The antibody drug conjugate VLS-101 targeting ROR1 is effective in CAR T-resistant mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare, aggressive and incurable subtype of non-Hodgkin's B-cell lymphoma. The principal barrier is frequent clinical relapse to multiple lines of therapies, including new FDA-approved biologics and cell therapy. Brexucabtagene autoleucel, the first and only FDA approved chimeric antigen receptor (CAR) T product in MCL, demonstrated unprecedented efficacy in overcoming resistance to Bruton's tyrosine kinase inhibitors. However, relapses have inevitably occurred and once relapsed these patients display a very poor clinical outcome. Currently, there is no optional therapy specifically designed for these patients. The development of tailored and more efficacious therapies is therefore critical and represents a new medical need. We found that while the receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed across most of the MCL cells, it is significantly elevated in CAR T-relapsed MCL tumors. To see whether this aberrant ROR1 expression contributed to CAR T resistance, we targeted ROR1 using VLS-101, a monomethyl auristatin E conjugated anti-ROR1 antibody. VLS-101 showed potent anti-MCL activity in vitro in ROR1-expressing MCL cell lines and ex vivo in primary patient samples. Importantly, VLS-101 safely induced tumor regression in PDX models resistant to CAR T-cell therapy, ibrutinib and/or venetoclax. These data advocate for targeting ROR1 as a viable approach in the treatment of ROR1-positive MCL tumors, especially those with failure to prior therapies. These data also provide strong evidence for future enrollment of post-CD19 CAR T-cell relapsed MCL patients in a first in-human phase 1b VLS-101 trial. The upcoming testing in a clinical setting will provide important insights on this new therapeutic development aiming to overcome the CAR T resistance via targeting ROR1, which is a rising unmet clinical need in MCL.

Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade.

Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the KrasLSL-G12D/+;p53f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.

CAR T-cell therapy for triple-negative breast cancer: Where we are.

Triple-negative breast cancer (TNBC) is the most complex and challenging breast cancer subtype to treat, and chemotherapy remains the standard of care. Clinically, TNBC has a relatively high rate of recurrence and poor prognosis, which leads to a significant effort to discover novel strategies to treat patients with these tumors. Currently, chimeric antigen receptor (CAR) T cell-based immunotherapy redirects the patient's immune system directly to recognize and eradicate tumor-associated antigens (TAAs) expressing tumor cells being explored as a treatment for TNBC. A steadily increasing research in CAR T-cell therapy targeting different TAAs in TNBC has reported. In this review, we introduce the CAR technology and summarize the potential TAAs, available CARs, the antitumor activity, and the related toxicity of CARs currently under investigation for TNBC. We also highlight the potential strategies to prevent/reduce potential "on target, off tumor" toxicity induced by CAR T-cell therapy. This review will help to explore proper targets to expand further the CAR T-cell therapy for TNBCs in the clinic.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

A fully chimeric IgG antibody for ROR1 suppresses ovarian cancer growth in vitro and in vivo.

BACKGROUND: Over-expression of Receptor-tyrosine-kinase-like Orphan Receptor 1 (ROR1) in cancer cells has been reported in the context of several tumors (including ovarian cancer) and is associated with poor prognosis. The aim of this study was to construct a fully chimeric anti-ROR1 IgG antibody (ROR1-IgG) and investigate its antitumor activity against ovarian cancer cells, bothin vitro and in vivo. METHODS: A fully chimeric anti-ROR1 IgG antibody (ROR1-IgG) eukaryotic expression vector was constructed and ROR1-IgG antibody was expressed in CHO cells. The characteristics of ROR1-IgG were investigated by ELISA, SPR, Western blotting, FACS and fluorescence staining analyses. CCK8 and wound healing assays were performed to determine inhibition and migration capacity of ovarian cancer cells after treatment with ROR1-IgGin vitro. Further, the antitumor activity of ROR1-IgG was assessed in vivo using tumor-mice xenograft model. RESULTS: The results showed that ROR1-IgG could specifically bind to ROR1-positive cells (HO8910 and A2780) with a high affinity. Functional studies revealed that ROR1-IgG inhibited the malignant behavior of ROR1-positive cells (HO8910 and A2780) in a time- and dose-dependent manner. These effects were not observed in ROR1-negative lose386 cells. The tumor inhibition rates following treatment with low, medium, and high concentrations of ROR1-IgG were approximately 47.72%, 53.79%, and 60.51%, respectively. In addition, the expression of Bcl-2 was obviously reduced while that of Bax was distinctly elevated in xenografts. CONCLUSIONS: Collectively, our findings suggest that ROR1-IgG may be a novel therapeutic agent for patients with ROR1-positive ovarian cancer.

