Characterization of anti-canine CD20 antibody 4E1-7-B_f and comparison with commercially available anti-human CD20 antibodies

Abstract

This study characterizes the previously reported anti-canine CD20 antibody 4E1-7-B_f and compares this with commercially available anti-human CD20 antibodies, rituximab and an obinutuzumab biosimilar. While the obinutuzumab biosimilar exhibited binding to canine CD20 in a CD20-transduced cell line, canine B-cell lymphoma cell line (CLBL-1/luc), and canine CD21 + B cells from healthy dogs, functional assays revealed the superiority of 4E1-7-B_f in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities over those of the obinutuzumab biosimilar. Epitope analysis suggested an extracellular region on canine CD20 targeted by 4E1-7-B_f. Furthermore, the lipid raft localization of CD20 in CLBL-1/luc cells by treatment with 4E1-7-B_f classified this antibody as a type II anti-CD20 antibody which works with strong ADCC activity, similar to the obinutuzumab biosimilar, unlike rituximab, a type I anti-CD20 antibody, whose main action is CDC activity. These findings underscore the potential clinical utility of 4E1-7-B_f, emphasizing the specificity, potency, and therapeutic promise in canine lymphoma treatment.

Fig 1. Reactivity of anti-human CD20 antibodies to canine CD20.

(a) NRK cell lines overexpressing canine CD20 (NRK/cCD20-FL), bovine CD20 (NRK/bCD20-FL), feline CD20 (NRK/fCD20-FL), human B-cell line, Ramos, and canine B-cell line (CLBL-1/luc) were stained with isotype control (histogram in red), 4E1-7-B_f, rituximab, and obinutuzumab biosimilar (histogram in blue), followed by appropriate secondary antibodies. Stained cells were analyzed via flow cytometry. (b) Obinuzutumab biosimilar bound to canine CD20 overexpressed in NRK cells. NRK/cCD20-FL was stained by serially diluted amounts of 4E1-7-B_f or obinutuzumab biosimilar; 0 µg/mL indicates isotype control. (c) Rituximab and obinutuzumab biosimilar did not bind to canine CD21 + B cells. Canine PBMCs were isolated from three dogs and stained with anti-canine CD21 antibody and rituximab, obinutuzumab biosimilar, or 4E1-7-B_f. Human IgG and dog IgG were used as isotype controls. Gating strategy is shown in S1 Fig in S1 File.

Fig 2. Epitope analysis of 4E1-7-B_f antibody.

(a) Comparison of amino acid sequences of canine, human, murine, feline, and bovine CD20. * indicates the identical amino acid. Shaded boxes in pink indicate the extracellular domains of CD20 of each species. (b) Comparison of schematic structures of canine and human CD20 molecules. Green, light blue, and purple amino acid regions indicate the epitopes of ofatumumab, rituximab, and obinutuzumab, respectively. Amino acids in red in dog CD20 represent amino acids substituted into human CD20, and M1 to M5 indicate that only that portion of canine CD20 was replaced by human CD20 amino acids. (c) Expression of mutant forms of cCD20 (M1 to M5) in NRK cell lines as assessed via western blotting analysis. WT, cCD20-M1, M2, M3, M4, and M5 were stably expressed in NRK cells. Whole cell lysates were extracted from transduced cells and analyzed via western blotting with an anti-FLAG antibody; actin was used as a loading control. For transparency, the full-length images of the cropped western blots are shown in S2 Fig in S1 File. Representative results from two independent experiments are shown. Replicates of the full dataset are provided in S3 Fig in S1 File. (d) Expression of mutant forms of cCD20 (M1-M5) in NRKs via flow cytometry and comparison of the dissociation constant (Kd) values of an antibody across five different cell types (FL, M2, M3, M4, M5). Cells were stained with 4E1-7-B_f antibody at concentrations prepared by a 4-fold serial dilution, starting from 40 µg/ml down to 0.04 µg/ml, followed by a secondary antibody. Stained cells were analyzed by flow cytometry. One representative histogram results of several independent experiments is shown on the left side. Individual colored dots in right graph represent independent experimental measurements of Kd value, and bars indicate the mean Kd values for each cell type. Statistical analysis was performed using the Tukey Kramer multiple comparison analysis, with significant differences indicated by p-values. Significant differences were observed among multiple groups, as shown in the figure.

Fig 3. In vitro functional analysis of 4E1-7-B_f and obinutuzumab biosimilar against canine lymphoma cell line, CLBL-1/luc.

(a) The ADCC assay was performed by incubating A20/cCD20/luc cells with serially diluted antibodies in the presence of effector cells, NK-92/cCD16γ. * indicates statistically significant difference (p < 0.05) between control human IgG antibody and 4E1-7-B_f or obinutuzumab by Tukey Kramer multiple comparison analysis. (b) The CDC assay was performed by incubating CLBL-1/luc cells with serially diluted antibody in the presence of rabbit complement. * indicates statistically significant difference (p < 0.05) between control dog IgG antibody and 4E1-7-B_f by Tukey Kramer multiple comparison analysis.

Fig 4. Lipid raft translocation of CD20 in Ramos and CLBL-1/luc cells.

Ramos cells were stimulated with human IgG1, rituximab, or obinutuzumab biosimilar, and CLBL-1/luc cells were stimulated with dog IgG or 4E1-7-B_f for 5 min. After stimulation, soluble and insoluble fractions of cell lysates were extracted for western blotting with anti-CD71, anti-Lyn. and anti-CD20 antibody. CD71 and Lyn were used as controls for soluble and insoluble fractions, respectively. For transparency, the full-length images of the cropped western blots are shown in S4 Fig in S1 File. Representative results from two independent experiments are shown. Replicates of the full dataset are provided in S5 Fig in S1 File.