Finding targets that are expressed on tumor cells and not or underexpressed on normal cells is critical for tumor immunity. Ror1 is a very promising drug target, and it has differentiated high expression in a variety of solid tumors and hematologic malignancies, but is not expressed in ** healthy tissues. This means that Ror1 is expected to become a new drug target with broad-spectrum anti-cancer potential.
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) 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 recognized for many years. Ror family members are now known to be receptors for the Wnt family signaling molecules Wnt5A B and Wnt16, with Wnt5A as the primary ligand.
As shown in the figure below, Ror1 is a single-shot transmembrane receptor that consists of three main parts: extracellular, transmembrane, and intracellular. The extracellular region (ECD) can be further divided into three domains: immunoglobulin-like domain (IG-like), coiled domain (FZD), and cyclic domain (KD). FZD binds to the ligand WNT5A to modulate non-canonical WNT signaling. KD mediates the interaction of Ror1 with other receptors such as Ror2. The intracellular domain (ICD) contains a tyrosine kinase-like domain, two serine-rich threonine-rich domains, and a proline-rich domain.
figure 1 the structure of ror1[1]
Ror1 is highly expressed during early embryonic development and can participate in the regulation of cell**, proliferation, and migration activities, as well as the generation of organs such as nerves, bones, and blood vessels. As the fetus progresses, the expression of Ror1 gradually decreases. In the childhood and ** stages, Ror1 is expressed with low or no expression in almost all normal tissues. In contrast, ROR1 is expressed in a variety of cancers, including hematologic cancers such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and solid tumors (ovarian, breast, prostate, lung, melanoma, and colorectal cancer, among others). The expression properties of Ror1 make this protein an ideal drug target for cancer**.
Studies have shown that the high expression of Ror1 in tumor cells can help improve the survival rate of tumor cells and promote the proliferation, migration and chemotaxis of tumor cells. Ror1-expressing hematologic and solid tumors have a high potential for self-renewal, exhibit higher survival and migration, and are associated with poor prognosis.
Wnt5A-Ror1 is involved in and upregulates the expression of YAP TAZ transcription and/or polycomb complex protein BMI-1 through the atypical Wnt pathway, which plays an important role in tumorigenesis and drug resistance. WNT5A binds to Ror1 to induce intracellular signaling, activate AKT, and then phosphorylate BMI-1, promoting genomic instability, cancer cell proliferation, and drug resistance. Upon binding to the Ror1 FZD complex, Wnt5A activates RHOA by G1213 binding, inhibiting LAT122 activity, resulting in YAP TAZ dephosphorylation and nuclear translocation. YAP TAZ binds to TEADS to induce stem cells and transcription of tumorigenesis-related genes. The increase in YAP TAZ transcription, in turn, upregulates the expression of Ror1.
figure 2 ror1 signaling pathway and tumor [2]
Antibody-based immunization** currently in development targeting Ror1 includes RoR1 monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies (BITES), and chimeric antigen receptor (CAR) T cells. Of course, in addition to immunity**, there are also small molecule inhibitors targeting ROR1, and here we focus on the clinical research progress of targeting ROR1 immunity**.
3.1 Antibody-drug conjugates (ADCs).
ADC is a targeted** drug that conjugates a small cytotoxic molecule to a monoclonal antibody through a chemical linker. Antibody-drug conjugates selectively deliver toxic substances to antigen-positive cancer cells.
zilovertamab vedotinvls-101
The original company of zilovertamab vedotin is velosbio, which was approved by Merck on November 5, 2020 with 27$500 million acquisition. Zilovertamab vedotin is a Ror1-targeting ADC drug conjugated to monomethyl auristatin E (MMAE) by a proteolytically cleavable linker targeting Ror1. MMAE is a drug that inhibits cells by blocking tubulin polymerization, and the highest R&D status of MMAE - the "group pet" toxin capacity of ADC zilovertamab vedotin is currently Phase III, with a mean DAR of 4, for patients with hematologic malignancies and solid tumors. At the 2023 ASH meeting, Merck disclosed updated results from the Phase 2 W**eline-004 study of zilovertamab vedotin*** or refractory diffuse large B-cell lymphoma (DLBCL). In this updated W**eline-004 result, zilovertamab vedotin still demonstrated clinically meaningful anti-tumor activity in patients with R r DLBCL. These patients are predominantly patients with Rr DLBCL who have progressed after autologous stem cell transplantation (ASCT) or chimeric antigen receptor (CAR-T) cells** or who are not eligible for ASCT and/or CAR T cells**,** with limited options. The safety profile of zilovertamab vedotin is manageable and consistent with that of known MMAE-containing agents.
lcb71 (cs5001)
LCB71 was originally developed by Legochem Biosciences and ABL Bio, two South Korean biopharmaceutical companies, with an upfront payment of $10 million +3US$53.5 billion milestone amount for the introduction of LCB71. LCB71 is an ADC consisting of a human monoclonal antibody targeting Ror1. At present, the highest stage of development is Phase I, targeting indications including solid tumors and hematologic malignancies.
