HUGO-Light™: Fully Human Common Light Chain Antibody Mice

The common light chain antibody mouse is a genetically engineered mouse model designed to produce fully human antibodies that share a common light chain. By modifying the genome so that all antibodies generated utilize the same light chain, this model facilitates the development of bispecific or multispecific antibodies. The HUGO-Light™ Fully Human Common Light Chain Antibody Mouse can generate fully human antibodies with high affinity and low immunogenicity in vivo, significantly accelerating antibody discovery and new drug development. Its performance has been validated by multiple multinational pharmaceutical companies, biotech companies, and academic research institutions.

Strain Name: Fully Human Common Light Chain Antibody Mouse
Strain Abbreviation: HUGO-Light™
Genetic Background: C57BL/6N, BALB/c
Coat Color: Black, White
Application: Development of fully human fixed light chain monoclonal antibodies

Schematic Diagram of Antibody Gene Structure in HUGO-Light™ Fully Human Common Light Chain Antibody Mouse

In this strain:

(i) The heavy chain utilizes the heavy chain from the HUGO-Mab™ mouse, with the variable region replaced by the full-length human VDJ variable region sequence;
(ii) The kappa light chain is engineered using TurboKnockout® ES cell targeting technology, fixing the mouse kappa light chain to express only one VJ variable region combination;
(iii) The lambda light chain is completely deleted.

Figure 1. Schematic Diagram of Antibody Gene Structure in HUGO-Light™ Fully Human Common Light Chain Antibody Mouse

Based on the pairing frequency between heavy and light chains, eight light chain genes were selected. In the HUGO-Light™ mice, the light chain expresses only one fixed human light chain VJ sequence, resulting in the construction of eight HUGO-Light™ mouse models. Additionally, Cyagen has developed another type of model by inserting four high-frequency light chain V genes along with one J gene into the mouse light chain locus, resulting in two additional HUGO-Light™ mouse models. In total, there are ten mouse models across the two product types (see Table 1).

Table 1. Common Light Chain Antibody Mouse Products

Strain Features of HUGO-Light™ Fully Human Common Light Chain Antibody Mice

• Fixed light chain genes, allowing variability only in the heavy chain
• Fully functional immune system
• Normal B cell function
• Preserved natural antibody functionality
  
  

Immunization Recommendations for HUGO-Light™ Fully Human Common Light Chain Antibody Mice

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

Advantages of the HUGO-Light™ Fully Human Common Light Chain Antibody Mouse Model

• Based on Cyagen’s proprietary TurboKnockout^® ES cell targeting technology.
• Strains cover the eight genes with the highest heavy and light chain pairing frequencies, offering a comprehensive product library.
• Heavy chains consist of fully human antibody sequences with rich diversity.
• Available in both C57BL/6N and BALB/c backgrounds, suitable for the development of a wide range of targets.
  
  

Validation Data of the HUGO-Light™ Fully Human Common Light Chain Antibody Mouse Model

HUGO-Light™ mice can generate fully humanized antibodies in vivo with high affinity and low immunogenicity, demonstrating superior functional activity compared to FDA-approved standard therapies.

1. Antibody Sequence Diversity Analysis

Taking the IGKV1-39 gene model as an example, spleens were collected from naive mice. Total RNA was extracted from the spleen samples and subjected to quality control. After passing quality checks, libraries were constructed, and high-throughput sequencing technology was employed to comprehensively assess the diversity of the immune system. The sequencing data underwent quality control and background filtering using specialized software. The resulting sequences were aligned to the IMGT immunoglobulin gene database to identify corresponding V(D)J gene segments. Precise VDJ fragments and sequence loci were identified, and statistical analyses were performed on VDJ gene usage frequency, clonal frequency distribution, the number of unique clone sequences, and CDR3 length distribution.

Figure 2. Detection of Heavy Chain Antibody VDJ Rearrangement Expression in Splenic B Cells of HUGO-Light™ 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 showed that the HUGO-Light™ mice exhibit rich diversity in their heavy chain antibody variable region sequences, and the usage frequencies of different families are similar to those observed in human heavy chain antibody families.

2. Immunophenotyping Analysis

Taking the HUGO-Light™ (Vk1-39) gene model as an example: spleen and peripheral blood samples were 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, and cells were incubated on ice for 20–30 minutes in the dark. After incubation, cells were washed with PBS buffer to remove unbound antibodies. Laser and filter parameters were set to match the antibody fluorescence. The stained cell samples were then analyzed using a flow cytometer, and data were acquired and saved for subsequent analysis.

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

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

3. Antigen Immunization

Mice were immunized subcutaneously at multiple sites with 25–50 μg per mouse of human recombinant protein emulsified with Freund’s adjuvant, with booster immunizations administered every two weeks. Wild-type mice were used as the control group. One week after the second, third, and fourth immunizations, serum samples were collected via retro-orbital bleeding, and antibody levels in the serum were measured using the ELISA method.

Figure 4. Immune Response of HUGO-Light™ 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-Light™ mice exhibited antigen-specific serum titers comparable to those of wild-type C57BL/6N (WT-C57) mice. The data shown represent immune sera against two different therapeutic target antigens, A and B.

4. Single B Cell Antibody Screening Based on Microfluidics

After immunization with the VEGF165 antigen, spleen, lymph node, and bone marrow cells were collected from HUGO-Light™ mice. Following CD138 bead enrichment, 2 × 10⁶ plasma cells were encapsulated in microdroplets and cultured for 2 hours. Antibodies secreted by individual B cells bound to antigens and secondary antibodies within the microdroplets. Positive droplets were sorted using a microdroplet sorter, and next-generation sequencing (NGS) was performed to obtain 1,444 paired heavy and light chain antibody sequences, including 189 unique pairs.

Figure 5. Positive B Cell Screening from HUGO-Light™ Mice.

Positive droplets containing plasma cells were sorted using a microfluidic device, and antibody sequences were obtained through next-generation sequencing (NGS).
(Note: This data was generated through a collaboration between Cyagen and Biointron.)

5. Activity Assessment of Candidate Antibody Molecules

Twenty unique antibody sequences were selected from the 189 identified pairs for synthesis and expression. After purification, the resulting proteins were subjected to ELISA to evaluate their binding activity.

Figure 6. Binding Activity Analysis of Anti-VEGF165 Antibodies.

VEGF165 antigen was coated onto plates, and candidate antibodies were serially diluted from a starting concentration of 100 nM at a 5-fold gradient. Binding activity between the antibodies and antigen was detected using an anti-human IgG HRP secondary antibody. The results demonstrated that the antibodies generated from HUGO-Light™ mice exhibited high binding affinity.

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