ROR1 upregulation after CD19-CAR T-cell relapse in mantle cell lymphoma identifies a targetable vulnerability for ROR1-directed bispecific modalities

BTK inhibition and CD19-directed chimeric antigen receptor (CAR) T-cell therapy represent essential treatments for relapsed/refractory (R/R) mantle cell lymphoma (MCL) and have achieved impressive response rates [ 1 ]. Unfortunately, most patients eventually relapse due to therapy resistance, immune evasion, or waning efficacy [ 2 ]. Bispecific antibodies are being developed for relapsed disease, with multiple approved or clinically advanced candidates [ 3, 4 ]. The receptor tyrosine kinase-like orphan receptor 1 (ROR1), an oncofetal antigen, is highly expressed across hematologic and solid tumors and further upregulated at relapse [ 5, 6 ]. By binding to Wnt5a, ROR1 promotes cancer cell survival, proliferation, metastasis, and activation of transcriptional programs associated with cancer stemness [ 7 ]. ROR-enriched cancer stem-like cells exhibit intrinsic resistance to targeted and adoptive cell therapies, thereby sustaining minimal residual disease and relapse [ 8, 9, 10 ]. Aberrant ROR1 expression in MCL [ 11 ] promotes cell growth through complex formation with CD19 in a B-cell receptor-independent manner [ 12 ]. These findings highlight ROR1 as a compelling therapeutic target in MCL [ 7 ]. Several ROR1‑directed bispecific antibodies have been developed and are currently undergoing clinical evaluation for patients with R/R B‑cell malignancies (ClinicalTrials.gov identifiers: NCT04763083 and NCT05607498). To address treatment resistance and relapse in MCL, we developed ROR1-directed bispecific T-cell-engaging antibodies as a novel option for patients who may not respond to current targeted therapy or CAR T-cell therapy. To define the therapeutic rationale for targeting ROR1, we examined its expression in diverse MCL patient cohorts, tissue microarrays (TMAs), and established MCL cell lines. Across these datasets, ROR1 expression was markedly elevated in patient tissue sections, as evidenced by increased immunohistochemistry staining in TMAs (Supplementary Fig. 1A ). By reanalyzing previously generated single-cell RNA-seq datasets from a cohort of patients with differential responses to BTK inhibition and/or CD19-CAR T therapy, we observed a progressive enrichment of ROR1⁺ tumor cells with disease progression. Compared to normal B cells, ROR1 expression cumulatively increased across tumor specimens stratified by therapeutic responses, particularly in BTKi- and CAR T-dual-resistant MCL (Fig. 1A ). Strikingly, within the CAR T-cell therapy cohort, ROR1 expression substantially increased in CD19-CAR T-resistant specimens ( p < 2.22e-16) (Fig. 1B ). To our knowledge, this association between ROR1 upregulation and CAR T‑cell resistance in MCL has not been previously reported. Next, relative cell-surface ROR1 expression was evaluated in primary MCL samples from CAR T-naïve and relapsed patients. Consistently, ROR1 expression is significantly elevated in the relapsed cohort, as determined by flow cytometric immunofluorescence staining ( p < 0.001) (Fig. 1C ). Further, heterogeneous, yet consistently elevated cell surface ROR1 expression was confirmed by flow cytometry across MCL cell lines (Supplementary Fig. 1B ). Together, these findings demonstrate a strong association between escalated ROR1 levels and relapses following CAR T-cell therapy, establishing ROR1 as an actionable therapeutic target for immune-based treatment in R/R MCL. To leverage this therapeutic vulnerability, we developed a ROR1- and CD3-directed bispecific T-cell-engaging antibody (ROR1-bsAb) in a Fab-scFv-KiH format (scFv, single chain variable fragment; KiH, “knob-into-hole”) within a human IgG1 framework, creating a full human IgG-like antibody (Fig. 1D; Supplementary Fig. 2A ). Human ROR1 and CD3ε antigen-binding specificity and T-cell-target engagement were confirmed by antibody-based flow cytometry (Supplementary Fig. 2B–E ), and its functional properties were demonstrated by enhanced T-cell activation, as evidenced by increases in the expression of T cell activation markers (CD25, CD69, CD71) (Supplementary Fig. 2F ). Affirming its functional capacity, activated T cells engaging target tumor cells triggered robust release of target-specific type I cytokines, including IFN-γ and TNF-α (Fig. 1E–H ) and Granzyme B (Supplementary Fig. 2G, H ). To evaluate its antitumor activity, we performed in vitro cytotoxicity assays using ROR1 high and BTK inhibitor-resistant MCL cell lines as the targets. Notably, at 10 and 50 nM, ROR1-bsAb effectively redirected T cells to potent cytotoxic effectors, resulting in robust tumoricidal activity against these targets (Fig. 1I, J ). Fig. 1: ROR1 upregulation following targeted therapy or CAR T-cell relapse defines an actionable therapeutic target for ROR1-directed bispecific antibody in relapsed/refractory MCL. Full size image A Box plot showing ROR1 mRNA expression in scRNA-seq data from samples classified as normal ( n = 2; 2 patients), BTK inhibitor-sensitive (S) ( n = 12; 4 patients), BTK inhibitor-resistant (R) ( n = 9; 8 patients), and BTKi-CAR T-dual-resistant (Dual) ( n = 5; 4 patients). Comparisons of mRNA expression between groups were performed at the per-cell level following batch correction, with adjusted p-values indicated on the plots. Immune cells clustered primarily by cell type rather than patient origin, indicating minimal residual batch effects, whereas tumor cells exhibited patient-specific clustering. B Box plot illustrating ROR1 mRNA expression levels in samples stratified by CAR T therapy response, including CAR T-sensitive ( n = 6; 5 patients), and CAR T-resistant groups ( n = 4; 3 patients). C Relative ROR1 expression, corresponding to median fluorescence intensity (MFI), was assessed by flow cytometry in CAR T-therapy-naïve ( n = 19; 7 patients) and CAR T-relapsed ( n = 17; 7 patients) specimens from MCL patients. A scatter plot shows MFI for individual specimen in each group with mean and error bars. D A stylized structural representation of ROR1-bsAb. E – H