anti-ROR1 Monoclonal Antibody Research Articles

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.

Retraction: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells.

Retraction: Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells.

ROR1 and ROR2 expression in pancreatic cancer

BackgroundThe Wnt receptors ROR1 and ROR2 are generating increased interest as cancer therapeutic targets but remain understudied in pancreatic ductal adenocarcinoma (PDAC). Compared to canonical Wnt/ β-catenin signalling, the role of noncanonical Wnt signalling in PDAC remains largely unknown. Only one study has investigated the prognostic significance of the noncanonical Wnt signalling receptor, ROR2 in PDAC. No studies have investigated the prognostic role of ROR1 in PDAC.MethodsHere, we performed analysis of ROR1 and ROR2 mRNA expression in three publicly available datasets ICGC-PACA-AU (n = 81), TCGA-PAAD (n = 150) and CPTAC-PDAC (n = 137). ROR1 and ROR2 protein expression from the CPTAC-PDAC discovery cohort were also analysed. Immunohistochemistry (IHC) using the validated anti ROR1 monoclonal antibody (4A5) was performed on the Australian Pancreatic Cancer Genome Initiative (APGI) cohort of PDAC samples (n = 152). Association between ROR1 cytoplasmic staining intensity and clinicopathological parameters including stage, grade and overall survival (OS) was investigated.ResultsHigh ROR1 mRNA expression levels correlated with a favourable OS outcome in all of the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. ROR1 protein expression was not associated with stage, grade or OS in the APGI cohort.ConclusionROR1 and ROR2 have potential as prognostic markers when measured at the mRNA level in PDAC. Our IHC cohort did not support ROR1 protein expression in predicting OS, and highlighted the discrepancy of prognostic biomarkers when measured by MS, IHC and RNAseq.

The ROR1 antibody-drug conjugate huXBR1-402-G5-PNU effectively targets ROR1+ leukemia

AbstractAntibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody–based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

Abstract 1062: Inhibition of ovarian and endometrial cancer cell proliferation by an anti-ROR1 monoclonal antibody

Abstract The non-canonical Wnt signalling receptor ROR1 has been shown to be aberrantly expressed in numerous cancers, including ovarian and endometrial cancer (EC). Cirmtuzumab is a humanised monoclonal antibody against ROR1 that blocks Wnt5a-induced ROR1 signalling. It has demonstrated safety and efficacy in several Phase I/II clinical trials for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Her2-negative breast cancer. The aim of this study was to investigate any anti-proliferative effect of cirmtuzumab in combination with commonly-used gynaecological cancer therapies (cisplatin, paclitaxel and the PARP inhibitor, olaparib) on high grade serous ovarian cancer (HGSOC) and EC cell lines including models of platinum resistance. The ROR1 positive HGSOC cells lines CaOV3, CaOV3CisR PEO1, PEO4 and EC cell lines Ishikawa, KLE were used in this study. First, IC50 for cisplatin, paclitaxel and olaparib in each cell line at 72h was determined using the cell counting kit 8 (CCK-8). Then, cells were seeded in 12-well plates and treated with cirmtuzumab at 25µg/ml or 50µg/ml for 4h prior to the addition of the chemotherapeutic agents at IC70 concentration. The effect of cirmtuzumab +/- agents was quantified using the IncuCyte S3 Live Cell Analysis System. Phase contrast cell images were obtained using a 10x objective lens within the instrument every 3h for 72h in total. The average confluence of each well was calculated and normalised against the baseline (time 0). Two-way ANOVA followed by Bonferroni post-test was performed to evaluate the effect of treatments. RNA and protein were extracted at the end of the incubation for qRTPCR and Western blot analysis. Single dose cirmtuzumab at both 25µg/ml and 50µg/ml significantly inhibited proliferation of CaOV3, CaOV3cisR, PEO1 and Ishikawa cells. 50µg/ml of cirmtuzumab decreased proliferation of KLE. Compared to paclitaxel alone, addition of 50µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3, CaOV3CisR, PEO1 and PEO4. Compared to cisplatin alone, addition of 25µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3CisR, PEO4; addition of 50µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3, CaOV3CisR, PEO4. Compared to olaparib alone, addition of 25µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3CisR; addition of 50µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3 and CaOV3CisR. No significant change in ROR1 or ROR2 expression levels was observed following cirmtuzumab treatment, however treatment did result in alterations to markers of epithelial-mesenchymal transition (EMT). Cirmtuzumab alone inhibited proliferation of ovarian cancer and EC cells in vitro, and could enhance the activity of commonly-used chemotherapeutic agents. This study supports the potential of cirmtuzumab or other ROR1 targeting therapies for treating women with HGSOC and EC. Citation Format: Dongli Liu, Gunnar F. Kaufmann, James B. Breitmeyer, Kristie-Ann Dickson, Deborah J. Marsh, Caroline E. Ford. Inhibition of ovarian and endometrial cancer cell proliferation by an anti-ROR1 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1062.

