolate clones with improved on-rates (k_on) and overall tighter binding (reduced K_D).

Sequential maturation cycles can routinely lower K_D values from 10⁻⁷ M into the 10⁻⁹–10⁻¹⁰ M range, suitable for demanding therapeutic and diagnostic applications.

Humanization and Developability Assessment

For clinical translation, nanobody immunogenicity and biophysical liabilities must be minimized:

1. Framework Humanization
   • Swap camelid-specific framework residues (e.g., hallmark positions in FR2 such as 37, 44, 45) for corresponding human VH germline residues (commonly IGHV3 family).
   • Preserve CDR loops to maintain antigen affinity.
2. In Silico Liability Scanning
   • Use computational tools (e.g., AggScore, PatchFinder2) to identify hotspots for oxidation (methionine), deamidation (Asn-X-Ser/Thr motifs), or aggregation (exposed hydrophobic patches).
   • Introduce conservative amino acid substitutions to mitigate these liabilities without compromising binding.

A well-executed humanization strategy yields nanobodies with ≥ 85 % sequence identity to human VH domains, low predicted immunogenicity, and retained high-affinity binding—ready for downstream expression, formulation, and in vivo evaluation.

Deciding on Nanobody Format

Monovalent V_HH

For single-domain applications—ELISA, Western blot, pull‐down assays—express your V_HH in E. coli with a C-terminal His₆ tag. Typical yields exceed 20 mg/L in periplasmic preparations, and purity >95 % by IMAC/SEC.

Bivalent or Multispecific Constructs

Link two (or more) V_HH domains via a flexible (G₄S)_n peptide. This format increases apparent affinity by 10–100× and enables simultaneous targeting of distinct epitopes. Expression remains compatible with bacterial systems, though yields may vary with linker length and domain orientation.

Nanobody–Fc Fusions

Fusion to a human IgG₁ Fc extends half-life via FcRn recycling and recruits effector functions (complement, ADCC). These ~75 kDa constructs require transient or stable expression in mammalian cells (HEK293 or CHO) and purification by Protein A/SEC, with typical yields of 50–100 mg/L in optimized systems.

Site-Specific Conjugates

Introduce engineered tags (sortase, unique cysteine) for covalent attachment of fluorophores, cytotoxins, or PEG. This approach affords precise control over conjugation stoichiometry—essential for quantitative imaging and targeted drug delivery—and can be performed on either bacterial or mammalian-expressed V_HH or Fc-fused formats.

Quality Control and Characterization

Even with an optimized sequence, rigorous QC is essential. Key evaluations include:

Purity & Yield

• Bacterial V_HH: Target ≥ 20 mg/L in periplasmic E. coli. Purify via Ni–IMAC followed by SEC. Aim for > 95 % purity by SDS–PAGE.
• Fc-Fused Constructs: Transiently express in HEK293 or CHO at 1 L scale. Expect 50–100 mg/L in clarified supernatant. Purify by Protein A chromatography and SEC.

Biophysical Stability

• Differential Scanning Calorimetry (DSC): Confirm T_m ≥ 60 °C.
• Dynamic Light Scattering (DLS): Ensure monodispersity (PDI < 0.2).

Binding Kinetics

• SPR or BLI: Measure K_D, k_on, and k_off against the native antigen. Single-digit nanomolar K_D is preferred.

Functional Assays

• Cell-Based Binding: Use flow cytometry or cell-surface ELISA to verify binding to target-expressing cells.
• Activity Tests: For receptor blockade or neutralization, perform relevant cell-based inhibition assays.

Expression Optimization

• Codon Usage: Optimize V_HH gene for your host organism.
• Signal Peptides: Compare leaders (e.g., pelB vs. OmpA in bacteria; IL-2 vs. native signal in mammalian cells) to improve secretion and yield.

Each QC step should confirm that your nanobody meets the required biochemical, biophysical, and functional specifications before downstream applications.

Putting It All Together

The in vivo generation of nanobodies, while methodical, ultimately parallels conventional antibody engineering workflows.

By immunizing camelids, isolating V_HH transcripts, cloning into phage‐display libraries, and conducting iterative panning and optimization, researchers can reliably obtain high‐affinity single-domain binders.

Rigorous QC, encompassing expression yield, purity, biophysical stability, and functional assays, ensures each candidate meets the stringent requirements for downstream applications, from diagnostics to therapeutics.

In Silico Nanobody Development (Ziab)

Ziab’s computational platform replaces the entire animal‐based and phage‐display pipeline with a fully in silico workflow.

Beginning with target‐structure modelling, it generates virtual nanobody libraries, applies AI‐driven affinity maturation, and predicts developability metrics, all in a matter of days.

Stay tuned for our upcoming deep-dive article In Silico Nanobody Generation with Ziab - insert link to article when live).