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S.C. Oostindie et al.
mAbs employ various mechanisms to eliminate cancer cells, such as induction of programmed cell death or Fc- mediated effector functions, including antibody-depen- dent cell-mediated cytotoxicity (ADCC), antibody-depen- dent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC), which can be increased by Fc engineering.7 ADCC and ADCP, for example, can be enhanced by improving FcγR binding through Fc glyco- engineering or amino acid modifications.8-11 Likewise, C1q binding and CDC can be increased by amino acid substi- tutions in Fc domains.12,13 CDC is initiated when mem- brane-bound antibodies bind the hexavalent C1q mole- cule, which together with C1r and C1s forms the C1 com- plex, the first component of the classical complement pathway. C1 activation triggers an enzymatic cascade that leads to covalent attachment of opsonins to target cells, and the generation of potent chemoattractants, anaphyla- toxins and membrane attack complexes (MAC).14 IgG antibodies bound to cell surface antigens assemble into ordered hexamers, providing high avidity docking sites to which C1 binds and is activated.15 IgG hexamer formation and complement activation can be enhanced by single point mutations in IgG Fc domains, such as E430G, which increase interactions between Fc domains of cell-bound IgG.16 The hexamerization-enhanced (Hx) CD20-targeting mAb 7D8 displayed strongly enhanced CDC of B cells from patients with chronic lymphocytic leukemia (CLL), which often demonstrate complement resistance due to low CD20 and high membrane complement regulatory protein (mCRP) expression.16-18
In polyclonal antibody responses, antibodies against dis- tinct epitopes or antigens are thought to cooperate result- ing in increased effector functions against target cells. This increase can be mimicked in mAb combinations or cock- tails. For example, mAbs targeting epidermal growth fac- tor receptor (EGFR) do not induce CDC in vitro, but com- binations of mAbs against multiple EGFR epitopes induced potent CDC.19,20
CD37, which is abundantly expressed on B cells, repre- sents a promising therapeutic target for the treatment of B- cell malignancies.21,22 Currently known CD37 mAbs in clin- ical development, however, are generally poor inducers of CDC.23-27 Here we show that introducing Hx mutations into CD37 mAbs strongly potentiated CDC of CLL cells, and that combinations of CD20 and CD37 targeting mAbs could further enhance CDC of tumor cell lines and primary patient cells. We investigated the mechanism behind the synergistic CDC activity of CD20 and CD37 mAbs, and found that the mAb combinations activate complement cooperatively. The two mAbs formed mixed hexameric antibody complexes consisting of both antibodies each bound to their cognate targets, which we termed hetero- hexamers. The concept of hetero-hexamer formation and the use of Fc-Fc interaction enhancing mutations could serve as a tool to enhance cooperativity, and thereby the tumor killing capacity, of mAb combinations.
Methods
Cells
Daudi, Raji and WIL2-S B-lymphoma cell lines were obtained from the American Type Culture Collection (ATCC n. CCL-213, CCL-86 and CRL-8885, respectively). All primary patient cells used in this study were obtained after written and informed consent
and stored using protocols approved by the institutional review boards in accordance with the Declaration of Helsinki (Online Supplementary Methods).
Antibodies and reagents
mAb IgG1-CD20-7D8, IgG1-CD20-11B8, IgG1-CD37 clone 37.3 and IgG1-gp120 were recombinantly produced at Genmab.18,28-30 The HIV-1 gp120 mAb b12 was used to determine assay background signal. Mutations to enhance or inhibit Fc-Fc interactions were introduced in expression vectors encoding the antibody heavy chain by gene synthesis (GeneArt). Rituximab (MabThera®), ofatumumab (Arzerra®), and obinutuzumab (Gazyvaro®) were obtained from the institutional pharmacy (UMC Utrecht). See Online Supplementary Methods for details on reagents used.
CDC assays
CDC assays with CLL patient cells were performed with human complement as described.31 CDC assays with B-lymphoma cell lines and patient-derived B-lymphoma cells were per-formed using 100,000 target cells incubated [45 minutes (min) at 37˚C] with a mAb concentration series and pooled normal human serum (NHS, 20% final concentration) as a complement source. Killing was cal- culated as the percentage of propidium idodide (PI) or 7-AAD pos- itive cells determined by flow cytometry. See Online Supplementary Methods for details on cell markers used to define cell populations.
Expression analysis
Expression levels of cellular markers were determined using an indirect immunofluorescence assay (QIFIKIT®, BioCytex) accord- ing to the manufacturer‘s instructions (Online Supplementary Methods).
Confocal microscopy
Raji cells were opsonized with A488 labeled Hx-CD20-7D8 and A594 labeled Hx-CD37 mAbs (2.5 μg/mL final concentrations), and incubated for 15 min at room temperature. After washing, cells were placed on a poly-D lysine-coated slide and images were captured with a Zeiss AxioObserver LSM 700 microscope using plan-Apochromat 63X/1.40 Oil DIC M27 objective lenses and acquired/processed using Zen software.
Förster resonance energy transfer analysis
Proximity-induced Förster resonance energy transfer (FRET) analysis was determined by measuring energy transfer between cells incubated with A555-conjugated donor and A647-conjugat- ed acceptor mAbs using flow cytometry (Online Supplementary Methods). The dynamic range of FRET analysis by flow cytome- try was determined using control mAbs (Online Supplementary Figure S1).
Data processing and statistical analyses
All values are expressed as the mean±Standard Deviation of at least two independent experiments. Graphs were generated and analyzed using GraphPad Prism 7.0 (CA, USA). Differences
C1q binding and CDC efficacy
Daudi cells (3x106 cells/mL) were incubated with 10 μg/mL mAb and a concentration series of purified human C1q for 45 min at 37˚C. After washing, cells were incubated with FITC-labeled rabbit anti-human C1q antibody for 30 min at 4˚C and analyzed on a FACS Canto II flow cytometer (BD Biosciences, CA, USA). The efficiency of C1q binding and subsequent CDC was assessed as described above using fixed mAb concentrations, a concentra- tion series of purified C1q and 20% C1q depleted serum.
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haematologica | 2019; 104(9)