Evaluation of ROR1 Targeted Antibody Drug Conjugates in Acute and Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is a developmentally restricted receptor tyrosine kinase that is expressed on the surface of multiple hematological and solid malignancies but not on normal adult tissues. Recently, there has been an increased interest in ROR1 as a potential therapeutic target in multiple ROR1 positive leukemia and lymphomas. In this study, we present novel anti-ROR1 monoclonal antibody drug conjugates (ADCs) based on chimeric rabbit/human mAb “XBR1-402” or its humanized version “huXBR1-402”, that show both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) leukemic cells.Methods: From our studies, we saw no difference in activity between XBR1-402-PNU and huXBR1-402-PNU. XBR1-402-PNU was used for our studies in pre B cell ALL and huXBR1-402-PNU was used for our studies in MCL and CLL. For ALL in vitro experiments, we monitored cytotoxicity in Kasumi2 and 697 cell lines. For MCL in vitro experiments we monitored cytotoxicity in Jeko and Mino cell lines. For CLL in vitro experiments, we monitored cytotoxicity in primary CLL samples. For ALL in vivo experiments we monitored overall survival in a murine disseminated 697 model. For CLL in vivo experiments we monitored ROR1+ leukemic cell depletion in human ROR1 transgenic mice engrafted with human ROR1+TCL1 leukemia.Results: We observed a decrease in viability for all XBR1-402-PNU and huXBR1-402-PNU treated ROR1 positive leukemic cell lines and primary cells in vitro . In the in vitro model of pre B cell ALL we observed a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU(EC50 = 77 and 1 ng/mL, respectively) when compared to treatment with the isotype-matched control ADC Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL, respectively). In the in vitro model of MCL, we observed a decrease in viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 and Jurkat cell lines when compared to the unconjugated huXBR1-402 antibody or the control Tras-PNU. Additionally, we tested primary CLL samples confirmed to be positive for ROR1 and found a significant decrease in viability over 96 hours when treated with the antibody-drug-conjugate huXBR1-402 (78.6 + 6.1%)as compared to the unconjugated antibody (100% + 2.4; p=0.022, N=7) and the control Tras-PNU (87.4% +/- 6.1; p=0.038). We have also detected promising in vivo activity with XBR1-402-PNU and huXBR1-402-PNU in ALL and CLL models, respectively. In vivo studies on a murine disseminated 697 ALL model showed that treatment with XBR1-402-PNU increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days after implantation respectively, p=0.021, n=6). In addition, in preliminarily experiments, we have tested depletion of human ROR1+ leukemic cells and normal B cells in vivo using human ROR1 transgenic mice engrafted with human ROR+TCL1 leukemia and observed a preferential depletion of ROR1+TCL1 leukemic cells upon treatment with huXBR1-402-PNU compared to control Tras-PNU.Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration in patients with ROR1 positive leukemia and lymphomas. Current long-term studies are ongoing to evaluate the in vivo cytotoxic effects of huXBR1-402-PNU on total tumor burden, disease progression, and overall survival.(EH is supported by Graduate Pelotonia Fellowship) DisclosuresWaldmeier:NBE-Therapeutics Ltd: Employment. Beerli:NBE-Therapeutics Ltd: Employment. Grawunder:NBE-Therapeutics Ltd: Employment. Byrd:The Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding.

Cirmtuzumab blocks Wnt5a/ROR1 stimulation of NF-κB to repress autocrine STAT3 activation in chronic lymphocytic leukemia

AbstractCoculture of nurse-like cells (NLCs) with chronic lymphocytic leukemia (CLL) cells induced leukemia cell phosphorylation of STAT3 (pSTAT3), which could be blocked by anti-Wnt5a antibodies or the anti-ROR1 monoclonal antibody, cirmtuzumab. Time-course studies revealed Wnt5a could induce activation of NF-κB within 30 minutes, but required more than 3 hours to induce pSTAT3. Culture of isolated CLL cells for 24 hours revealed Wnt5a-induced expression of interleukin 6 (IL-6), IL-8, CCL2, CCL3, CCL4, and CXCL1, which in turn could induce pSTAT3 in unstimulated CLL cells within 30 minutes. We found that Wnt5a could induce CLL cell expression of NF-κB target genes, including IL-6, and that this effect could be blocked by cirmtuzumab or drugs that inhibit NF-κB. Examination of CLL cells and plasma collected from patients treated with cirmtuzumab revealed reduced levels of phosphorylated p65 and diminished expression of NF-κB and STAT3 target genes in CLL cells, as well as lower plasma levels of IL-6, in the samples after therapy. Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling contributes to CLL cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pSTAT3. As such, this study demonstrates that cirmtuzumab can inhibit leukemia cell activation of both NF-κB and STAT3 in patients with CLL.

