Liver cancer is one of the six most common cancers worldwide and falls into three main types, namely, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, and mixed cell carcinoma[27]. HCC is the most prevalent and lethal variety, representing approximately 90% of all liver cancers. Liver tumors may also be derived from metastasis of other malignancies, such as lung tumors. Due to its high mortality rate and ease of metastasis, HCC is a subject of significant interest in cancer research[28].

A study by Cetin et al[29] assessed ROR1 in HCC, demonstrating its presence in both human and mouse cells, without limitation to metastatic or mesenchymal cell lines. Induction of EMT by transforming growth factor-β (TGF-β) reduced ROR1 levels, while ROR1 knockdown inhibited both the proliferation and migration of HCC cells and increased resistance to apoptosis. Chemotherapeutic agent uptake was modified in ROR1 knockdown epithelial-type HCC cells, leading to resistance to chemotherapy-induced apoptosis. ROR1 has been shown to promote HCC progression through the modulation of various pathways, and changes in ROR1 expression may be useful in diagnosis and prognostic prediction. Meng et al[30] reported that ROR1 expression is substantially increased in acid-treated cancer cells and significantly promotes invasion and migration of HCC. ROR1 knockdown through siRNA successfully inhibited acid-induced tumor cell migration, invasion, and EMT. Nevertheless, the underlying regulatory actions of ROR1 in HCC are not yet clear.

Gastric cancer (GC) is a heterogeneous disease with a high mortality rate. It is known that infection with Helicobacter pylori is both necessary and insufficient for the development of GC[31] which appears to involve a multifactorial and multistep pathogenesis[32].

There is limited information on ROR1 in GC. Kotoh et al[33] found an association between Wnt5a and GC invasion and metastasis, showing that Wnt5a bound to FZD/ROR1 and was linked to various signaling pathways including β-catenin-TCF/LEF, Dishevelled–RhoA–Rho-associated protein kinase (DVL–RhoA–ROCK), and mitogen-activated protein kinase kinase kinase 7–nuclear factor-B (MAP3K7–NF-κB) in specific contexts. ROR1 also associates with c-Src, promoting its phosphorylation. C-Src is a non-RTK and known oncogene that has been linked to the development of numerous human cancers, including colon, gastric, lung, breast, and prostate cancers. Once activated, c-Src can regulate a variety of processes, both normal and cancer-associated, including cell survival, proliferation, motility, differentiation, and angiogenesis[34,35].

Colorectal cancer (CRC) is among the most prevalent cancer types globally, ranking fifth in incidence in China. Advanced CRC is associated with both recurrence and drug resistance, reducing patient survival. Thus, investigation of the mechanisms underlying CRC is important to improve survival and develop new drugs. Zhou and colleagues investigated expression of ROR1 in CRC[36], observing markedly increased levels in CRC tissues and in approximately 94% of patients with CRC. However, the molecular mechanisms underlying these increases remain unknown.

Wnt signaling is closely associated with regulation of morphogenesis during embryonic development and repair processes. The Wnt ligands are key to these pathways, and are known to bind different receptors, including proteins of the FZD family and ROR1. The interactions trigger downstream cascades involving multiple pathways to influence the arrangement of the cytoskeleton, transcription of genes associated with proliferation and cell growth, and the behavior of intracellular organelles. Dysregulated Wnt signaling has been linked to various malignancies, including CRC, through the modulation of cells associated with the tumor microenvironment (TME)[37].

Pancreatic cancer is an especially lethal form of cancer. Despite significant progress in elucidating its molecular pathology, the prognosis remains extremely poor, mostly due to treatment resistance and the formation of distant metastases in the early stages of the disease[28]. Therefore, uncovering the underlying molecular mechanisms of pancreatic cancer to create new targeted therapeutic strategies remains a pressing research priority.

Yamazaki et al[38] demonstrated that ROR1 promotes the proliferation of pancreatic ductal adenocarcinoma (PDAC) by activating E2F via c-Myc and inducing expression of aurora kinase B. It was also found that transcription of ROR1 was dependent on the binding of YAP/BRD4 to its promoter region. Prevention of this binding reduced ROR1 expression and inhibited PDAC growth. These findings suggest that increased levels of ROR1 are closely involved in tumorigenesis, highlighting its importance in PDAC progression and its potential as a target for treating the cancer.

