CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering

Abstract

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

Figure 1.

Hexamerization-enhancing mutations in CD20 and CD37 mAbs substantially enhance complement-dependent cytotoxicity (CDC) of chronic lymphocytic leukemia (CLL) B cells. (A and B) CDC of B cells obtained from patient A with CLL. Cells were opsonized with different concentrations of CD20 mAb 7D8 as wild type (IgG1-CD20-7D8) or with a hexamerization-enhancing mutation (Hx-CD20-7D8) (A); or CD37 mAb 37.3 as wild type (IgG1-CD37) or with a hexamerization-enhancing mutation (Hx-CD37) (B) in the presence of 50% pooled normal human serum (NHS), heat-inactivated (HI) NHS, NHS + EDTA or medium. Representative examples of three replicate experiments are shown. (C-E) CDC of B cells obtained from 12 different CLL patients (patient B-M). CLL B cells were opsonized with 16 μg/mL (C), 2 μg/mL (D) or 0.25 μg/mL (E) Hx-CD20-7D8 or Hx-CD37. The dashed line represents 95% cell lysis. CDC induction is expressed as the percentage lysis determined by the fraction of TO-PRO-3 positive cells and data shown are mean and Standard Deviation of duplicate measurements.

Figure 2.

CD20 and CD37 mAbs synergistically induce complement-dependent cytotoxicity (CDC) of malignant B cells. (A and B) CDC on Daudi cells opsonized with 30 μg/mL wild-type (WT) type I CD20 mAb 7D8 (IgG1-CD20-7D8) (A) or type II CD20 mAb 11B8 (IgG1-CD20-11B8) (B), CD37 mAb 37.3 (IgG1-CD37), or a combination thereof (15 + 15 μg/mL) in the presence of 20% NHS. CDC induction is expressed as the percentage lysis determined by the fraction of propidium iodide (PI)-positive cells. Data shown are mean and Standard Deviation of triplicate measurements. (C) 8 x 8 CDC dose response matrix plot for the combination of hexamerization-enhanced CD37 mAb Hx-CD37 (0-0.8 μg/mL) with hexamerization-enhanced CD20 mAb Hx-CD20-11B8 (0-8 μg/mL), tested on Daudi cells and categorized as a color gradient from green (0% lysis) to yellow (50% lysis) to red (100% lysis). HIV gp120-specific mAb b12 (IgG1-gp120) was used as a negative control human mAb. (D) Loewe additivity-based combination index (CI) values calculated by CompuSyn for the CDC dose response matrix as described in (C) and categorized as synergistic (<1, red), additive (1, white) and antagonistic (>1, blue). Representative examples of two replicate experiments are shown. (E) CDC and CD37 expression analysis on Daudi, Raji and WIL2-S cells. For the CDC assay, cells were opsonized with Hx-CD37 (10 μg/mL), different CD20 mAb variants (10 μg/mL) or combinations thereof (10 + 10 μg/mL). Data show the mean of nine replicates collected from three independent experiments. Expression levels were determined using QIFIKIT analysis. The number of antibody molecules per cell was calculated from the antibody-binding capacity (mean fluorescence intensity, MFI) normalized to a calibration curve, according to the manufacturer’s guidelines. Expression data show the mean of four replicates collected from two independent experiments. ****P<0.0001.

Figure 3.

Enhanced binding and use of C1q by combinations of hexamerization-enhanced CD20 and CD37 mAbs. The capacity to bind C1q (A, C) and the efficiency to bind C1q and promote Complement-dependent cytotoxicity (CDC) (B and D) was assessed using Daudi cells opsonized with 10 μg/mL of hexamerization-enhanced variants of type I CD20 mAb-derived Hx-CD20-7D8 (A-B) or type II CD20 mAb-derived Hx-CD20-11B8 (C and D), CD37 mAb 37.3-derived Hx-CD37, or a combination thereof (5 + 5 μg/mL). Binding was detected using a FITC-labeled rabbit anti-human C1q secondary antibody and is expressed as mean fluorescence intensity. CDC induction was assessed in C1q-depleted serum by calculating the percentage of propidium idodide (PI)-positive cells as determined by flow cytometry. Representative examples of three replicate experiments are shown.

Figure 4.

CD20 and CD37 mAbs colocalize on B cells. (A) Confocal fluorescence microscopy analysis to detect colocalization of cell-bound CD20 and CD37 mAbs. Raji cells were opsonized with hexamerization-enhanced A488-conjugated CD20 mAb 7D8-derived Hx-CD20-7D8 (image 1, green) and hexamerization-enhanced A594-conjugated CD37 mAb 37.3-derived Hx-CD37 (image 2, red), and incubated for 15 minutes (min) at room temperature. Images were captured in PBS imaging medium at ambient temperature using a Zeiss Axi-oObserver LSM 700 microscope with Plan-Apochromat 63X/1.40 Oil DIC M27 objective lenses and acquired/processed using Zen software. Two excitation lasers were used at 488 and 555 nm. In the merged image, overlap of red and green produces orange or yellow. A representative example of two replicate experiments is shown. (B and C) FRET analysis to detect the molecular proximity of (B) WT type I CD20 mAb 7D8 (IgG1-CD20-7D8) or (C) WT type II CD20 mAb 11B8 (IgG1-CD20-11B8), WT CD37 mAb 37.3 (IgG1-CD37) or a combination thereof on the cell membrane of Daudi cells. (D and E) FRET analysis to detect the molecular proximity of hexamerization enhanced variants of (D) type I CD20 mAb 7D8-derived Hx-CD20-7D8 or (E) type II CD20 mAb 11B8-derived Hx-CD20-11B8, CD37 mAb 37.3-derived Hx-CD37 or a combination thereof on the cell membrane of Daudi cells. Daudi cells were opsonized with 10 μg/mL A555-conjugated-and 10 μg/mL A647-conjugated antibody variants for 15 min at 37°C. FRET was calculated from mean fluorescence intensity values as determined by flow cytometry. Data shown are mean and Standard Deviation of six replicates collected from three independent experiments. ****P<0.0001.

