Receptor tyrosine kinase-like orphan receptor 1 (ROR1) has gained increasing attention. In recent years, Boehringer Ingelheim and Merck both added ROR1-directed antibody-drug conjugates (ADCs) to their pipeline via acquisition, highlighting the importance of ROR1 in oncology and its promising potential as an ADC target.

Here, we analyze the structural and functional features of ROR1 and explore the ROR1 ADC pipelines.

ROR1: Structure and Expression

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR receptor family, which contains two closely related type I transmembrane proteins, ROR1 and ROR2. The ROR family belongs to the Wnt signaling pathway and is closely related to MuSK (muscle-specific kinase) and Trk (tropomyosin) family receptors.

ROR1 consists of an extracellular region with one immunoglobulin (Ig), one frizzled (Fz) and one kringle (Kr) domain and an intracellular region harboring a pseudokinase domain.

Figure 1. Schematic domain structure of ROR1. Source: Reference [1]

ROR1 can play an important role in a variety of physiological processes, including the regulation of cell division, proliferation, migration, and chemotaxis, by mediating the signaling of non-canonical Wnt pathways, especially Wnt5a. Wnt5a is a typical activator of non-canonical Wnt signaling pathway, which is involved in the phosphorylation of NF-κB subunit p65, activates NF-κB pathway in tumor cells, and promotes cell migration and invasion, EMT, and cancer metastasis.

As a receptor for Wnt5a, ROR1 is involved in the activation of the tumor cell NF-κB pathway, which is responsible for the inflammatory response and immune regulation, and is constitutively activated in a wide range of tumor types. As shown in Figure 2, Wnt5a activates the receptors ROR1 or FZD5, leading to activation of Dvl2/3 and phosphorylation of Akt, which in turn promotes the phosphorylation of IKKα to activate the IKK complex, an entity responsible for the degradation of IκBα and the phosphorylation of the NF-κB subunit p65. Phosphorylated p65 is transferred to the nucleus to promote the transcription and expression of target genes, including Wnt5a, whose secretion promotes a new round of autonomic feedback loop. Activation of the ROR1/Akt/p65 pathway in the autonomic feedback loop further promotes the secretion of pro-inflammatory factors (e.g., IL-6) and chemokines (e.g., CCL2).

​Figure 2. The Wnt5a/NF-κB signaling pathway. Source: reference [3]

Role of ROR1 in Malignant Tumors

ROR1 is highly expressed during early embryonic development and is involved in the regulation of cell division, proliferation and migration, and in the generation of organs such as nerves, bones and blood vessels. Along with the process of fetal development, the expression of ROR1 gradually decreases. In childhood and adult stages, ROR1 expression is low or absent in almost all normal tissues.

In contrast to normal tissues, ROR1 is highly expressed in a variety of cancers, including hematological cancers such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and solid tumors (ovarian, breast, prostate, lung, melanoma, and colorectal cancers, etc.).

Immunohistochemical staining showed high expression of ROR1 in pancreatic cancer specimens, with positive staining localized in the cytoplasm as well as in the nucleus of the cancer cells (Figures C and D); in normal pancreatic tissue samples, ROR1 was expressed exclusively in pancreatic islet cells; ROR1 expression was also observed in the stomach region (Figure J); and ROR1 was not detected in normal tissue samples from the heart (Figure E), liver (Figure F), brain (Figure G), kidneys (Figure H) or lungs (Figure I).

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Figure 3. Immunohistochemistry staining of human tissues. Source: reference [4]

In lung cancer, ROR1 is involved in the activation of c-Src and MET, leading to the inhibition of apoptosis in tumor cells, and ROR1 has also been found to be a scaffolding protein for cavin-1 and caveolin-1, which activate AKT in lung adenocarcinoma.

In breast cancer, ROR1 promotes activation of the PI3K/AKT pathway, and high ROR1 expression is associated with more severe disease progression. In conclusion, the aberrant expression of ROR1 and associated pro-growth signaling events observed in many types of malignant tumors make ROR1 an attractive therapeutic target for anti-cancer drug development.

Advances in ROR1-targeted ADCs

Several companies are currently developing anti-cancer therapies targeting ROR1, including monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, and CAR-T therapies, as well as other therapeutic modalities. Among them, ADC drugs are progressing faster and show good application prospects.

Table. ROP1 ADC under investigation

Zilovertamab vedotin (MK-2140,VLS-101)

MK-2140 is one of the fastest progressing ROR1 ADCs and was previously developed by VelosBio, which was acquired by Merck in November 2020 for $2.75 billion in cash.

MK-2140 conjugates UC-961, a monoclonal antibody targeting ROR1, to Monomethyl auristatin E (MMAE), a microtubule-targeting agent , via proteolytically cleavable linker mc vc PAB. UC-961 is a humanized antibody that binds to the intradomain epitope of ROR1 with high affinity (Kd=2nM) and has direct cytotoxic activity against ROR1-positive tumor cells by blocking the binding of Wnt5a to ROR1.

Due to the unique epitope recognized by UC-961, this antibody triggers significant internalization of ROR1 upon binding, facilitating the delivery of a toxic payload specifically to ROR1-positive tumor cells. MK-2140 has been shown to be effective at inducing cell cycle arrest and apoptosis in an in vitro model, and effective at blocking tumor growth in vivo in a xenograft model.

