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 Expression of IFN- γ and TNF-α were assessed using BD OptEIA™ Human IFN-γ or TNF-α ELISA Set (BD Biosciences) in healthy donor T cells co-cultured with ROR1 + JeKo-1 BTK-KD or Maver-1 cells at an effector-to-target ratio of 3:1 with or without ROR1-bsAb in 96-well plates for 24 hours. I, J Antigen-specific tumoricidal activity of ROR1-bsAb-guided T cells was evaluated using luminescence-based cytotoxicity assays in expanded healthy donor T cells (effector) cocultured with ROR1 high JeKo-1 BTK-KD or Maver-1 cell lines at an effector-to-target ratio of 3:1 for 24-48 h with or without the antibody. To evaluate its therapeutic potential in overcoming CAR T-cell therapy relapses, we assessed the antitumor effect of ROR1-bsAb using ROR1-positive primary MCL cells derived from patients who had relapsed after CAR T-cell treatment. Critically, CD19-CAR T cells alone exhibited limited target-specific tumoricidal activity. In contrast, engagement with the ROR1‑directed bispecific antibody (20–50 nM) elicited robust and dose-dependent killing of these CAR T-resistant cells (Fig. 2A–D ). These findings have substantial clinical relevance for MCL, where therapeutic options after CAR T‑cell relapse remain limited. At present, CD20‑directed bispecific modalities (mosunetuzumab, glofitamab, and epcoritamab) represent the major treatment category available in the clinic for patients who relapse following CAR T‑cell therapy. CD20-directed bispecific antibodies have shown modest activity with limited durability in patients relapsing after CAR T therapy [ 13 ]; however, no direct comparative analyses were performed in this study. Thus, the robust ROR1‑bsAb-mediated killing observed in ROR1 high CAR T‑resistant MCL supports a strong rationale for exploring ROR1‑targeted strategies as a potential next‑line therapeutic approach in this population. Fig. 2: ROR1-directed bispecific modalities alone or in combination with CD19-CAR T cells elicit potent anti-lymphoma activity against ROR1 high CAR T-resistant MCL in vitro and in vivo. Full size image A–D The therapeutic potential of ROR1-bsAb in overcoming CAR T-cell therapy relapse was evaluated using an LDH-based cytotoxicity assay in primary MCL cells derived from CAR T-cell therapy-relapsed patients, co-cultured either with CD19-CAR T cells or with untransduced healthy T cells (UTC) combined with varying concentrations of ROR1-bsAb. E:T, effector-to-target ratio. E Antigen-specific antitumor cytotoxicity was evaluated in CD19-CAR T cells cocultured with either CD19 low Z-138-R cell line and patient-derived primary MCL cells, with or without ROR1-bTCE. F, G Target-specific induction of cytokine release (IFN- γ and TNF-α) was assessed in ROR1-bTCE-guided CD19-CAR T cells cocultured with CD19 low Z-138-R or primary MCL cells, with or without Fc-less ROR1-CD3-bTCE-secreting engager-T cells in the upper chambers of Transwell inserts. H Antigen-specific antitumor cytotoxicity of ROR1-bTCE-guided T cells evaluated in untransduced T cells (UTC) or CD19-CAR T cells cocultured with CD19 low primary MCL with or without engager-T cells in the upper chambers of Transwell inserts. I A CDX model was established by implanting NSG mice with luciferase-expressing Maver-1 cells. On day 7 post-inoculation, tumor-bearing mice were stratified based on model-specific tumor burden criteria and randomized into four groups ( n = 5 per group). Three groups were reconstituted with T cells from healthy donors, of which two groups received the bispecific antibody (50 and 400 µg/kg, respectively) daily for 7 consecutive days, followed by twice-weekly dosing for an additional two weeks. Tumor growth was monitored by BLI imaging weekly, and growth curves were generated by plotting the mean BLI radiance (ROI) for each group over the course of treatment. Error bars indicate mean ± standard error of the mean (SEM). J Overall survival of the treated mice in the CDX model was assessed using Kaplan-Meier survival curve analysis (GraphPad Prism 10, Dotmatics), with statistical significance determined by the Log-rank (Mantel-Cox) test across all models in this study. P value: * P < 0.05; ** P < 0.01; *** P < 0.001. Given the emergence of CD19-dependent and independent CAR T cell resistance [ 14 , 15 ], incorporating an alternative antigen‑targeted strategy represents a rational approach to mitigate immune escape. To leverage this approach, we engineered a ROR1‑directed bispecific T‑cell engager (ROR1-bTCE) in a tandem-linked scFv‑Fc format, designed to primarily facilitate tumor‑antigen recognition and T‑cell engagement. The bispecific T-cell-engaging construct demonstrated functional capability through specific binding to both ROR1 and CD3, induction of T‑cell activation, and