ALBERT

Description: Abstract 3603 Background: The receptor tyrosine kinase Ror1 is a tumor-associated molecule over-expressed in chronic lymphocytic leukemia (CLL) and a variety of other malignancies with potential implication for targeted immunotherapy. Little is known about the functional role of Ror1 in the leucomogenesis of CLL. Aims: To assess spontaneous T cell response against Ror1 in CLL patients. Methods: Autologous T cell response against Ror1 was studied in 9 CLL patients and 6 healthy subjects expressing the HLA-A2 allele. Four synthetic 9-mer HLA-A2 restricted peptides selected from different parts of the Ror1 molecule were loaded on dendritic cells and co-cultured with enriched autologous T cells. T cell response was determined by enumeration of the frequency of IFN-γ, IL-5 and IL-17A secreting T cells by ELISPOT. Cell proliferation was determined by 3H-thymidine incorporation. Results: Our results demonstrated a significantly higher frequencies of IFN-γ producing T cells (p

Description: Development of targeted therapies against B-CLL is dependent on the identification of molecules that are essential for the proliferation and survival of the leukemic cells. One such molecule investigated by us as a putative leukemia-associated target is Fibromodulin (FIM). FIM is an extracellular matrix molecule belonging to the leucin-rich proteoglycan family. Our laboratory studies have verified that expression of FIM is upregulated in B-CLL cells. Moreover, this molecule is specifically overexpressed in B-CLL cells and not on normal peripheral blood mononuclear cells. Analysis of FIM expression on various other hematological tumor cells have revealed that with the exception of mantle cell lymphoma, FIM is not expressed in other hematological malignancies. RNAi technology has recently emerged as a powerful method to specifically silence the expression of a gene. We have generated three siRNA against various segments of FIM and tested the effects of these siRNA on B-CLL cells. Transfection of B-CLL cells with these siRNA significantly diminished, or completely abrogated the expression of FIM mRNA as detected by RT-PCR. The figure below shows silencing of the FIM gene following siRNA transfection, as assayed by RT-PCR. “Untrans” and “ctrl” represents untransfected and control siRNA-transfected B-CLL cells respectively. As noted, transfection with each of the three siRNA against FIM completely abrogated the expression of FIM siRNA. 24–48 hours after siRNA transfection, a substantial fraction (30–70%) of the B-CLL cells, compared to cells transfected with control non-silencing siRNA, went into apoptosis as assayed by Annexin-V-propidium iodide staining. Time kinetic studies revealed that siRNA mediated silencing and apoptosis occurred between 18 and 48 hours. No such effect was noted when peripheral blood mononuclear cells of healthy donors or FIM-positive fibroblast cell lines were transfected with siRNA. In reconstitution experiments, siRNA treated B-CLL cells were co-cultured with various numbers of FIM-positive fibroblasts. The presence of these fibroblasts greatly diminished the apoptosis of B-CLL cells induced after siRNA treatment. Our results indicate that overexpression of FIM in B-CLL cells are critical for their survival and FIM may serve as a leukemia-specific target for B-CLL therapy. Figure Figure

Description: The idiotypic structures expressed on the myeloma Ig protein may serve as tumor specific antigens and can be used as target structures for Id (Id) vaccination. In this study, 28 patients with IgG myeloma stage I-II were repeatedly immunized intracutaneously during a 110 week period with the autologous M-protein, either in combination with interleukin-12 (IL-12) (n=15), or with IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (n=13). An Id-specific T cell response, as assessed by [3H]-thymidine incorporation, ELISPOT assay and delayed type hypersensitivity (DTH) reaction and strictly defined by 3 specified criteria, developed in 4/15 patients (27%) in the IL-12 group and 11/13 patients (85%) in the GM-CSF/IL-12 group (p=0.003). Three of 15 patients (20%) showed a gradually increasing Id-specific T cell response during the study whereas 10/15 patients (67%) showed an initial response which then disappeared rapidly; the latter pattern was frequently associated with subsequent progressive disease. In two patients (both in the IL-12 group) a reduction of the M-protein concentration by 〉 50% and 〉25%, respectively, was observed 18 and 21 months after the last immunization. These results indicate that Id immunization of myeloma patients using IL-12 and GM-CSF may more frequently induce a specific T cell response than with IL-12 alone, but also that repeated immunizations may result in a non-reversible state of tolerogenic T cells.

