Popis: |
Background Lymphocyte activation gene 3 (LAG3) (CD223) is a T cell inhibitory receptor that promotes tumor cell immune escape, is associated with signaling pathways in multiple immune disorders and cancer, and is considered as a potential target for cancer diagnostic and immunotherapeutic applications. Targeting LAG3 towards these ends requires developing additional new antibodies. Here we compare the binding characteristics of a new anti-LAG3 rabbit antibody, the SP464 clone, with the thirty-year old and extensively used anti-LAG3 mouse antibody, the 17B4 clone. Methods The three orthologous techniques of automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) were used to assess the relative binding properties of each clone for their shared immunogen and its sub-fragments and to map the relative location of their respective epitopes within the immunogen. Alanine scanning substitution of the SP464 epitope was used to further refine the epitope boundaries and to identify amino acids essential for binding. Results The rabbit SP464 clone exhibited between 20x to 30x greater binding to the target than did the mouse 17B4 clone. The minimal epitopes of the SP464 and 17B4 clones were localized to separate, but overlapping, sub-fragments within the amino-terminal fifteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Further application of this orthologous approach towards quantifying the effects of sequential alanine substitutions along the minimal epitope of the rabbit SP464 clone were in close agreement and identified two amino acids within the SP464 epitope that are essential for binding and four additional amino acids that likely contribute towards binding. Conclusions Our results show that the combined application of ACE, SPR, and IHC constitute a powerful orthologous approach both for comparing antibody-binding characteristics and for fine mapping of linear epitopes within an immunogen. The results of this approach indicate that the rabbit clone SP464 may serve as an additional useful antibody for assessing LAG3 expression. |