Identifying targets that are expressed on tumor cells but not or lowly expressed in normal cells is crucial for tumor immunity. ROR1 is a highly promising drug target, exhibiting differentiated high expression in various solid tumors and hematologic malignancies, while not expressing in adult healthy tissues. This suggests that ROR1 holds the potential to become a broad-spectrum anti-cancer drug target.
1. Structure and Expression of ROR1
ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a type I transmembrane protein belonging to the receptor tyrosine kinase-like orphan receptor (ROR) family. The family is named because its ligands have not been identified for many years. Now, it is known that the ROR family members are receptors for Wnt family signaling molecules Wnt5a/b and Wnt16, with Wnt5a being the primary ligand.
As shown in Figure 1, ROR1 is a single-pass transmembrane receptor consisting of three main parts: the extracellular domain (ECD), transmembrane domain, and intracellular domain. The extracellular domain (ECD) can be further divided into three structural domains: immunoglobulin-like domain (Ig-Like), frizzled-like domain (FZD), and kringle domain (KD). FZD can bind to the ligand Wnt5a to regulate non-classical Wnt signaling. KD can mediate the interaction of ROR1 with other receptors such as ROR2. The intracellular domain (ICD) contains a tyrosine kinase-like domain, two serine/threonine-rich domains, and a proline-rich domain.
Figure 1 The structure of ROR1 [1]
ROR1 is highly expressed during early embryonic development, playing a role in the regulation of cell division, proliferation, and migration. It also contributes to the formation of organs such as the nervous system, skeleton, and vasculature. As embryonic development progresses, the expression of ROR1 gradually decreases. In children and adults, ROR1 is expressed at very low levels or is not expressed in almost normal tissues. In contrast, ROR1 is expressed in various cancers, including hematological malignancies such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and solid tumors (ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, etc.). The expression characteristic of ROR1 makes it an ideal drug target for cancer therapy.
2. ROR1 Signaling and Tumorigenesis
Studies indicate that the high expression of ROR1 in tumor cells contributes to increased cell survival, promoting tumor cell proliferation, migration, and chemotaxis. Blood cancers and solid tumors expressing ROR1 exhibit high self-renewal potential, demonstrating higher survival and migration rates, and are associated with poor prognosis.
The Wnt5a-ROR1 pathway plays a crucial role in tumorigenesis and drug resistance by engaging in the non-canonical Wnt pathway and upregulating YAP/TAZ transcription and/or the multi-subunit complex protein BMI-1. Following the binding of Wnt5a to ROR1, intracellular signal transduction is induced, activating AKT, subsequently phosphorylating BMI-1, promoting genomic instability, cancer cell proliferation, and drug resistance. Upon binding of Wnt5a to the ROR1/FZD complex, Gα12/13 activation of RhoA occurs, inhibiting Lats1/2 activity, leading to YAP/TAZ dephosphorylation and nuclear translocation. The binding of YAP/TAZ to TEADs induces the transcription of genes associated with stem cells and tumorigenesis. The increased transcription of YAP/TAZ, in turn, upregulates the expression of ROR1.
Figure 2 ROR1 signaling pathway and tumor [2]
3. ROR1 and Cancer Immunotherapy
Current antibody-based immunotherapies targeting ROR1 include ROR1 monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific T-cell engagers (BiTEs), and chimeric antigen receptor (CAR) T-cell therapies. Apart from immunotherapies, there are also small molecule inhibitors targeting ROR1. Here, we will focus on understanding the clinical research progress of ROR1-targeted immunotherapies.
| Drug Type | Name | Other Names | Stage | Company |
| ROR1 ADC | Zilovertamab vedotin | MK-2140; VLS-101 | Phase III | Merck |
| LCB-71 | CS-5001; LCB-71; ABL-202 | Phase I | CStone Pharmaceuticals | |
| NBE-002 | BI-3702025; NBE-002 | Phase II | NBE-Therapeutics | |
| CAR-T | LYL-797 | LYL-797 | Phase I | Lyell Immunopharma Inc |
| PRGN-3007 | PRGN-3007 | Phase I | Precigen Inc | |
| ONCT-808 | ONCT-808 | Phase II | Oncternal Therapeutics Inc | |
| BiTE | NVG-111 | NVG-111 | Phase II | NovalGen Ltd |
| EMB-07 | EMB-07 | Phase I | EpimAb | |
| Tetra-specific | GNC-035 | GNC-035 | Phase I | Systimmune |
| Monoclonal antibody | Zilovertamab | UC-961 | Phase III | Oncternal Therapeutics Inc |
3.1 ADC
ADC is a targeted therapeutic drug that links cytotoxic small molecules to monoclonal antibodies through a chemical linker. Antibody-drug conjugates selectively deliver toxic substances to antigen-positive cancer cells.
