Receptor 1 Research Articles

Revolutionizing pediatric neuroblastoma treatment: unraveling new molecular targets for precision interventions.

Neuroblastoma (NB) is the most frequent solid tumor in pediatric cases, contributing to around 15% of childhood cancer-related deaths. The wide-ranging genetic, morphological, and clinical diversity within NB complicates the success of current treatment methods. Acquiring an in-depth understanding of genetic alterations implicated in the development of NB is essential for creating safer and more efficient therapies for this severe condition. Several molecular signatures are being studied as potential targets for developing new treatments for NB patients. In this article, we have examined the molecular factors and genetic irregularities, including those within insulin gene enhancer binding protein 1 (ISL1), dihydropyrimidinase-like 3 (DPYSL3), receptor tyrosine kinase-like orphan receptor 1 (ROR1) and murine double minute 2-tumor protein 53 (MDM2-P53) that play an essential role in the development of NB. A thorough summary of the molecular targeted treatments currently being studied in pre-clinical and clinical trials has been described. Recent studies of immunotherapeutic agents used in NB are also studied in this article. Moreover, we explore potential future directions to discover new targets and treatments to enhance existing therapies and ultimately improve treatment outcomes and survival rates for NB patients.

Abstract 857: Identify ROR1 expression in healthy human tissues using single-cell RNA sequencing

Abstract Introduction: The Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) is a receptor for Wnt5a and is considered a tumor-specific antigen. Researchers are actively exploring the development of antibodies and adoptive cell therapies targeting ROR1, while the potential "on-target, off-tumor" toxicity is still a major concern. Previous studies have provided inconsistent results regarding the expression of ROR1 in normal tissues, highlighting the complexity of its expression profile. Therefore, we are conducting this study to address this challenge through the detection of ROR1 mRNA expression via single-cell RNA sequencing (scRNA-seq) and the evaluation of ROR1 protein levels by immunohistochemistry (IHC). Methods: We retrieved the Gene Expression Omnibus (GEO) database and downloaded the scRNA-seq data of each organ. We used the Seurat package to normalize data, perform dimensionality reduction and conduct clustering. The differentially expressed genes of each cell cluster were identified, and the cell clusters were then manually annotated. We further conducted the IHC on the tissue microarray (TMA), and evaluated the ROR1 immunostaining. Results: We analyzed the scRNA-seq data of 908,941 cells from 14 types of healthy human organs, and 132,837 (14.61%) of cells expressed ROR1. Cell clusters in which the organ's ROR1 positive rate was >1% included the human Heart (Overall 25.99%, Ventricular cardiomyocyte 70%, Atrial cardiomyocyte 50%, Mesothelial 37% and Adipocytes 27%), Pancreas (Overall 9.36%, Beta cell 12.4%, Alpha cell 9.1%, Duct cell 7.3%, Acinar cell 7.2% and Delta cell 7.2%), Colon and small intestine (Overall 6.09%, Paneth 14.9%, Intermediate absorptive enterocyte 14.4%, Transit amplifying 13.8% and Enteroendocrine cell 12.5%), Lung (Overall 3.86%, Alveolar type I 23%, Ciliated cells 5.8% and Alveolar type II 5.4%), Bladder (Overall 2.73%, Fibroblasts 6.8% and Myofibroblast 6.4%), Esophagus (Overall 1.93%, Epithelial suprabasal 5% and Epithelial basal 4.4%) and Stomach (Overall 1.31%, Gland mucous cell 3% and Pit mucous cell 1.9%). The ROR1 expression was found relatively low (<1%) on Adipose, Kidney, Ileum, Liver, Spleen, Bone marrow and PBMC. For the IHC, ninety normal tissues were prepared and used. Consistent with the scRNA-seq results, we found relatively high ROR1 protein on the Heart, Lung, Stomach, Duodenum and Colon and low ROR1 protein on Liver and Ileum. However, ROR1 protein was not detected on Bladder, Pancreas or Esophagus during the IHC process. Conclusion: Our findings suggest that some healthy human tissues may be at risk of "on-target, off-tumor" toxicity when utilizing ROR1-targeted therapies, including Heart, Colon and small intestine, Lung and Stomach. Close monitoring of possible toxicities related to the high ROR1 expressing organs should be considered in future clinical trials. Citation Format: Mingyang Feng, Sirui Tan, Yue Chen, Qiu Li, Yongsheng Wang. Identify ROR1 expression in healthy human tissues using single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 857.

