bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Cathpsin L by the Serpin Protein C Inhibitor.
Authors: Fortenberry Yolanda , Bialas Ryan , Mitchell Candace , Church Frank C.
Published: October 2019

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation and fibrinolysis including thrombin and activated protein C (APC). The physiological role of PCI, however, remains under investigation since PCI both inhibits and promotes thrombin generation. The cysteine protease, cathepsin L, has been shown to play a role in many physiological processes including cardiovascular disease, atherosclerosis, blood vessel remodeling, and tumor cell invasion. Recently, several serpins have been described to inhibit both serine and cysteine proteases and they are termed “cross-class” inhibitors. The goal of this project was to determine if PCI inhibits cathepsin L activity and if so, does this inhibition process mimic the mechanism of serine proteases. Previous studies have shown that the prototypical serpin, antithrombin (AT), inhibits the cysteine proteases papain and cathepsin L. We found that PCI is a more efficient inhibitor of cathepsin L than AT with an inhibition rate (k2) of 1.5 × 106 M−1min−1 compared to 5.2 × 104 M−1min−1 for AT. Also, PCI is a more efficient inhibitor of cathepsin L than either thrombin or APC whose inhibition rates are 5.7 × 105 M−1min−1 and 3.4 × 104 M−1min−1, respectively. In contrast to AT, PCI does not inhibit papain. Thrombin inhibition by AT and PCI is accelerated in the presence of glycosaminoglycans such as heparin and heparan sulfate. The inhibition of cathepsin L by PCI is not accelerated in the presence of heparin suggesting either that cathepsin L does not bind heparin or that heparin is not required for accelerated inhibition of cysteine proteases. Interestingly, a reactive site P1 mutant (R354A) of PCI does not inhibit thrombin but does inhibit cathepsin L at rates comparable to wild-type PCI. This implies that the P1 residue of PCI does not determine specificity for inhibition of cathepsin L unlike for thrombin and APC. We believe that the specificity is primarily determined by the hydrophobic Phe residue located at the P2 position since other serpins that inhibit cathepsin L contain either a Phe or Val at the P2 position. Mutating the P14 residue (T341R; mutation in the hinge region) of PCI results in the conversion of PCI from an inhibitor to a substrate. As expected, the PCI-P14 mutant does not inhibit either thrombin or cathepsin L. Another characteristic of the serpin inhibition mechanism is formation of a bi-molecular SDS stable complex. We found that wild-type PCI and PCI-P1 mutant both form a stable complex with cathepsin L under non-reducing conditions. Lastly, the wild-type PCI-cathepsin L interaction has a stoichiometry of inhibition (SI) value of 1.6. This indicates that PCI is an effective and possibly a physiologically relevant inhibitor of cathepsin L. Regulating cathepsin L by serpins like PCI may be a novel and new pathway of regulation of hemostasis-thrombosis, cardiovascular and metastatic diseases.
Mobilization Studies in Immunodeficient Mice Support a Role of Complement in Modulating the Trafficking of Hematopoietic Stem Cells (HSC) - A Pivotal Role of C3 Cleavage Fragments in Retention and C5 Fragments in Mobilization/Egress of HSC.
Authors: Reca Ryan , Wysoczynski Marcin , Magda Kucia , Janowska-Wieczorek Anna , Ratajczak Janina , Ratajczak Mariusz Z.
Published: October 2019

