Consequences of CD20 ligation in diffuse large B cell lymphoma

About the Project

The addition of the monospecific chimeric CD20 antibody rituximab to chemotherapy transformed outcomes for patients with DLBCL without additional significant toxicities (1). Furthermore, bispecific CD20xCD3 antibodies show markedly enhanced activity alone and in combination with chemotherapy (2). However, up to 30% of patients either fail to respond or relapse despite most expressing abundant CD20 at the cell surface, indicating inherent resistance. The mechanisms underlying resistance to CD20 antibodies remain unknown. Prospective identification of patients destined to relapse would allow rational introduction of experimental therapies.

Given that CD20 antibodies have been administered to millions of patients worldwide, surprisingly little is known about CD20’s functions in health and disease. CD20 interacts directly with the B-cell receptor for antigen (BCR) and is thought to segregate BCR and CD19 nanoclusters, thereby acting as a gatekeeper, preventing constitutive BCR activation (3). Ligation of CD20 by bivalent antibodies induces several signalling cascades whose consequences are not fully understood (4).

Better understanding of both the organisation of CD20 at the cell surface and the consequences of CD20 ligation are necessary to develop new targeted therapies.

We have shown that:-

• DLBCL cell lines vary hugely in their sensitivity to CD20 antibodies; some expressing large amounts of CD20 (50,000 molecules/cell) are inherently resistant whereas others expressing 500 molecules/cell retain sensitivity (5)
• Proteins that coimmunoprecipitate with CD20 vary considerably from cell line to cell line (6)
• That CD20 ligation and cross-linking unexpectedly results in downregulation of CD19 (7).

The molecular basis to the above observations remains unknown. We propose to:-

• Establish the cell surface proteome in CD20 resistant/sensitive DLBCL cell lines before and after CD20 ligation using methods developed in our lab (6,7)
• Investigate the nanoscale organisation of CD20 in DLBCL cell lines of defined sensitivity to CD20 ligation using super-resolution microscopy and proximity-based ligation imaging techniques.
• Extend the CD20 interactome with further immunoprecipitations from resting DLBCL cells and from cells exposed to CD20 and BCR cross-linking antibodies.
• The functional importance of CD20 interacting proteins in determining sensitivity to CD20 ligation will be determined by CRISPR experiments.

Collectively, these data will determine mechanisms of resistance to CD20-based therapies and may have clinical application.

Our group based in the Ernest and Helen Scott Haematological Research Institute (EHSHRI) situated in the Henry Wellcome building (HWB) on the University of Leicester campus, has a long-standing interest in antibody therapy of lymphoid malignancies, most recently in terms of CD20xCD3 BsAb (5 and PhDs of Drs Joshua Bray and Gethin Thomas). We have had long-standing industrial collaborations with Roche, Isogenica and Bioinvent amongst others both in the lab and in clinical trials. Moreover, we have a large tissue bank of well characterised primary cases and an extensive panel of derived cell lines. In collaboration with Dr Yolanda Markaki (also based in the HWB) we have access to cutting-edge super-resolution imaging techniques. Similarly, with Professor Don Jones (van Geest MultiOmics Facility in the adjacent Hodgkin building) we have access to cutting-edge proteomics.

Enquiries

Project Enquiries to hw191@le.ac.uk / yolanda.markaki@leicester.ac.uk

To apply please refer to

https://le.ac.uk/study/research-degrees/research-subjects/cancer-studies