ROR1 and ROR2-novel targets for neuroblastoma.

Background: Despite advances in immunotherapeutic strategies for neuroblastoma (NBL), relapse remains a significant cause of mortality for high risk patients. The discovery of novel tumor associated antigens to improve efficacy and minimize the toxicities of immunotherapy is therefore warranted. Receptor Tyrosine Kinase-like Orphan Receptor-1 and 2 (ROR1 and ROR2) have been found to be expressed in several malignancies with limited expression in healthy tissues. Objectives: Given their role in tumor migration and proliferation and the fact that they were originally cloned from a NBL cell line, we hypothesized that ROR1 and ROR2 could serve as potential targets for anti-ROR1 and anti-ROR2 based immunotherapies in NBL. Methods: We characterized the mRNA and protein expression of ROR1 and ROR2 in NBL cell lines and tissue microarrays of patient samples. To explore the potential of ROR1 targeting, we performed in vitro cytotoxicity assays against NBL using NK92 cells as effector cells. Results: Both ROR1 and ROR2 are expressed across all stages of NBL. In patients with non-MYC amplified tumors, expression of ROR1/ROR2 correlated with survival and prognosis. Moreover, in a proof-of-concept experiment, pretreatment of NBL cell line with anti-ROR1 antibody showed additive cytotoxicity with NK92 cells. Conclusions: ROR1 and ROR2 could serve as novel targets for immunotherapy in NBL. The additive effect of anti-ROR1 antibodies with NK cells needs to be explored further to evaluate the possibility of combining anti-ROR1 antibodies with immune effectors such as NK92 cells as a potential off-the shelf immunotherapy for NBL and other ROR1 expressing malignancies.

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

Coculture 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.

ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models.

Solid tumors impose immunologic and physical barriers to the efficacy of chimeric antigen receptor (CAR) T cell therapy that are not reflected in conventional preclinical testing against singularized tumor cells in 2-dimensional culture. Here, we established microphysiologic three-dimensional (3D) lung and breast cancer models that resemble architectural and phenotypical features of primary tumors and evaluated the antitumor function of receptor tyrosine kinase-like orphan receptor 1-specific (ROR1-specific) CAR T cells. 3D tumors were established from A549 (non-small cell lung cancer) and MDA-MB-231 (triple-negative breast cancer) cell lines on a biological scaffold with intact basement membrane (BM) under static and dynamic culture conditions, which resulted in progressively increasing cell mass and invasive growth phenotype (dynamic > static; MDA-MB-231 > A549). Treatment with ROR1-CAR T cells conferred potent antitumor effects. In dynamic culture, CAR T cells actively entered arterial medium flow and adhered to and infiltrated the tumor mass. ROR1-CAR T cells penetrated deep into tumor tissue and eliminated multiple layers of tumor cells located above and below the BM. The microphysiologic 3D tumor models developed in this study are standardized, scalable test systems that can be used either in conjunction with or in lieu of animal testing to interrogate the antitumor function of CAR T cells and to obtain proof of concept for their safety and efficacy before clinical application.

[The Construction of ROR1 Targeting Chimeric Antigen Receptor Modified T Cells and Its Killing Effect for ROR1-positive Tumor Cells].