Abstract 4495: Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells

Abstract Background: The identification of novel compounds that specifically target cancer cells while sparing normal tissues is crucial. The orphan tyrosine kinase-like receptor 1 (ROR1) is one such target expressed exclusively in various human malignancies, including pancreatic cancer, but not in normal adult tissues. Inhibiting ROR1 signaling shows promise in halting pancreatic cancer cell proliferation and invasion. In this study, lead compounds CSF1R-IN-1 and MCULE-8715589337 were found as potential inhibitors of the ROR1 and were further evaluated for its potential effect on pancreatic cancer cell viability. Method: We curated a compound library of 6 million from PubChem, 2 million lead-like compounds from ZINC15, and 2 million compounds from diverse vendors. Employing Glide’s HTVS followed by XP, we refined the selection based on docking scores lower than the reference molecule, Ponatinib. Compounds were shortlisted by pre-simulation MMGBSA calculations with Schrödinger Prime MM-GBSA module, focusing on compounds with energies below -100 kcal/mol. Then we performed MD simulation for 100ns. Post-simulation MMGBSA on the last 1 ns of simulation assessed change in ligands’ binding energies. The selected leads underwent further validation utilizing two pancreatic cancer cell lines, MiaPaCa-2 and PANC-1. Half-maximal inhibitory concentration (IC50) was assessed using MTS cell viability assay. Result: Comparing pre- and post-simulation MMGBSA energies, the reference compound showed increased, while CSF1R-IN-1 and MCULE-8715589337 exhibited decreased energies. The reference ligand's RMSD showed stability with fluctuations of 3 to 3.5 Å, whereas CSF1R-IN-1 and MCULE-8715589337 had stable and lower RMSD values (approximately 3 Å and 2.4 Å). Test compounds, CSF1R-IN-1 and MCULE-8715589337 showed better molecular interactions with ROR1 compared to reference. The reference compound formed hydrogen bonds with ILE555 and ASP633, along with pi-pi stacking with PHE552. CSF1R-IN-1 forms a hydrogen bond with ILE555 and ASP633, pi-pi stacking with PHE552, and interaction with SER632 through a water bridge. Additionally, compound MCULE-8715589337 forms hydrogen bonds with ASP633, SER632, LYS506, and GLU523. CSF1R-IN-1 notably inhibited proliferation in MiaPaCa-2 cells (IC50: 1.29 μM) while no efficacy in PANC-1 cells (IC50: 78.41 μM). MCULE-8715589337 showed relatively similar IC50 values of 39.06 μM and 37.73 μM in MiaPaCa-2 and PANC-1 cells, respectively. Conclusion: The reduction in MMGBSA energies, low RMSD values, and a higher number of interactions highlight the better characteristics of both compounds compared to the reference. Our findings show CSF1R-IN-1 and MCULE-8715589337 have efficacy in pancreatic cancer cells, but further studies are required to validate and to understand the mechanism of action. Citation Format: Indrakant Kumar Singh, Shradheya RR Gupta, Pooja Mittal, Shivani Soni, Jae Ho Lo, Francesca Battaglin, Yan Yang, Lesly Torres-Gonzalez, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Heinz Josef Lenz. Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4495.

How receptor tyrosine kinase-like orphan receptor 1 meets its partners in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in western societies, recognized by clinical and molecular heterogeneity. Despite the success of targeted therapies, acquired resistance remains a challenge for relapsed and refractory CLL, as a consequence of mutations in the target or the upregulation of other survival pathways leading to the progression of the disease. Research on proteins that can trigger such pathways may define novel therapies for a successful outcome in CLL such as the receptor tyrosine kinase-like orphan receptor 1 (ROR1). ROR1 is a signaling receptor for Wnt5a, with an important role during embryogenesis. The aberrant expression on CLL cells and several types of tumors, is involved in cell proliferation, survival, migration as well as drug resistance. Antibody-based immunotherapies and small-molecule compounds emerged to target ROR1 in preclinical and clinical studies. Efforts have been made to identify new prognostic markers having predictive value to refine and increase the detection and management of CLL. ROR1 can be considered as an attractive target for CLL diagnosis, prognosis, and treatment. It can be clinically effective alone and/or in combination with current approved agents. In this review, we summarize the scientific achievements in targeting ROR1 for CLL diagnosis, prognosis, and treatment.

ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling.

This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in triple-negative breast cancer (TNBC). ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR. ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis. The migration, invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined. ROR2 expression was high in metastatic TNBC tissues. ROR2 knockdown suppressed the migration, invasion and chemoresistance of TNBC cells. ROR2 overexpression in MDA-MB-435 cells promoted the migration, invasion, and chemoresistance. Moreover, ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin. ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells. ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.

Quantitative Investigation of Neuroprotective Role of ROR1 in a Cell Culture Model of Alzheimer's Disease.

Cytoskeletal and microtubule atrophy are major hallmarks of Alzheimer's disease (AD). A method to investigate endogenous proteins that can interact/stabilize the cytoskeleton (under pathological cues) is rare. Here, we describe how receptor tyrosine kinase-like orphan receptor 1 (ROR1), a receptor tyrosine kinase (RTK), can act as a neuroprotective molecule by promoting neurite outgrowth, stabilizing cytoskeletal components, and altering the dynamics of actin assembly in a cell culture model of AD.