target‑dependent T‑cell proliferation. Upon engaging ROR1‑expressing cancer cells, the activated T cells produced increased levels of IFN‑γ and TNF‑α (Supplementary Fig. 3A–D ). We next evaluated its ability to redirect CD19‑CAR T cells toward MCL cells with diminished CD19 expression. Strikingly, the inclusion of ROR1‑bTCE in effector-target co‑cultures substantially enhanced CD19‑CAR T‑cell antitumor activity against the CD19‑low Z‑138‑R variant and primary MCL from patients who relapsed after CD19‑CAR T therapy, which similarly exhibited low CD19 expression by flow cytometry (Fig. 2E ). These findings support the ability of ROR1‑bTCE to redirect functional T cells and circumvent antigen‑loss-mediated resistance. To further evaluate its therapeutic potential, we engineered T cells to secrete a tandemly linked anti‑ROR1 and anti‑CD3ε bi-scFv T-cell engager (Fc-less ROR1-CD3-bTCE), thereby generating ROR1‑directed T-engager secreting-T cells (“engager T cells”). The T-cell-secreted engager remains confined to the local immune cell milieu and promotes bystander effector recruitment. Consistently, antibody exposure via a Transwell system from ROR1‑bTCE‑secreting T cells markedly increased cytokine production (Fig. 2F, G ) and, critically, restored effector cytotoxicity against patient-derived CAR T-resistant MCL cells (Fig. 2H ), demonstrating that ROR1‑bTCE enables CAR T-cell redirection to an alternative tumor antigen independent of CD19 expression. Notably, in engager T cells, Fc-less ROR1-bTCE functions as an antigen-targeting module that redirects CAR T cells targeting a distinct antigen, with the CAR T cells themselves constituting the primary effector population. While this strategy is promising, further feasibility studies and rigorous preclinical validation are required. To validate the therapeutic potential of the ROR1‑bsAb in a preclinical setting, we established cell line-derived xenograft (CDX) models in NSG mice using MCL cell lines. Tumor-bearing mice were reconstituted with human T cells and treated with two doses of ROR1-bsAb. Notably, treatment of T-cell-reconstituted mice with ROR1-bsAb elicited robust anti-lymphoma efficacy, as evidenced by pronounced suppression of tumor growth compared to the vehicle or T‑cell - only group (Fig. 2I and Supplementary Fig. 3E ) and by significantly prolonged survival, with the higher dosage (400 μg/kg) conferring a significant survival advantage (Fig. 2J ). To further validate the antitumor potential of ROR1 targeting, we next evaluated the anti-lymphoma efficacy of a complementary ROR1‑directed approach. In two independent experiments, co-administration of ROR1-bTCE and human T cells consistently elicited potent tumor-controlling effects, resulting in markedly reduced tumor burden compared to T-cell treatment alone (Supplementary Figs. 4A–C and 5A, B ). This antitumor effect was accompanied by significantly prolonged survival of tumor-bearing mice (Supplementary Figs. 4D and 5C ), with two treatment groups achieving a 100% survival rate at day 60 post‑treatment (Supplementary Fig. 4D ). Notably, no overt toxicity was observed across the tested dose range in all three models, with no significant weight loss or other adverse clinical signs in treated animals. These results demonstrate that ROR1‑directed engagement provides robust in vivo antitumor activity and supports the therapeutic potential of ROR1 targeting in R/R MCL. Taken together, our findings demonstrate that CD19‑CAR T‑cell relapse in MCL is characterized by ROR1 upregulation and can be effectively targeted using anti‑ROR1 bispecific antibody modalities. Importantly, ROR1‑bsAb-mediated engagement of CD19‑CAR T cells induced robust, dose‑dependent cytotoxicity against ROR1 high CAR T‑resistant primary cells, providing a compelling rationale for incorporating ROR1‑targeted strategies as next‑line therapeutic options in this setting. Among these modalities, the ROR1 bispecific antibody in the Fab-scFv-KiH format provides enhanced developability and therapeutic window, supporting clinical translation and combination with CAR T cells to overcome antigen escape and relapse, whereas the Fc-silent ROR1-bTCE is primarily used for functional and validation studies. The limitations of this study include the modest sample size, the single-center design, and the preclinical nature of the supporting evidence. Accordingly, our findings should be viewed as hypothesis-generating and warrant validation in larger cohorts. Despite these limitations, our results support the continued investigation of ROR1-directed bispecific approaches, alone or in combination with CAR T-cell therapy, as a promising strategy to circumvent antigen escape and improve outcomes in patients with MCL relapsing after CD19-CAR T-cell therapy.