We postulate that mobilization of hematopoietic stem cells (HSC) is part of a immune-response and to support this notion i) complement (C) cascade cleavage fragments modulate trafficking of HSC (Blood2003;101,3784; Blood2004;103,2071) and ii) patients suffering from severe combined immunodeficiency (SCID) are poor mobilizers (Blood1996;88, 1104). To shed more light on mechanisms involved in C-mediated trafficking of HSC we studied mobilization in several strains of immune-deficient mice mobilized by employing various mobilization protocols. We noticed that mobilization of HSC is always preceded by C activation - occuring by the classical (G-CSF, CY) or alternative (polysaccharides, chemokines) activation pathways. Accordingly, the classical pathway of C activation after G-CSF or CY administration is a result of proteolytic modification of the BM environment leading to the exposure of a neo-antigen in BM tissue, and binding of natural IgM antibodies to this neo-epitope triggers activation of the C cascade. In support of this, IgM-deficient mice (SCID, RAG2null and Jh) are poor mobilizers and G-CSF-induced mobilization in these mice was restored after supplementation with purified C-activating inmmunoglobulins. On the other hand the mobilization in these mice (SCID, RAG2null and Jh) was normal in response to polysaccharides or chemokines that activate complement by the alternative IgM-independent pathway. To further support an important role of C in HSC mobilization, we studied HSC mobilization in mice deficient for various C components (C3−/− and C5−/− mice). We noticed that C3−/− mice are easy mobilizers. In contrast mobilization was very poor in C5−/− mice. This suggests that C3 and C5 cleavage fragments differently control the mobilization of HSC. We will present evidence that C3 cleavage fragments directly interact with HSC and prevent their uncontrolled egress from BM during mobilization by increasing their SDF-1-dependent marrow retention. In contrast C5 cleavage fragments activate neutrophils and BM-endothelium and thus are crucial for the egress of HSC. Thus the mobilization of HSC is i) dependent on C activation by the classical or alternative pathway, ii) modified differently by C3 cleavage fragments which enhance retention and C5 cleavage fragments which promote egress of HSC. Thus modulation of C activation in BM may help to develop new more efficient strategies for both HSC mobilization and their homing/engraftment.
Differential Expression of Type I MAGE in New and Relapsed Multiple Myeloma: Evidence for Association with Proliferation and Progression of Disease.
Authors: Cho Hearn J. , Ely Scott , Austin Wayne R. , Niesvizky Ruben , Pearse Roger , Coleman Morton , Bhardwaj Nina , DiLiberto Maurizio , Chen-Kiang Selina , Old Lloyd J. , Jungbluth Achim A.
Published: October 2019

The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p<0.0002). Expression of CT7 and CT10 (MAGE-C2), a type I MAGE not previously associated with myeloma, was stable between newly-diagnosed and relapsed patients (76.0% of new samples vs. 77.1% of relapsed for CT7, 48.5% vs. 50.0% for CT10). In contrast, MAGE-A3 was detected in a significantly greater percentage of relapsed patients (77.1%) compared to newly diagnosed (35.6%, p=0.0003). The link between MAGE expression and unrestricted proliferation was further supported by in vitro studies with human myeloma cell lines. Proliferating myeloma cells were metabolically labeled with the nucleotide analog bromodeoxyuridine (BrdU) followed by intracellular staining and flow cytometry. This assay demonstrated that proliferating myeloma cells that incorporated BrdU into their genomic DNA expressed higher levels of type I MAGE protein compared to non-proliferating cells. Arresting cells at the G1-S interface by double thymidine blockade lead to the accumulation of cells expressing high levels of MAGE, which rapidly entered S phase and incorporated BrdU upon release from block. These results strongly suggest that expression of CT7 and CT10 are relatively early and stable events in the pathogenesis of myeloma, whereas activation of MAGE-A3 expression is associated with disease progression. Furthermore, MAGE expression is correlated with abnormal proliferation in vitro and in vivo, suggesting a potential functional role in the dysregulation of the cell cycle that is a hallmark of this disease. These results support the further exploration of the role of type I MAGE expression in myeloma and the development of therapeutic agents targeting them, especially tumor vaccines.
Targeting PKC in Multiple Myeloma: In Vitro and In Vivo Effects of the Novel, Orally Available Small-Molecule Inhibitor Enzastaurin (LY317615.HCl).
Authors: Raab Marc S. , Podar Klaus , Zhang Jing , McMillin Douglas , Breitkreutz Iris , Tai Yu-Tzu , Lin Boris , Ghobrial Irene , Munshi Nikhil , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: October 2019

