Investigating CD20 antigen escape mechanisms, Ang et al. identified four mRNA isoforms (V1-4) of CD20 with unique 5’-UTRs. V1 and V3 were the most abundant, and while V3 correlated with CD20 protein expression, V1 did not, and was found to contain features that inhibited polysome recruitment and limited translation. V1 yielded sufficient CD20 to be recognized by CD20-targeted CAR T cells, but allowed for escape from monoclonal (rituximab) and bispecific (mosunetuzumab) antibody therapies. Splice-switching Morpholino oligomers or reconstituting CD20 KO cells with V3 recovered CD20 expression. Evidence of a V3–V1 shift was also identified in relapse after treatment with mosunetuzumab.
Contributed by Lauren Hitchings
ABSTRACT: Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5' untranslated regions (5'-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.
Author Info: (1) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (2) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (3) Children's Hospit
Author Info: (1) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (2) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (3) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (4) Children's Hospital of Philadelphia, Wyomissing, Pennsylvania, United States. (5) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (6) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (7) The Wistar Institute, Philadelphia, Pennsylvania, United States. (8) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (9) The Wistar Institute, Philadelphia, Pennsylvania, United States. (10) Children's Hospital of Philadelphia, United States. (11) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (12) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (13) Children's Hospital of Philadelphia, United States. (14) Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (15) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (16) Children's hospital of Philadelphia, Philadelphia, Pennsylvania, United States. (17) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (18) Children's Hospital of Philadelphia and University of Pennsy. (19) Children's Hospital of Philadelphia & University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States. (20) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (21) The Wistar Institute, Philadelphia, Pennsylvania, United States. (22) University of Pennsylvania, Philadelphia, Pennsylvania, United States. (23) Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania, United States. (24) Children's Hospital of Philadelphia, United States.