Bone marrow-derived mesenchymal stromal cells obstruct AML-targeting CD8+ clonal effector and CAR T-cell function while promoting a senescence-associated phenotype

Bone marrow mesenchymal stromal cells (MSCs) have been described as potent regulators of T-cell function, though whether they could impede the effectiveness of immunotherapy against acute myeloid leukemia (AML) is still under investigation. We examine whether they could interfere with the activity of leukemia-specific clonal cytotoxic T-lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells, as well as whether the immunomodulatory properties of MSCs could be associated with the induction of T-cell senescence. Co-cultures of leukemia-associated Wilm’s tumor protein 1 (WT1) and tyrosine-protein kinase transmembrane receptor 1 (ROR1)-reactive CTLs and of CD123-redirected switchable CAR T cells were prepared in the presence of MSCs and assessed for cytotoxic potential, cytokine secretion, and expansion. T-cell senescence within functional memory sub-compartments was investigated for the senescence-associated phenotype CD28−CD57+ using unmodified peripheral blood mononuclear cells. We describe inhibition of expansion of AML-redirected switchable CAR T cells by MSCs via indoleamine 2,3-dioxygenase 1 (IDO-1) activity, as well as reduction of interferon gamma (IFNγ) and interleukin-2 (IL-2) release. In addition, MSCs interfered with the secretory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, tumor necrosis factor alpha, and IL-2. Abrogated T cells were shown to retain their cytolytic activity. Moreover, we demonstrate induction of a CD28loCD27loCD57+KLRG1+ senescent T-cell phenotype by MSCs. In summary, we show that MSCs are potent modulators of anti-leukemic T cells, and targeting their modes of action would likely be beneficial in a combinatorial approach with AML-directed immunotherapy.

Receptor tyrosine kinase-like orphan receptor 1 inhibitor strictinin exhibits anti-cancer properties against highly aggressive androgen-independent prostate cancer

Aim: It is important to identify anti-cancer compounds that can inhibit specific molecular targets to eradicate androgen-receptor negative (ARneg), androgen-independent (AI) prostate cancer, which is an aggressive form of prostate cancer with limited treatment options. The goal of this study was to selectively target prostate cancer cells that have high levels of oncogenic protein Receptor tyrosine kinase-like orphan receptor 1 (ROR1) by using strictinin, a small molecule ROR1 inhibitor. Methods: The methods performed in this study include western blots, methyl thiazolyl tetrazolium (MTT) proliferation assays, phosphatidylserine apoptosis assays, apoptosis flow cytometry (Annexin V, caspase 3/7), migration scratch assays, Boyden chamber invasion assays, and cell cycle flow cytometry. Results: Strictinin was most lethal against PC3 [half-maximal drug inhibitory concentration (IC50) of 277.2 µmol/L], an ARneg-AI cell type that expresses the highest levels of ROR1. Strictinin inhibited ROR1 expression, downstream phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-glycogen synthase kinase 3beta (GSK3β) pro-survival signaling, and epithelial-to-mesenchymal transition markers in PC3 cells. Additionally, strictinin decreased PC3 cell migration and invasion, while increasing S-phase cell cycle arrest. In ARneg-AI DU145 cells, strictinin inhibited ROR1 expression and modulated downstream AKT-GSK3β signaling. Furthermore, strictinin exhibited anti-migratory, anti-invasive, but minimal pro-apoptotic effects in DU145 cells likely due to DU145 having less ROR1 expression in comparison to PC3 cells. Throughout the study, strictinin minimally impacted the phenotype of normal prostatic epithelial cells RWPE-1 (IC50 of 658.5 µmol/L). Strictinin was further identified as synergistic with docetaxel [combination index (CI) = 0.311] and the combination therapy was found to reduce the IC50 of strictinin to 38.71 µmol/L in PC3 cells. Conclusions: ROR1 is an emerging molecular target that can be utilized for treating prostate cancer. The data from this study establishes strictinin as a potential therapeutic agent that targets ARneg-AI prostate cancer with elevated ROR1 expression to reduce the migration, invasion, cell cycle progression, and survival of prostate cancer.

Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1)

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Defining Signaling Interactomes Involved in Non-Canonical Wnt Signaling By ROR1 or ROR2 in Hairy Cell Leukemia That Can Influence Cell Morphology and Migration

