Journal: Frontiers in Oncology

Article Title: A novel anti-CD20, concabody, enhances immunotherapy efficacy by targeting MPZL1 and augmenting antibody-induced cell death

doi: 10.3389/fonc.2026.1748576

Figure Lengend Snippet: Concabodies, anti-CD20 antibodies fused with the MPZL1-binding lectin Con A, enhance direct cell death and antibody-dependent cellular cytotoxicity. (A) Schematic design of concabody and SDS-PAGE analysis of purified concabodies. These recombinant Con A–fused antibodies carry a 4×G4S linker(~2kDa) between the Fc region and Con A (~28 kDa). O, obinutuzumab; T-C, isotype control (trastuzumab)-Con A; R-C, rituximab-Con A; O-C, obinutuzumab-Con A. (B) Dose-dependent induction of direct cell death (DCD) by wild-type antibodies (TRA, RTX, OBI) and concabodies (T-C, R-C, O-C) in Raji cells (n=3). (C) Cell death induced by TRA-Con A or free Con A in Raji cells(n=3). (D) DCD induced by OBI and OBI-Con A in CD20 knockout Raji cells. (E) Comparison of DCD induced by wild-type anti-CD20 antibodies alone, free Con A plus antibody co-treatment, and concabodies in Raji cells (n=3). (F) MPZL1 dependency of R-C– and O-C–induced DCD assessed in doxycycline-inducible MPZL1 knockdown cells (DOX− vs . DOX+) in Raji cells(n=3). (G) Internalization efficiency of concabodies compared to wild-type antibodies in Raji cells (n=4). (H) DCD induced by concabodies in MPZL1 Y241F mutant cells, in which phosphorylation-dependent signaling is disrupted in Raji cells (n=3). EV, empty vector. (I) Lysosomal membrane permeabilization(LMP) (left) and direct cell death(DCD) (right) assays were conducted in Raji cells using wild-type antibodies (T, TRA; R, RTX; O, OBI) and concabodies (T-C, TRA-Con A; R-C, RTX-Con A; O-C, OBI-Con A)(n=3). (J) Lysosomal membrane permeabilization(LMP) (left) and Direct cell death(DCD) (right) assays were conducted in Ramos cells using wild-type antibodies (T, TRA; R, RTX; O, OBI) and concabodies (T-C, TRA-Con A; R-C, RTX-Con A; O-C, OBI-Con A)(n=3). (K) Increased antibody-dependent cellular cytotoxicity (ADCC) by concabodies (T-C, R-C, O-C) compared to wild-type antibodies(n=3). ADCC was measured by co-incubating 1×10 4 Raji cells with 5×10 4 healthy donor PBMCs. (L) Enhanced B-cell depletion by concabodies compared to wild-type antibodies in ex vivo assays using PBMCs from patients with chronic lymphocytic leukemia (CLL). Data are presented as mean ± SD. Statistical significance was determined using Welch’s t-test for comparisons between parental controls and specific groups (G, H) .For comparisons between multiple conjugate groups, One-way ANOVA followed by Tukey’s post-hoc test was employed. (F–J) Significance is indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

Article Snippet: CHO-K1, HEK293T and Ramos cells were purchased from the Korean Cell Line Bank.

Techniques: Binding Assay, SDS Page, Purification, Recombinant, Control, Knock-Out, Comparison, Knockdown, Mutagenesis, Phospho-proteomics, Plasmid Preparation, Membrane, Ex Vivo