The ADC drug has a unique design in which LCB71 combines a unique -glucuronide linker and a pyrrolobenzodiazepine (PBD) protoxin dimer through directional conjugation technology. Both linkers and protoxins can be cleaved by lysosome-glucuronidase, which is overexpressed in many cancer cells. As a result, LCB71 is cleaved by its linker to release the PBD protoxin only after reaching the tumor, and then the PBD protoxin is activated within the tumor cells, thereby killing the tumor cells. This "dual control" mechanism of linker plus protoxin effectively solves the typical toxicity problems associated with traditional PBD loading, and has a better safety profile.
A multi-regional Phase I clinical trial of LCB71 has advanced with dose escalation in the U.S. and Australia and has demonstrated favorable safety and tolerability. On April 24, 2023, CStone announced that it has completed the first patient enrollment in the multi-regional Phase I clinical trial of LCB71 in China. Dr. Yang Jie, Chief Executive Officer of CStone Pharmaceuticals, said, "This marks our Pipeline 20 Another milestone in the strategy. With the addition of the China Research Center, the pace of this important project will be further accelerated.
nbe-002
NBE-002 was originally developed by NBE-Therapeutics, and on December 10, 2020, Boehringer Ingelheim announced the acquisition of NBE-Therapeutics for approximately $1.5 billion**, resulting in NBE-002. NBE-002 is an ADC consisting of a novel anti-human Ror1 monoclonal antibody antibody XBR1-402 and a derivative of the anthracycline PNU-159682. HUXBR1-402 is a rabbit-human chimeric monoclonal antibody screened from the rabbit antibody library by phage display.
NBE-002 has shown significant antitumor activity in patient-derived xenograft models of breast, lung, ovarian, and sarcoma. The drug is currently being conducted in a first-in-human Phase 1 2 study in patients with advanced solid tumors in the U.S., designed to evaluate the recommended dose, safety, tolerability, anti-tumor activity, immunogenicity, pharmacokinetics and pharmacodynamics of NBE-002 for further clinical development in patients with advanced solid tumors.
3.2 CAR-T cells for ROR1**.
Constructing CAR-T cells** targeting Ror1 can also play a role in targeted killing of Ror1-positive cancer cells.
lyl-797
LYL-797 is a ROR1-targeting CAR-T cell** developed by Lyell ImmunoPharma, which combines genetic and epigenetic reprogramming techniques GEN-R and EPI-R to overcome the barrier of CAR-T cells** in solid tumors. The ROR1-specific CAR contains a single-stranded variable fragment (SCFV) derived from the R12 rabbit monoclonal antibody that highly specifically recognizes and binds to human Ror1.
Gen-R is an ex vivo gene reprogramming technique that engineers CAR-T cells to overexpress C-Jun. The dysregulation of activator protein 1 (AP-1) is associated with CAR-T cell exhaustion, and studies have shown that overexpression of C-Jun can make CAR-T cells less susceptible to depletion, thereby enhancing anti-tumor efficacy and persistence in preclinical models of hematological malignancies and solid tumors. EPI-R is a proprietary, optimized manufacturing process that maintains the stem cell phenotype and function of T cell products. In preclinical studies, lyl797 cells reprogrammed with GEN-R and EPI-R showed improved functional activity in the presence of Ror1+ tumor cells compared to conventional ROR1 CAR-T cells [3].
LYL797-101 is currently enrolled in a Phase I clinical trial (NCT05274451) to evaluate the safety, pharmacokinetics (PK) and antitumor activity of LYL797 in ROR1+ triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC).
prgn-3007
PRGN-3007 is a multigenic, autologous CAR-T cell generated by Precigen based on the non-viral UltraCAR-T** platform. PRGN-3007 is engineered using a single multigene transposon plasmid to co-express novel mechanistic gene expression targeting the ROR1 chimeric antigen receptor, membrane-bound interleukin-15 (MBL15), control switches, and intrinsically blocking PD-1 gene expression using the Sleeping Beauty Platform (SB System). Currently, PRGN-3007 is in Phase 1 1B clinical trials in the U.S. for ROR1+ hematologic malignancies and solid tumors to test the drug's tolerability.
onct-808
ONCT-808 is an autologous CAR-T cell** targeting Ror1 developed by Oncternal Therapeutics, Inc., which is currently in a Phase 1 2 clinical trial (NCT05588440) in patients with *** or refractory** aggressive B-cell lymphoma, including those who have previously failed CD19 CAR T**. This clinical trial is designed to evaluate the safety and efficacy of ONCT-808 in patients with aggressive BCL. The study consists of two distinct phases: Phase I dose escalation and Phase II dose escalation, which is currently in a state of enrollment.