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) and 2 (ROR2) are highly conserved oncoembryonic surface proteins expressed on neoplastic cells of patients with a variety of human cancers, but not on most normal postpartum tissues. ROR1 and ROR2 are each a receptor for non-canonical Wnt factors that direct migration and planar cell polarity, which is essential for organ development. We developed humanized monoclonal antibodies (mAbs) each specific for ROR1 (UC-961) or ROR2 (6E6-70) and found the leukemia cells of patients with classic hairy cell leukemia (cHCL) or variant HCL (vHCL) express both ROR1 and ROR2 and that these receptors function in HCL non-canonical Wnt signaling (Widhopf, G. et al). We hypothesized that ROR1 and ROR2 each may associate with overlapping and distinctive sets of proteins, which may contribute to ROR1/2, ROR1, or ROR2 signaling in HCL. To test this hypothesis, we optimized conditions for generating immune precipitates (i.p.) with either UC-961 or 6E6-70, and examined the proteins of each i.p. via data dependent acquisition mass spectrometry. Consistent with the assumed capacity of each of these mAb to disrupt ROR1/2 heteroligomerization, we identified ROR1, but not ROR2, in i.p. generated using UC-961 and ROR2, but not ROR1, in i.p. generated using 6E6-70 on cell lysates of leukemia cells isolated from a patient with cHCL. In the anti-ROR1 i.p. we identified 1,414 proteins, 849 (60%) of which also were found in the anti-ROR2 i.p.. In the anti-ROR2 i.p. we identified 2,523 proteins, a third of which also were found in the ROR1 i.p.. Among proteins common to both anti-ROR1 and anti-ROR2 i.p. we noted hematopoietic cell-specific protein-1 (HS1 or HCLS1), which we found associated a SH3-binding motif that was dependent on a proline residue at position 841 of activated ROR1 in chronic lymphocytic leukemia (CLL); we found this SH3-binding motif was critical for ROR1-dependent Wnt5a-induced chemokine-directed migration ( Leukemia 12:2615, 2017). Also found common to both ROR1 and ROR2 in HCL were proteins that drive actin polymerization and cytoskeletal dynamics that influence cell morphology and migration. To validate that ROR2 itself was involved in binding HS1 and in enhancing migration, we generated mutant forms of ROR2, each harboring a proline-to-alanine mutation in one of several proline residues found in the proline-rich domain of ROR2. We transduced MEC1 cells, which lack ROR1 or ROR2, with lentivirus vectors encoding wild-type ROR2 or each one of several P-A mutants of ROR2. Flow cytometry confirmed that each of the transduced cells had comparable expression of surface ROR2. We generated anti-ROR2 i.p. using lysates of each and found that all but one of the mutant forms of ROR2 could bind to HS1 by immunoblot analysis. In contrast to wild-type ROR2, or other mutant forms of ROR2, expression of this mutant form of ROR2 could not enhance chemokine-directed migration relative to that of wild-type MEC1 cells. Collectively, our studies define signaling interactomes involved in non-canonical Wnt signaling by ROR1 and/or ROR2 in hairy cell leukemia and define a residue within ROR2 that is necessary for binding HS1 and facilitating cellular migration.

Development and Testing of a Glycoengineered Anti-ROR1 Antibody with Enhanced Capacity for Directing Antibody-Dependent Cellular Cytotoxicity (ADCC) of Chronic Lymphocytic Leukemia Cells

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic surface antigen expressed on the neoplastic cells of patients with chronic lymphocytic leukemia (CLL) or other types of cancer, but not on virtually all normal postpartum tissues (Fukuda T., et al., PNAS, 105:3047, 2008). Zilovertamab (also known as UC-961 or cirmtuzumab) is a humanized anti-ROR1 monoclonal antibody (mAb) currently under clinical investigation; this mAb is capable of blocking ROR1-signaling, which can contribute to cancer-cell migration, proliferation, survival, and self-renewal. We generated a glycoengineered form of zilovertamab (GE- zilovertamab) and examined whether it could mediate enhanced antibody-dependent cellular cytotoxicity (ADCC) against neoplastic cells that express ROR1. Initial studies used Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, San Diego, CA, USA) which express high-affinity CD16A (FcγRIIIA, V158 allotype) as effector cells (EC), and the CLL-cell line MEC1, or MEC1 cells transduced to express ROR1 (MEC1-ROR1), as target cells (TC), which each also express CD20. Co-culture of Jurkat-Lucia cells for 6 hours with the anti-CD20 mAb rituximab and MEC1 or MEC1-ROR1 cells induced Jurkat-Lucia cells to express a luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements; this endowed the Jurkat-Lucia cells with high luminescence activity that was not observed in co-cultures of EC and TC without added mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat-Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat-Lucia cells co-cultured with MEC1-ROR1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab. We generated an orthogonal system with which to examine the relative ADCC activity of each of these mAb, using EC generated from the NK cell line, NK92, which we transduced with a lentivirus encoding a newly-generated high-affinity variant of CD16A (CD16v), which has been modified to block its cleavage by ADAM17. NK92 or NK92-CD16v were used as EC to effect cytolysis of Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL cells in a chromium-release assay. Addition of rituximab to co-cultures of EC with Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL caused mAb-dose-dependent ADCC of each of these TC by NK92-CD16v that was significantly greater than that by parental NK92 cells. Again, neither zilovertamab nor GE-zilovertamab could direct ADCC by NK92-CD16v or NK92 ADCC of MEC1 cells, which lack ROR1. However, GE-zilovertamab directed dose-dependent ADCC of MEC1-ROR1 or primary ROR1 + CLL cells by either NK92-CD16v or NK92 at levels comparable to that directed by rituximab for either EC population; such levels were significantly greater than that directed by zilovertamab. ROR1 + CLL cells harboring del(17p) or mutations in TP53 (del(17p)/m TP53) and/or that were resistant to targeted therapies (e.g., inhibitors of BTK or BCL2), were as susceptible to GE-zilovertamab-directed ADCC as were CLL cells without del(17p)/m TP53 from patients who had not had prior therapy. These data demonstrate that GE-zilovertamab can direct high-level ADCC lysis of ROR1-expressing neoplastic cells with greater activity than zilovertamab, encouraging development of clinical studies to evaluate GE-zilovertamab for therapy of patients with CLL or other ROR1-positive cancers.