In multiple myeloma (MM), protein kinase C (PKC) overexpression has been reported including the prognostic adverse patient group with t (4;14) translocation. Importantly, PKC signaling pathways have been implicated in MM cell proliferation, apoptosis, and migration. Here, we investigated the novel, orally available PKC inhibitor Enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits membrane, cytosolic, and nuclear phosphorylation of homologous PKC isoform residues, as well as associated kinase activity, induced by the major PKC activator TPA (Tumor-promoting phorbol ester). Consequently, it also abrogates TPA-induced phosphorylation of signaling molecules downstream of PKC including MARCKS and PKCm. In MM, Enzastaurin inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs); co-stimulation with the extracellular matrix protein fibronectin, VEGF or IL-6; as well as MM patient serum. Phosphorylation of downstream signaling molecules was also abrogated, including cytoplasmic and nuclear ERK, JNK, ribosomal protein S6 and GSK3b as well as nuclear cMyc. Enzastaurin inhibits both proliferation and survival of MM cell lines and MM cells isolated from multidrug- resistant patients; as well as overcomes MM cell growth triggered by tumor cell binding to BMSCs and endothelial cells at a low micromolar range equivalent to the concentrations achieved in patient plasma during clinical trials. Importantly, synergistic cytotoxicity is observed when Enzastaurin is combined with bortezomib. Besides proliferation and survival, Enzastaurin inhibits MM cell adhesion, as well as VEGF- and IGF-1-triggered MM cell migration. It also blocks VEGF- triggered signaling pathways in endothelial cells, thereby inhibiting tubule formation. Finally and most importantly, tumor growth, survival, and angiogenesis are abrogated by Enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor Enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve patient outcome in MM.
NSC 338258 Represents a Novel Anti-Neoplastic Agent for the Treatment of Multiple Myeloma.
Authors: Tian Erming , Landowski Terry , Stephens Owen , Yaccoby Shmuel , Barlogie Bart , Shaughnessy John D.
Published: October 2019

We have shown that high-risk multiple myeloma (MM) can be linked to amplification and overexpression of the cell cycle regulator CKS1B. With this in mind we correlated high expression of this gene in the NCI60 cell line panel with IC50 of over 10,000 anticancer compounds. NSC 338258 (EPED3) was identified in this search. EPED3 is a highly stable, hydrophilic derivative of ellipticine. In vitro, this synthetic compound exhibits drastic cytotoxic activity on myeloma cells, which is unique among most ellipticine derivatives. This efficacy of EPED3 was examined in myeloma cells co-cultured with bone marrow stromal cells. Despite the protective influence of stromal cells, micromolar concentrations of EPED3 were highly effective at killing myeloma cells; however, this lethal activity was exclusive of stromal cells. In co-culture, EPED3-induced cell cycle arrest and massive apoptotic progression appears to be a consequence of its instant impact on cytoplasmic organelles, particularly mitochondria. Disruption of mitochondrial and endoplasmic distribution of cytochrome c initiated the intracellular proteolytic cascade through the intrinsic apoptotic pathway. Effects of EPED3 treatment were further evaluated in myeloma cell lines with selective tolerances to doxorubicin, dexamethasone, and Velcade; EPED3 overcame these acquired drug resistances. In addition, the potency of EPED3 was tested on mononuclear cells isolated from peripheral blood of healthy donors. Under mitogenic stimulation, EPED3 had no significant growth inhibition effects within a range of concentrations killing myeloma cells. Collectively, our current data suggest that EPED3 is an extraordinary agent that, in vitro, targets mitochondrial function to rapidly deplete chemical energy and to initiate apoptosis in myeloma cells at low concentration, while leaving healthy stromal and mononuclear cells unharmed.
ARRY-797, a Potent and Selective Inhibitor of p38 Map Kinase, Inhibits LPS-Induced IL-6 and In Vivo Growth of RPMI-8226 Human Multiple Myeloma Cells.
Authors: Wright Dale , Winski Shannon L. , Anderson Deborah , Lee Patrice , Munson Mark , Winkler James
Published: October 2019

