ALBERT

Description: Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Description: Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-κB (NF-κB) complex and also through phosphoinositide-3 (PI3)–kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.

Description: Abstract 3476 The development of reduced intensity conditioning (RIC) regimens has increased the use of allogeneic hematopoietic stem cell transplantation (alloHSCT) in patients with chronic lymphocytic leukemia (CLL). Despite decreased rates of transplant related mortality, RIC regimens confer slower rates of donor engraftment and increased relapse post-transplant. It has been reported that faster rates of conversion to donor chimerism after RIC alloHSCT result in lower rates of CLL relapse1. We hypothesized that systematic targeted host T-lymphocyte depletion (TLD) to a target level by serial administration of standard chemotherapy prior to RIC alloHSCT will result in more rapid donor engraftment and full donor chimerism, thereby enhancing graft versus leukemia activity. We evaluated the effects of TLD on host CD4 lymphocyte depletion, disease response, and toxicity as well as donor engraftment, non-relapse mortality, and disease control in 27 patients undergoing RIC alloHSCT for CLL from 1999 to 2010. All patients underwent TLD receiving 1–3 cycles of EPOCH-F±R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, fludarabine ± rituximab) prior to RIC (fludarabine/cyclophosphamide) and matched-related (N=17) or unrelated (N=10) donor transplantation followed by calcineurin inhibitor-based GVHD prophylaxis. The number of cycles administered (max of three cycles) was titrated to achieve an absolute CD4 count ≤ 100 cells/μL prior to RIC. The median number of EPOCH-F±R cycles given was 2. The median CD3+, CD4+, and CD8+ lymphocyte counts at baseline were 819, 443, and 269 cells/μL, respectively. The median CD3+, CD4+ and CD8+ lymphocyte counts after TLD were 124, 97, and 52 cells/μL, respectively. The overall response rate to EPOCH-F±R was 50% (9% CR, 41% PR), including 44% of patients who were refractory to their last treatment. The incidence of grade 3 and 4 non-hematologic toxicity with TLD was 72% and 32%, respectively. No patients failed to engraft. The median days to full donor peripheral blood lymphoid and myeloid chimerism were 28 (14-60) and 21 (14-180), respectively. Development of full lymphoid chimerism by day 14 (first evaluation point) versus day 28 or greater trended towards association with development of acute GVHD (90% vs. 35%, P=0.014) but not chronic GVHD (50% vs. 41%, P=0.71). Achievement of full lymphoid chimerism on day 14 as compared to day 28 or greater trended towards association with decreased CLL cells identified in the bone marrow by multicolor flow cytometry at day 100 (median 0% vs. 8.5%, P=0.016) but was not associated with (P=0.077) improved overall survival (OS). There was no association between rates of development of full myeloid chimerism and acute or chronic GVHD or disease control. The rates of acute (grade 2–4) and chronic GVHD were 52% and 44%, respectively. Non-relapse related mortality at one and two years was 21% and 30%, respectively. The rate of complete remission was 54% and the rate of minimal residual disease negativity after transplantation as assessed by multicolor flow cytometry was 55%. The incidence of disease progression after transplant was 33%. The three year event-free and OS probabilities were 56% and 57%, respectively. The median OS was 5.99 years (95% CI, 2.11-NR). In summary, the use of TLD resulted in a high rate of early donor engraftement after RIC alloHSCT in patients with CLL. Rapid donor engraftment was associated with better disease control and occurence of acute GVHD, but it did not appear to alter overall survival. These data suggest that methods resulting in faster donor chimerism may improve CLL control after RIC alloHSCT. Separation of antileukemic effects from severe GVHD remain a challenge. 1. Brown JR et al. Biol Blood Marrow Transplant. 2006 Oct;12(10):1056-64. Table 1: Patient characteristics at study entry N 27 Median age (range) 56 (37–71) Number male 21 (78%) Rai stage 3/4 16 (59%) Poor risk cytogenetics1 (n=25) 12 (44%) Bulky disease ≥5 cm (n=25) 6 (24%) ≥4 Prior regimens 16 (59%) Refractory to last regimen 14 (52%) Median time from diagnosis to HSCT (range) 58 m (17–205) Sorror comorbidity score2     0-2 20 (74%)     3+ 7 (26%) 1. Defined by deletion of 17p or 11q. 2. Sorror et al. Blood 2005;106:2912-2919. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3262 Relapsed acute lymphoblastic leukemia (ALL) remains a difficult challenge for both pediatric and adult patients. Chimeric antigen receptors (CARs) are genetically engineered molecules expressed in transduced T lymphocytes. CARs express both a target binding motif and TCRzeta signals needed for T cell activation, with or without other costimulatory domains. CARs that target CD19 (expressed on most ALL blasts) are being studied in several clinical trials in adults and are planned for pediatric relapsed/refractory ALL. To date, CARs have been expanded ex vivo using antigen-independent techniques. This project sought to develop antigen specific expansion of cells transduced with a CD19-specific CAR by employing artificial APCs (aAPCs) and to explore whether the method of expansion impacted functionality of CD19-CAR T cells. aAPCs used in these studies express the high affinity Fc receptor (CD64) and the costimulatory