Dual Fc optimization to increase the cytotoxic activity of a CD19-targeting antibody

Abstract

Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions.

Keywords: ADCC; ADCP; CD19; CDC; Fc engineering; antibody hexamerization; antibody therapy.

Figure 1

Generation and binding characteristics of CD19-DEK. (A) Schematic illustration of a CD19-antibody with a dual engineered Fc part for improved FcγR binding and effector cell recruitment (DE-variant: S239D/I332E, pink) and efficient recruitment of the complement system (C1q) via E345K amino acid substitution (green) to favor antibody hexamerization on the target cell surface resulting in improved CDC. IgG model structure based on pdb file provided by Dr. Mike Clark (63) and Hexamer model structure based on crystal structure of IgG1-b12 (1HZH) provided by Dr. Rob de Jong was modified using Discovery Studio Visualizer (Biovia). (B) CD19-DEK and CD19-DE were analyzed by size exclusion chromatography. (C) SDS-PAGE under reducing and non-reducing conditions and Coomassie blue staining validated the purity and molecular mass of the dual-engineered CD19 antibody compared to CD19-DE. (D) Binding specificity of CD19-DEK and CD19-DE was tested via flow cytometry on CD19-positive cell lines SEM and Nalm-6. The CD19-negative T-ALL cell line Jurkat was used as control. The binding capacity of the optimized Fc part to FcγRIIa was investigated by flow cytometry on stably transfected cells. Data show representative results of three independent experiments. (E) Concentration dependent binding of CD19-DEK and CD19-DE compared to isotype control antibodies (ctrl-DE and crtl-DEK) was tested with the CD19-positive BCP-ALL cell line Nalm-6 via flow cytometry. (F) Concentration dependent binding of the optimized Fc part of CD19-DEK and CD19-DE to FcγRIIIa was analyzed by flow cytometry on stably transfected BHK cell line (BHK-CD16a). A control antibody with a silent Fc domain lacking FcγR binding (ctrl-FcKO) was used as a negative control. Mean values ± SEM of three independent experiments, *P<0.05%, ns, not significant. Two-way ANOVA with Bonferroni post-test.

Figure 2

The dual Fc-optimized antibody CD19-DEK triggers FcγR-mediated effector functions comparable to CD19-DE. (A) Chromium release assays were performed to analyze ADCC. CD19-positive tumor cell lines SEM and Nalm-6 were used as target cells and PBMC of healthy donors at an Effector : Target (E:T) ratio of 40:1 were applied. The tumor cell lysis triggered by CD19-DEK and CD19-DE was compared to control antibodies (ctrl-DEK and ctrl-DE). (B) The antibody-dependent cell-mediated phagocytosis (ADCP) was measured for 4 h by high-throughput fluorescence microscopy. CD19-positive cell lines were labelled with a pH-sensitive red-fluorescent dye and were incubated at an E:T ratio of 1:1 with polarized M0 macrophages and 10 µg/ml of the indicated antibodies. Phagocytosis is depicted as the relative red object counts per image. Data represent mean values ± SEM of three independent experiments, *P<0.05%, ns, not significant. CD19-DEK vs. CD19-DE, Two-way ANOVA with Bonferroni post-test.

Figure 3

CD19-DEK efficiently triggers antibody-dependent complement deposition on target cells and CDC. (A) Antibody-dependent complement deposition on CD19-positive B-ALL cell line SEM was analyzed via flow cytometry. Target cells were incubated with the respective antibodies (10 µg/ml) and 25% v/v human serum of healthy donors supplemented with eculizumab. Mean values ± SEM of three independent experiments are presented, *P<0.05% CD19-DEK vs. CD19-DE, One-way ANOVA with Bonferroni post-test. (B) CDC of the tumor cell lines SEM and Nalm-6 was performed in chromium release assays with increasing antibody concentrations and 25% v/v human serum of healthy donors. The tumor cell lysis was tested for CD19-DEK and CD19-DE and was compared to the control antibodies ctrl-DEK and ctrl-DE. Mean values ± SEM of three independent experiments are presented, *P<0.05% CD19-DEK vs. CD19-DE, Two-way ANOVA with Bonferroni post-test. (C) Target antigen specific CDC was tested in chromium release assays with CD19-positive (Nalm-6, SEM) and CD19-negative (MOLT-16, CEM) tumor cells at an antibody concentration of 50 µg/ml and 25% v/v human serum of healthy donors. The tumor cell lysis mediated by CD19-DEK was compared to CD19-DE. Mean values ± SEM of three independent experiments are presented, * P<0.05%, ns, not significant. CD19-DEK vs. CD19-DE, two-tailed t-Test with Mann-Whitney test.

Figure 4

The dual-engineered antibody CD19-DEK showed improved cytotoxic activity compared to CD19-DE using whole blood as effector source. (A, B) Chromium release assays with a concentration of 2 µg/ml of the respective antibodies and 25% v/v whole blood of healthy donors was performed to analyze the combined anti-tumor effect of CD19-DEK via the complement system (CDC) and via recruitment of effector cells (ADCC). For inhibition of tumor cell lysis via the complement system the blood was supplemented with 50µg/ml eculizumab. Mean values ± SEM of three (SEM cells) or seven (Nalm-6 cells) independent experiments, *P<0.05% CD19-DEK vs. CD19-DE, One-way ANOVA with Bonferroni post-test. (B) For inhibition of tumor cell lysis via FcγRIII (CD16) or FcγRIIa (CD32a) expressing effector cells the blood was preincubated with 100µg/ml specific blockade antibodies with an silenced Fc-part, lacking FcγR binding. (C, D) Chromium release assays with the B-ALL cell line SEM and increasing concentrations of the respective antibodies and 25% v/v whole blood of healthy donors was performed. For inhibition of tumor cell lysis via the complement system the blood was supplemented with 50µg/ml eculizumab. Mean values ± SEM of three independent experiments, *P<0.05%, ns, not significant. One- or Two-way ANOVA with Bonferroni post-test.