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.
Heparanase Influences Expression and Shedding of Syndecan-1, and Its Expression by the Bone Marrow Environment Is a Bad Prognostic Factor in Multiple Myeloma.
Authors: Mahtouk Karene , Hose Dirk , Reme Thierry , Hundemer Michael , Jourdan Michel , Jourdan Eric , Pantesco Veronique , Baudard Marion , De Vos John , Larroque Marion , Moehler Thomas , Rossi Jean-Francois , Goldschmidt Hartmut , Klein Bernard
Published: October 2019

The heparan sulfate (HS) proteoglycan (PG) syndecan-1 plays a major role in multiple myeloma (MM), in part by concentrating HS-binding growth factors on the surface of MM cells (MMC). The function of HSPG is regulated by extracellular enzymes that modulate the structure of HS-chains. One such enzyme is heparanase (HPSE), an endo-glucuronidase that cleaves HS chains at only a few sites, resulting in fragments of 10–20 sugar units long that are biologically active.

Using Affymetrix microarrays and real-time RT-PCR, we showed that the gene encoding HPSE was expressed by 11/19 myeloma cell lines (HMCLs). In HSPE-positive HMCLs, syndecan-1 mRNA expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Downregulation of HPSE by HPSE-siRNA resulted in a decrease of syndecan-1 mRNA expression and soluble syndecan-1 production, without affecting membrane syndecan-1. Contrary to HMCLs, HPSE was expressed in only 4/39 primary MMC samples at a low level, whereas it was highly expressed in 36/39 whole bone marrow (WBM) microenvironment samples. In the latter, HPSE was expressed at a median level in polynuclear cells and T cells, at high level in monocytes and osteoclasts, and it was not expressed in BM stromal cells. In order to investigate the connection of clinical parameters with HPSE gene expression, 30/39 myeloma patients treated with high dose chemotherapy and autologous stem cell transplantation where classified into two groups: 15 patients with the lowest or the highest HPSE expression in the WBM. The HPSEhigh group had an increased percentage of patients with elevated C-reactive protein and β2-microglobulin. According to the international staging system (ISS), the HPSEhigh group included a significant higher frequency of patients with stage III MM and a lower frequency of patients with stage I MM. HPSEhigh patients had a shorter event free survival (EFS) (p=.017) and overall survival (OAS) (p=.023) than HPSElow patients. Classifying these 30 patients into two groups according to HPSE expression in MMC did not yield to any EFS or OAS significant differences between the two groups.

Altogether, those data provide evidence that HPSE not only modulate syndecan-1 activity through the cleavage of HS chains but also regulates syndecan-1 mRNA expression and shedding. We report for the first time that the expression of a gene in the BM, i.e. HSPE, is an indicator of poor prognostic for MM patients. Therefore, HPSE inhibitors like PI-88, which is currently in clinical trial, will be of interest for the treatment of patients with MM.
Cancer Germline Antigen-Specific T Cells Are Present and Functional in Many Patients with Multiple Myeloma and Paraproteinaemia.
Authors: Goodyear Oliver , Piper Karen , Khan Naeem , Starczynski Jane , Mahendra Prem , Pratt Guy , Moss Paul
Published: October 2019

The expression of Cancer Germline Antigens (CGAgs) is normally restricted to the pre-meiotic spermatogonia cells of the testis. The testis is an immunologically privileged site and so immunological tolerance to CGAg is not established. However, CGAg expression is also detected in many types of malignant disease including plasma cells from patients with multiple myeloma. CGAg expression has been shown to prime a T cell immune response in many patients with solid tumours and this may offer a novel target for immunotherapy in patients with myeloma.