Abstract 1541: Evaluation of ROR1 targeted antibody drug conjugate in ROR1 positive leukemia

Abstract Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is expressed on the surface of multiple blood and solid tumors. There have been increased interest in ROR1 as a potential therapeutic target in ROR1 positive leukemia and lymphomas. In this study, we present a novel first-in-class anti-ROR1 monoclonal antibody drug conjugate (ADC) based on chimeric rabbit/human mAb “XBR1-402” or its humanized version “huXBR1-402”. Conjugated to a highly potent anthracycline derived toxin, PNU-159682 (PNU) via a non-cleavable peptide/amide linker, it shows both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) cells. Results: We saw a decrease in viability for all ADC treated ROR1 positive leukemic cell lines. We saw a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU (EC50 = 77 and 1 ng/mL) when compared to treatment with isotype-matched control Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL). In in vitro models of MCL, we saw decreased viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 cell line when compared to Tras-PNU. While we were unable to observe significant direct cytotoxicity in primary CLL cells, we show that PNU ADCs mediates antibody dependent cellular cytotoxicity and phagocytosis in vitro. We also show that PNU ADCs mediates a G2/M cell cycle arrest in affected cell lines and hypothesize that this is necessary for direct cytotoxicity. As ex vivo primary CLL cells are non-proliferative, we postulate that PNU would target actively dividing cells in proliferation centers (e.g. secondary lymphoid organs). To test this hypothesis, we set up in vivo studies to test with XBR1-402-PNU and huXBR1-402-PNU in disseminated and aggressive ROR1+ ALL and CLL models, respectively. In vivo studies on a murine 697 ALL model suggested XBR1-402-PNU treatment increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days post implantation, p=0.021, n=6). In vivo CLL studies with engrafted murine human-ROR1 expressing TCL1 leukemic cells showed that huXBR1-402-PNU treatment (3 times/week for 1 week) both suppressed leukemia burden and increased overall survival when compared with Tras-PNU control (Median survival of 62 and 41 days post implantation, p=0.0028, n=4). Ongoing proliferation and cell cycling studies will confirm the mechanism of the in vivo cytotoxicity of huXBR1-402-PNU. Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration as either a single agent or combination therapy in patients with ROR1 positive leukemia and lymphomas. Citation Format: Eileen Hu, Priscilla Do, Rajeswaran Mani, Frank Frissora, Rebecca Pearson, Gerard Lozanski, Haiyong Peng, Lorenz Waldmeier, Roger Beerli, Christoph Rader, Ulf Grawunder, John Byrd, Natarajan Muthusamy. Evaluation of ROR1 targeted antibody drug conjugate in ROR1 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1541.

ROR1 Expression and Its Functional Significance in Hepatocellular Carcinoma Cells.

Background: Hepatocellular carcinoma (HCC) is a common and deadly cancer; however, very little improvement has been made towards its diagnosis and prognosis. The expression and functional contribution of the receptor tyrosine kinase ROR1 have not been investigated in HCC before. Hence, we investigated the expression of ROR1 in HCC cells and assessed its involvement in hepatocarcinogenesis. Methods: Recombinant bacterial ROR1 protein was used as an immunogen to generate ROR1 monoclonal antibodies. ROR1 transcript levels were detected by RT-qPCR and the protein expression of ROR1 in HCC was assessed by Western blotting by using homemade anti-ROR1 monoclonal antibodies. Apoptosis, cell cycle, trans-well migration, and drug efflux assays were performed in shRNA-ROR1 HCC cell clones to uncover the functional contribution of ROR1 to hepatocarcinogenesis. Results: New ROR1 antibodies specifically detected endogenous ROR1 protein in human and mouse HCC cell lines. ROR1-knockdown resulted in decreased proliferation and migration but enhanced resistance to apoptosis and anoikis. The observed chemotherapy-resistant phenotype of ROR1-knockdown cells was due to enhanced drug efflux and increased expression of multi-drug resistance genes. Conclusions: ROR1 is expressed in HCC and contributes to disease development by interfering with multiple pathways. Acquired ROR1 expression may have diagnostic and prognostic value in HCC.

Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody

Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer.

Wnt5a induces ROR1 to recruit DOCK2 to activate Rac1/2 in chronic lymphocytic leukemia

AbstractReceptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on chronic lymphocytic leukemia (CLL) that can serve as a receptor for Wnt5a, which can promote leukemia cell migration, proliferation, and survival. We found Wnt5a could induce ROR1 to complex with DOCK2 (dedicator of cytokinesis 2) and induce activation of Rac1/2; these effects could be blocked by cirmtuzumab, a humanized anti-ROR1 monoclonal antibody. We find that silencing DOCK2 specifically impaired the capacity of Wnt5a to induce activation of Rac1/2 or enhance CLL cell proliferation. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for Wnt5a to induce ROR1 to complex with DOCK2 and activate Rac1/2 in the CLL cell-line MEC1. We introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at potential binding sites for the Src-homology 3 domain of DOCK2. In contrast to wild-type ROR1, or other ROR1 P→A variants, ROR1P808A was unable to recruit DOCK2 in response to Wnt5a. Moreover, unlike MEC1 cells transfected with wild-type ROR1 or ROR1 with P→A substitutions at positions 784, 826, or 841, MEC1 cells transfected to express ROR1P808A did not have a growth advantage over MEC1 cells that do not express ROR1. This study reveals that the recruitment of DOCK2 may be critical for the capacity of Wnt5a to enhance CLL proliferation, which may contribute to the observed increased tendency for disease progression in patients who have CLL cells that express high levels of ROR1.

Production and evaluation of a 67Ga-labeled anti-Ror1 monoclonal antibody in a mouse model of breast cancer

Receptor tyrosine kinase Ror1 is widely expressed during embryogenesis but it is absent within most mature tissues. However, expression of Ror1 has been reported in multiple human malignancies including breast cancer. High level expression of Ror1 in breast adenocarcinoma was associated with aggressive disease. Breast cancer radioimmunoscintigraphy targeting Ror1 expression is an attractive object in molecular imaging especially nuclear medicine research. In this study, we evaluated production, quality control and preclinical qualifications of a gallium-67 labeled anti-Ror1 antibody to see if it has potential to be used as a SPECT tracer for diagnosis and monitoring of Ror1 expressed tumors.

Dishevelled proteins are significantly upregulated in chronic lymphocytic leukaemia.

Dishevelled (DVL) proteins are components of the Wnt signalling pathways, and increased expression is associated with various malignancies. Information on DVLs in chronic lymphatic leukaemia (CLL) is limited. The aim of the present study was to investigate the role of DVLs in CLL cells and association with Wnt pathways downstream of ROR1. DVL1, 2 and 3 were exclusively expressed in CLL cells as compared to normal peripheral blood mononuclear cells (PBMCs). The expression of DVL1 and DVL3 proteins was significantly more pronounced in progressive than in non-progressive disease (p < 0.01), whereas the level of DVL2 was significantly higher in non-progressive as compared to progressive disease (p < 0.001). Treatment of CLL cells with anti-ROR1 specific monoclonal antibodies induced dephosphorylation of ROR1 as well as of tyrosine and serine residues of both DVL2 and DVL3. However, gene silencing of DVLs in the CLL cell line (EHEB) did not induce detectable apoptosis. Non-progressive CLL patients had a different protein activity pattern with regard to Wnt signalling pathway proteins as GSK-3β, β-catenin and AKT as compared to progressive disease. The DVL2 protein may play a role in the activation of signalling pathways in CLL during early stages of the disease, while DVL1 and 3 may have a role in later phases of the leukaemia.

Cirmtuzumab (UC-961), a First-in-Class Anti-ROR1 Monoclonal Antibody: Planned Interim Analysis of Initial Phase 1 Cohorts