Targeting B-Cell Malignancies with Anti-ROR1 CAR T-Cell Therapy

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has been shown to induce high complete response rates in the majority of B-cell lymphoma patients. However, over 50% of patients relapse within one year. A major cause of failure appears to be due to loss of CD19 expression on the tumor cell surface. This indicates that novel therapeutic strategies are still needed in clinic. Receptor tyrosine kinase like orphan receptor 1(ROR1) is an oncofetal receptor for Wnt5α that is expressed in multiple embryotic tissues but absent in virtually all adult tissues. However, ROR1 is expressed at high levels in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse Large B-Cell Lymphoma (DLBCL), and several solid tumors. The high expression level of ROR1 in tumor cells is associated with adverse clinical prognosis. In addition, a small percentage of cancer cells with characteristics of undifferentiated leukemia cells or cancer stem cells were found to overexpress ROR1. Using hybridoma technology, we generated an anti-ROR1 monoclonal antibody that specifically recognized ROR1 protein. The antibody bound to ROR1-expressing MCL cell lines but not to normal donor T cells, B cells, or monocytes, indicating the specificity of the antibody. Lentiviral transduction of a CAR molecule derived from the anti-ROR1 antibody into primary human T cells redirected their specificity against B-cell lymphoma cell lines. We found the anti-ROR1 CAR T cells specifically lysed B- cell lymphoma tumor cells at high efficiency but not normal B cells. At an effector: target ratio of 1:1, the anti-ROR1 CAR T cells lysed over 95% of B-cell lymphoma tumor cells during 3-days of coculture. To determine the optimal CAR construct, we evaluated various hinge transmembrane, and costimulatory domains in the CAR molecule. We found that incorporation of the CD28 hinge and transmembrane domain in the CAR construct dramatically enhanced the in vitro lysis efficiency of anti-ROR1 CAR-T cells. In conclusion, we developed a novel and potent anti-ROR1 CAR T-cell therapy product that may be used for treatment of various B-cell malignancies and ROR1-expressing solid tumors.

Time Limited Exposure to a ROR1 Targeting Bispecific T Cell Engager (NVG-111) Leads to Durable Responses in Subjects with Relapsed Refractory Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL)

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is absent or expressed at low levels in normal adult tissues but overexpressed in a range of malignancies including CLL and MCL. NVG-111 is a humanized first in class, tandem scFv, ROR1xCD3 bispecific T cell engager. NVG-111 mediates potent killing of ROR1 + tumors by engaging a unique epitope on the Frizzled domain of ROR1 and redirecting T cell activity via the CD3 binder engineered for attenuated cytokine release. Methods: In this phase 1, first-in-human, dose-escalation study (ClinicalTrials.gov identifier: NCT04763083), NVG-111 was evaluated in relapsed/refractory CLL and MCL subjects with an ECOG PS of 0-2 who received ≥2 prior lines of treatment including a Bruton tyrosine kinase inhibitor (BTKi). Bayesian continual reassessment method with overdose control was implemented to guide escalation over a dose range of 0.3-45ug/day. Each subject received at least one dose of NVG-111, administered as a continuous intravenous infusion (cIV) over 21 days followed by 7 days off drug (=1 cycle). NVG-111 was administered in combination (N=8) with ibrutinib to subjects who had achieved a partial response to >1 year of ibrutinib therapy, or as monotherapy in subjects that progressed after covalent BTKi/B-cell lymphoma 2 inhibitor (BCL2i) (N=4). Primary objective was safety, with the secondary objectives being efficacy (overall response rates [ORR], minimal residual disease (MRD) measured by ERIC-compliant flow cytometry and duration of response [DoR]). Serum cytokine levels were determined using a human high sensitivity cytokine premixed magnetic Luminex assay and T cell function was longitudinally evaluated using cytometry by time of flight (CyTOF) analysis. Results: Between May 2021 and July 2023 12 subjects (10 males and 2 females, median age 60 years) completed a maximum of 6 cycles of treatment with NVG-111 (median=3 cycles [range 1-6 cycles]). Pharmacokinetic and pharmacodynamic data demonstrated systemic NVG-111 exposure and exposure-dependent NVG-111 targeting of ROR1 on circulating tumor cells. Adverse events (AEs) related to NVG-111 occurred in 10 subjects (83%), with the most common being nausea (58%), headaches (67%), and fatigue (33%), majority of which were grade 1 or 2. Grade 1(71%) or 2(29%) cytokine release syndrome (CRS) occurred in 58% of subjects during week 1 of cycle 1 except in one subject who had a second grade 1 CRS in cycle 2. Grade 3 dose limiting toxicities (DLTs) occurred in three subjects (25%) consisting of transient elevation of liver enzymes (ALT and AST) in one subject dosed at 3ug/day, immune effector cell-associated neurotoxicity syndrome-like symptoms (ICANS) in one subject dosed at 30ug/day and headache in one subject dosed at 45ug/day. All AEs were fully reversible with no late emergent grade 3 or greater toxicities. Evidence of T cell activation was observed in all 12 subjects with peak cytokine levels at the 30µg/day dose levels (mean±SEM): TNFα, 43±17pg/ml; IL6, 566±541pg/ml; IFNɣ, 18±12pg/ml; and IL10, 103±56pg/ml. Furthermore, T cell profiling showed an increase in cytotoxic CD8 + T cell activation marker expression during each NVG-111 treatment cycle without evidence of T cell exhaustion. Efficacy was evaluable in 11 subjects with objective clinical responses observed in 55% (6/11), including two subjects with clear evidence of single agent activity. Amongst these, three CLL subjects were rendered MRD negative in peripheral blood and one MCL subject achieved complete metabolic response by the Lugano criteria. Two subjects had stable disease but in one of these subjects there was a >50% reduction in peripheral blood lymphocytosis and a 40% reduction in lymphadenopathy. Despite time limited exposure to NVG-111, the restricted mean survival time for DoR is currently 13.6 months (SEM 3.07), the median is not yet calculable with response ongoing in four subjects. The median progression-free survival currently is 18.7 months (95% CI 2.6 - not calculable). Conclusion: These data provide clinical proof of concept for selective targeting of ROR1 with a TCE leading to objective evidence of antitumor activity with encouraging response durability even in CLL patients known to have defective T cell function. The safety profile was consistent with the mechanism of action. Further evaluation of this promising molecule is ongoing.