3.3 Bispecific antibodies.
nvg-111
NVG-111 is a humanized Ror1 and CD3 bit developed by Novalgen, which is a bispecific antibody concatenated from SCFV of Ror1 and CD3. NVG-111 targets RoR1-positive malignant cells and harnesses the intrinsic cytotoxic effects of T cells, thereby inducing tumor cell death in vivo and in vitro. Preclinical studies have shown that NVG-111 has favorable T cell-mediated cytotoxicity in CLL and solid tumors. Novalgen presented results from a first-in-human dose escalation study of NVG-111 in Phase I at 2023 ASH. The published data provides a clinical proof of concept for TCE selectively targeting Ror1, providing objective evidence of anti-tumor activity with durable response even in CLL patients with known T-cell dysfunction. Safety is consistent with the mechanism of action.
emb-07
EMB-07 is a T cell redirecting bispecific antibody developed by EPIMAB targeting Ror1 and CD3. EMB-07 is a bispecific antibody based on Epimab's proprietary FIT-IG (Fabs-In-Tandem immunoglobulin) technology. EMB-07 binds to TAA on the surface of tumor cells to recruit and activate CD3-expressing T lymphocytes, thereby promoting the cytotoxicity of T lymphocytes. In preclinical trials, EMB-07 has shown great potential for efficacy and safety. It is currently in the stage I stage of ** lymphoma.
3.4 Quadrispecific antibodies.
GNC-035 is an octavalent tetraspecific antibody developed by Systimmune, targeting PD-L1 CD3 4-1BB Ror1 and is currently in Phase I clinical trials. Systimmune presented preclinical results of GNC-035 in the 2023 AACR demonstrating the potential of GNC-035 to mediate RR1+ cancer regression, overcome TCR-based immune evasion, and reverse T cell immunosuppression in the tumor microenvironment [4].
3.5 Monoclonal antibody drugs.
Zilovertamab (formerly known as Cirmtuzumab or UC-961) is a monoclonal antibody drug targeting Ror1 developed by Oncternal Therapeutics, which, when it binds to Ror1, blocks WNT5A signaling, thereby inhibiting tumor cell proliferation, migration and survival, and inducing tumor cell differentiation.
Oncternal Therapeutics has been evaluated in a Phase 1 2 clinical study of zilovertamab in combination with imbruvica**MCL and patients with chronic lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL) (NCT03088878). In addition, there are two ongoing clinical trials of zilovertamab, a Phase 2 clinical trial of zilovertamab in combination with the BCL-2 inhibitor venetoclax**r r cll (NCT04501939).The other is a Phase 1B clinical trial of zilovertamab in combination with docetaxel*** in metastatic castration-resistant prostate cancer (NCT05156905).
In April 2023, Oncternal Therapeutics announced a strategic prioritization alignment in light of the rapidly changing business landscape of Bruton tyrosine kinase inhibitors (BTK inhibitors), the Phase 3 study of Zilo-301 in combination with ibrutinib and the Phase 1 study CIRM-001* will be closed in order to extend the company's cash runway through 2025 and focus on the clinical development of ONCT-808 and ONCT-534. The Company emphasizes that the decision was not based on any concerns about the safety or efficacy of zilovinatomab.
DMA is a biotechnology company focused on providing preclinical R&D products and services for druggable targets, with innovative functional membrane protein development, single B cell lead antibody discovery, antibody engineering and functional validation platforms. DMA now offers a full range of products and services for ROR1 targets, including a variety of active proteins, flow-proof monoclonal antibodies and reference antibodies. The service covers humanization, affinity maturation, and PTM risk site removal. In addition, in order to accelerate the development of Ror1 target drugs, DMA has also prepared a B cell seed bank for Ror1, which can complete the screening of lead antibody molecules in 20 days at the earliest.
Recombinant active proteins.
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 Active 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 validation reference 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 reference antibodies:
anti-ror1 (zilovertamab biosimilar) mab (bme100073)
References: 1]Zhao **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 n**igation control (gnc) molecule development for treatment of ror1+ malignancies. cancer res 1 april 2023; 83 (7_supplement): 5679.