Figure 4. MK-2140 mechanism, source: Vaisitti Tiziana. Blood, 2021

An open-label, single-arm Phase II clinical trial (waveLINE-004, NCT05144841) enrolling 40 patients who had received at least second-line therapy (as of 2022.11.16) was designed to evaluate the efficacy and safety of MK-2140 (2.5 mg/kg) monotherapy for the treatment of patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). The results showed that after treatment with MK-2140, patients had an ORR of 30% (6/20), including 2 CRs and 4 PRs.

In a phase I study of hematologic malignancies (NCT03833180), MK-2140 was well tolerated and effective in patients with advanced mantle cell lymphoma (MCL) or diffuse large B-cell lymphoma (DLBCL), with an ORR of 47% (7/15) in the MCL group, including 3 CRs and 4 PRs, and an ORR of 80% (4/5) in the DLBCL group, including 2 CRs and 2 PRs.

A Phase 2 study (NCT04504916) in patients with solid tumors, including breast and lung cancer, is also currently underway.

NBE-002

On December 11, 2020, Boehringer Ingelheim announced the acquisition of all shares of NBE-Therapeutics for €1.18 billion ($1.43 billion, including clinical and regulatory milestone payments), taking the company's unique iADC technology platform and corresponding pipeline.

NBE-002, NBE-Therapeutics' fastest progressing ADC drug targeting ROR1, is produced by enzymatic conjugation of a humanized antibody (huXBR1-402) to the anthracycline PNU-159682 via sortasea A-mediated site-specific transpeptidation reaction at a predefined drug-antibody ratio (DAR), resulting in a purer and more homogenous product with more stable biophysical and pharmacokinetic profiles. NBE-002 not only has direct anti-tumor activity, but also increases T-cell infiltration into tumors and converts "cold" tumors into "hot" tumors, causing them to respond to checkpoint inhibitors against PD-1 and CTLA-4. 

Figure 5. NBE-002 pipeline, source: https://nbe-therapeutics.com/

NBE-002 is currently in a Phase 1/2 clinical trial (NCT04441099) to evaluate safety and tolerability in patients with advanced solid tumors, particularly triple-negative breast cancer.

LCB71 (CS5001)

LCB71 is being developed by two South Korean biopharmaceutical companies, LegoChem Biosciences and ABL Bio. In October 2020, CStone Pharmaceuticals introduced LCB71 for an upfront payment of $10 million and milestone payments of up to $353 million and additional tiered royalties. Under the agreement, CStone obtains the exclusive global right to lead development and commercialization of LCB71 outside the Republic of Korea.

LCB71 (CS5001) is an ADC consisting of a human monoclonal antibody targeting ROR1. This ADC drug is uniquely designed to incorporate a unique β-glucuronide linker and a  pyrrolobenzodiazepine (PBD) prodrug through site-specific conjugation technology known as ConjuALL™.  Only after reaching the tumor, the linker and prodrug are cleaved to release the PBD toxin, resulting in lethal DNA cross-links in cancer cells. The use of the linker plus PBD prodrug effectively helps addressing the toxicity problem associated with traditional PBD payloads, leading to a better safety profile.

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Figure 6. CS5001, source: https://www.cstonepharma.com/en/  

CS5001 is currently in Phase I clinical trial in the United States, Australia, and mainland China, with preliminary data anticipated before the end of 2023. CS5001 demonstrated significant anti-tumor activity in both Jeko-1 (mantle cell lymphoma) and MDA-MB-231 (breast cancer) xenograft tumor models in a dose-dependent manner. It  is a promising drug candidate with precision therapeutic potential in both hematological tumors and malignant solid tumors with high ROR1 expression.

Conclusion

ROR1 is widely expressed in various solid and hematological tumor cells, making it an attractive and important target for ADC drug development. Due to the unique site-specific conjugation strategy, both NBE-002 and LCB71 are homogeneous ADCs with a defined DAR, which facilitates manufacturing. In addition, the stable linkers in both ADCs provide a better pharmacokinetic profile. Although the product of VLS-101 is not homogeneous, a phase 1 study has demonstrated safety and clinical efficacy in patients with MCL and DLBCL, and it is the leading anti-ROR1 ADC currently in a phase 2 clinical trial. Regardless, all 3 anti-ROR1 ADCs look very promising, and the 3 recent major commercial deals may facilitate the rapid development of multiple therapeutic modalities targeting ROR1.
 

References:
[1] Zhao Y, Zhang D, Guo Y, Lu B, Zhao ZJ, Xu X, Chen Y. Tyrosine Kinase ROR1 as a Target for Anti-Cancer Therapies. Front Oncol. 2021 May 28;11:680834. doi: 10.3389/fonc.2021.680834. PMID: 34123850; PMCID: PMC8193947.
[2] Peng H. Perspectives on the development of antibody-drug conjugates targeting ROR1 for hematological and solid cancers. Antib Ther. 2021;4(4):222-227. Published 2021 Oct 15. doi:10.1093/abt/tbab023
[3] Lopez-Bergami, P., Barbero, G. The emerging role of Wnt5a in the promotion of a pro-inflammatory and immunosuppressive tumor microenvironment. Cancer Metastasis Rev 39, 933–952 (2020). https://doi.org/10.1007/s10555-020-09878-7
[4] Gohil SH, Paredes-Moscosso SR, Harrasser M, Vezzalini M, Scarpa A, Morris E, Davidoff AM, Sorio C, Nathwani AC, Della Peruta M. An ROR1 bi-specific T-cell engager provides effective targeting and cytotoxicity against a range of solid tumors. Oncoimmunology. 2017 May 17;6(7):e1326437. doi: 10.1080/2162402X.2017.1326437. PMID: 28811962; PMCID: PMC5543882.

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