Expression of Wnt5a and ROR2, Components of the Noncanonical Wnt-Signaling Pathway, is Associated with Tumor Differentiation in Hepatocellular Carcinoma

BackgroundWnt5a is the key ligand of the noncanonical Wnt pathway, and receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a receptor associated with Wnt5a. The association between the noncanonical Wnt-signaling pathway and carcinogenesis in hepatocellular carcinoma (HCC) is unclear. This study investigated the significance of ROR2 expression in HCC.MethodsThe study examined ROR2 expression in liver cancer cell lines. Immunohistochemical staining of ROR2 was performed on 243 resected HCC specimens. The study investigated ROR2 expression and its association with clinicopathologic factors and prognosis.ResultsFindings showed that ROR2 was expressed in well-differentiated Huh7 and HepG2 cells, but not in poorly differentiated HLE and HLF cells. Expression of ROR2 was positive in 147 (60.5%) and negative in 96 (39.5%) HCC specimens. A significant association was shown between ROR2 negativity and high alpha-fetoprotein (AFP) level (P = 0.006), poor differentiation (P = 0.015), and Wnt5a negativity (P = 0.024). The 5-year overall survival (OS) rate for the ROR2-negative group (64.2 %) tended to be worse than for the ROR2-positive group (73.8%), but the difference was not significant (P = 0.312). The 5-year OS rate was 78.7% for the ROR2+Wnt5a+ group, 71.3 % for the ROR2+Wnt5a− group, 80.8% for the ROR2−Wnt5a+ group, and 60.5 % for the ROR2−Wnt5a− group. The OS in the ROR2−Wnt5a− group was significantly poorer than in the ROR2+Wnt5a+ group (P = 0.030). The multivariate analysis showed that Wnt5a−ROR2− was an independent prognostic factor (hazard ratio, 2.058; 95% confidence interval, 1.013–4.180; P = 0.045).ConclusionsThe combination of ROR2 and Wnt5a may be a prognostic indicator for HCC. The Wnt5a/ROR2 signal pathway may be involved in the differentiation of HCC. This pathway may be a new therapeutic target for HCC.

ROR2 deficit may induce the tetralogy of Fallot via down-regulating of β-catenin/SOX3/HSPA6 in vitro and in vivo.

Tetralogy of Fallot (TOF) is the highly conventional appearance of cyanotic congenital heart disease. Our study aimed to assess the involvement of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in TOF and elucidate the specific mechanism. Upon investigation of human tissue samples, we observed a decrease in ROR2 expression in TOF patients compared to healthy control individuals. Transcriptome analysis revealed diminished ROR2 expression in TOF pathological samples relative to normal tissues. Of the 2246 genes that exhibited altered expression, 886 were upregulated, while 1360 were down-regulated. KEGG analysis and GO analysis of the differentially expressed genes indicated that these genes were significantly enriched in the Wnt signalling pathway, apoptosis and cardiac development function. Importantly, ROR2 was the only gene shared among the three pathways. Furthermore, interference with ROR2 promotes apoptosis and curtails cell proliferation in vitro. The knockdown of the ROR2 gene in AC16 cells resulted in a significant decrease in Edu-positive cells. Flow cytometry studies indicated an increase in the percentage of cells in the S phase. In contrast, the G2/M cell cycle transition was blocked in the ROR2-knockdown group, leading to a significant increase in apoptosis. Moreover, the CCK-8 cell viability assay demonstrated a reduced proliferation in the ROR2-knockdown group. Furthermore, both in vivo and in vitro data indicated that the expression of HSPA6 (Recombinant Heat Shock 70 kDa Protein6), an essential gene enriched in cardiac tissue and associated with apoptosis, was down-regulated following ROR2 knockdown mediated by the β-catenin/SOX3 signalling pathway. In conclusion, low expression of ROR2 plays a crucial role in the occurrence and development of TOF, which may be related to the downregulation of HSPA6 through the β-catenin/SOX3 signalling pathway.

Identification and validation of a small molecule targeting ROR1 for the treatment of triple negative breast cancer.

