Single site solution: Engineering novel antibodies for the treatment of ITP
What is this research about?
Canadian Blood Services’ researchers have made significant advances toward developing a new therapy for a bleeding disorder. Immune thrombocytopenia (ITP) is an autoimmune disease in which the body generates antibodies against its own blood platelets leading to platelet destruction. Platelets are important cells in the bloodstream that form clots to stop bleeding. When platelets are destroyed, bruising and bleeding can readily occur. A drug called IVIG can be used to treat ITP.
However, IVIG is derived from human plasma, is costly and alternative therapies are needed. In ITP, autoimmune antibodies bind to platelets and trigger phagocytosis, a defence mechanism by which an immune cell engulfs or eats another cell to destroy it. Macrophages, the immune cells involved in platelet destruction in ITP, have proteins on their surface called Fc receptors. Using these receptors, macrophages recognize platelets that have antibodies bound to them, then engulf and destroy them.
A new fusion protein could provide a safe, costefficient alternative therapy for patients with immune thrombocytopenia.
One of the major Fc receptors involved in recognizing platelets is called Fc-gamma-RIII. Blocking this receptor could be a potential treatment for ITP. Although autoimmune antibodies cause ITP, other antibodies generated by researchers treat ITP. An Fcgamma-RIII blocking antibody called 3G8 successfully decreased platelet destruction in more than 50 per cent of patients with ITP who did not respond to other therapies. However, some patients experienced unwanted side effects, such as vomiting, nausea and fever. In an attempt to prevent these side effects, a similar, but modified antibody was generated that also improved platelet counts in patients with ITP.
However, this treatment reduced levels of certain white blood cells, and this could potentially cause anaphylactic shock, a severe and life-threatening allergic reaction. Most antibodies are Y-shaped proteins. Due to their shape, antibodies are ‘bivalent’; they can bind to two sites. Our researchers speculated whether antibody crosslinking was occurring in previous studies. Crosslinking is when a bivalent antibody binds to multiple receptors and acts as a bridge between them. This may result in uncontrolled consequences and could be the cause of undesired side effects seen in previous studies. This theory led the team of researchers to design and generate a potentially superior therapy to combat ITP.
What did the researchers do?
Researchers engineered proteins that were a fusion of antibodies against Fc-gamma-RIII and albumin, a plasma protein. Unlike the previously examined antibodies, these fusion proteins were monovalent; they had only a single binding site. A human fusion protein and a similar mouse fusion protein were generated.
Researchers conducted in vitro (outside of the body) tests to evaluate whether the proteins could bind specifically to Fc-gamma-RIII and whether they had similar binding properties compared to antibodies in previous studies. They tested whether the mouse protein could treat antibody-induced ITP in a clinicallyrelevant mouse model of ITP. They evaluated whether the fusion protein would cause unwanted side-effects by monitoring the body temperature and white blood cell levels following administration. Lastly, they generated a crosslinked version of the protein to test if crosslinking was the actual cause of unwanted side effects associated with earlier antibody therapies.
What did the researchers find?
The monovalent human fusion protein bound to human Fc-gamma-RIII in a similar manner to the previous bivalent antibody (3G8). The monovalent mouse fusion protein exhibited superior qualities compared to the previous antibody:
Superior qualities were mostly due to its larger size and the extended life-span in the body (because of fusion with albumin).
Using this protein, the researchers were able to improve Fc-gamma-RIII antibody-induced ITP.
The fusion protein was specific to the Fc-gamma-RIII receptor as it could not improve ITP that relied on other Fc receptors.
There were no observed adverse effects (based on no changes in body temperature).
White blood cell numbers were unaltered, indicating this protein did not cause anaphylactic shock Treatment with a crosslinked version of the fusion protein caused a drop in body temperature. This indicated crosslinking was the source of the unwanted side effects, confirming the study’s hypothesis.
How can you use this research?
There are few effective therapies for ITP and response rates vary from patient to patient. While blocking the Fc receptor to treat ITP has long been considered, the unwanted side effects of previous therapies meant they were not suitable for patients. In contrast, this novel monovalent protein fused to albumin targets Fc-gammaRIII but does not cause side effects, providing a first-in-class therapy for antibody-mediated ITP. Recently, albumin-coupled drugs have been approved for the treatment of other disorders, paving the way for this novel fusion protein to be developed as a therapy for ITP. IVIG, a current treatment for ITP, is derived from human plasma.
However, plasma is a limited resource. IVIG is also extremely costly, and some patients do not respond well to IVIG treatment. If developed into a therapy, this fusion protein could be a safer treatment for patients, and a more suitable alternative treatment for ITP. It could reduce the use of IVIG for ITP, which is very expensive for the health care system. This new therapy may also be beneficial for other Fc-gamma-R-mediated diseases.
About the research team
Dr. Xiaojie (Ben) Yu is a Canadian Blood Services postdoctoral fellow in the laboratory of Dr. Alan Lazarus. Dr. Lazarus is a Canadian Blood Services’ scientist and a professor in medicine and laboratory medicine and pathobiology at the University of Toronto. Dr. Lazarus is a member of the Toronto platelet immunology group at the Keenan research centre for biomedical science at St. Michael’s Hospital, Toronto. Dr. William Sheffield is associate director, researcher and senior scientist at the centre for innovation, Canadian Blood Services and a professor in the department of pathology and molecular medicine, McMaster University, Hamilton. The research team included members of Drs. Lazarus and Sheffield’s laboratories, and collaborators from the immunology research group, Budapest, Hungary.
This research unit is derived from the following publication(s)
Acknowledgements: This research received financial support from Canadian Blood Services, funded by the federal government (Health Canada), provincial and territorial ministries of health, and the Canadian Institutes of Health Research. The views expressed herein do not necessarily represent the views of governments. Dr. Xiaojie Yu is a recipient of a Canadian Blood Services postdoctoral fellowship. This Research Unit was prepared by Dr. Naadiya Carrim, a Canadian Blood Services’ postdoctoral fellow based in Toronto.
Keywords: Immune thrombocytopenia, platelet, Fc receptor, antibody, albumin, treatment, drug.