The application of anti-ROR1 mAbs to block binding between the receptor and the Wnt5a ligand prevents activation of downstream signaling and activates the immune system to promote removal of tumor cells[39]. Zilovertamab, also known as cirmtuzumab or UC-961, is an mAb targeting ROR1. Zilovertamab binding to ROR1 blocks Wnt5a signaling, thus inhibiting tumorigenic behavior, such as cell growth and survival, and inducing differentiation. This mAb has shown significant antitumor efficacy against both hematological and solid malignancies, such as GC and pancreatic cancer, in Phase I clinical trials, and is currently the only antibody on the market that has undergone clinical evaluations[40]. A phase I/II trial of a combination of zilovertamab and paclitaxel for treating breast cancer is currently underway.

Chen et al[24] reported that the Wnt5a/ROR1 axis can activate the NF-κB pathway in tumor cells, resulting in the production of interleukin-6 and signal transducer and activator of transcription 3 phosphorylation. Zilovertamab has the potential to inhibit these processes. According to Hasan et al[15], Wnt5a promotes complexation between ROR1 and DOCK2, leading to the activation of Rac1/2. Silencing of DOCK2 specifically reduces Wnt5a-mediated activation of Rac1/2 and tumor cell proliferation. The proline-rich domain (PRD) of ROR1 has been implicated in the Wnt5a interaction with DOCK2 and activation of Rac1/2 in MEC1 CLL cells. This effect is blocked by zilovertamab, which also has the advantages of having a long half-life in the plasma and no dose-limiting toxicity. The use of this antibody showed reductions in the activities of RhoA and HS1, while transcriptome analysis indicates reductions in the expression of stemness genes in CLL in vivo. It is apparent that zilovertamab is both safe and effective for the inhibition of ROR1 in various tumor cells[40].

ADCs involve linkage between mAbs and small cytotoxic molecules via specific chemical linkers. ADCs targeting ROR1 are able to transfer cytotoxic agents and regulatory RNAs to ROR1-expressing tumor cells.

VLS-101 (MK-2140) is an ADC consisting of a lead antibody to effectively target ROR1, for treating hematological malignancies and solid tumors. VLS-101 represents a combination of zilovertamab, a lysyl iminohexene-valine-citrulline p-aminobenzoate linker, and a cytotoxic agent monomethyl auristatin E (MMAE) that targets microtubules. Within cells, MMAE is released from the linker to block microtubule polymerization, thus adversely affecting mitosis and cell division[41]. The drug also safely promotes tumor regression in patient-derived xenograft models previously found to be resistant to CAR-T cell, ibrutinib, and/or venetoclax treatment[42]. These findings provide valuable insights into the increasing clinical demand for VLS-101 in gastrointestinal cancers.

LCB71 (CS5001) is an ADC based on an mAb that targets ROR1. LCB71 has a distinctive design involving a unique β-glucosidic acid linker and a pyrrolobenzodiazepine (PBD) pre-toxin dimer. Both the linker and the pretoxin are cleaved by lysosomal β-glucuronidase, an enzyme significantly overexpressed in many types of cancer cells. Thus, LCB71 can eliminate cancerous cells after arrival at the tumor site and cleavage of the linker to release the PBD pre-toxin, which is subsequently activated within the tumor cell. The dual control mechanism of the linker plus precursor toxin resolves typical toxicity issues associated with traditional PBD loads, resulting in an improved safety profile. LCB71 has shown notable dose-dependent antitumor activity in several xenograft tumor models of condylomatous lymphoma and breast cancer and is thus a promising drug candidate with potential for precision therapy in both hematological and solid malignant tumors expressing high levels of ROR1. The ADC NBE-002 is composed of a humanized antibody, huXBR1-402, conjugated to the anthracycline derivative, PNU-159682. HuXBR1-402 is a chimeric rabbit/human mAb identified from a rabbit antibody library using phage display. NBE-002 has been shown to be effective in xenograft models of various human tumors[43]. The drug is currently in a clinical Phase I study. Chiang et al[44] connected an anti-ROR1 mAb to a regulatory miRNA (miR-29b). This ADC treatment reduced the expression of the miR-29 targets, DNA methyltransferase (DNMT) 1 and DNMT3A, in cells expressing ROR1. It also led to alterations in global DNA methylation patterns, downregulation of SP1, and upregulation of p21. Another ADC used an mAb against ROR1 linked to nanoparticles containing OSU-2S, a sphingosine analog with anti-tumor capabilities, that activated protein phosphatase 2A and induced SHP1 phosphorylation and nuclear translocation[45], resulting in apoptosis.

