RenMice-Drug Discovery Engine

RenNano®

RenNano mice generate heavy chain-only antibodies (HCAbs) that do not require in vitro humanization, making the process faster and more cost-effective. RenNano-derived HCAbs demonstrate sequence diversity, optimal affinity and favorable PK properties.
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  • Key Features of RenNano® Mice
  • Validation Data of RenNano® Mice
  • Next-Gen RenNano® Mice

Publication

    Key Features of RenNano® Mice
    1. Fully human heavy chain variable region and modified constant region
    Fully human heavy chain variable region and modified constant region
    2. RenNano mice demonstrate robust immune responses against multiple antigens
    TFR1, 4-1BB, and ALB img

    RenNano mice' immune responses against TFR1, 4-1BB, and ALB. Sera from RenNano mice immunized with TFR-1 and 4-1BB (membrane antigens) were diluted and incubated with antigen-expressing CHO cells. Fluorochrome-conjugated secondary antibodies were used to label CHO-bound HCAbs, and mean fluorescence intensity (MFI) was measured using flow cytometry to indicate the antigen-specific HCAb titer. Sera from RenNano mice immunized with ALB (secreted antigen) were diluted and incubated with fluorochrome-conjugated secondary antibodies. OD450 was measured using ELISA to indicate the antigen-specific HCAb titer.

    3. RenNano-derived antigen-specific HCAbs showed great diversity
    RenNano-derived antigen-specific HCAbs img

    HCAbs derived from RenNano mice are diverse and many contain longer CDR3 regions. Antigen-specific HCAbs were analyzed by their heavy chain germline gene usage and CDR3 length. Analysis indicates broad IGHV germline diversity. Additionally, most CDR3 lengths observed were longer than 12 AA, with some longer than 17 AA.

    Validation Data of RenNano® Mice
    Binding affinity

    The affinity of 68 purified RenNano HCAbs for 8 targets (4-1BB, TROP2, IL3RA, OX40, Nectin-4, ROR1, PSMA and DLL3) were tested by SPR. For most HCAbs, KD were between 10-6 ~ 10-10 M.

    B cells (mCD19+ mCD3-) and Spleen FO/MZ-B

    B cell development and differentiation patterns in RenNano mice. RenNano mice have comparable B cell proportions in peripheral lymphoid organs (spleen, SP and lymph nodes, LN) to WT mice or RenMab mice. Spleens from RenNano mice also have comparable percentages of follicular B (FO-B) cells and marginal zone B (MZ-B) cells to WT mice or RenMab mice.

    RenNano-derived HCAbs demonstrate outstanding developability

    RenNano HCAbs' physiochemical property analysis. RenNano HCAbs show high purity after one-step purification (A). HCAbs exhibit short retention time in HIC-HPLC and CIC-HPLC, indicating superb hydrophilicity and specificity (B, C). RenNano HCAbs also exhibit favorable thermostability (D).

    Next-Gen RenNano® Mice:
    Combining Fully Human and FR2-Camelized (FERF) Human V Genes to Maximize VHH 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 is designed to maximize discovery success and broaden structural epitope coverage.
    Combining Fully Human and FR2-Camelized (FERF) Human V Genes to Maximize VHH Discovery Potential
    FR2 Engineering for Improved Screening Success and Developability
    In human heavy chains, FR2 residues at positions 37, 44, 45, and 47 (Martin numbering scheme) 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 within conventional heterotetrameric antibodies. (Sang et al., 2022, Structure)
    In a heavy-chain-only or VHH format, however, these positions become more solvent-exposed. Naturally occurring camelid heavy-chain-only antibodies therefore feature more hydrophilic residues at these positions, most notably the characteristic FERF (Phe-Glu-Arg-Phe) motif. (Sang et al., 2022, Structure)
    By replacing exposed hydrophobic residues with more hydrophilic ones, FR2 camelization helps reduce unwanted hydrophobic interactions, thereby mitigating aggregation and improving overall candidate developability.
    Comparative studies have shown that humanized libraries incorporating the FERF motif can achieve higher screening success rates for isolating viable lead candidates than libraries based on the human VGLW motif. (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.
    Strategic Advantages of Next-Generation RenNano®
    • Reduced Aggregation Risk & Enhanced Developability: 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.
    • Dual-Engine Sequence Diversity: Combines intact human V genes (VGLW) with engineered FR2-camelized human V genes (FERF), expanding candidate diversity across hard-to-target epitopes.
    • Preserved Fully Human Potential: Retains the original human V gene repertoire, allowing discovery campaigns to continue generating fully human VHHs when unmodified human sequences are preferred.
    • In Vivo Affinity Maturation: Enables natural in vivo affinity maturation to generate high-affinity VHH leads, streamlining downstream optimization.
    FR2 Camelization Improves Soluble VHH Expression in E. coli
    highter success rate img
    Greater Yield img
    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 VHH-Fcs Show Significantly Enhanced Hydrophilicity
    HIC img
    Hydrophobic interaction chromatography (HIC) analysis demonstrates that FR2-camelized VHH-Fcs exhibit significantly shorter retention times, indicating markedly enhanced surface hydrophilicity and a more favorable solubility profile. RenNano®-derived VHH-Fcs 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 img
    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.