HUGO-Mab™: Fully Human Monoclonal Antibody Mice

In response to the growing demand for innovative fully human antibody drug development, Cyagen has leveraged its strong technological innovation capabilities and proprietary TurboKnockout® ES targeting technology to develop the HUGO-Mab™ Fully Human Monoclonal Antibody Mouse. By employing a large-fragment in situ replacement strategy, the mouse’s endogenous VH, VK, and VL genes are replaced with human gene sequences.

This mouse model can produce fully human antibodies in vivo with high affinity and low immunogenicity, significantly accelerating the processes of antibody discovery and new drug development. Its effectiveness has been validated by numerous multinational pharmaceutical companies, biotech companies , and academic research institutions.

Strain Name: Fully Human Monoclonal Antibody Mouse
Strain Abbreviation: HUGO-Mab™
Genetic Background: C57BL/6N, BALB/c, SJL
Coat Color: Black, White
Application: Development of fully human monoclonal antibodies

HUGO-Mab™ Fully Human Monoclonal Antibody Mouse – Schematic of Antibody Gene Structure

Using TurboKnockout® ES targeting technology, the following genetic modifications are made:

• The mouse heavy chain variable region is replaced with the full-length human VDJ variable region sequence.
• The mouse kappa light chain variable region is replaced with the full-length human VJ variable region sequence.
  
  

The mouse lambda light chain is replaced with the full-length VJ variable region and constant region.

Figure 1. Schematic Diagram of Antibody Gene Structure in HUGO-Mab™ Fully Human Monoclonal Antibody Mouse

The HUGO-Mab™ mouse was developed by Cyagen on a C57BL/6N background through the following genetic modifications:

• The mouse antibody heavy chain and kappa light chain variable region sequences are replaced in situ with human variable region gene sequences. The mouse no longer expresses murine V(D)J sequences but retains the murine constant region coding genes and regulatory elements to support antibody class switching.
• The entire variable and constant region sequences of the mouse lambda light chain are replaced with human gene sequences, eliminating expression of murine lambda light chain sequences.
• As a result, the HUGO-Mab™ mouse contains the full repertoire of human antibody variable region sequences with high diversity. Upon antigen stimulation, it is capable of mounting a robust immune response comparable to that of wild-type mice, generating high-titer and diverse antibodies.

Key Features of the HUGO-Mab™ Fully Human Monoclonal Antibody Mouse Strain

• Fully humanized V(D)J variable region sequences
• Intact and functional immune system
• Normal B cell function
• Retains natural antibody functionalities

Immunization Recommendations for HUGO-Mab™ Fully Human Monoclonal Antibody Mice

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

Advantages of the HUGO-Mab™ Fully Human Monoclonal Antibody Mouse Model

• Developed based on Cyagen’s proprietary TurboKnockout® ES targeting technology.
• Full-length in situ replacement of the heavy chain, kappa light chain, and lambda light chain variable region genes enables expression of complete human immunoglobulin genes.
• Preserves natural antibody functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependend cellular phagocytosis (ADCP)and complement-dependent cytotoxicity (CDC).
• Directly generates fully human antibodies without the need for subsequent humanization, thereby shortening the drug development timeline.
• Available in three configurations: [Heavy chain + kappa light chain only], [Heavy chain + lambda light chain only], and [Heavy chain + both kappa & lambda light chains].
• Offered on C57BL/6, BALB/c, and SJL backgrounds, suitable for a wide range of target development applications.

Validation Data for the HUGO-Mab™ Fully Human Monoclonal Antibody Mouse Model

HUGO-Mab™ mice are capable of producing fully human antibodies in vivo with high affinity and low immunogenicity. In terms of functional activity, the antibodies generated outperform those of standard FDA-approved therapies.

1. Growth Curve

After weaning and individual housing, the mice are weighed weekly at the same time each week. Stable environmental conditions—such as temperature, humidity, and lighting—are maintained throughout the process to minimize external interference with growth. Based on the recorded time and body weight data, growth curves are generated using data analysis software.

Figure 2. Growth Curve of HUGO-Mab™ Mice

As the mice age, their body weight increases steadily, following a growth trend consistent with that of C57BL/6N mice. The weight range and physical appearance are also comparable to those of the C57BL/6N strain.

2. Antibody Sequence Diversity Analysis

Spleens are collected from naive HUGO-Mab™ mice, and total RNA is extracted. After passing quality control, RNA samples are used for library construction. High-throughput sequencing is then employed to comprehensively assess immune repertoire diversity.

The resulting sequencing data undergo quality control and background filtering using specialized software. The filtered sequences are then aligned to the IMGT immunoglobulin gene database to identify corresponding V(D)J gene segments. The analysis includes:

• Identification of precise V(D)J gene segments and sequence locations
• Statistical analysis of V(D)J gene usage frequency
• Clone frequency distribution
• Number of unique clone sequences
• CDR3 length distribution

These results provide a comprehensive view of the antibody diversity in HUGO-Mab™ mice.

Figure 3. VDJ Rearrangement Analysis of Heavy Chain Antibody Sequences in Splenic B Cells of HUGO-Mab™ Mice

RNA from the spleens of naive HUGO-Mab™ mice was used for library construction and sequencing to analyze the diversity of heavy chain variable region antibody sequences. The results show that HUGO-Mab™ mice possess a rich diversity in heavy chain variable region sequences. Moreover, the usage frequency of each gene family closely resembles that observed in the human heavy chain antibody repertoire.