INTRODUCTIONChronic lymphocytic leukemia (CLL) cells of nearly all patients (pts) express ROR1 (Receptor tyrosine kinase-like Orphan Receptor 1), an orphan-receptor tyrosine-kinase-like protein that is normally expressed during embryogenesis, but not by normal post-partum tissues. ROR1 is a receptor for Wnt5a, which can induce non-canonical Wnt signaling to enhance CLL-cell survival and/or proliferation. We have developed antibodies that bind to epitopes that span the extracellular portion of human ROR1, and selected one mAb that had the most potent activity in inhibiting such signaling. We fully humanized this antibody to generate UC-961 (cirmtuzumab). GLP-compliant studies demonstrated that this antibody does not cross-react with normal post-partum tissues. Preclinical pharmacology/toxicology studies in Sprague-Dawley rats showed no evidence of toxicity for cirmtuzumab at doses up to 400 mg/kg by IV administration with an apparent half-life exceeding 7 days. Studies in cynomolgus monkeys again showed no apparent toxicity with an antibody half-life of approximately 14 days. Based on these IND-enabling studies, we have initiated a first-in-human phase 1 dose escalation study to determine the safety and tolerability of cirmtuzumab in the treatment of pts with relapsed or refractory CLL. Here, we report results of planned interim safety analysis of pts in the initial phase 1 cohorts. METHODSKey eligibility criteria included relapsed or refractory CLL and indication for therapy according to working group guidelines (iwCLL). The starting dose was 15 mcg/kg, based on preclinical modeling of 30% target saturation. Cirmtuzumab was administered every 14 days for a total of 4 doses, with intra-patient dose escalation in the absence of toxicity. Patients were enrolled in cohorts of 3 to 6 patients, with the most recently completed cohort receiving doses of 0.5 mg/kg to 1 mg/kg. Pre-planned analysis of safety and tolerability was conducted at the completion of a 56-day dose-limiting toxicity observation period. RESULTSBetween August 2014 and July 2015, 10 pts have received cirmtuzumab. The median age of the treated pts was 72 (range 58 to 81); the median number of prior therapies was 4 (ranging from 1-9). Nine pts had at least 1 high-risk prognostic factor, including leukemia-cell expression of ZAP-70 (4 pts), unmutated IGHV (5 pts), del(17p) or complex karyotype (5 pts). Cirmtuzumab was well tolerated. We have not observed ≥ grade 2 drug-related adverse events (AE) in any of the treated pts. The only grade 1 drug-related AE that occurred in more than 1 pt was anemia (3 pts), all of which resolved. One patient came off study due to continued disease progression after receiving a single dose of 15 mcg/kg; the rest of the pts received all four doses of cirmtuzumab, as per protocol. The low dose administered to patients in the first few cohorts precluded us from detecting sufficient cirmtuzumab in the plasma for pharmacokinetic studies. However, cirmtuzumab was detected in the plasma of pts in later cohorts, allowing us to estimate a half-life for cirmtuzumab of approximately 14 days. Response was assessed as per iwCLL criteria 2 months after the final dose of cirmtuzumab. 3 pts have not yet reached that time point, and 1 pt was not evaluable due to progresison and early study discontinuation. Of the 6 pts evaluable for response, 2 experienced continued disease progression and 4 met criteria for stable disease. With a median follow-up of 103 days after the completion of cirmtuzumab, 2 pts that had disease progression have required additional therapy; the other 4 have yet to require additional treatment. CONCLUSIONSCirmtuzumab is a first-in-class anti-ROR1 monoclonal antibody. In a phase 1 dose-escalation trial, pts with relapsed or refractory CLL have tolerated cirmtuzumab well, without any drug-related grade 2 or higher AEs. This is consistent with the preclinical profile and lack of target expression in normal tissues. The ongoing phase 1 study will evaluate the safety and tolerability of higher doses to determine the maximum tolerated dose or biologically optimal dose of cirmtuzumab. DisclosuresChoi:Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Jamieson:J&J: Research Funding; GSK: Research Funding. Kipps:Gilead: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding.

Abstract 4406: ROR1 targeted delivery of OSU-2S, a non-immunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle cell lymphoma in-vitro and in-vivo