- Zilovertamab vedotin (VLS-101)
Zilovertamab vedotin was originally developed by VelosBio, which was acquired by Merck for $2.75 billion on November 5, 2020. Zilovertamab vedotin is an ADC drug targeting ROR1. It involves a monoclonal antibody targeting ROR1 conjugated to monomethyl auristatin E (MMAE) through a protease-cleavable linker. MMAE is a drug that inhibits cell division by blocking microtubule polymerization. For more information about MMAE, click here>>
Zilovertamab vedotin is currently in Phase III with a mean DAR (Drug-to-Antibody Ratio) of 4. It is intended for the treatment of patients with hematologic malignancies and solid tumors. In the 2023 ASH (American Society of Hematology) conference, Merck presented the latest results from the Phase II Waveline-004 study of Zilovertamab vedotin in relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients. In this updated Waveline-004 data, Zilovertamab vedotin exhibited clinically meaningful anti-tumor activity in R/R (Relapsed or Refractory) DLBCL patients, mainly those with disease progression after autologous stem cell transplantation (ASCT) or chimeric antigen receptor T-cell (CAR-T) therapy or those not eligible for ASCT and/or CAR T-cell therapy. The safety of Zilovertamab vedotin was manageable and consistent with the known safety of MMAE-containing drugs.
- LCB71 (CS5001)
LCB71 was initially developed by two South Korean biopharmaceutical companies, LegoChem Biosciences and ABL Bio. On October 29, 2020, CStone Pharmaceuticals acquired LCB71 for an upfront payment of $10 million and milestone payments totaling $353.5 million. LCB71 is an ADC composed of a human monoclonal antibody targeting ROR1. It is currently in Phase I, targeting indications including solid tumors and hematologic malignancies.
This ADC features a unique design. Through targeted coupling technology, LCB71 combines a distinctive β-glucosidic acid linker and a pyrrolobenzodiazepine (PBD) dimer of the prodrug. Both the linker and the prodrug can be cleaved by lysosomal β-glucuronidase, which is overexpressed in many cancer cells. This enables LCB71 to release the PBD prodrug only after reaching the tumor, where the linker is cleaved, activating the PBD prodrug within tumor cells and effectively killing them. This “dual-control” mechanism of linker and prodrug addresses typical toxicity issues associated with traditional PBD payloads, providing improved safety.
Multi-regional Phase I clinical trials of LCB71 have progressed with dose escalation in the United States and Australia, demonstrating good safety and tolerability. On April 24, 2023, CStone Pharmaceuticals announced the completion of the first patient enrollment in the multi-regional Phase I clinical trial of LCB71 in China. Dr. Yang Jie, CEO of CStone Pharmaceuticals, stated that this marked another milestone in their Pipeline 2.0 strategy, and with the inclusion of the Chinese research center, the progress of this significant project will accelerate further.
- NBE-002
NBE-002 was initially developed by NBE-Therapeutics, and on December 10, 2020, Boehringer Ingelheim announced the acquisition of NBE-Therapeutics for approximately $1.5 billion, gaining control of NBE-002. NBE-002 is an ADC composed of a novel anti-human ROR1 monoclonal antibody (HuXBR1-402) and a derivative of the anthracene class drug PNU-159682. HuXBR1-402 is a rabbit/human chimeric monoclonal antibody selected from a rabbit antibody library through phage display.
NBE-002 has demonstrated significant anti-tumor activity in patient-derived xenograft models of breast cancer, lung adenocarcinoma, ovarian cancer, and sarcoma. The drug is currently undergoing a Phase 1/2 clinical study in the United States for late-stage solid tumor patients. The study aims to evaluate recommended doses for further clinical development, safety, tolerability, anti-tumor activity, immunogenicity, pharmacokinetics, and pharmacodynamics of NBE-002 in late-stage solid tumor patients.
3.2 ROR1 CAR-T Cell Therapy
The development of CAR-T cell therapy targeting ROR1 serves as a targeted approach to selectively eliminate ROR1-positive cancer cells.
- LYL-797
LYL-797 is a CAR-T cell therapy targeting ROR1 developed by Lyell Immunopharma. It incorporates both genetic and epigenetic reprogramming technologies, namely Gen-R and Epi-R, to overcome challenges associated with CAR-T cell therapy in solid tumors. The ROR1-specific CAR includes a single-chain variable fragment (scFv) derived from the R12 rabbit monoclonal antibody, providing high specificity for recognizing and binding human ROR1.
