Purpose: Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1protein expression. Experimental Design: Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation,Western blotting, and ELISA. Results: ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1was determined to be in the range of 10 3 to 10 4 molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL. Conclusions: The restricted, uniform, and constitutive cell surface expression of ROR1protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.

Chronic lymphocytic leukemia (CLL) is IntroductionThe most common leukemia among white adults, B-cell chronic lymphocytic leukemia (CLL), remains incurable and its pathogenesis poorly defined. 1 Currently no system permits differentiation and long-term growth of CLL cells in vitro; therefore, an in vivo animal model that reproducibly supports engraftment and growth of human CLL cells would help elucidate key features of CLL cell biology and lead to better treatments.Previous attempts to engraft human CLL cells into mice have been hampered for 2 reasons. First, xenogeneic recipients were not sufficiently immune deficient to prevent human cell rejection. [2][3][4][5] Although Dü rig et al 5 successfully transferred CLL cells into nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, apparently the level of CLL cell growth was not sufficient to correlate kinetics with essential interactions with different cell subpopulations. Second, optimal engraftment and growth may have been impaired by the inability of a murine microenvironment to support CLL cells in vivo. Indeed, in vitro studies suggest at least 3 cell lineages are involved in CLL survival and growth: lymphoid (T cells 6,7 ), myeloid (monocytes and monocyte-derived nurse-like cells 8 ), and mesenchymal ("stromal cells" 9,10 ).To provide a more physiologic microenvironment for CLL cells within highly immune incompetent recipients, we introduced precursors of human hematopoietic and mesenchymal lineages into NOD/Shi-scid,␥c null (NSG) mice, a NOD/SCID-derived strain that lacks the IL-2 family common cytokine receptor gamma chain gene (␥c), rendering animals completely deficient in lymphocytes, including natural killer (NK) cells. We found activated autologous T cells were essential for leukemia cells to successfully engraft, survive, and proliferate in vivo and to recapitulate cardinal features of human CLL cells: kinetics, CD38 expression, and growth in secondary lymphoid tissues. This adoptive transfer model may facilitate the definition of leukemic and nonleukemic elements involved in the interactions and kinetics of CLL cells in patients. Methods Patients and samplesThe Institutional Review Board and the Institutional Animal Care and Utilization Committee of the North Shore-LIJ Health System sanctioned these studies. After obtaining informed consent, in accordance with the Submitted December 10, 2010; accepted February 17, 2011. Prepublished online as Blood First Edition paper, March 8, 2011 DOI 10.1182 DOI 10. /blood-2010 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on June 7, 2019. by guest www.bloodjournal.org From Declaration of Helsinki, we collected blood from 37 CLL patients for whom clinical information, la...

Monoclonal antibodies and T cells modi- IntroductionB-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) are common B-cell malignancies that respond to chemotherapy but are rarely cured. Allogeneic hematopoietic stem cell transplantation (HCT) enables a T cell-mediated graft-versus-leukemia (GVL) effect and induces durable remissions in a subset of patients with chemotherapy-refractory B-CLL and MCL, demonstrating that these malignancies are susceptible to recognition and elimination by T cells. 1,2 In a previous study, we identified tumor-reactive CD8 ϩ T cells directed against minor histocompatibility (H) and tumor-associated antigens (TAA) expressed by B-CLL in patients with sustained tumor regression after allogeneic HCT. 3 These results have encouraged the development of T cell-adoptive immunotherapy to augment the GVL effect after HCT. However, major challenges for therapy with ␣␤ ⌻-cell receptor (TCR)-bearing T cells include the need to identify antigens with restricted expression on malignant cells to avoid graft-versus-host disease, and the population distribution and requirement for human leukocyte antigen (HLA)-restriction for both minor H antigens and TAA. 4 An approach that could overcome these challenges and also enable T-cell therapy for B-CLL and MCL in the nontransplant setting is to genetically modify T cells to express a chimeric antigen receptor (CAR) that is specific for a cell surface protein expressed by malignant cells. CARs consists of a single-chain antibody fragment (scFv) that is derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb) linked to the TCR CD3 chain that mediates T-cell activation and cytotoxicity. 5 Costimulatory signals can also be provided through the CAR by fusing the costimulatory domain of CD28 or 4-1BB to the CD3 chain. 5,6 CARs are specific for cell surface molecules independent from HLA, thus overcoming the limitations of TCR-recognition including HLA-restriction and low levels of HLA-expression on tumor cells. B-cell lineage differentiation molecules such as CD19 and CD20 are retained on most B-cell tumors, and T cells modified with CD19-and CD20-specific CARs are currently being evaluated in clinical trials. 7,8 However, targeting B-cell lineage-specific antigens with immunotherapy has the disadvantage of eliminating normal mature B cells, which can increase the risk of infection. 9,10 Here, we evaluate a strategy to selectively eliminate malignant B cells without damaging the mature normal B-cell compartment by targeting the receptor tyrosine kinase-like orphan receptor 1 (ROR1). ROR1 was identified as a highly expressed gene in B-CLL by expression profiling and it has been shown that ROR1-protein is uniformly expressed on the cell surface of B-CLL. 11-14 The ROR1-gene encodes a 105-kDa protein with a Submitted May 3, 2010; accepted August 4, 2010. Prepublished online as Blood First Edition paper, August 11, 2010; DOI 10.1182 DOI 10. /blood-2010 An Inside Blood analysis of this article appears at...