Introduction: Breast cancer is the most common cancer in women, with roughly 10-15% of new cases classified as triple-negative breast cancer (TNBC). Traditional chemotherapies are often toxic to normal cells. Therefore, it is important to discover new anticancer compounds that target TNBC while causing minimal damage to normal cells. Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is an oncofetal protein overexpressed in numerous human malignancies, including TNBC. This study investigated potential small molecules targeting ROR1. Methodology: Using AutoDock Vina and Glide, we screened 70,000 chemicals for our investigation. We obtained 10 representative compounds via consensus voting, deleting structural alerts, and clustering. After manual assessment, compounds 2 and 4 were chosen for MD simulation and cell viability experiment. Compound 4 showed promising results in the viability assay, which led us to move further with the apoptosis assay and immunoblotting. Results: Compound 4 (CID1261330) had docking scores of -6.635 and -10.8. It fits into the pocket and shows interactions with GLU64, ASP174, and PHE93. Its RMSD fluctuates around 0.20nm and forms two stable H-bonds indicating compound 4 stability. It inhibits cell proliferation in MDA-MB-231, HCC1937, and HCC1395 cell lines, with IC50 values of approximately 2μM to 10μM, respectively. Compound 4 did not kill non-malignant epithelial breast cells MCF-10A (IC50 > 27μM). These results were confirmed by the significant number of apoptotic cells in MDA-MB-231 cells (47.6%) but not in MCF-10A cells (7.3%). Immunoblot analysis provided additional support in the same direction. Discussion: These findings collectively suggest that compound 4 has the potential to effectively eliminate TNBC cells while causing minimal harm to normal breast cells. The promising outcomes of this study lay the groundwork for further testing of compound 4 in other malignancies characterized by ROR1 upregulation, serving as a proof-of-concept for its broader applicability.

Evaluation of the prognostic significance of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in lung carcinoma and its relation to lymphangiogenesis and epithelial mesenchymal transition

Exploring the carcinogenic mechanisms of lung carcinoma helps to discover novel prognostic biomarkers and develop new therapeutic options to improve patient’s survival. Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1), a transmembrane protein, contributes to cancer progression and metastasis; via stimulation of epithelial mesenchymal transition (EMT) and promotion of angiogenesis. This makes ROR1 an important target for tumor therapy. This study aimed to evaluate expression of ROR1, E-cadherin (a marker of EMT), and D2–40 (a marker of lymphangiogenesis) in lung carcinoma and associate their expressions with the available clinicopathological parameters and patients’ survival. Immunohistochemical staining using ROR1, E-cadherin, and D2–40 was performed for 78 cases of lung carcinoma. Kaplan-Meier survival curves and Cox-regression analysis were done. High ROR1 expression was detected in 46.2% of cases. Significant relations were found between high ROR1 expression and larger tumor size (P < 0.001), poorly differentiated tumors (P = 0.001), advanced tumor stages (P < 0.001), positive lymph nodal status (P < 0.001), decreased E-cadherin expression (P < 0.001), and high lymphovascular density (LVD) (P < 0.001). Patients’ progression free survival (PFS) and overall survival (OS) were shorter with high ROR1 expression. High ROR1 expression, high LVD, large tumor size, and adenocarcinoma histopathological type were independent risk factors for OS in lung carcinoma patients. High ROR1 expression is associated with poor prognostic parameters in lung carcinoma patients including higher grade, advanced stages, high LVD, epithelial mesenchymal transition, as well as decreased PFS and OS.

A phase 2 open-label study of conditionally active biologic ozuriftamab vedotin (BA3021) in failed PD-1/L1 treatment of patients with recurrent or metastatic squamous cell carcinoma of the head and neck.

TPS6107 Background: Ozuriftamab vedotin (BA3021) is a conditionally active biologic (CAB) anti-receptor tyrosine kinase orphan receptor 2 (ROR2) humanized monoclonal antibody (IgG1) conjugated to monomethyl auristatin E (MMAE) using a cleavable linker (CAB ROR2 ADC) (Short, et al., 2014, Chang, et al. 2021). Conditional and reversible binding by CABs is designed to reduce off-tumor toxicity and immunogenicity, avoid tissue-mediated drug deposition, and improve pharmacokinetics (PK). ROR2 is a cell-surface transmembrane receptor protein tyrosine kinase highly expressed in several tumor types including head and neck squamous cell cancer (HNSCC). Increased ROR2 expression has been associated with tumor resistance to chemotherapy, programmed death-1 (PD-1) inhibitors, molecular targeted therapy, and radiation therapy. A pronounced unmet need exists as the majority of patients with recurrent or metastatic HNSCC experience disease progression with existing therapies (Chow 2020). Methods: BA3021-002 is a multi-center, open-label, single-arm Phase 2 study (n=40) designed to evaluate the efficacy and safety of BA3021 in patients previously treated with PD-(L)1 with ROR-2 expressing recurrent or metastatic HNSCC. Eligible patients include those with measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 criteria. Failure to prior treatment is defined as documented disease progression after no more than one PD-1/L1 inhibitor either administered as monotherapy or in combination therapy. Pharmacokinetic (PK), pharmacodynamic (PD), immunogenicity, and biomarker assessments will be performed. Enrollment is ongoing (NCT05271604)

CRI-CCTG-0003/IND.240: An immunotherapy platform study in platinum-resistant high grade serous ovarian cancer (IPROC).