Bispecific antibodies (BiAbs) are synthetic antibodies created through cell fusion or recombinant DNA technology. They are formed by two single-chain antibody fragments, or single-chain fragment variable (scFvs), together with different variable regions composed of both heavy and light chains and a common constant region, or Fc. Bispecific T-cell conjugates (BiTes) are fusion proteins composed of two separate scFvs without an Fc. One arm can bind to the Kringle domain on ROR1 while the other interacts with the CD3 subunit of the T cell receptor, thus recruiting polyclonal T cells to the site of the tumor. BiTEs do not rely on antigen presentation and can activate T-cell binding, resulting in the killing of neighboring target cells. Both BiAbs and BiTes can bind simultaneously to two different antigens or epitopes on the same antigen. Due to their specificity and bifunctionality, the use of BiAbs and BiTes is a trending research topic in antibody engineering and has a broad range of applications in tumor therapy and autoimmune diseases.

Qi et al[46] co-conjugated multiple anti-ROR1 single-chain antibodies to a single-chain antibody against CD3 with glycosylated IgG1-Fc, generating various BiAbs. They found that targeting scFv using ROR1 with the proximal epitope R11 of the membrane was the most effective one for recruiting T cells to ROR1-positive solid cancer cell lines. This BiAb exhibited promising antitumor activity both in vitro and in vivo. In a similar investigation, Wang et al[47] also used a BiAb composed of an scFv and Fc that targeted both ROR1 and CD3; this was termed R11×v9-biAb. When tested on multiple ROR1-positive solid cancer cell lines, the observed cytotoxicity was found to be dependent on the R11×v9-biAb dosage. The BiAb targeted ROR1-positive cancerous cells, induced tumor cell death, and recruited significant numbers of CD4+ and CD8+ T cells to the tumor through the secretion of T-cell-derived proinflammatory factors and promoting perforin production by granzyme B and CD8+ T cells.

NVG-111 is a unique humanized BiTE recognizing both ROR1 and CD3, and is composed of tandem scFvs against ROR1 and CD3. NVG-111 is the first BiAb that utilized the inherent cytotoxicity of T cells. Ongoing clinical trials of NVG-111 have shown promising results in both CLL and solid tumors, with favorable cytotoxicity[48]. Another bispecific T-cell splicer, SFG. ROR1-BITE, includes scFvs targeting the cysteine-rich coiled-coil structural domain of ROR1 and CD3, and showed strong cytotoxicity against pancreatic cancer cells expressing ROR1 at low concentrations, as well as significant tumor reduction in a mouse model of pancreatic tumors[49].

The lack of clearly defined kinase activity in ROR1 has limited the identification of small-molecule inhibitors that could target this activity. Although some catalytic activity was observed in immunoprecipitated ROR1 suggesting the possibility of autophosphorylation in vivo, this was found to be negligible[50,51].

Nevertheless, Hojjat-Farsangi et al[52] identified a promising inhibitor, KAN0439834, against ROR1 by screening a library of 110000 small molecules. This compound targeted the TK domain, preventing phosphorylation of ROR1 by Wnt5a and thus blocking the activation of downstream proteins such as Src, Akt, protein kinase C, and MAPK. Further research demonstrated that KAN0439834 was more effective in inducing apoptosis in cancer cells than mAbs with a potency equivalent to that of venetoclax, an inhibitor of Bcl-2 used for the treatment of patients with CLL. Clinical trials have also shown that combinations of KAN0439834 with erlotinib and ibrutinib, small-molecule inhibitors of epidermal growth factor receptor and Bruton's tyrosine kinase, act synergistically in the treatment of patients with pancreatic cancer[53]. The same group screened KAN04415711C, a second-generation compound targeting ROR1, which was found to be effective against tumors in a zebrafish model[54]. KAN04415711C was also effective in vitro when combined with venetoclax. Its mechanism of action, however, requires further investigation.