Abstract Treatment of mantle cell lymphoma (MCL), an uncommon non-Hodgkin's lymphoma, remains challenging despite numerous therapeutic advances. We have previously shown the preclinical effect of FTY720 (Fingolimod) against MCL through down modulation of cyclin D1, the protein almost uniformly over-expressed in this disease. Herein, we describe the potent direct cytotoxicity of OSU-2S, a novel non-immunosuppressive FTY720 derivative in MCL cells and evaluate tumor directed lipid based nanoparticle formulation of OSU-2S designed to selectively deliver to ROR1+ MCL cells. OSU-2S is a FTY720 derivative that does not traffic T cells and exhibits potent cytotoxicity in MCL cell lines and in MCL patient-derived primary cells (p = 0.0049). Exploratory studies aimed to identify the best combination therapies identified induction of cell surface CD74 in primary MCL cells treated with OSU-2S. Similarly, induction of cell surface CD74 was also confirmed in JeKo and Mino cells. Evaluation of OSU-2S and anti-CD74 antibody, milatuzumab in cell lines and primary MCL cells revealed enhanced cytotoxicity compared to either of the agents alone. B-cell malignancy restricted expression of receptor tyrosine kinase ROR1 in MCL, chronic lymphocytic leukemia (CLL) and subset of pediatric acute lymphocytic leukemia (ALL) has been reported. Consistent with this, JeKo, Mino and primary MCL cells but not normal B cells expressed ROR1. As tumor directed delivery of cytotoxic cargo offers the potential to further enhance the therapeutic index of cancer therapeutics such as OSU-2S, we developed a lipid-based OSU-2S nanoparticle (OSU-2S-LP) tethered with an anti-ROR1 mouse monoclonal antibody (2A2) to form 2A2-OSU-2S-ILP that mediated selective cytotoxicity of MCL. Testing of immunonanoparticle formulation on JeKo, Mino cell lines and primary lymphoma cells showed selective cytotoxicity of 2A2-OSU-2S-ILP in MCL (p&amp;lt;0.001) compared to other controls, including non-targeted formulation (OSU-2S-LP), non-specific antibody (IgG-OSU-2S-ILP) or empty targeted formulation (2A2-Empty-ILP). In-vivo treatment with 2A2-OSU-2S-ILP in Mino cell line xenografted NOD-SCID-Gamma−/− mice revealed significantly diminished tumor growth compared to the control (p = 0.0001). These findings describe the novel OSU-2S molecule as a successor of FTY720 that is active against MCL. OSU-2S enhanced CD74 expression can be exploited in combination therapy with milatuzumab which is in human clinical trials. Importantly, the tumor antigen ROR1 directed delivery system for increasing the payload selectively to the cancer cells thus obviating the exposure of chemotherapeutics and targeted therapeutics to other unintended target cells has broad implications in diverse ROR1+ malignancies. Citation Format: Rajeswaran Mani, Chi-Ling Chiang, Frank W. Frissora, Ribai Yan, Xiaokui Mo, Sivasubramanian Baskar, Christoph Rader, Mitch Phelps, Ching-Shih Chen, Robert Lee, John Byrd, Robert Baiocchi, L James Lee, Natarajan Muthusamy. ROR1 targeted delivery of OSU-2S, a non-immunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle cell lymphoma in-vitro and in-vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4406. doi:10.1158/1538-7445.AM2015-4406

Abstract 206: Depletion of ovarian cancer initiating cells by a monoclonal antibody against ROR1

Abstract ROR1 is a type I orphan-receptor tyrosine-kinase-like surface protein that is expressed during embryogenesis and by various cancer cells, but not on normal adult tissues. Its expression is associated with less-well-differentiated tumors with high metastatic potential (Zhang et al Am J Pathol, 2012, PMCID 3509760; Cui et al Cancer Res, 2013, PMCID 3832210). We evaluated the expression of ROR1 in human ovarian cancers and examined the activity of newly developed anti-ROR1 monoclonal antibodies (mAbs, e.g. UC-961) found to have activity against ROR1-expressing breast cancer cell lines and B-cell leukemias. We examined primary human ovarian cancer cells engrafted into immune-deficient NOD/scid mice (PDX) for human ROR1 expression and for phenotypic and functional characteristics of cancer-stem cells (CSC). In each tumor specimen, we observed different proportions of ROR1-expressing cells. Expression of ROR1 associated with markers of CSC, such as aldehyde dehydrogenase 1 (ALDH1). Moreover, the ROR1+ cells from any given PDX sample had a greater capacity to form spheroids in vitro and to engraft immune-deficient mice than did ROR1-negative cells isolated from the same tumor population. Treatment of ovarian cancer cells with the anti-ROR1 mAb UC-961, but not control IgG, reproducibly impaired their ability to form spheroids, and to engraft into immune-deficient NOD/scid mice. In subsequent studies, we isolated ALDH1+ and ALDH1- cells from the same PDX samples. As noted in prior studies (Ginestier et al Cell Stem Cell, 2007, PMCID 2423808), ALDH1+ cells engrafted more effectively than ALDH1- cells. However, treatment of ovarian cancer cells with the UC-961 mAb abrogated the difference in the capacity of ALDH1+ versus ALDH1- cells to engraft NOD/scid mice. Analysis of the few tumors that did develop in mice engrafted with UC-961-treated, but not control IgG-treated, tumor cells revealed that, compared to the original PDX tumor sample, these tumors had lower proportions of ALDH1+ cells and reduced expression of the polycomb ring finger oncogene, BMI1, and changed the expression level of markers associated with the epithelial-mesenchymal transition (e.g. E-Cadherin and vimentin). Collectively, these studies indicate that expression of ROR1 is associated with CSC and that anti-ROR1 mAbs such as UC-961 can interfere with CSC function and self-renewal, suggesting that such biologics may be useful in the treatment of patients with ovarian cancer. Citation Format: Suping Zhang, Bing Cui, Hsien Lai, Grace Liu, Christina Wu, George Widhopf, Rongrong Wu, Fitzgerlad Lao, Richard Schwab, Dennis Carson, Thomas J. Kipps. Depletion of ovarian cancer initiating cells by a monoclonal antibody against ROR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2014-206