Gen-R is an ex vivo gene reprogramming technology designed to modify CAR-T cells for overexpression of c-Jun. Dysregulation of activating protein 1 (AP-1) is associated with CAR-T cell exhaustion, and studies suggest that overexpression of c-Jun makes CAR-T cells less prone to exhaustion, enhancing anti-tumor efficacy and persistence in preclinical models of hematologic and solid tumors. Epi-R is a proprietary optimization manufacturing process aimed at maintaining the stem cell phenotype and functionality of T cell products. In preclinical studies, LYL797 cells reprogrammed with Gen-R and Epi-R exhibited improved functional activity in the presence of ROR1+ tumor cells compared to traditional ROR1 CAR-T cells [3].
LYL797-101 is currently in a Phase I clinical trial (NCT05274451) aimed at evaluating the safety, pharmacokinetics (PK), and anti-tumor activity of LYL797 in ROR1+ triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). The trial is currently in the recruitment phase.
- PRGN-3007
PRGN-3007 is a multi-gene, autologous CAR-T cell therapy developed by Precigen using the non-viral UltraCAR-T™ treatment platform. Engineered design involves a single multi-gene transposon plasmid, utilizing the Sleeping Beauty (SB) system to co-express a chimeric antigen receptor targeting ROR1, membrane-bound interleukin-15 (mbIL15), a control switch, and a novel mechanism gene expression inhibiting PD-1. Currently, PRGN-3007 is undergoing a Phase 1/1b clinical trial in the United States to assess its tolerability for ROR1+ hematologic and solid tumors.
- ONCT-808
ONCT-808 is an autologous CAR-T cell therapy targeting ROR1 developed by Oncternal Therapeutics. It is currently undergoing a Phase 1/2 clinical trial (NCT05588440) for the treatment of relapsed or refractory aggressive B-cell lymphoma patients, including those who have previously failed CD19 CAR-T therapy. The clinical trial aims to assess the safety and efficacy of ONCT-808 in aggressive BCL patients. The study comprises two distinct stages: the first stage involves dose escalation, and the second stage involves dose expansion. The trial is currently in the recruitment phase.
3.3 BiTE antibody
- NVG-111
NVG-111 is a humanized ROR1 and CD3 BiTE developed by NovalGen. It is a bispecific antibody composed of ROR1 antibody and single-chain variable fragments (scFv) targeting CD3. NVG-111 targets malignant cells that express ROR1 and utilizes the inherent cytotoxicity of T cells, inducing tumor cell death in vivo and in vitro. Preclinical studies have demonstrated that NVG-111 exhibits potent T cell-mediated cytotoxicity in CLL and solid tumors. NovalGen presented Phase I first-in-human dose-escalation study results for NVG-111 at the 2023 ASH meeting. The disclosed data provide clinical proof-of-concept for selective targeting of ROR1 by TCE, demonstrating objective evidence of anti-tumor activity even in CLL patients with known T cell functional defects, with sustained responses. Safety is consistent with the proposed mechanism of action.
- EMB-07
EMB-07 is a T cell-redirection bispecific antibody targeting ROR1 and CD3, developed by EpimAb. Designed using EpimAb’s proprietary FIT-Ig (Fabs-In-Tandem Immunoglobulin) technology, EMB-07 binds to tumor-associated antigens (TAA) on the surface of cancer cells, recruiting and activating CD3-expressing T lymphocytes to enhance T cell cytotoxicity. In preclinical experiments, EMB-07 has demonstrated promising efficacy and safety. It is currently in Phase I for the treatment of lymphomas.
3.4 Tetra-Specific Antibody
GNC-035, developed by Systimmune, is an octavalent Tetra-specific antibody targeting PD-L1/CD3/4-1BB/ROR1. Currently undergoing Phase I clinical trials, preliminary results presented at the 2023 AACR indicate that GNC-035 holds promise in mediating the regression of ROR1+ cancers, overcoming TCR-based immune escape, and reversing T cell immunosuppression in the tumor microenvironment [4].
