FR2-Camelized RenNano® Mice
In human heavy chains, FR2 residues at positions 37, 44, 45, and 47 (Martin numbering) form part of the VH-VL interface. These residues are typically hydrophobic and are represented by the VGLW (Val-Gly-Leu-Trp) motif, which is normally buried through pairing with the light chain (Sang et al., 2022, Structure). In a heavy-chain-only or VHH format, however, these positions become more solvent-exposed. The exposed hydrophobic surface can increase unwanted interactions and aggregation, potentially reducing soluble expression and complicating candidate development.
FR2 camelization replaces the human VGLW motif with the camelid-associated FERF (Phe-Glu-Arg-Phe) motif, making the exposed interface more hydrophilic to reduce aggregation risk and improve candidate developability.
Comparative studies have shown that humanized libraries incorporating FERF can achieve higher screening success rates for viable lead candidates than libraries based on human VGLW (Arras et al., 2023, mAbs). Our in-house data further show that clinical-stage VHHs retaining camelid FR2 signatures exhibit higher expression yields and greater thermostability than those carrying the human VGLW motif.
Combining Fully Human and FR2-Camelized Human V Genes to Maximize HCAb Discovery Potential
Building on our proprietary RenNano® mouse platform for human heavy-chain-only antibody (HCAb) discovery, next-generation RenNano® mice retain the original, fully intact human V gene repertoire while incorporating dozens of engineered framework region 2 (FR2)-camelized human V gene segments.
This expanded repertoire increases sequence and framework diversity and is designed to improve discovery success and broaden epitope coverage.
Strategic Advantages of FR2-Camelized RenNano® Mice
The more hydrophilic FERF framework helps reduce unwanted hydrophobic interactions and aggregation risk, supporting improved solubility, lower nonspecific binding, better overall developability, and higher lead identification success.
Combines intact human V genes (VGLW) with engineered FR2-camelized human V genes (FERF), expanding candidate diversity across hard-to-target epitopes.
Retains the original human V gene repertoire, allowing discovery campaigns to continue generating fully human VHHs when unmodified human sequences are preferred.
Enables natural in vivo affinity maturation to generate high-affinity VHH leads, streamlining downstream optimization.
Immune Phenotype and Antibody Repertoire
Preserved B-Cell Development and Immune Cell Profiles
FR2-camelized RenNano® mice showed B-cell development and splenic B-, T-, and NK-cell proportions comparable to those of the fully human RenNano® mice, supporting an expanded V gene repertoire while maintaining similar immune cell profiles.
Diverse V(D)J Rearrangements and Broad CDR3 Length Distribution
NGS analysis of peripheral blood from unimmunized FR2-camelized RenNano® mice showed that nearly all FR2-camelized V gene segments participated in V(D)J recombination. D and J gene usage and CDR3 length distributions were similar to those observed in the fully human RenNano® mice.
FR2 Camelization Improves Soluble VHH Expression
Higher Success Rate
Proportion of VHHs successfully expressed in soluble form in E. coli
FR2 camelization significantly improves soluble VHH expression in E. coli, achieving a 100% expression success rate and markedly higher expression yields.
VHHs derived from RenNano® mice were expressed in E. coli and purified in-house. Data are presented as the median and interquartile range.
FR2-Camelized Human HCAbs Show Significantly Enhanced Hydrophilicity
Hydrophobic interaction chromatography (HIC) analysis demonstrates that FR2-camelized HCAbs exhibit significantly shorter retention times, indicating markedly enhanced surface hydrophilicity and a more favorable solubility profile. RenNano®-derived HCAbs were expressed in CHO cells and purified in-house. Data are presented as the median and interquartile range.
FR2-Camelized VHHs Exhibit Markedly Improved Monomer Purity
Percentage of VHHs Meeting Monomer Purity Thresholds
FR2-camelized VHHs demonstrate improved monomer purity, with a significantly higher proportion of clones meeting stringent purity thresholds (>80%, >90%, and >95%). RenNano®-derived VHHs were expressed in CHO cells or yeast and purified in-house. All 11 fully human VHHs were expressed in yeast; among the 60 FR2-camelized VHHs, 10 were expressed in yeast and 50 in CHO cells.
Case Studies: Expanding VHH Discovery with FR2 Camelization
FR2-camelized RenNano® mice retain the intact fully human V gene repertoire while incorporating engineered FR2-camelized human V gene segments. Screening enriched FR2-camelized binders for CD28 and fully human binders for CD3, highlighting how the combined repertoires support candidate discovery across different targets.
Both case studies used FR2-camelized RenNano® mice for immunization.
Case Study 1 | CD28
CD28: Enrichment of FR2-Camelized Binders
FR2 Camelization Confers an Advantages in CD28 HCAb Discovery
Phage display screening of libraries derived from FR2-camelized RenNano® mice revealed strong enrichment of FR2-camelized sequences among CD28 binders (genetic ratio: 1:1).
FR2-camelized sequences increased from 27.0% in the initial library to 82.3% among antigen-positive clones after panning, despite an initial 1:1 germline ratio. FR2-camelized binders showed generally higher thermal stability and favorable HIC profiles, supporting the value of FR2 camelization in obtaining stable, binding-competent VHHs.
Frequently Asked Questions (FAQs) About RenNano
1. What is a heavy-chain-only antibody (HCAb)?
A heavy-chain-only antibody (HCAb) recognizes and binds an antigen without a light chain. Antigen recognition is mediated by its heavy-chain variable domain, known as a
VHH, which can also function independently as a compact single-domain antibody. An HCAb includes additional heavy-chain regions, whereas an isolated VHH consists only of the antigen-binding domain.
2. What is RenNano®, and how does it generate fully human HCAbs?
RenNano® is Biocytogen’s fully human heavy-chain-only antibody (HCAb) discovery platform within the RenMice® family. It replaces mouse heavy-chain variable region with human V(D)J segments and engineers immunoglobulin loci to enable antibody production without light chains. Following immunization, RenNano® generates diverse fully human HCAbs that undergo natural recombination and immune selection directly in vivo.
3. How do RenNano®-derived VHHs differ from camelid-derived VHHs?
Camelid-derived VHHs originate from naturally occurring heavy-chain-only antibodies in animals such as llamas and alpacas. RenNano®-derived VHHs are generated in genetically engineered mice carrying a human heavy-chain variable repertoire. This enables RenNano® mice to directly generate fully human VHH antigen-binding sequences in vivo without the need for downstream antibody humanization.
4. How does RenNano® support stable HCAb production while maintaining B-cell development?
RenNano® combines constant-region and light-chain locus engineering to remove light-chain dependency while preserving the immunoglobulin functions required for B-cell receptor signaling, antibody assembly, and production. This enables stable HCAb production while maintaining overall normal B-cell development in RenNano® mice.
5. How does RenNano® generate diverse and high-affinity HCAb candidates?
RenNano® combines a fully human V(D)J repertoire with natural gene recombination to generate diverse HCAbs. Following immunization, somatic hypermutation and in vivo immune selection refine antibody binding through affinity maturation, supporting the discovery of high-affinity candidates. For suitable targets, RenNano® target-gene knockout mice can help overcome immune tolerance and expand epitope diversity.
6. Why can extended CDR3 regions help HCAbs recognize difficult-to-access epitopes?
The CDR3 forms part of an antibody’s antigen-binding site. In HCAbs, the absence of a paired light chain can allow longer CDR3 loops to extend into recessed pockets or otherwise difficult-to-reach epitopes. This helps VHH domains recognize challenging targets, including
GPCRs and ion channels.