Description: Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γcnull mice under the influence of activated CLL-derived T lymphocytes. By cotransferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4+ T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Description: The idiotypic structure of the monoclonal immunoglobulin (Ig) in multiple myeloma (MM) might be regarded as a tumor-specific antigen. The present study was designed to identify T-cell epitopes of the variable region of the Ig heavy chain (VH) in MM (n = 5) using bioinformatics and analyze the presence of naturally occurring T cells against idiotype-derived peptides. A large number of human-leukocyte-antigen (HLA)–binding (class I and II) peptides were identified. The frequency of predicted epitopes depended on the database used: 245 in bioinformatics and molecular analysis section (BIMAS) and 601 in SYFPEITHI. Most of the peptides displayed a binding half-life or score in the low or intermediate affinity range. The majority of the predicted peptides were complementarity-determining region (CDR)–rather than framework region (FR)–derived (52%-60% vs 40%-48%, respectively). Most of the predicted peptides were confined to the CDR2-FR3-CDR3 “geographic” region of the Ig-VH region (70%), and significantly fewer peptides were found within the flanking (FR1-CDR1-FR2 and FR4) regions (P 〈 .01). There were 8– to 10–amino acid (aa) long peptides corresponding to the CDRs and fitting to the actual HLA-A/B haplotypes that spontaneously recognized, albeit with a low magnitude, type I T cells (interferon γ), indicating an ongoing major histocompatibility complex (MHC) class I–restricted T-cell response. Most of those peptides had a low binding half-life (BIMAS) and a low/intermediate score (SYFPEITHI). Furthermore, 15- to 20-aa long CDR1-3–derived peptides also spontaneously recognized type I T cells, indicating the presence of MHC class II–restricted T cells as well. This study demonstrates that a large number of HLA-binding idiotypic peptides can be identified in patients with MM. Such peptides may spontaneously induce a type I MHC class I– as well as class II–restricted memory T-cell response.

Description: Abstract 1778 Background: Phosphorylation of receptor tyrosine kinases (RTK) plays an important role for many aspects of cell life, as cell-cycle progression, differentiation, apoptosis, intercellular communication and cell survival. RTKs as e.g. EGFR, HER2/neu, VEGFR, IGFR etc. are important structures contributing to the malignant phenotype of many tumors. Targeting RTKs by monoclonal antibodies (MAb) or small inhibitory molecules has been successful for the treatment of various cancers. ROR is one of the twenty RTK families and consists of two members, ROR1 and ROR2. ROR1 and ROR2 have important functions during embryogenesis. ROR1 is uniquely expressed in CLL cells compared to normal tissues. ROR1 siRNA transfection of CLL cells induced specific apoptosis of the leukemic cells. Aims: To study ROR1 protein variants in CLL, ROR1 phosphorylation and relation to clinical activity of the disease. Methods: Phosphorylation of primary human CLL cells from patients with non-progressive and progressive disease, and a series of cell lines were studied applying immunoprecipitation (IP) of the ROR1 molecule, using anti ROR1 and irrelevant MAbs by Western blotting as well as in intracytoplasmic staining of the cells by flowcytometry, using an anti-phospho ROR1 (pROR1) MAb (a MAb specifically recognizing a phospho-peptide of the cytoplasmic TK domain of ROR1) and semi quantification of the staining intensity as well as by p-tyrosine and p-serine MAbs. Results: IP of the ROR1 molecule of fresh leukemic cells using ROR1 specific antibodies and subsequent Western blot with anti ROR1 MAbs, showed various protein bands. Two bands had the size of 105 and 130 KDa probably representing unglycosylated or partially glycosylated ROR1 and the fully glycosylated glycoform respectively. One of the two bands dominated in individual patients. A band of 260 KDa could also be detected in a large number of patients probably representing dimerized monomers. Finally a band of 64 KDa could also be noted which may represent an intracellular part of ROR1, as has been described in fetal and adult human CNS, leukemia and lymphoma cell lines (Reddy UR et al, Oncogene. 1996; 13:1555–9). The 64, 105 and 130 KDa bands were constitutively phosphorylated in all patients both at tyrosine and serine residues. The intensity of phosphorylation of the 130 KDa band was significantly higher in patients with progressive disease vs. non-progressive disease (p=0.0001). There was no relation between the pattern of the different ROR1 protein bands and disease activity. Using 9 cell lines of different hematologic malignancies including CLL, similar protein bands and phosphorylation status as for fresh CLL cells were noted. We have also produced specific mouse monoclonal antibodies against defined epitopes of the extracellular parts of ROR1. One of those MAbs (anti-KNG, IgG1) induced a high degree of specific apoptosis of the CLL cells (ASH Annual Meeting Abstracts, Nov 2010; 116: 916) and could also be shown to induce a specific dephosphorylation of the cytoplasmic TK domain which was noted already after 20 min and gradually increased up to 4h. No effects were noted using irrelevant MAbs or healthy donor lymphocytes. Conclusion: Our results showed that the ROR1 molecule in CLL cells are expressed in various protein variants probably representing different glycosylation patterns (105–130 KDa), dimerized monomers (260 KDa) and a truncated protein variant (64 KDa). ROR1 was constitutively phosphorylated (64, 105 and 130 KDa) both at serine and tyrosine residues. Treatment of CLL cells in vitro with a ROR1 specific antibody induced specific apoptosis as well as rapid dephosphorylation of ROR1. Collectively the data suggest that phosphorylated ROR1 might be an important structure for the growth potential of CLL cells and an interesting structure to target in a therapeutic intervention. Disclosures: Mellstedt: Kancera AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hojjat-Farsangi:Kancera AB: Equity Ownership. Rabbani:Kancera AB: Equity Ownership.