Abstract 4770: Anti-ROR1 monoclonal antibodies induce apoptosis in pancreatic cancer cells via the PI3-kinase/AKT/mTOR pathway

Abstract Background: The PI3-kinase/AKT/mTOR pathway is a frequently dysregulated pathway in cancer and of importance for tumor cell growth, differentiation and survival. This pathway may be activated in various cancers due to aberrant activation of receptor tyrosine kinases (RTKs). The PI3-kinase/AKTmediated activation of mTOR, stimulates cell proliferation and regulation of protein translation. In pancreatic cancer, PI3-kinase and AKT are constitutively activated. ROR1 is a type I transmembrane RTK belonging to one of the twenty families of RTKs. ROR1 is overexpressed and constitutively phosphorylated in pancreatic carcinoma. A unique anti-ROR1 mAb directed against the CRD (cysteine-rich domain) of the extracellular region of ROR1 was capable of inducing apoptosis as well as dephosphorylating the ROR1 molecule in CLL cells. Aim: To investigate the apoptotic effect of the anti-ROR1 CRD mAb and effects on the downstream signaling pathways PI3-kinase/AKT/mTOR using the ROR1+ pancreatic cancer cell line PaCa-2. Methods: The MTT assay and Annexin V/PI methods were used to detect apoptosis in a 24-96 hr assay. Cell lysates were prepared from antibody treated and untreated samples and subjected to Western blot analysis for identification of the signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. The total and phosphorylated levels of the following signaling proteins were analysed: ROR1, p-ROR1, PI3-kinase, p-PI3-kinase, AKT, p-AKT, mTOR, p-mTOR, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 30 min-2 h. Results: ROR1 was expressed on the surface of 40-45% of the pancreatic carcinoma cell line PaCa-2. ROR1 was constitutively phosphorylated. The frequency of apoptotic cells after incubation with the anti-ROR1 mAb ranged from 10% at 24 hr to 40% at 96 hr. Western blot analysis showed decreased levels of phosphorylated ROR1 and the PI3-kinaseδ isoform (p110δ) in treated compared to untreated samples. Phosphorylation intensity of AKT and mTOR also decreased in treated compared to untreated cells. There was no change in the phosphorylation levels of ERK and PKC proteins. Conclusion: Incubation of pancreatic cancer cells with an anti-ROR1 CRD mAb induced apoptosis. Apoptosis was preceded by dephosphorylation of ROR1, PI3-kinaseδ isoform (p110δ), AKT and mTOR proteins indicating deactivation of these signaling proteins by the anti-ROR1 mAb. Activation of mTOR molecule was related to activation of the PI3-kinase/AKT pathway. This pathway might be a survival signal in pancreatic cancer cells associated with an aberrant expression of ROR1. Further studies are warranted to better understand the signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs, which might be a potential therapeutic option. Citation Format: Amir Hossein Daneshmanesh, Mohammad Hojjat-Farsangi, Ali Moshfegh, Eva Mikaelsson, Abdul Salam Khan, Anders Österborg, Håkan Mellstedt. Anti-ROR1 monoclonal antibodies induce apoptosis in pancreatic cancer cells via the PI3-kinase/AKT/mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4770. doi:10.1158/1538-7445.AM2014-4770

Tumor Antigen ROR1 Targeted Delivery Of FTY720 Derivative OSU-2S Prolongs Survival In ROR1 Engineered Mouse Model Of Chronic Lymphocytic Leukemia

