Fig. 4: Assessing the structure-function relationship of anti-CD20 antibodies.
From: Resolving the structural basis of therapeutic antibody function in cancer immunotherapy with RESI
a Schematic representation of CD20-CD3 TCE configurations: classical (c-TCE) and inverted (i-TCE), featuring an anti-CD20 monoclonal antibody (mAb) based on OBZ and an anti-CD3 Fab fragment at two different positions in the molecule. b, c RESI images of mAb-CD20 clusters when treated with c-TCE (b) and i-TCE (c). d, e Nearest-Neighbor Distance (NND) analysis of CD20 clusters treated with c-TCE (d) and i-TCE (e), showing increased frequencies of non-random sub-25-nm distances in the second and third NND histograms for i-TCE. Least-squares fit (solid lines) of monomers, dimers, trimers and tetramers shows an increased trimer- and tetramerization for i-TCE compared to c-TCE. f Bar graph depicting the percentage of CD20 oligomers in monomeric, dimeric, and higher-order forms (trimers and tetramers) for OBZ, c-TCE, and i-TCE. The frequency of trimers and tetramers is increased for i-TCE compared to c-TCE. The bars and error bars represent the mean and standard deviation, respectively. The number of biological replicates is n(OBZ) = 3, n(c-TCE)=n(i-TCE) = 4. Statistical significance was tested using a two-way ANOVA, adjusting for multiple comparisons (p = 0.0027). Source data are provided as a Source Data file. g Direct cell killing assay of CD20-positive Raji cells in untreated condition, and upon treatment with untagged versions of Rituximab (RTX), Obinutuzumab (OBZ), c-TCE, and i-TCE. i-TCE has a reduced killing efficiency compared to c-TCE, approaching values for Type I-RTX. The number of biological replicates is n = 3. The height of the bar and error bars represent mean and standard deviation, respectively. Statistical significance was tested using a one-way ANOVA, adjusting for multiple comparisons (p < 0.0001). Source data are provided as a Source Data file. h Graph depicting cell binding affinity in Raji cells across a range of mAb concentrations (0.01 to 100 nM) for untagged versions of RTX, OBZ, c-TCE, and i-TCE, measured as Mean Fluorescence Intensity (MFI) geometric mean. The results show an increased binding for i-TCE compared to c-TCE. The data points and error bars represent mean and standard deviation, respectively. The number of biological replicates is n = 3. Source data are provided as a Source Data file. Created with the help of BioRender (https://BioRender.com/y76v9f4 and https://BioRender.com/5575210).