Figure 4. VJ Rearrangement Analysis of Kappa Light Chain Antibody Sequences in Splenic B Cells of HUGO-Mab™ Mice

RNA from the spleens of naive HUGO-Mab™ mice was used for library construction and sequencing to analyze the diversity of kappa light chain variable region antibody sequences. The results demonstrate that HUGO-Mab™ mice exhibit a rich diversity in kappa light chain variable region sequences, with gene family usage frequencies closely mirroring those observed in the human kappa light chain antibody repertoire.

Figure 5. VJ Rearrangement Analysis of Lambda Light Chain Antibody Sequences in Splenic B Cells of HUGO-Mab™ Mice

RNA from the spleens of naive HUGO-Mab™ mice was used to construct sequencing libraries for the analysis of lambda light chain variable region antibody sequence diversity. The results indicate that HUGO-Mab™ mice possess a high level of diversity in lambda light chain variable regions, with gene family usage frequencies closely resembling those found in the human lambda light chain antibody repertoire.

Figure 6. CDR3 Length Distribution of Heavy Chain Variable Region Antibody Sequences in Splenic B Cells of HUGO-Mab™ Mice

RNA extracted from the spleens of naive HUGO-Mab™ mice was used to construct sequencing libraries for the analysis of CDR3 length in heavy chain variable region sequences. The results show that the CDR3 length distribution in HUGO-Mab™ mice follows a normal distribution pattern, consistent with the typical CDR3 length distribution observed in humans.

3. Immune Cell Profiling

Spleens from naive HUGO-Mab™ mice are collected, and the cells are incubated in a solution containing blocking antibodies (e.g., Fc Block) to prevent non-specific binding. Fluorescently labeled antibodies are added at recommended concentrations according to the antibody datasheets. The cells are incubated on ice for 20–30 minutes, protected from light.

After incubation, cells are washed with PBS buffer to remove unbound antibodies. The flow cytometer is configured with appropriate laser and filter settings to match the fluorescence profiles of the antibodies used. The stained cell samples are then loaded onto the flow cytometer, and fluorescence signals are collected based on the preset parameters. Data is acquired using flow cytometry software and stored for subsequent analysis.

Figure 7. Normal Proportions of B, T, NK, and Macrophage Cells in the Spleen of HUGO-Mab™ Mice

Representative flow cytometry immunophenotyping and statistical analysis were performed on spleen tissues from HUGO-Mab™ mice to assess the composition of T cells, B cells, NK cells, and macrophages. The results show that the proportions of B cells (CD3⁻CD19⁺), T cells (CD3⁺CD19⁻), NK cells (CD3⁻CD335⁺), and macrophages (CD11b⁺F4/80⁺) in HUGO-Mab™ mice are comparable to those in wild-type (WT) mice, indicating normal immune cell distribution.

4. Antigen Immunization

HUGO-Mab™ mice and wild-type control mice are immunized via subcutaneous multi-site injections with recombinant protein emulsified in Freund’s adjuvant. Booster immunizations are administered every two weeks. One week after the second, third, and fourth immunizations, mouse serum is collected. Antibody titers in the serum are then measured using ELISA assays.

Figure 8. Immune Response in HUGO-Mab™ Mice

Antigens were coated on plates, and mouse sera were serially diluted starting at 1:1000. Detection was performed using an anti-mouse IgG Fc secondary antibody. HUGO-Mab™ mice exhibited antibody titer levels comparable to those of wild-type C57BL/6N mice. The results shown represent immune titer measurements against four different therapeutic target antigens: A, B, C, and D.

5. Kinetic Affinity Analysis of Anti-PD-L1 Antibodies

The binding kinetics between fully human anti-PD-L1 antibodies and human PD-L1 protein were characterized using the ForteBio molecular interaction system. Human PD-L1-His protein was immobilized on HIS1K biosensors, and the test antibodies were diluted to specific concentrations for kinetic analysis.

Figure 9. Dynamic Affinity of Anti-PD-L1 Antibodies

The results show that the fully human antibody molecules generated by HUGO-Mab™ mice exhibit affinity levels comparable to those of Atezolizumab.

6. In Vitro Functional Assay

Dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) are co-cultured at defined concentrations. Various concentrations of the test antibodies are added to the culture. After 5 days of incubation, the supernatant is collected for the quantification of IL-2 and IFN-γ levels.

Figure 10. Anti-PD-L1 MLR Assay

The results indicate that the fully human antibody molecules generated by HUGO-Mab™ mice possess the functional capability to activate T cells.

7. In Vivo Functional Validation

Severely immunodeficient mice are used for in vivo validation. NCI-H358 tumor cells are implanted subcutaneously into the right flank of each mouse. One day prior to tumor inoculation, PBMCs are administered via tail vein injection. Antibody treatment is administered twice per week, and tumor size is measured twice weekly to evaluate therapeutic efficacy.

Figure 11. Anti-PD-L1 Tumor Suppression Model Validation

The results demonstrate that the fully human antibody molecules generated by HUGO-Mab™ mice are capable of inhibiting tumor cell growth in vivo.

To learn more about the validation data for HUGO-Mab™ Fully Human Monoclonal Antibody Mice, we invite you to download and explore the brochure:“HUGO-Ab^TM Humanized Antibody Mouse Models”.