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CD20 and CD37 antibodies show synergy in CDC
potential of IgG1 mAbs can be enhanced by introducing mutations that improve hexamerization by Fc-mediated clustering and thereby increase CDC activi- ty.15,16 In the present study, we introduced a Hx mutation, E430G, into CD20 and CD37 mAbs and observed an impressive increase in CDC activity in primary CLL sam- ples. Moreover, combinations of CD20 and CD37 mAbs showed enhanced and synergistic CDC activity, including combinations of Hx-CD37 mAbs with the approved mAbs rituximab, ofatumumab and obinutuzumab. With several CD20 and CD37 mAbs approved or in clinical develop- ment, it is attractive to study the mechanism behind the cooperativity between mAbs targeting these two antigens.34,35 It was recently reported that expression levels of CD20 and CD37 mRNA and protein are correlated on lymphoma B cells.36 Here, using confocal microscopy and FRET analysis we show that CD20 and CD37 mAbs colo- calize on surfaces of B cells and that enhancing Fc-Fc inter- actions increases mAb colocalization. The observed syner- gistic CDC activity of CD20 and CD37 mAbs was sup- ported by increased C1q binding and increased CDC effi- cacy, as illustrated by enhanced CDC at relatively low C1q concentrations. Synergy in complement activation was most evident for CD37 mAbs in combination with type II CD20 mAbs, than with type I CD20 mAbs which are already effective at clustering as WT mAbs.
De Winde et al.37 recently suggested that the organiza- tion of the B-cell plasma membrane is shaped by dynamic protein-protein interactions and that this organization might be altered by targeted mAb therapies. It has previ- ously been described that membrane proteins can cluster into lipid rafts or tetraspanin-enriched microdomains (TEM), enabling efficient signal transduction.38,39 We hypothesized that the synergistic interactions in CDC between CD20 and CD37 mAbs could be driven by clus- tering of both target-bound mAbs into oligomeric com- plexes. By introducing Fc-Fc inhibiting mutations, we were able to demonstrate that CD20 and CD37 mAbs do not only permit the formation of homo-hexamers con- sisting of mAbs bound to either single target separately, but also allow the formation of hetero-hexamers com- posed of alternating CD20 and CD37 mAbs, each bound to their own cognate target, explaining the synergistic effects. Other hetero-hexamer variants may occur, although the presence of such alternative variants remains to be demonstrated (see Figure 7). We therefore propose a model for Fc-mediated clustering of synergistic mAb combinations on malignant B cells (Figure 7). Upon binding of mAbs targeting two different coexpressed antigens on the plasma membrane that are able to colo- calize, hetero-hexameric complexes are formed, provid-
ing a docking site for C1q binding and CDC induction. Introducing hexamerization-enhancing mutations can increase Fc-mediated clustering of mAbs into both homo- and hetero-hexameric complexes, thereby increasing the number of hexamers and further potentiating CDC. Increasing the therapeutic potency of mAb combinations, driven by hetero-hexamerization, could be of clinical rel- evance, as illustrated by a combination of Hx-CD20 and Hx-CD37 mAbs that showed strong CDC of tumor B cells obtained from patients with different B-cell malig- nancies. Hicks et al.40 recently reported that the antitumor activity of IMGN529, a CD37-targeting antibody-drug conjugate in clinical development, was potentiated in combination with rituximab in vivo in B-NHL xenograft models, which was associated with increased CD37 internalization rates. Other mechanisms of synergy between CD20 and CD37 have also been reported, such as upregulation of CD20 expression in Daudi cells after treatment with the radiolabeled anti-CD37 mAb
References
1. Deans JP, Li H, Polyak MJ. CD20-mediated apoptosis: signalling through lipid rafts. Immunology. 2002;107(2):176-182.
2. Gopal AK, Press OW. Clinical applications of anti-CD20 antibodies. Transl Res. 1999; 134(5):445-450.
3. Leget GA, Czuczman MS. Use of rituximAb, the new FDA-approved anti- body. Curr Opin Oncol. 1998;10(6):548-551.
4. Beurskens FJ, Lindorfer MA, Farooqui M, et al. Exhaustion of cytotoxic effector systems may limit mAb-based immunotherapy in cancer patients. J Immunol. 2012; 188(7):3532-3541.
5. Taylor RP, Lindorfer MA. Analyses of CD20 Monoclonal Antibody–Mediated Tumor Cell Killing Mechanisms: Rational Design of Dosing Strategies. Mol Pharmacol. 2014;86(5):485-491.
6. Weiner GJ. Building better monoclonal antibody-based therapeutics. Nat Rev Cancer. 2015;15(6):361-370.
7. Wang X, Mathieu M, Brezski RJ. IgG Fc engineering to modulate antibody effector functions. Protein Cell. 2018;9(1):63-73.
8. Lazar GA, Dang W, Karki S, et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A. 2006;103(11):4005-4010.
9. Richards JO, Karki S, Lazar GA, Chen H, Dang W, Desjarlais JR. Optimization of antibody binding to Fc RIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther. 2008;7(8):2517-2527.
10. Shields RL, Namenuk AK, Hong K, et al.
41 177Lu‐lilotomab.
The concept of antibody hetero-hexamer formation may hold relevance for a broader range of targets and effector mechanisms. An emerging therapeutic approach is the development of designer polyclonals, consisting of multiple mAbs in one product of which several are in clinical development, such as MM-151, targeting three epitopes on EGFR and Sym013, a mixture of six mAbs targeting all three HER family members (EGFR, HER2 and HER3).42,43 One could speculate that enhancing Fc-mediat- ed antibody clustering involving different coexpressing targets on hematologic or solid tumors may induce syner- gistic efficacy, providing a rationale for application in designer polyclonals. Whether effector mechanisms other than CDC, such as ADCC or ADCP are also enhanced by combinations of CD20 and CD37 mAbs remains to be elucidated. One may speculate that the engagement of two mAbs binding coexpressed targets allows for higher total antibody binding on the cell surface, allowing more efficient engagement of FcγRs on effector cells.
In the present work, we have demonstrated that syner- gy in CDC induced by combinations of CD20 and CD37 mAbs is likely driven by Fc-mediated clustering into het- ero-hexameric antibody complexes on the cell surface. Enhancing hetero-hexamerization between mAb combi- nations using Fc engineering represents a powerful tool to increase the therapeutic efficacy of mAb combinations directed against hematologic and other tumor targets.
Acknowledgments
The authors would like to thank Joost Bakker (SCICOMVI- SUALS) for designing Figure 7.
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