Description: Abstract 916 Methods: A peptide-based mouse monoclonal antibody generation method was used for producing MAbs against the three extracellular domains of Ror1. Twenty CLL patients (10 with progressive and 10 with non-progressive disease) were enrolled in this study. Flow cytometry was used for surface staining of Ror1. Annexin V and propidium iodide (flow cytometry) as well as PARP cleavage (Western blot) was used for detection of apoptosis. Results: Six monoclonal antibodies of different isotypes (IgG, IgM) were produced against Ror1. The frequency of Ror1 positive cells were in the range of 24–89%. Patients with progressive disease had a significantly higher number of Ror1 positive cells as compared to those with non-progressive disease as well as in patients with unmutated compared to mutated IgVH genes. All six antibodies alone induced apoptosis (Annexin V and PARP cleavage) of CLL cells. A higher frequency of apoptotic CLL cells was induced by the antibodies against the CRD region (ligand binding site for Wnt proteins) as well as one antibody against the kringle domain (also a binding region for regulatory proteins) . Apoptosis induced by these three antibodies alone was significantly higher than that induced by Rituximab (p

Description: Fibromodulin is an extracellular matrix protein normally produced by collagen-rich tissues; the fibromodulin gene has been found to be the most overexpressed gene in B-cell chronic lymphocytic leukemia. In this study, fibromodulin was expressed at the gene level (reverse transcription-polymerase chain reaction [RT-PCR]) in all patients with B-CLL (n = 75) and in most (5 of 7) patients with mantle cell lymphoma (MCL). No mutations in the fibromodulin gene were detected. Fibromodulin was also detected at the protein level in the cytoplasm of the B-CLL cells and in the supernatant after in vitro cultivation, but not at the cell surface. Fibromodulin was not found in patients with T-cell chronic lymphocytic leukemia (T-CLL), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), hairy cell leukemia, follicular lymphoma, lymphoplasmacytic lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), or chronic myelogenous leukemia (CML) or in 36 hematologic cell lines. Normal blood mononuclear cells (T and B lymphocytes, monocytes), tonsil B cells, and granulocytes did not express fibromodulin. Activation (phorbol 12-myristate 13-acetate [PMA]/ionomycin) of normal T and B lymphocytes induced weak fibromodulin gene expression, but not to the extent seen in freshly isolated B-CLL cells. The reason for the exclusive ectopic expression of fibromodulin in B-CLL and MCL is unknown. However, its unique protein expression makes it likely that fibromodulin is involved in the pathobiology of B-CLL and MCL. (Blood. 2005;105:4828-4835)

Description: Abstract 694 Background: Small leucine rich proteoglycans (SLRPs) are a family of glycosylated proteins normally expressed in the extracelluar matrix (ECM) of collagen rich tissues. The biological role of the SLRPs is multifactoral, but mostly these proteins are secreted and bind to membrane receptors or ECM proteins affecting cell proliferation and cell migration. Some SLRPs (eg. Decorin, Lumican) have been reported to be expressed in cancer, but the expression as well as the glycosylation pattern differ. Microarray studies have revealed that the SLRP family member fibromodulin (FMOD) gene is overexpressed in chronic lymphocytic leukemia (CLL). We later reported the specific expression of FMOD in CLL and mantle cell lymphoma (MCL). FMOD is located on chromosome 1q32 adjacent to two other members of the SLRP family, proline/arginine-rich end leucine-rich repeat protein (PRELP) and opticin (OPTC). Aims: To analyse 1) the expression of PRELP and OPTC in CLL and other hematological malignancies. 2) the glycosylation pattern of PRELP and OPTC, and cellular localization of OPTC in CLL cells. Methods: PRELP: Gene expression was tested by RT-PCR and realtime-PCR. Protein expression was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the PRELP protein. OPTC: Cell fractionation was performed using four different methods. Protein expression (molecular weight and cellular location) was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the OPTC protein. Results: PRELP was expressed at the gene level (RT-PCR) in all CLL patients tested (n=30) and in 3/5 patients with mantle cell lymphoma, but not in normal leukocytes (n=10) or in other hematological malignancies (7 different types, n=35). The PRELP protein was detected in all CLL samples tested but not in leukocytes of healthy donors (western blot). Molecular analysis of the CLL PRELP protein revealed a unique unglycosylated 38 kDa core protein, with an intact signal peptide. This protein was not detected in serum that, in combination with the uncleaved signal peptide, suggests cellular retention. A “normal” OPTC protein (50 kDa) was detected in cell lysates of both CLL tumor cells (n=30) and normal leukocytes (n=10). However, the CLL cells also expressed a 37 kDa OPTC that was not detected in healthy controls. This 37 kDa OPTC was detected in all CLL samples and molecular analysis revealed a unique unglycosylated core protein that was located in the cell nucleus and endoplasmatic reticulum of the CLL cells. Conclusion: The unique expression of the SLRPs PRELP and OPTC in CLL (in addition to the previously reported FMOD) is unexpected and merits further studies since the specific expression of three closely related SLRP genes may indicate a role in the pathobiology of the disease. Disclosures: No relevant conflicts of interest to declare.