CAR-T therapy involves the transfer of structural domains that specifically recognize antigens and genetic material promoting T cell activation signals into T-cells. This enables the T cells to target the specific antigens on the surfaces of cancer cells, leading to the release of cytotoxic compounds such as perforin and granzyme B. Cytokines may also be released to recruit endogenous immune cells for cotreatment. Additionally, the formation of memory T cells can establish a long-lasting antitumor mechanism in vivo[55-57].

ROR1 is currently considered a promising target in the design of CAR-T cell therapy. Hudecek et al[58] and Hudecek et al[59] designed a range of CAR-T cells targeting ROR1, featuring a concise “hinge-only” extracellular spacer, that was found to be highly effective in destroying ROR1-positive tumor cells and inducing T cell effector functions. The safety of this therapy is currently being evaluated due to concerns about cross-reaction with normal tissues by the scFv[60,61]. Lee et al[62] developed two CAR-T cell constructs targeting ROR1 by utilization of the antigen-recognition domains of zilovertamab and different hinge regions on the antibody. It was found that the construct with a truncated IgG4 hinge fragment was the most successful in terms of expression and in vitro functionality. Additionally, it effectively controlled tumor growth in xenograft mouse models with no significant toxicity. This study demonstrates the potential of Zilovertamab-derived ROR1 CAR-T for the clinical treatment of solid tumors.

The vast majority of candidate CAR targets in solid tumors, such as HER2, GD2, and mesothelin, are co-expressed in a variety of normal tissues, making off-tumor toxicity a significant risk for immunotherapies targeting these molecules. This makes it crucial to study the toxicity of ROR1 targeted therapy for gastrointestinal tumors, as ROR1 is no exception. SNIP CARs provide a solution to CAR-T therapy toxicities by utilizing a regulated protease-based system controlled by an FDA-approved small molecule. They offer improved potency and safety in various models, enabling remote control over CAR activity for treating solid tumors[63].

A study by Srivastava and colleagues[64] found that ROR1 CAR-T therapy led to fatal bone marrow failure in mice due to the recognition of ROR1-positive stromal cells. To enhance selectivity, they integrated synthetic Notch (synNotch) receptors specifically recognizing epithelial cell adhesion molecule or B7-H3 into T cells. These receptors were expressed on ROR1-positive cancer cells but not on stromal cells expressing ROR1. This induced a selective ROR1 CAR-T effect, leading to successful tumor ablation without toxicity in normal ROR1-positive cells. The study demonstrated that the use of synNotch receptors for combined antigen sensing can prevent CAR-T-cell-mediated lethal toxicity to normal tissue, provided that the tumor and normal tissue were spatially separated. However, if they are highly colocalized at the same site, this approach may not be effective. Moreover, the same group further discovered that ROR1 CAR-T cells showed poor tumor infiltration and malfunctioned. The inclusion of oxaliplatin, however, in the regimen allowed the recruitment of T cells by activated tumor macrophages[65], improving CAR-T-cell infiltration to modify the TME and increasing the sensitivity of the tumor to immunotherapy. Thus, the ROR1 CAR-T combination therapy provides an alternative strategy for the enhancement of CAR-T cell efficacy in clinical settings.

Some metabolites and small-molecule drugs can boost the anticancer actions of CAR-T cells. It was found that some short-chain fatty acids, such as butyrate and valerate, could enhance the actions of cytotoxic T lymphocytes and CAR-T cells through both epigenetic and metabolic means[66]. Furthermore, SD-208, a kinase inhibitor, could enhance the cytolytic activity, cytokine production, viability, and proliferation of ROR1-CAR T cells by blocking TGF-β receptor signaling[67]. These findings underscore the significance of novel combination therapies in enhancing the efficacy of ROR1 CAR T cells for treating gastrointestinal cancers.