Advantages of Therapeutic Hu-VHH

Owing to their ability to recognize antigen with a single VHH (variable heavy domain of heavy chain) domain and construct into HcAb (Heavy Chain Only antibody) format, VHH 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 VHH in HcAb facilitates the manufacturing process, which is crucial if the VHH will be assembled into complex modalities such as a bispecific/multispecific antibody or cell therapy.

RenNano^®：Biocytogen’s Fully Human VHH (Hu-VHH) Antibody Platform

Discovery of novel therapeutic VHH platform from natural VHH-producing species (e.g., camel, shark) is a costly and time-intensive process. To de-risk the process of VHH discovery, Biocytogen engineered Hu-VHH 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.

Hu-VHH Platform Overview

Fully human HCAbs can be obtained directly from immunized RenNano mice and developed into Hu-VHH 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 proportion in peripheral lymphoid organs (spleen, SP and lymph nodes, LN) to WT mice or RenMab mice. RenNano mice also have comparable percentages of follicular B (FO-B) cells and marginal zone B (MZ-B) cells in the spleens 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). They exhibit short retention time in HIC-HPLC and CIC-HPLC, indicating superb hydrophilicity and specificity (B, C). RenNano HCAbs also exhibit favorable thermostability (D).

Hu-VHH Platform 100 Project

• Using RenNano mice, Biocytogen is creating a fully human VHH (Hu-VHH) antibody 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 Hu-VHH development abilities with collaborators’ expertise in Hu-VHH -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/Hu-VHH platform, please refer to our posters, webinar, and contact BD-Licensing@biocytogen.com.

Posters

CNS Delivery Summit 2023: Discovery of RenNano^®-derived human heavy-chain-only TFR1 antibodies that can efficiently cross the blood-brain barrier and deliver drug payload

AACR 2023: RenNano^® Mice: A Heavy-Chain-Only Antibody Platform for the Generation of Nanobody Therapeutics

AACR 2023: Discovery of RenNano®-Derived Human Heavy-Chain-Only Antibodies That Cross the Blood-Brain-Barrier

Webinar

Jan 24, 2024: Maximize Your Drug Design Possibilities with RenNano-derived Fully Human Nanobodies