OBJECTIVE: To test the killing effect of type Ⅰ receptor tyrosine kinase-like orphan receptor (ROR1) chimeric antigen receptor T cell (CAR-T) on several ROR1-expressing tumor cells in vitro. METHODS: The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid, and BamHⅠ/EcoRⅠ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry (FCM). The killing effect of CAR-T on positive cells was detected by FCM, the LDH assay and ELISA. RESULTS: The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells, more interferonI-γ (FN-γ) could be released during the CAR-T killing process than control T (P<0.05). CONCLUSION: We successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ, providing an experimental basis for clinical application.

Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells.

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.

Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1.

T cell-engaging bispecific antibodies (biAbs) present a promising strategy for cancer immunotherapy, and numerous bispecific formats have been developed for retargeting cytolytic T cells toward tumor cells. To explore the therapeutic utility of T cell-engaging biAbs targeting the receptor tyrosine kinase ROR1, which is expressed by tumor cells of various hematologic and solid malignancies, we used a bispecific ROR1 × CD3 scFv-Fc format based on a heterodimeric and aglycosylated Fc domain designed for extended circulatory t1/2 and diminished systemic T cell activation. A diverse panel of ROR1-targeting scFv derived from immune and naïve rabbit antibody repertoires was compared in this bispecific format for target-dependent T cell recruitment and activation. An ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activity in vitro, in vivo, and ex vivo and emerged as a prime candidate for further preclinical and clinical studies. To elucidate the precise location and engagement of this membrane-proximal epitope, which is conserved between human and mouse ROR1, the 3D structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-Å resolution.

Detecting Receptor Tyrosine Kinase ROR1 Using a Developed Anti-ROR1 Polyclonal Antibody.

Receptor tyrosine kinase ROR1 has been introduced as an interesting prognostic cancer marker in histopathology. The aim of this study was to produce a polyclonal antibody (PAb) against recombinant human ROR1 protein to be used as a tool for investigation of ROR1 expression in human cancer tissue blocks. The extracellular part of human ROR1 recombinant protein was expressed using pET-28b(+) plasmid in Escherichia coli Bl21(DE3) host. The recombinant ROR1, as a candidate immunogen, was purified and injected to a New Zealand rabbit. Followed by raising the titration of antibody, polyclonal anti-ROR1 antibody was purified through affinity chromatography column. After determining the purity of PAb anti-ROR1, its specific reactivity was assessed through various assessments. Flow cytometry analysis showed that PAb anti-ROR1 specifically recognizes ROR1 molecule in a number of positive and negative cell lines. Results obtained from detection of ROR1 in paraffin-embedded breast adenocarcinoma tissue blocks (n = 11) also demonstrated that PAb anti-ROR1 can effectively be used in immunohistochemistry. In conclusion, the developed anti-ROR1 PAb can be used as a tool for determining the prognostic value of ROR1 in histopathology of cancer tissues.

Inhibition of tumor growth by mouse ROR1 specific antibody in a syngeneic mouse tumor model.

INTRODUCTION: Immunotherapy with tumor-associated antigens (TAAs) is a potentially powerful approach to eradicate tumor cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) plays a crucial role for survival of tumor cells and is overexpressed in various malignancies. In the present study, we developed a syngeneic mouse tumor model to assess anti-tumor effect of mouse ROR1 specific polyclonal antibody (pAb) in vivo. MATERIALS AND METHODS: Mouse ROR1 specific antibody was produced in rabbit using recombinant ROR1 protein. Tow mouse tumor cell lines, (4T1 and CT26), were transfected with full length mouse ROR1 construct and stable clones were selected and characterized by immunocytochemistry, Western blot and flow cytometry. In vitro and in vivo anti-tumor activities of anti-ROR1 antibody were assessed by XTT and syngeneic BALB/c mouse model, respectively. RESULTS: We successfully established two mouse ROR1-overexpressing tumor cell lines. The in vitro results indicate that the ROR1pAb did not significantly inhibit growth of ROR1+ cell lines. One of these cell lines (CT26-ROR1) was implanted in syngeneic BALB/c mice to assess anti-ROR1 tumor inhibitory activity in vivo. The tumor size was significantly reduced in mice treated with ROR1 specific pAb. CONCLUSION: Our results demonstrated for the first time tumor inhibitory effect of mouse ROR1 specific antibody in a syngeneic mouse tumor model. This model is a promising tool for preclinical assessment of ROR1 therapeutics and investigation of the underling molecular mechanisms.