TPS5617 Background: Identifying new treatments for patients with platinum-resistant high grade serous ovarian cancer (HGSC) is a priority. Although several biological features of HGSC suggest inherent immunogenicity, immunotherapy has had limited efficacy to date. IPROC is an adaptive platform trial designed to evaluate multiple immunotherapy combinations selected and informed by an expert panel. IPROC accommodates both biomarker-agnostic and biomarker-directed combinations. Protocol and sub study specific translational studies are conducted on archived diagnostic tissue and serial prospective blood and tumor sample collection. Methods: IPROC is an open-label, multi center, phase II platform trial. Eligibility includes HGSC histology; progression within 6 months of the last platinum regimen; &lt;1 prior line of chemotherapy for platinum resistant disease (unlimited prior lines for platinum sensitive); prior treatment with immunotherapy allowed (provided not discontinued for toxicity), ECOG 0/1. Patients must be willing and able to undergo study procedures. The primary endpoint is ORR (RECIST1.1). Secondary endpoints include iRECIST ORR, RECIST/iRECIST progression free and overall survival and safety. Exploratory objectives include whole exome sequencing and immune profiling of tumor and blood samples to identify biomarkers and new immunotherapy strategies/combinations for future evaluation. Tumor biopsy occurs at baseline, cycle 2 and progression (voluntary) with blood and plasma collected at each cycle and on progression. All sub studies use Simon 2-stage design with 10 patients (pts) enrolled in each stage. At the end of stage 1, in the absence of response, a modified cohort based on biomarker selection may be created through protocol amendment. In each substudy a null hypothesis that the objective response rate (ORR) is ≤5% is tested against a one-sided alternative that the ORR is ≥ 25%. A combination would be accepted as active if ≥4/20 pts respond with α=0.02 and power of 0.76. As of January 19, 2023, 2 sub studies are open and 12 patients have accrued. Sub study A is evaluating the combination of durvalumab and BA3011, a conditionally active anti-AXL humanized monoclonal antibody (IgG1) conjugated to monomethyl auristatin E (MMAE) in AXL overexpressing HGSC. Sub study B is evaluating durvalumab plus BA3021, a CAB anti-receptor tyrosine kinase orphan receptor 2 (ROR2) humanized monoclonal antibody (IgG1) conjugated to MMAE in ROR2 overexpressing HGSC. Further sub studies are planned (NCT04918186). Clinical trial information: NCT04918186 .

WaveLINE-007: Phase 2 study of zilovertamab vedotin (ZV) in combination with cyclophosphamide, doxorubicin, and prednisone plus rituximab (R-CHP) in previously untreated diffuse large B-cell lymphoma (DLBCL).

TPS7589 Background: The preferred first-line regimen for DLBCL is rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP); but novel therapies are needed. A recent phase 3 study showed that replacing vincristine with an antibody-drug conjugate (ADC) is a viable approach (Tilly H et al. N Engl J Med. 2022;386:351-363). Receptor tyrosine kinase–like orphan receptor 1 (ROR1) is an oncofetal protein that is minimally expressed in adult tissues and overexpressed in DLBCL. ZV is an ADC comprising a humanized IgG1 monoclonal anti-ROR1, a proteolytically cleavable linker, and the antimicrotubule agent, monomethyl auristatin E. The single-arm, open-label, phase 2 waveLINE-007 study (NCT05406401) will investigate ZV combined with R-CHP in patients with previously untreated DLBCL. Part 1 is being conducted to determine safety and tolerability and recommended phase 2 dose (RP2D) of ZV in combination with R-CHP. Part 2 will be conducted to investigate efficacy of ZV at the RP2D with R-CHP. Methods: Eligible patients will be ≥18 years of age and have previously untreated histologically confirmed DLBCL, positron emission tomography (PET)–positive disease verified by blinded independent central review (BICR), an ECOG PS of 0 or 1, and adequate organ function. Patients diagnosed with primary mediastinal B-cell lymphoma, with a history of transformation of indolent disease to DLBCL, or active central nervous system lymphoma will be excluded. Approximately 60 patients will be enrolled (part 1, n = 45; part 2, n = 15). Part 1 will use a modified toxicity probability interval design to establish the RP2D of ZV when administered with R-CHP. The starting dose of ZV will be 1.75 mg/kg (modified to 1.5, 2.0, 2.25, or 2.5 mg/kg) administered as an intravenous infusion every 3 weeks (Q3W) in combination with R-CHP. In part 2, an additional 15 patients will receive ZV at RP2D plus R-CHP Q3W for up to 6 cycles until disease progression per Lugano 2014 criteria, unacceptable toxicity, or withdrawal. Disease response assessment, computed tomography, and PET will occur at baseline and cycles 3 and 6. Adverse events (AEs) will be monitored up to 30 days after cessation of treatment (90 days for serious AEs, or 30 days if new anticancer therapy is initiated). AEs will be graded per National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0. Primary end points are safety and tolerability and RP2D for ZV in combination with R-CHP, and complete response rate per Lugano 2014 criteria as assessed by the investigator. Secondary end points are objective response rate and duration of response per Lugano 2014 criteria by investigator review. Exploratory end points include progression-free survival per Lugano 2014 criteria by BICR and overall survival. Recruitment is currently underway. Clinical trial information: NCT05406401 .