B-cell malignancies define a subset of hematological cancers, including the B-cell proliferative disorders B-cell chronic lymphocytic leukemia (B-CLL) and B-cell non Hodgkin lymphoma’s (B-NHL) and the plasma cell proliferative disorder multiple myeloma (MM). Survival rates can vary widely for the various (sub)types as well as the stage of the disease. Although antibodies are the cornerstone of therapy for both B-CLL and B-NHL (the CD20-targeting rituximab) as well as MM (the CD38-targeting daratumumab), patients that relapse or are refractory still have a very poor prognosis. The advances in antibody engineering over the last decades has allowed the development of different antibody-based therapies. In chapter 1 we describe the various Fc-engineering strategies and bispecific antibody formats, and introduce two novel antibody technologies: the DuoBody and HexaBody. The DuoBody technology is based on a controlled-fab arm exchange, which is mimicked from IgG4 antibodies that dynamically exchange half-molecules and applied to IgG1 antibodies via single matched point mutations at the IgG1 antibody Fc-tail. The HexaBody technology introduces point mutations in the IgG1 antibody Fc-tail that stimulate Fc:Fc interactions upon target binding and induce antibody-hexamerization, thereby facilitating C1q binding and inducing complement-dependent cytotoxicity (CDC). In this thesis we focused on the application and preclinical evaluation of several antibodies that were developed by the DuoBody and HexaBody platforms, for the treatment of B-cell malignancies. In chapter 2 we explored the possibility of redirecting CD3+ T-cells to CD20+ B-cell lymphoma cells using epcoritamab (DuoBody-CD3xCD20, GEN3013), a bispecific IgG1 antibody created using the DuoBody platform. We demonstrate that targeting CD20 with a bispecific antibody can be effective even in samples from patients who had (recently) become refractory to previous CD20-targeting therapies. We also found that the expression of the immune checkpoint molecule herpesvirus entry mediator (HVEM) can negatively impact epcoritamab-mediated T-cell activation. In chapter 3 we introduced the E430G HexaBody-mutation in CD20- and CD37-targeting antibodies and found that antibody hexamerization significantly enhanced CDC levels, even for CD37-targeting antibodies that do not naturally have the capacity to activate the complement pathway. We observed that CD20- and CD37-targeting HexaBodies co-localize on the cell surface and synergistically induce CDC. In chapter 4 part I we evaluated various CD37-HexaBody variants, and found that a combination of dual-epitope targeting and antibody hexamerization most potently induces CDC. We therefore combined the DuoBody and HexaBody platforms to develop DuoHexaBody-CD37, a biparatopic bispecific hexamerization-enhancing IgG1 antibody. In part II of chapter 4 we evaluated the preclinical efficacy of DuoHexaBody-CD37 in samples from patients with B-CLL and B-NHL. We observed high cytotoxicity across the different subtypes, even when tumor cells highly expressed complement regulatory proteins. In concordance with the data presented in chapter 2, we found that simultaneous targeting of CD37 and CD20, here by DuoHexaBody-CD37 and rituximab, enhanced CDC. In addition to CDC induction, the HexaBody technology can be employed to induce receptor clustering. In chapter 5 we applied the E430G HexaBody mutations to a mixture of two non-competing death receptor 5 (DR5) targeting IgG1 antibodies, termed HexaBody-DR5/DR5. Receptor (hyper)clustering of DR5 is required to induce extrinsic apoptotic pathway activation. In our preclinical ex vivo evaluation, we found that HexaBody-DR5/DR5 was especially effective in recently treated relapsed/refractory patients. Important for future clinical trial design is our observation that HexaBody-DR5/DR5 can be combined in therapy with both immunomodulatory drugs as well as proteasome inhibitors, presumably due to the fact that HexaBody-DR5/DR5 can induce direct apoptosis as well as antibody-dependent cellular cytotoxicity (ADCC). To conclude, in this thesis we demonstrate the potential of DuoBody-CD3xCD20, DuoHexaBody-CD37 and HexaBody-DR5/DR5 for the treatment of B-cell malignancies, and aimed to provide the tools for successful clinical application, to move cancer therapy forward.
|Qualification||Doctor of Philosophy|
|Award date||2 Nov 2021|
|Place of Publication||Amsterdam|
|Publication status||Published - 2 Nov 2021|
- Antibody therapy, engineered antibodies, B-cell malignancies, Fc-engineering, Bispecific antibodies, lymphoma, multiple myeloma, complement-dependent cytotoxicity, apoptosis, antibody-dependent-cellular cytotoxicity