The discovery of predominantly inactive phosphatases in a variety of cancers and the potential for phosphatase targeted therapy as an alternative to kinase inhibitors especially in situations where the efficacy of the kinase inhibitors are compromised due to resistance mechanisms attributed to mutations and single nucleotide polymorphisms of the drug targets prompted us to evaluate potential activators of phosphatases in chronic lymphocytic leukemia (CLL) and other B cell malignancies. We have recently identified cytotoxic activity of OSU-2S, a novel non-immunosuppressive FTY720 derivative and PP2A activator against CLL. OSU-2S induced cytotoxicity was associated with PKC dependent phosphorylation of Serine 591 (S591) of tumor suppressor phosphatase SHP1 and its nuclear translocation consistent with a potential role for S591 phosphorylation. Here in, we demonstrate the molecular mechanisms and a rational approach for developing this novel agent for preclinical and clinical studies. In-vitro kinase assay demonstrated OSU-2S increased activity of purified PKC directly (p<0.0001) and also in CLL-B cells (N=5; p<0.05). Further, OSU-2S induced phospho SHP1S591 is inversely correlated with viability in CLL-B cells (N=20; rs= -0.64; p=0.0026). To elucidate the role of nuclear phospho SHP1S591, we performed gene expression studies by microarray analysis of RNA isolated from OSU-2S treated CLL cells revealing at least 260 genes that have changed by two fold (p<0.0005). Ingenuity pathway analysis (IPA) of the top 40 genes included some of B cell receptor (BCR) signaling candidates such as PI3Kγ, PLCγ, MAP2K6. Consistent with this, OSU-2S treatment reduced BCR activation of CLL cells stimulated with goat F(ab')2 against human IgA+IgG+IgM (H+L), as identified with reduced activation and viability. Moreover, with relevant to CLL disease Tcl1A expression that was identified to be down regulated in response to OSU-2S in the gene expression profile was independently confirmed to be significantly down regulated both at the mRNA (N=7; p=0.0159) and protein levels with the corresponding up regulation in cFOS and FRA2 two known inhibitory targets of Tcl1A. To overcome the limitations associated with non specific activity on unintended target cells and normal counterparts, we made OSU-2S immunoliposome (2A2-OSU-2S-ILP) formulation targeting malignant B cell specific tumor antigen receptor tyrosine kinase-like orphan receptor (ROR1). ROR1 is an orphan receptor tyrosine kinase that is expressed exclusively in malignant B but not normal B cells. We have used a non-cytotoxic anti-ROR1 monoclonal antibody 2A2 to formulate immunoliposome 2A2-ILP which showed selective binding and internalization in ROR1+ CLL B cells but not ROR1- normal B cells from healthy donors. To demonstrate the chemotherapeutic efficiency in a more relevant CLL model in-vivo, we have generated Eµ-hROR1 transgenic mouse which expresses B cell specific human ROR1. Crossing the Eµ-hROR1 mouse with Eµ-Tcl1 CLL mouse resulted in generation of Eµ-hROR1-Tcl1 mouse that exhibit CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Ex-vivostudies using CLL primary B cells or Eµ-hROR1-Tcl1 double transgenic mouse B cells showed selective toxicity of leukemic B cells by 2A2-OSU-2S-ILP compared to 2A2-Empty-ILP which does not have OSU-2S. Further, administration of 2A2-OSU-2S-ILP in Eμ-hROR1 transgenic mice resulted in selective depletion of ROR1 positive B cells and prolonged survival in Eµ-hROR1-Tcl1 spleen engrafted mouse model of CLL (N=11 for 2A2-OSU-2S-ILP and N=9 for 2A2-Empty-ILP; p<0.001). The novel OSU-2S, its delivery formulation, and the mouse models described here provide the tools for further development of OSU-2S formulations for B cell malignancies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

e22198 Background: The receptor tyrosine kinase ROR1 has important functions during normal embryogenesis. It is overexpressed and of importance for the survival of different malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. There is however little information on the expression and function of ROR1 in melanoma. Here, we investigated ROR1 expression, phosphorylation and the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on seven melanoma cell lines. Methods: ROR1 expression and phosphorylation was analysed in a series of malignant melanoma cell lines using RT-PCR, immunocytochemistry, flow cytometry and western blot. Annexin V/PI staining, MTT assay, PARP and caspase 8 and 9 cleavage as well as MCL-1 protein were used for detection of apoptosis induced by anti-ROR1 mAbs. The cytotoxic effects of anti-ROR1 mAbs were evaluated in the absence or presence of complement (CDC) or immune effector cells (ADCC). Transfection of melanoma cell lines using ROR1 siRNA was used for gene silencing. Results: ROR1 was shown to be overexpressed to a varying degree at protein level. ROR1 was phosphorylated at tyrosine and serine residues. Three out of four anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in the presence of complement or immune effector cells. ESTDAB081 and 094 cell lines were resistant to direct apoptosis of the four anti-ROR1 mAbs alone compared to the other melanoma cell lines, but not to complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC). ROR1 siRNA transfection induced down-regulation of the ROR1 expression both at the mRNA and protein levels proceeded by a significant apoptosis of the melanoma cells. Conclusions: The results indicate that ROR1 may play an important role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable targeted for mAb therapy.