Advantages of Therapeutic Nanobodies
Owing to their ability to recognize antigen with a single VHH (variable heavy domain of heavy chain) domain, nanobodies are half the size of scFv (single-chain fragment variable) proteins, which allows for better penetration of solid tumors or the blood-brain barrier. Additionally, the simple structure of functional nanobodies facilitates the manufacturing process, which is crucial if the nanobody will be assembled into complex modalities such as a bispecific/multispecific antibody or cell therapy.
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RenNano®：Biocytogen’s Fully human nanobody platform
Discovery of novel therapeutic nanobodies from natural nanobody-producing species (e.g., camel, shark) is a costly and time-intensive process. To de-risk the process of nanobody discovery, Biocytogen engineered fully human nanobody mice (RenNano® mice) as a more feasible alternative. 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.
Overview of the RenNano mouse heavy chain locus, which contains the full human antibody heavy chain variable regions and a modified constant region for the development of heavy-chain-only antibodies (HCAbs) or nanobodies.
Nanobody Platform Overview
Fully human HCAbs can be obtained directly from immunized RenNano® mice and developed into nanobodies or used as building blocks for other modalities.
RenNano®️ mice exhibit normal B cell development and differentiation
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®️ mice demonstrate robust immune responses against multiple antigens
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.
RenNano®️-derived antigen-specific HCAbs showed great diversity
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.
RenNano®️-derived HCAbs show high 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.
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).
Nano 100 Project
Using RenNano® mice, Biocytogen is creating a nanobody library for 100+ high potential therapeutic targets including tumor associated antigens, GPCRs, immune-checkpoints, cytokine/chemokines, and factors related to neurological diseases.
Biocytogen is actively seeking collaborations with global pharmaceutical and biotech companies to combine its large-scale nanobody development abilities with collaborators’ expertise in nanobody-related therapeutics, such as cell therapies, bispecific and multi-specific antibodies, ADCs and RACs, to develop novel drugs with first-in-class and/or best-in-class potentials.
For in vitro and in vivo functional data of RenNano-derived HCAbs/nanobodies, please refer to our posters, webinar, and contact [email protected].