Antibody Humanization for Infectious Disease Serological Assay Development

Development of Humanized Antibodies for Virology Research

In a recent paper, researchers at the United Kingdom Health Security Agency (UKHSA) sought to compare different humanized antibodies targeting Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a tick-borne pathogen with a global case fatality rate of 11.7% in humans with acute infection. The World Health Organization has classified it as a priority disease for research and development due to its epidemic potential. (Dowall et. al. 2022) The authors set out to identify an antibody that can be used in serological standards for diagnostic assays, and that can be made readily available for global public use.

As part of this research, the team reviewed 16 different humanized antibody variants to determine if they can be used as a positive control for serological assays. The variants were engineered and manufactured by Absolute Antibody, using our Prometheus™ antibody humanization service.

What is Antibody Humanization?

Antibody humanization is an engineering process that involves transferring critical non-human amino acids onto a human antibody framework. This primarily includes the grafting of amino acids in the complementarity-determining regions (CDRs) but also other framework amino acids critical for the variable heavy:variable light (VH:VL) interface and CDR orientation.

Figure. Progressive humanization of antibodies.
A schematic representation of the advancement from fully mouse antibodies, represented by red domains, to fully human antibodies, represented by blue domains.

Keys for successful humanization:

• Introduce as much human content as possible to reduce the risk of immunogenicity
• Retain enough non-human content to maintain the original binding activity of the parent antibody

Additionally, manufacturability needs to be taken into consideration. Manufacturing properties to consider include:

• Expression titer
• Aggregation
• Stability
• Solubility

Absolute Antibody’s Prometheus™ humanization service was designed with increased antibody manufacturability in mind. Our team of expert scientists will work with you to design a variant guaranteed to have comparable activity to the parent antibody.

How Does the Process Work?

How do you start with a mouse (or rat, rabbit, camelid) antibody and end with a humanized version? The process is technical and requires a team of experienced antibody engineers. A shortened version of the process looks like this:

1. The antibody-producing hybridoma is sequenced
2. Sequences are grafted onto preferred germline backbones selected for key manufacturability properties (learn more about we identified those preferred properties in our research poster)
3. Variable heavy and light chains are cloned into vectors
4. Plasmid DNA for transfection is generated
5. Cells are transiently transfected
6. Humanized antibodies are expressed and purified

Antibody Humanization and Diagnostics Development

For the recent UKHSA paper, Absolute Antibody successfully created 16 different humanized antibody variants. To assess their function, the researchers performed an indirect CCHFV ELISA to compare the binding of the fully humanized antibodies to that of a chimeric control. As reported, “The IC50 values were calculated with four of the humanized antibodies showing activity within two-fold of the chimeric antibody.”

These results show promising potential for use in serological diagnostic tests. Serological assays based on humanized antibodies could help, according to the authors, “address a shortfall in available tests that meet regulatory diagnostic standards and to aid surveillance activities to extend knowledge on the distribution of CCHFV”.

The team at Absolute Antibody believes the future of serological assays is recombinantly manufactured antibodies, whether humanized, chimeric, or in the original species. This technology is free from issues that regularly plague hybridoma antibody production, including:

• Genetic drift
• Contamination
• Cell line death

Recombinant antibodies are manufactured using synthetic genes expressed in a defined in vitro cell line. This animal-free production ensures:

• Batch-to-batch reproducibility
• No genetic drift
• High purity
• Steady supply chain

These elements are important when designing and manufacturing serological assays. Recombinant technology enables you to control the antibody supply chain and maintain assay reproducibility. Consistently produced and easily accessible recombinant antibodies can continue to aid in the development of standardized serology tests, especially for under-represented diseases. In addition, antibody engineering, from humanization in this recent example to more straightforward species or isotype switching, enables assay developers to reformat an antibody and overcome assay design problems.

Contact our team of experts for more information and to ensure the best outcome for your project.