The humble lab mouse is the workhorse for immunology research, especially antibody discovery. Immunizing mice with pathogens or recombinant antigens triggers an antibody response by mouse B cells, which researchers then screen for powerful and potent antibody therapy candidates. This pipeline has allowed quick development of mouse monoclonal antibodies that are now in trials or approved for use in humans.
However, antibodies developed in mice have clear limitations in clinical use: despite impressive similarity to human antibodies, they are still fundamentally mouse proteins. This means that a human immune system can and does reject these proteins.
One notable example of this is therapies for Epstein-Barr virus (EBV). Most known for causing infectious mononucleosis (‘mono’ aka the kissing disease), EBV has also been implicated in the development of several cancers, multiple sclerosis, lupus, rheumatoid arthritis, and long COVID. In immunocompromised patients, the risks from EBV are even higher.
Therefore, using mAbs to protect against EBV is a long sought-after goal. One pilot study in pediatric liver transplant recipients suggested that that neutralizing mAb therapy may be protective against EBV acquisition. However, there was a huge catch: the therapeutic mAb used in the study was a monoclonal antibody originally isolated from a mouse. Due to the fundamental difference between human and mouse proteins, each patient eventually developed an immune response against the drug. One patient developed severe hypersensitivity against the mouse mAb, leading the study authors to discontinue treatment in all patients and recommend against its future therapeutic use.
So, how can we find human monoclonal antibodies that can protect vulnerable patients from EBV? This question was recently tackled by the McGuire Lab in the Vaccine and Infectious Disease Division.
“Monoclonal antibodies (mAbs)…may have utility as therapeutics against EBV-associated diseases, particularly posttransplant lymphoproliferative disease (a deadly EBV-driven B cell lymphoma affecting transplant patients),” explains Dr. Crystal Chhan, who recently defended her PhD in the McGuire Lab. She is the lead author of a recent study in Cell Reports Medicine that details an exciting new discovery pipeline to find new anti-EBV mAbs with therapeutic potential.
Human mAbs have the potential to show the benefits “with minimal anti-drug reactogenicity previously seen with non-human mAbs,” says Dr. Chhan. However, it is challenging to find these drug candidates, especially for a virus as complicated as EBV.
One method to discover human mAbs involves using the antigenic protein as a bait to fish out the antigen-binding, antibody-producing B cells. They then sequence the gene encoding the antibody which allows cloning of a recombinant mAb.
However, a major challenge to EBV antibody discovery is that the virus infects the very B cells that produce antibodies. The virion attaches to B cells via two important glycoproteins: gp350, which binds complement receptors present on B cells, and gp42, which binds human leukocyte antigens (HLA) molecules. Complement receptors and HLA proteins play essential roles in signaling during immune responses, and both are constitutively expressed on B cells. Therefore, screening for B cells that bind gp350 or gp42 will overwhelming produce false positives – B cells that bind the viral proteins via complement or HLA do not necessarily produce antibodies targeting the virus.
To get around this, Chhan and colleagues turned to a humanized mouse model. There are many ways to humanize mice, so the details are important here. The model used in this study is called ATX-GK from Alloy Therapeutics. These mice have a completely murine immune system except for their antibody repertoire, as all murine antibody genes have been completely replaced with human genes.
With this model, the authors didn’t need to account for gp350 and gp42 binding to the murine B cells, since the murine version of their receptors don’t bind those glycoproteins. However, the antibody repertoire generated by these mice is entirely human, allowing the authors to screen for human antibodies that target gp350 and gp42.
The authors began by immunizing the ATX-GK mice with recombinant glycoproteins gp350 and gp42. In collaboration with the Antibody Technology Shared Resource, they harvested splenic B cells from the mice post-immunization and combined them with myeloma cells to generate hybridomas, which are long-lived, immortalized cells that produce massive amounts of monoclonal antibodies.
From the hybridoma cultures, they isolated 2 antibodies targeting gp350 and 8 antibodies targeting gp42. They performed binding assays to screen for the most potent binders and neutralization assays to determine which of these antibodies could block EBV infection in (human) epithelial and B cells. They found that their human antibodies were just as potent at blocking EBV infection as the mouse mAb previously used in clinical trials.
In collaboration with the Pancera Lab in VIDD, the then authors performed structural analyses of their most potent mAbs to characterize binding between antibody and antigen. They found that both gp350-targeting antibodies (ATX-350-1 and -2) block gp350 binding to the complement receptor, and two of the gp42-targeting antibodies (ATX-42-2 and -1.1) could block gp42 binding to HLA.
Dr. Chhan and colleagues then asked: how well do these antibodies protect against EBV in vivo? To answer this, they needed a mouse model of EBV infection. Not just any mouse can serve as this model, as another complication of EBV is that it is highly specific to humans.
They therefore switched to another humanized mouse system to model EBV infection. In this system, the mice are highly immunodeficient until they are engrafted with human CD34+ cord blood cells, which confer susceptibility to EBV.
"Some of the mice we used for evaluating the mabs in challenge studies were provided by the Kiem lab," says Dr. McGuire, highlighting an example of productive collaboration across Fred Hutch Divisions.
After transplant, the mice recovered for 8 weeks before injection with either ATX-350-2 or ATX-42-2 mAbs and subsequent infection with EBV. They found that the ATX-42-2 antibody potently blocked EBV infection in the spleen, but that the ATX-gp350-2 mAb only partially protected the animals from EBV infection. They suspect this may be because gp350 is not required for infection of human B cells.
The next steps of this project will focus on improving the pharmacokinetics and dosing regimen of these therapeutic mAbs rather than the single injection used in this study. The authors are also interested in combining these mAbs to improve protection through additive or synergistic mechanisms.
“These mAbs contribute to filling the gap of the very small number of genetically human mAbs against EBV’s surface glycoprotein gp42 and gp350,” Dr. Chhan concludes.
In the bigger picture, the lab is excited by the potential of the ATX-GK mouse model to rapidly discover antibodies against a wide range of human pathogens.
“The use of humanized mice to generate these mAbs offer a quick pipeline to generate genetically human mAbs with high potency that could be applicable to other diseases,” says Dr. Chhan.
This publication was also featured by the Fred Hutch News Service. Click here to check out their story.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Hans-Pieter Kiem and Andrew McGuire contributed to this research.
The spotlighted research was funded by the National Institutes of Allergy and Infectious Disease, the National Cancer Institute, the National Institute of General Medical Sciences, the National Institutes of Health, the Howard Hughes Medical Institute, the M.J. Murdock Charitable Trust, the Evergreen Fund from the Fred Hutchinson Cancer Center, The J. B. Pendleton Charitable Trust, the José Carreras/E. Donnell Thomas Endowed Chair for Cancer Research, and the Stephanus Family Endowed Chair for Cell and Gene Therapy.
Chhan CB, Lang K, Davis AR, Wan YH, Aldridge NT, Kher G, Scharffenberger SC, Hardy SR, Iureniev R, Giltiay NV, Edwards KR, Radtke S, Kiem HP, Pancera M, McGuire AT. 2026. Transgenic mouse-derived human monoclonal antibodies targeting EBV gp350 and gp42 provide basis for therapeutic development. Cell Rep Med. doi: 10.1016/j.xcrm.2026.102618.
Hannah Lewis is a postdoctoral research fellow with Jim Boonyaratanakornkit’s group in the Vaccine and Infectious Disease Division (VIDD). She is developing screens to find rare B cells that produce protective antibodies against human herpesviruses. She obtained her PhD in molecular and cellular biology from the University of Washington.