N-Methyl-D-aspartic acid (nmdaReceptors (nmdar) is an ionotype glutamate receptor widely expressed in the central nervous system and is involved in learning and memory processes. Anti-NMDAR autoantibodies can cause:NMDAR encephalitis, the most common form of autoimmune encephalitis, in which patients experience confusion, epilepsy, and autonomic dysfunction. In the early stages of the disease, NMDAR autoantibodies appear in the blood and cerebrospinal fluid, and these antibodies are produced by short-lived plasma blasts, which are constantly differentiated and renewed by memory B cells. The current standard** is to extensively clear all CD20+ B cells with the monoclonal antibody rituximab, but at the same time remove beneficial antibodies, which may lead to serious *** such as sepsis, thrombosis, etc., so new ones need to be developed**.
Recently, from the Charité Medical School in Berlin, Germanyharald prusswiths. momsen reinckeFirst author and co-correspondentThe research team is incellPublished on the article entitledchimeric autoantibody receptor t cells deplete nmda receptor-specific b cellsarticle,An NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) T cell was developed to selectively remove anti-NMDAR B cells and pathogenic autoantibodies, accurately recognize NMDAR autoantibodies produced in patients with NMDAR encephalitis, release effector molecules, proliferate and specifically kill antigen-specific target cells, and NMDAR-CAAR T cells can clear anti-NMDAR in mouse models B cells, and maintain low levels of autoantibodies without significant off-target toxicity
NMDAR is a heterotetrameric complex consisting of 2 GLUN1 and 2 GLUN2 or GLUN3 subunits, and most NMDARs are composed of 2 GLUN1 and 2 GLUN2 subunits. The extracellular portion of each subunit is composed of an amino-terminal domain (ATD) and a ligand-binding domain (LBD). To study the epitope of human NMDAR autoantibodies, the researchers:Three soluble fusion proteins of FC with the extracellular domain of GLUN1 or GLUN2B were designedFourteen monoclonal antibodies were obtained from the cerebrospinal fluid and B cells of patients with NMDAR encephalitis, and their binding capacity was tested, and it was found that all NMDAR autoantibodies detected could bind to the extracellular domain of NMDAR, and 13 and 14 could bind to the ATD of GLUN1 and GLUN2B. And then theyThese fusion proteins are fused to the CD8 transmembrane domain, the 4-1BB intracellular coactivation domain, and the CD3 domain to form NMDAR-CAAR, which was expressed in Jurkat T cells, and the antibody binding ability was detected, and it was found that CAARs containing GN2B ATD had a stronger ability to recognize NMDAR autoantibodies.
Next, they constructed a K562 cell line that mimics the B-cell receptor (BCR) by expressing a monoclonal anti-NMDAR IgG antibody on the cell surface. To investigate the functionality of NMDAR-CAAR T cells, they were transfected with lentivirus expressing NMDAR-CAAR in primary human T cells from healthy humans, and the expression of the early activation markers CD25 and CD69 was detected after co-culture with antibody-expressing K562 cells for 16-20 h, and it was foundNMDAR-CAAR T cells can be activated by autoantibodies and can secrete effector molecules
They then tested whether NMDAR-CAAR T cells could kill target cells, and using luciferase experiments, they first tested these 3 CAARs for expressing high-affinity antibodies 003-102, The killing of K562 cells of the medium-affinity antibody 008-218 and the low-affinity antibodies 007-124 and 007-142 showed that all NMDAR-CAAR could effectively kill K562003-102 cells, and for the low- and moderate-affinity antibodies, N1ATDN2BATTD-CAAR had the best killing ability. Since NMDAR encephalitis patients also have high levels of NMDAR autoantibodies in their blood, they also tested the ability of NMDAR-CAAR to kill target cells in the presence of soluble high-affinity 003-102 antibodies, and found that it can still effectively kill target cells. Since the proliferative ability of T cells is important for their function and sustained effects in vivo, they also tested the proliferative ability of NMDAR-CAAR T cells and found that they proliferate when they encounter target cells.
The above experimental results found that considering the breadth of antibody recognition, cytotoxicity and proliferative ability, N1ATDN2BADD-CAAR was the best combination, and then they experimented on mice and found that:N1ATDN2BATTD-CAAR T cells can effectively clear antibody levels in the brain, and the presence of a blood-brain barrier does not lead to downregulation of NMDAR or histopathological changes in hypothalamic neurons due to the presence of the blood-brain barrier, N1ATDN2BATTD-CAAR T cells could be detected in anatomical structures that were in direct contact with the brain, suggesting that they could enter the brain, and they tested for adverse effects and found no off-target toxicity markers at the histopathological level.
NMDAR-CAAR T cells clear anti-NMDAR B cells.
Overall, this study reveals:NMDAR-CAAR T cells can specifically target anti-NMDAR B cells to clear NMDAR autoantibodiesIt provides an important basis for NMDAR encephalitis.