Figure 5.

Hexamerization-enhanced CD20 and CD37 mAb cooperate in complement-dependent cytotoxicity (CDC) through Fc-mediated clustering in hetero-hexamers. The effect of introducing Fc-Fc inhibiting mutations S440K and K439E on the CDC induction of hexamerization-enhanced type II CD20 mAb 11B8-derived Hx-CD20-11B8 on Daudi cells (A) and WIL2-S cells (B), hexamerization-enhanced CD37 mAb 37.3-derived Hx-CD37 on Daudi (C) and WIL2-S cells (D) and the mAb combinations thereof on Daudi (E) and WIL2-S cells (F). Cells were opsonized with concentration series of Hx-CD20-11B8 and Hx-CD37 variants in the presence of 20% NHS. CDC induction is expressed as the percentage lysis determined by the fraction of propidium iodide (PI)-positive cells. Representative examples of two (WIL-2S) and three replicates (Daudi) are shown. (G) The effect of introducing Fc-Fc inhibiting mutations S440K and K439E on the molecular proximity of Hx-CD20-11B8 and Hx-CD37 variants on the cell membrane of Daudi cells. Daudi cells were incubated with 10 μg/mL A555-conjugated Hx-CD20-11B8 variants and 10 μg/mL A647-conjugated Hx-CD37 variants for 15 minutes at 37°C. FRET was calculated from the mean fluorescence intensity values as determined by flow cytometry. Data shown are mean and Standard Deviation of six replicates collected from three independent experiments. ****P<0.0001.

Figure 6.

Combinations of hexamerization-enhanced CD20 and CD37 monoclonal antibodies (mAbs) induce superior ex vivo complement-dependent cytotoxicity (CDC) of tumor cells obtained from patients with B-cell malignancies. (A) B cells obtained from 15 patients diagnosed with chronic lymphocytic leukemia (CLL) were opsonized with fixed concentrations of hexamerization-enhanced type I CD20 mAb 7D8-derived Hx-CD20-7D8 or hexamerization-enhanced CD37 mAb 37.3-derived Hx-CD37 (open symbols: 2.5 μg/mL, closed symbols: 2 μg/mL; each presented as 100%), or 1:1 mixtures thereof (open symbols: 0.625 μg/mL of each mAb, closed symbols: 0.5 μg/mL of each mAb; each represented as 50%) in the presence of 50% NHS. CDC induction is presented as the percentage lysis determined by the fraction of TO-PRO-3 positive cells. (B) B cells of a representative CLL patient sample (patient G) were opsonized with different total mAb concentrations of Hx-CD20-7D8 or Hx-CD37 (single agents indicated as 100%) and combinations thereof at different antibody ratios (indicated as 75%:25%, 50%:50% and 25%:75%) in the presence of 50% NHS. CDC induction is presented as the percentage lysis determined by the fraction of TO-PRO-3 positive cells. Data shown are mean and Standard Deviation of duplicate measurements. (C) B cells obtained from ten patients diagnosed with different B-cell malignancies [B-cell non-Hodgkin lymphoma (B-NHL) not otherwise specified (NOS), follicular lymphoma (FL), marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL)] were opsonized with 10 μg/mL of hexamerization-enhanced type II CD20 mAb 11B8-derived Hx-CD20-11B8 or Hx-CD37, and the combination thereof (5 + 5 μg/mL) in the presence of 20% NHS. CDC induction is presented as the percentage lysis determined by the fraction of 7-AAD positive B-lymphoma cells. (D) CDC assay with B-cell patient samples representative for B-NHL (NOS), FL, MZL and MCL as described in (C). Data shown are mean and Standard Deviation of duplicate measurements. *P<0.05; **P<0.01; ****P<0.0001.

Figure 7.

Model for Fc-mediated clustering of CD20 and CD37 monoclonal antibodies (MAbs) in hetero-hexamers upon binding to the cell surface. (A) mAbs naturally cluster into hexameric complexes upon antibody binding to a cognate antigen on a target cell, thereby providing a docking site for C1q binding and complement-dependent cytotoxicity (CDC) induction. (B) Upon binding of mAbs targeting two different coexpressed antigens on the plasma membrane that (are able to) colocalize, hetero-hexameric antibody complexes are formed consisting of both mAbs, providing a docking site for C1q binding and CDC induction. Introducing hexamerization-enhancing mutations can increase Fc-mediated clustering of mAbs, both into homo-and hetero-hexameric antibody complexes on the cell surface, thereby increasing the number of C1q docking sites and further potentiating CDC.