MicroRNA dysregulation to identify therapeutic target combinations for chronic lymphocytic leukemia.

Loss of miR-15/16 is the most common genetic lesion in chronic lymphocytic leukemia (CLL), promoting overexpression of BCL2, which factors in leukemia pathogenesis. Indeed, an inhibitor of Bcl2, venetoclcax, is highly active in the treatment of patients with CLL. However, single-agent venetoclcax fails to eradicate minimal residual disease in most patients. Accordingly, we were interested in other genes that may be regulated by miR-15/16, which may target other drivers in CLL. We found that miR-15/16 targets ROR1, which encodes an onco-embryonic surface protein expressed on the CLL cells of over 90% of patients, but not on virtually all normal postpartum tissues. CLL with high-level expression of ROR1 also have high-level expression of Bcl2, but low-to-negligible miR-15/16 Moreover, CLL cases with high-level ROR1 have deletion(s) at the chromosomal location of the genes encoding miR-15/16 (13q14) more frequently than cases with low-to-negligible ROR1, implying that deletion of miR-15/16 may promote overexpression of ROR1, in addition to BCL2 ROR1 is a receptor for Wnt5a, which can promote leukemia-cell proliferation and survival, and can be targeted by cirmtuzumab, a humanized anti-ROR1 mAb. We find that this mAb can enhance the in vitro cytotoxic activity of venetoclcax for CLL cells with high-level expression of ROR1, indicating that combining these agents, which target ROR1 and Bcl2, may have additive, if not synergistic, activity in patients with this disease.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.

Antiproliferative and Apoptotic Effects of Novel Anti-ROR1 Single-Chain Antibodies in Hematological Malignancies.

Receptor tyrosine kinase-like orphan receptor (ROR) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including cell survival, differentiation, cell migration, cell communication, cell polarity, proliferation, metabolism, and angiogenesis. ROR1 has recently been shown to be expressed in various types of cancer cells but not normal cells. Pharmacokinetics and pharmacodynamics of single-chain Fragment variable (scFv) antibodies provide potential therapeutic advantages over whole antibody molecules. In the present study, scFvs against a specific peptide from the extracellular domain of ROR1 were selected using phage display technology. The selected scFvs were further characterized using polyclonal and monoclonal phage enzyme-linked immunosorbent assay (ELISA), soluble monoclonal ELISA, colony PCR, and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies were also evaluated in lymphoma and myeloma cancer cell lines using MTT and annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the ROR1 peptide. Colony PCR confirmed the presence of full-length VH and Vκ inserts. The percentages of cell growth after 24 h of treatment of cells with individual scFv revealed that the scFv significantly inhibited the growth of the RPMI8226 and chronic lymphocytic leukemia (CLL) cells in comparison with the untreated cells ( p < 0.05). Interestingly, 24-h treatment with specific scFv induced apoptosis cell death in the RPMI8226 and CLL cells. Taken together, our results demonstrate that targeting of ROR1 using peptide-specific scFv can be an effective immunotherapy strategy in hematological malignancies.

[Function and modulation of type â ¡ innate lymphoid cells and their role in chronic upper airway inflammatory diseases].

Type Ⅱ innate lymphoid cells (ILC2) is a family of innate immune lymphocytes, which provide effective immune responses to cytokines. ILC2 are regulated by the nuclear transcription factor ROR alpha and GATA3, secreting cytokines IL-5 and IL-13, etc. Animal models have shown that ILC2 are involved in allergic diseases, such as asthma and atopic dermatitis, and also play a very important role in the metabolic balance. In addition, recent reports suggest that ILC2 not only play a role in the initial stages of the disease, but also can lead to chronic pathological changes in the disease, such as fibrosis, and may have an effect on acquired immunity. This paper mainly focus in the role and regulation of ILC2 cells, and review the research status of ILC2 in the field of chronic upper airway inflammatory diseases including allergic rhinitis and chronic rhinosinusitis.

Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.