Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia

Abstract

Cirmtuzumab is a humanized monoclonal antibody (mAb) that targets ROR1, an oncoembryonic orphan receptor for Wnt5a found on cancer stem cells (CSCs). Aberrant expression of ROR1 is seen in many malignancies and has been linked to Rho-GTPase activation and cancer stem cell self-renewal. For patients with chronic lymphocytic leukemia (CLL), self-renewing, neoplastic B cells express ROR1 in 95% of cases. High-level leukemia cell expression of ROR1 is associated with an unfavorable prognosis. We conducted a phase 1 study involving 26 patients with progressive, relapsed, or refractory CLL. Patients received four biweekly infusions, with doses ranging from 0.015 to 20 mg/kg. Cirmtuzumab had a long plasma half-life and did not have dose-limiting toxicity. Inhibition of ROR1 signaling was observed, including decreased activation of RhoA and HS1. Transcriptome analyses showed that therapy inhibited CLL stemness gene expression signatures in vivo. Cirmtuzumab is safe and effective at inhibiting tumor cell ROR1 signaling in patients with CLL.

Conflict of interest statement

DECLARATION OF INTERESTS

Cirmtuzumab was developed by Dr. Kipps and his laboratory and licensed by the University of California to Oncternal Therapeutics, Inc. M.Y.C., J.E.C., and T.J.K. received honoraria and research funding from Pharmacyclics. C.J. received research funding from Johnson & Johnson and Celgene.

Copyright © 2018. Published by Elsevier Inc.

Figures

Figure 1.. Study Design, Pharmacokinetics, and ROR1 Pharmacodynamics

(A) Eligible patients had relapsed or refractory chronic lymphocytic leukemia and progressive disease with an indication for therapy. The short duration of cirmtuzumab administration (four biweekly infusions) was sufficient to determine the primary aim of the study, which was to determine the maximum tolerated dose or biologically active dose of cirmtuzumab. (B) Average number of ROR1 molecules per CLL cell (as indicated on the y axis) for individual patients treated in cohorts 1 through 7 and the average number of ROR1 molecules per CLL cell for all patients (±SD) as indicated on the x axis. A dotted line indicates the threshold for defining high-level ROR1, which was found to be associated with adverse prognosis (Cui et al., 2016). (C) Concentration of cirmtuzumab in plasma of representative patients. Concentration (μg/mL) is indicated on the y axis, and time (days) is indicated on the x axis. Arrows indicate days of infusion of cirmtuzumab. Time points collected after the last infusion of cirmtuzumab indicate that significant levels are present for approximately 100–150 days, with a half-life of 32.4 days. Values indicated were determined by interpolation using a four-parameter logistic nonlinear regression model compared to a standard curve generated by serial dilutions of a known concentration of cirmtuzumab mAb. (D) Wnt5a present in the plasma of patients with CLL who enrolled in the clinical trial (n = 26) is significantly higher than age-matched controls (n = 4) (unpaired Student’s t test, p = 0.0007). (E) The relative levels of ROR1 on CLL cells of treated patients were normalized with respect to expression levels of ROR1 found on paired pre-treatment samples over time in days after the initial infusion, as indicated on the x axis.

Figure 2.. Inhibition of Rho-GTPase Activation

(A) Levels of phosphorylated HS1 and total HS1 were assessed via immunoblot analyses on the CLL cells of patients in cohort 6 (16 mg/kg). There were marked reductions in the ratios of pHS1/HS1 within 24 hr of dosing. The pHS1/HS1 ratios rose after several months when the levels of cirmtuzumab in the plasma became undetectable. Reduction in pHS1/HS1 again were noted within 24 hr after re-treatment months later. (B) Activated RhoA from patients in cohort 6 (16 mg/kg) was assessed by western blot after pull-down of GTP-Rho complexed with rhotekin-Rho binding domain beads. There was a reduction in Rho-GTPase activation noted 24 hr after dosing. (C) Gene set enrichment analysis (GSEA) plots of genes activated by Rac1 and/or RhoA on pre-treatment versus paired post-cirmtuzumab CLL cells at D28 of each patient in cohort 6 (n = 3). Gene set size (SIZE), enrichment score, normalized ES (NES), and FDR q value (FDR q) are indicated. (D) GSEA on the transcriptomes of paired pre- and post-treatment CLL cells at D28 evaluating for pre- and post-treatment differences in the expression of the 11,699 set of genes associated with stemness (Malta et al., 2018).

Figure 3.. Clinical Effects

(A) Waterfall plot of best absolute lymphocyte count (ALC) on trial relative to pre-treatment baseline. (B) Waterfall plot of the sum of lymph node products assessed by radiography 2 months after the final infusion of cirmtuzumab. Patients who discontinued therapy prior to the completion of four doses or who initiated other forms of treatment prior to the 2-month response assessment were not required to have imaging and were not included in the final response evaluation. (C) Time to next treatment after CLL progression. Despite entering the study with progressive disease requiring therapy, patients did not require subsequent therapy for an extended duration after receiving four doses of cirmtuzumab. The median time to next treatment after CLL progression was 262 days. Four patients were censored because they started subsequent therapy prior to CLL progression and prior to completing the scheduled doses of cirmtuzumab. One patient switched therapy upon approval of venetoclax by the FDA; one patient had an isolated increase in the ALC (consistent with a redistributive lymphocytosis) but had concerns about progression and requested to come off trial to pursue other treatment options; one patient who noted eligibility for new clinical trial requested to switch treatment; and one patient had stable but bulky lymphadenopathy and chose to come off trial to seek other treatment. Three patients have not started subsequent therapy at the time of this analysis.