HUGO-Nano™: Fully Human Heavy Chain Only Antibody Mice

Fully human nanobodies represent a new class of antibody technology characterized by their ultra-small molecular weight at the nanometer scale, fully human genetic origin, and high antigen affinity. These features enable fully human nanobodies to more easily penetrate tissues, including crossing the blood-brain barrier, allowing for precise targeting of diseases.

Compared with alpaca-derived nanobodies, the primary distinction of fully human nanobodies lies in their entirely human genetic source, which helps avoid potential immune reactions in humans and enhances the safety and efficacy of the antibodies. Additionally, fully human nanobodies are more amenable to industrial-scale production and exhibit high stability, maintaining biological activity even under extreme conditions.

These advantages position fully human nanobodies as a promising therapeutic approach for the treatment of various diseases, including cancer, infectious diseases, and inflammatory disorders, marking a significant breakthrough in the field of antibody drug development.

Fully Human Heavy Chain Only Antibody Mice are genetically engineered mouse models designed to produce fully humanized heavy chain-only antibodies. These antibodies consist solely of heavy chains and can bind antigens without the assistance of light chains, similar to single-domain antibodies found in camelids. Fully Human Heavy Chain Only Antibody Mice combine the clinical advantages of fully human antibodies with the structural features of heavy chain-only antibodies, offering high stability, small molecular size, and strong engineering potential. They are particularly suited for the development of therapeutics targeting complex disease pathways.

This technology has greatly expanded the applications of antibody drugs in new target discovery, bispecific antibody design, and the development of diagnostic tools.

Strain Name: Fully Human Heavy Chain Only Antibody Mice
Strain Abbreviation: HUGO-Nano™
Genetic Background: C57BL/6N
Coat Color: Black
Application: Development of fully human single-domain antibodies

Schematic Diagram of Heavy Chain Gene Structure in HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mouse

This model was generated using TurboKnockout^® ES cell targeting technology to:

(i) Modify the mouse heavy chain constant region to express an IgG1 gene lacking the CH1 domain;
(ii) Delete the kappa light chain;
(iii) Delete the lambda light chain.

Figure 1. Schematic Diagram of Heavy Chain Gene Structure in HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mouse

HUGO-Nano™ mice carry the full repertoire of human antibody variable region sequences. When paired with the heavy chain, the variable region skips the CH1 domain of the constant region, forming heavy chain-only antibodies lacking the CH1 structure. Additionally, these mice lack light chain expression.

Strain Features of HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mice

Fully humanized heavy chain VDJ variable region sequences; no light chain; production of single heavy chain only antibodies.

Immunization Recommendations for HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mice

• Recommended Immunization Age: 6–8 weeks
• Health Status: Specific Pathogen Free (SPF)

Advantages of the HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mouse Model

• Based on Cyagen’s proprietary TurboKnockout^® ES cell targeting technology.
• Enables the development of fully human germline-derived nanobodies.

Validation Data of the HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mouse Model

1. Antibody Sequence Diversity Analysis

Spleens were collected from naive mice, and total RNA was extracted. After passing quality control, downstream amplification primers targeting the CH2 region were used for library construction. High-throughput sequencing technology was applied to comprehensively assess immune system diversity. The sequencing data were processed through quality control software to remove background noise, followed by alignment with the IMGT immunoglobulin receptor database to identify corresponding V(D)J gene segments. Precise VDJ fragments and sequence loci were mapped, and statistical analyses were performed on VDJ gene usage frequency, clonal frequency distribution, and the number of unique clone sequences.

Figure 2. Detection of Heavy Chain Antibody VDJ Rearrangement Expression in Splenic B Cells of HUGO-Nano™ Mice.

Total RNA from the spleens of naive mice was used for library construction and sequencing analysis of heavy chain antibody variable region diversity. The results demonstrated that HUGO-Nano™ mice exhibit rich diversity in heavy chain antibody variable region sequences, and the antibody sequences lack the CH1 domain.

2. Immunophenotyping Analysis

Peripheral blood was collected from naive mice. Cells were incubated in a solution containing blocking antibodies (e.g., Fc Block) to prevent nonspecific binding. Fluorescently labeled antibodies were added at the recommended concentrations according to the antibody datasheets, followed by incubation on ice for 20–30 minutes in the dark. After incubation, cells were washed with PBS buffer to remove unbound antibodies. Laser and filter settings were adjusted to match the fluorescence characteristics of the antibodies. The stained cell samples were then analyzed using a flow cytometer, and data were collected and saved for subsequent analysis.

Figure 3. Normal Proportions of B, T, and NK Cells in the Peripheral Blood of HUGO-Nano™ Mice.

Representative flow cytometry immunophenotyping analysis and statistical comparison were performed on T, B, and NK cell populations from the peripheral blood of HUGO-Nano™ mice. The results showed that the proportions of B cells (CD3⁻CD19⁺), T cells (CD3⁺CD19⁻), and NK cells (CD3⁻CD335⁺) were comparable to those in wild-type (WT) mice, with no significant differences observed.

3. Antibody Activity Analysis

Anti-PD-L1 antibodies expressed in CHO-S cells were analyzed for binding activity to the PD-L1 antigen using ELISA.

Figure 4. Binding Activity Analysis of Fully Human Nanobodies.

ELISA results showed that the fully human anti-PD-L1 nanobodies exhibited varying levels of binding affinity to the PD-L1 antigen.

For more information on the validation data of HUGO-Nano™ Fully Human Heavy Chain Only Antibody Mice, please download and refer to the HUGO-Ab^TM  Humanized Antibody Mouse Models brochure.