molecule CD137L (aAPC-41BBL). We created an Fc-CD19 fusion protein that when loaded onto the aAPCs, engages the chimeric antigen receptor and induces antigen specific activation. Antigen-specific vs. non-specific approaches to activation and expansion of CAR expressing T cells were compared using three different expansion protocols (EPs). CD19-CAR transduced T cells were expanded with A) irradiated aAPC-41BBL loaded with anti-OKT3, B) irradiated aAPC-41BBL loaded with rFc-CD19 or C) irradiated allogeneic PBMC feeder cells with anti-OKT3. Briefly, OKT3 and IL-2 activated T cells were transduced with CD19CAR, control CAR (Her2-specific), or non-transduced (Mock). All cultures were maintained in IL-2. Functionality was assessed in a 4-hour 51Cr release assay against 4 distinct CD19+ ALL cell lines, K562 cells, and K562 stably transfected with CD19 (K562-CD19) or NGFR (K562-NGFR). Results demonstrate similar significant levels of CD19-specific cytotoxicity in the 4h assay at E:T ratios as low as 2.5:1, regardless of the expansion protocol used (20-40% lysis of all CD19+ targets when expanded by OKT3/aAPC, 40–60% lysis with rFc-CD19/aAPC, and 35–45% lysis with allogeneic feeders and OKT3). To evaluate cytotoxicity in long term culture, CD19-CAR T cells expanded using the three protocols were co-cultured with ALL cell targets for 4 days, then flow cytometry was performed to enumerate surviving ALL cells as determined by CD22+ staining. NALM6 and K562-CD19 cells were entirely eliminated even at an E:T ratio of 2.5:1 by CD19-CAR T cells, regardless of expansion protocol, and not by any of the 3 Mock EPs. Therefore, these results demonstrate that the cytolytic potential of CAR transduced T cells is similar, regardless of whether expansion occurs via CD3 signaling or via the chimeric receptor itself. Interestingly, we did observe substantial NK mediated killing in these assays, which correlated with CD56+ cell content and was eliminated by cold target inhibition using K562 cells. Studies are underway to determine whether differences in NK killing varies with expansion protocol. In summary, Fc-bearing artificial antigen presenting cells combined with CAR specific Fc fusion proteins provide a potential off-the-shelf reagent for antigen specific expansion of T cells with chimeric antigen receptors. This could overcome variable transduction efficiencies and allow administration of a more homogenous population of CAR specific T cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1349 Background: Clinical and translational studies suggest that allogeneic hematopoietic cell transplantation (alloHCT) may induce production of anti-tumor antibodies in the recipient after transplantation. We hypothesized that this phenomenon can serve as a platform for the generation of novel therapeutic monoclonal antibodies (mAbs). An important step to this goal is the identification of targeted antigens with defined ability to support antibody induced cell death. To address this challenge, we developed a chronic lymphocytic leukemia (CLL) membrane protein display system capable of discovering cell surface proteins targeted by serum antibodies and applied this tool to post-alloHCT patient samples. Methods: Total and mRNA was purified from peripheral blood mononuclear cells from six untreated CLL patients and used to generate cDNA. The patient cDNA was pooled, cloned into the retroviral vector pBMN, and expressed in the murine T-cell line Bw 5147 (Bw-CLL-Lib). Enrichment of Bw-CLL-Lib cells displaying membrane proteins of interest was performed via fluorescence activated cell sorting. The unselected Bw-CLL-Lib pool was blocked with recombinant human Fc followed by staining with 1:200 diluted post-alloHCT patient sera and by secondary staining with pooled Alexa Fluor 647 labeled goat-anti-human-lambda and -anti-kappa light chain specific antibodies. Bw-CLL-Lib cells positively binding to serum antibodies were collected and re-grown in culture to 1×106 cells. A second round of enrichment was performed, after which the Bw-CLL-Lib cells were placed in limiting dilution culture. Individual clones were screened for serum reactivity and the retroviral inserts in reactive Bw-CLL-Lib clones were rescued via PCR and sequenced. Proteins of interest were re-expressed in Bw 5147 cells and used to confirm reactivity of the patient serum with specific cell surface proteins. Results: The Bw-CLL-Lib cell pool was screened separately with serum from ten patients with CLL post unrelated donor alloHCT. Serum from three patients enriched positively binding Bw-CLL-Lib clones. Thus far, we have successfully identified serum antibodies to a membrane proximal epitope on a therapeutically relevant CLL cell surface protein. Conclusions: Here we demonstrate a methodology for identifying targets of anti-tumor antibodies in serum from patients after alloHCT. This technique yields a high degree of successful identification of antibody reactivity with cell surface proteins in post alloHCT serum samples. When combined with post-alloHCT antibody Fab phage display (Baskar et al., Blood 114, 4494–4502, 2009) this methodology forms a complete drug and target discovery platform for the generation of tumor specific mAbs derived from alloHCT patients. Disclosures: No relevant conflicts of interest to declare.

Description: 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

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: Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.