3.5 Monoclonal Antibody
Zilovertamab (formerly cirmtuzumab or UC-961), a monoclonal antibody developed by Oncternal Therapeutics, specifically targets ROR1. By blocking WNT5A signaling upon binding to ROR1, Zilovertamab inhibits tumor cell proliferation, migration, and survival, inducing tumor cell differentiation. Oncternal Therapeutics is conducting Phase 1/2 clinical studies evaluating Zilovertamab in combination with Imbruvica for MCL, CLL, and MZL patients. Additionally, ongoing clinical trials explore Zilovertamab in combination with Venetoclax for R/R CLL (Phase 2, NCT04501939) and with docetaxel for metastatic castration-resistant prostate cancer (Phase 1b, NCT05156905). In April 2023, Oncternal Therapeutics strategically adjusted priorities, closing the Phase 3 ZILO-301 and Phase 1/2 CIRM-001* studies to extend cash runway to 2025 and focus on the clinical development of ONCT-808 and ONCT-534, emphasizing that this decision was not based on safety or efficacy concerns for Zilovertamab.
4. DIMA’s ROR1 Products Accelerate Drug Development
DIMA Biotech is a biotechnology company committed to advancing preclinical research for potential drug targets. Specializing in cutting-edge functional membrane protein development, single B-cell lead antibody discovery, and advanced antibody engineering and functional validation platforms, DIMA offers a comprehensive array of products and services centered around the ROR1 target. This includes diverse active proteins, flow-validated monoclonal antibodies, and benchmark reference antibodies. Services encompass humanization, mature affinity development, and the removal of PTM risk sites. Additionally, DIMA expedites ROR1 target drug development with a prevalidated B-cell seed library, facilitating the screening of lead antibody molecules in as little as 20 days.
- Recombinant Protein
Human ROR1 Protein, His Tag (PME100399)
Figure 3. Validation data of purified Human ROR1 Protein, His Tag (PME100399). Human ROR1 Protein, His Tag on SDS-PAGE (left); ROR1 Protein (PME100399) can bind Anti-ROR1 Neutralizing antibody (BME100073) in a linear range of 0.64-16 μg/ml (right).
More Recombinant Proteins:
Human ROR1(838-937) Protein, hFc Tag ( PME101444)
Human ROR1 (312-391) Protein, hFc Tag ( PME100630)
Mouse ROR1 Protein, His Tag (PME-M100069)
- Recombinant Rabbit Monoclonal Antibody
Anti-ROR1 antibody(DM149); Rabbit mAb (DME100149)
Figure 4. Validation data of Anti-ROR1 antibody (DME100149); Human ROR1 protein (PME100399) can bind Rabbit anti-ROR1 monoclonal antibody (DME100149) in a linear range of 1-50 ng/ml (left); Flow cytometry analysis with Anti-ROR1 (DME100149) on Expi293 cells transfected with human ROR1 (Blue histogram) or Expi293 transfected with irrelevant protein (Red histogram), and Isotype antibody on Expi293 transfected with irrelevant protein (Orange histogram) (right).
- ELISA&FC Validated Benchmark Antibody
Anti-ROR1(NBE 002 biosimilar) mAb (BME100191)
Figure 5. Validation data of Anti-ROR1(NBE 002 biosimilar) mAb (BME100191); Anti-ROR1(NBE 002 biosimilar) mAb (BME100191) can bind Anti-ROR1(NBE 002 biosimilar) mAb (BME100191) in a linear range of 0.64–16 ng/mL (left); Flow cytometry analysis with 1μg/mL Anti-ROR1(NBE 002 biosimilar) mAb (BME100191) on Expi293 cells transfected with Human ROR1 protein (Blue histogram) or Expi293 transfected with irrelevant protein (Red histogram),and isotype antibody on Expi293 transfected with irrelevant protein (Orange histogram) (right).
More Benchmark Antibody:
Anti-ROR1 (zilovertamab biosimilar) mAb (BME100073)
Reference:
[1]Zhao Yuming, Zhang Dengyang, Guo Yao et al. Tyrosine Kinase ROR1 as a Target for Anti-Cancer Therapies. [J] .Front Oncol, 2021, 11: 680834.
[2]Karvonen Hanna, Barker Harlan, Kaleva Laura et al. Molecular Mechanisms Associated with ROR1-Mediated Drug Resistance: Crosstalk with Hippo-YAP/TAZ and BMI-1 Pathways. [J] .Cells, 2019, 8: undefined.
[3]Spencer Park, Courtney Simianer, Sydney Spadinger, et al; Abstract 2754: LYL797, a ROR1 CAR T-cell therapy with genetic and epigenetic reprogramming for solid tumors. Cancer Res 15 June 2022; 82 (12_Supplement): 2754.
[4]Jahan Salar Khalili, et al; Abstract 5679: Tetra-specific antibody GNC-035: guidance and navigation control (GNC) molecule development for treatment of ROR1+ malignancies. Cancer Res 1 April 2023; 83 (7_Supplement): 5679.