Monoclonal antibodies are an important class of immunotherapeutics for cancer and autoimmune diseases. According Antibody Society data from 2010 to 2020, over 39% of all antibody therapeutics approved by the FDA are fully-human antibodies, as opposed to chimeric/murine or humanized versions, illustrating the significance of fully-human antibodies as therapeutic agents.
Some important intrinsic benefits include a lower risk of immunogenicity, better biophysical properties and manufacturability, and desirable pharmacodynamics properties, such as affinity and specificity, as well as a reduced need for antibody engineering in the development process, shortened timelines for drug development, and better translation in clinical trials. Given these advantages, a strong interest persists in developing fully-human antibodies.
In vivo platforms are still the preferred method to discover human antibody hits
Two major approaches for fully-human antibody discovery exist: in vitro technologies, such as phage display and other surface expression techniques, and in vivo methods via immunization of transgenic animals, predominantly mice.
In vitro display technologies use vast immune libraries that produce great antibody diversity. However, one caveat of these discovery methods is the lack of natural pairing between heavy and light chains leading to unnatural antibody pairing and subsequently lower antibody affinity. Therefore, most antibodies produced from phage display may need to undergo further in vitro affinity maturation processes.1
Immunization of transgenic mice remains a steadfast productive means for fully-human antibody discovery.2 Technically, maturation inside these animals can harness the natural power for heavy and light chain pairing to ensure high affinity. In vivo methods based on hybridoma cell fusion have also been widely utilized in antibody discovery groups as a cost-effective technique since the inception of the method in the 1970s.3
Recent engineering successes in advanced, efficient, and high-throughput single B-cell technologies and instruments have also generated new momentum for in vivo antibody discovery using animal models. But despite technological advancement, only a handful of commercially accessible transgenic mice models are available, such as those from Biocytogen. This fledgling field has a lot to offer researchers and will continue to harness new discoveries to develop new, improved models.
A mouse model designed for a fully-human antibody repertoire
Biocytogen’s mission is to develop in vivo tools to expedite and improve the antibody discovery process. With the goal to provide a truly complete human antibody repertoire, the company employed a proprietary chromosome engineering technology to perform an in situ replacement of mouse antibody genes with their human counterparts to generate the RenMab mouse.
Sequencing of the immune repertoire of the RenMab mouse revealed a full human germline heavy chain VDJ utilization of the 46VH, 28DH, and 6JH genes. The subsequently produced RenMab antibodies exhibit human-like features as characterized by CDRH3 length, amino acid composition, and VDJ recombination pattern. Somatic hypermutation upon immunization has also been observed, leading to guaranteed diversification of the antibody repertoire after affinity maturation in the RenMab mouse model.
Case study: Leveraging the RenMab mouse for rapid fully-human antibody discovery
As part of the industry-wide endeavor amid the global COVID-19 pandemic, Biocytogen recently established a campaign to generate timely monoclonal antibody treatment of COVID-19 infections. Less than two months after the design of the immunization scheme, a panel of high-affinity antibody hits that functionally block Spike/hACE2 interaction were discovered by immunization of the RenMab mouse using spike protein antigens.
Biocytogen continues to actively work on blockade screening of pseudoviral infection in a hACE2-expressing cell line and pseudoviral infection in hACE2-transgenic mice. Investigation of protection against COVID-19 in cynomolgus monkeys is also planned.
The RenMab antibodies are proven to bind to the multiple epitopes on the receptor binding domain (RBD) of the COVID-19 spike protein with exquisite affinities in the sub-nanomolar to nanomolar range without any ex vivo optimization. This sets the stage for development of an effective virus-neutralizing monoclonal antibody treatment with the RenMab mouse as the source.
Discovery of antibody for difficult targets using “RenMab-knockout” mice
Amid its promising potential in fully-human antibody discovery, the RenMab mouse and other transgenic mice face limitations when it comes to certain classes of targets. Failure to efficiently generate antibodies from immunization may arise from either a low antigenic response when there is a high conservation or homology between the human and mouse antigen, or a lack of permissive preparation of soluble protein as immunogens for transmembrane proteins, such as GPCR.4
Various immunization protocols for delivering DNA vectors, viral-like particles, and viruses to the animal models have been explored as partial solutions. Cells with over-expression of target membrane proteins are a common boost to non-protein antigen immunizations.5
As an alternative method, researchers have deployed knockout mice from wild-type and transgenic mice to augment antibody generation for select targets.6,7 To further leverage the power of “knockout immunization,” Biocytogen has launched an extensive plan (Project Integrum) to tackle difficult targets based on the immunization of RenMab knockout mice (Figure 1).
To date, the company has successfully obtained several knockout mice with targets such as CCR4 in the GPCR family. Currently, access to these models is offered to the industry through exclusive partnerships or collaborations.
Coming next: Genetically engineered mice for bispecific discovery
Biocytogen continues to expand its in vivo platform for the discovery of therapeutic modalities based on fully-human antibodies. The RenLite mouse is a genetically-engineered model with complete humanization in the variable region of the heavy chains, while maintaining a fully-humanized common light chain strategically engineered into the antibody gene (Figure 2).
Similar to the generation of the RenMab model, a mega-base-pair-scale chromosome engineering technology is employed for the replacement of 2.4 Mb of mouse heavy chain genes with 1.0 Mb of human heavy chain genes. In this case, the light chain genes are strategically “fixed” to a common human sequence. The mouse model is now being extensively validated.
Early analyses of B-cell development, immune cell profiling, and Ig class switch are all highly comparable to the wild-type mouse. Further validations of the RenLite mouse are ongoing and are expected to be fully available later this year and readily accessible for researchers to utilize the model to venture into bispecific discovery.
Biocytogen focuses on gene-modified animal model production, generating unique small animal tools by replacing murine genes with their human counterparts, such as in the RenMab and RenLite models.
As experts in genetically-engineered animal development and maintenance with large-scale animal breeding capacity, antibody discovery, and preclinical pharmacology services, the company provides seamless integrated antibody discovery services to the biomedical community for a true one-stop solution from target to IND application.
1. Fitzgerald V and Leonard P, Methods, 2017, Mar 1;116:34-42. doi: 10.1016/j.ymeth.2016.11.006
2. Booth B, Forbes, https://www.forbes.com/sites/brucebooth/2017/05/11/human-antibody-discovery-of-mice-and-phage/#43dee2d17f26
3. Leavy O, Nature Immunology, 2016, 17, pageS13
4. Hrabovska A, Bernard V, Krejci E, PLoS One 2010 Sep 23;5(9):e12892
5. Lu ZJ et al., World J Biol Chem. 2012 Dec 26; 3(12): 187–196.
6. Chen WC and Murawsky CM, Front Immunol. 2018; 9: 460.
7. Hazen M et al., MAbs. 2014 Jan 1; 6(1): 95–107