We have used immunodominant peptide epitopes from a range of CGAgs to screen for CGAg-specific T cells in the blood of patients with multiple myeloma at various stages of their disease. Initial studies demonstrated that T cells from 15 out of 37 patients responded to one or more CGAg peptides and the magnitude of the CGAg-specific CD8+ T cell response ranged between 0.0004% and 0.1% of the total CD8+ T cell pool. Serial analysis showed that these immune responses were detectable in individual patients at multiple time-points during the course of their disease. A further 13 peptides have now been obtained including several CD4 peptide. We have subsequently cloned CD4 T cells specific to a MAGE 3 peptide and have shown them to be functional. In some patients we determined the membrane phenotype of the CGAg-reactive cells as CD45RA+ and CCR7−, an effector memory differentiation state. CGAg-specific responses have also been detected in patients with clinically benign forms of paraproteinaemia indicating that T cell immunity may play a role in the control of disease progression. Plasma cells are localised to bone marrow and we are now focussing on the study of immunity to CGAg at this site. Initial findings indicate a higher proportion of CGAg-specific T cells within bone marrow and the phenotypic profile of these cells is being determined. Functional T cells specific for CGAg are therefore present in a large proportion of patients with multiple myeloma and offer the possibility of a novel approach for immunotherapy in this disease.
Analysis of Varicella Zoster Virus Reactivation among Bortezomib-Treated Patients in the APEX Study.
Authors: Chanan-Khan Asher Alban , Sonneveld Pieter , Schuster Michael W. , Irwin David , Stadtmauer Edward A. , Facon Thierry , Harousseau Jean-Luc , Ben-Yehuda Dina , Lonial Sagar , Goldschmidt Hartmut , Reece Donna E. , Neuwirth Rachel B. , Anderson Kenneth C. , Richardson Paul G.
Published: October 2019

BACKGROUND: Bortezomib (btz, VELCADE®), a selective and reversible proteasome inhibitor, is approved for the second-line treatment of multiple myeloma (MM). There is a tendency for reactivation of varicella zoster virus (VZV;herpes zoster) among patients (pts) receiving immunosuppressive agents for cancer. Rates of VZV reactivation during btz therapy in phase 2 studies were reported to be 11% in SUMMIT and 13% in CREST. To investigate whether btz increases the incidence of VZV reactivation, data were reviewed from the phase 3 APEX trial of btz vs high-dose dexamethasone (dex) in pts with relapsed MM following 1–3 prior therapies.

METHODS: The APEX trial was described previously [Richardson et al. N Engl J Med2005;352:2487–98]. Use of prophylactic anti-virals and anti-bacterials was not mandated by this protocol.

RESULTS: Baseline characteristics, including age, prior ASCT, β2-microglobulin, and disease stage, were comparable between the btz and dex arms. The incidence of VZV reactivation was significantly higher with btz vs dex (13% [42/331] vs 5% [15/332], respectively; p=0.0002). Most VZV reactivation events in both arms were grade 1/2. Notably, the incidence of grade 3/4 events was similar between the two arms (1.8% [6/331] vs 1.5% [5/332]), as was the rate for serious adverse events (1.5% [5/331] vs 0.9%, [3/332], respectively). The incidence of other opportunistic infections, including VZV, was comparable between the btz and dex arms; 23% vs 20%, respectively. There were no deaths due to VZV reactivation in either arm. Median time to onset of VZV reactivation tended to be shorter in the btz arm vs dex, though this was not statistically significant (31 vs 51 days; p=0.221). At first assessment post VZV reactivation event, median ALC was significantly lower in the btz vs dex arm (1050 vs 1750 cells/μL; p=0.028). However, immediately prior to the event, median ALC was similar between arms (1030 vs 1100 cells/μL; p=0.529).

CONCLUSIONS: Btz was associated with a higher incidence of VZV reactivation events than dex. The reasons for this are unclear and require further study. Preliminary evidence from recent studies has shown that this adverse event of btz could be overcome with the use of prophylactic acyclovir [Mateos et al. Blood 2006; Epub ahead of print].
Lenolidamide (Revlimid), in Combination with Cyclophosphamide and Dexamethasone (CRD) Is an Effective Regimen for Heavily Pre-Treated Myeloma Patients.
Authors: Morgan Gareth J. , Schey Stephen , Wu Ping , Srikanth M. , Phekoo Karen J. , Jenner Matthew W. , Dines Sharon , Saso Radovan , Davies Faith E.
Published: October 2019

Lenolidamide (Revlimid®) is an oral immunomodulatory drug that has been shown to be effective for the treatment of relapsed refractory myeloma, and in-vitro laboratory studies suggest that its action may be synergistic with a number of conventional chemotherapeutic agents. The aim of this study is to assess the efficacy and toxicity profile when lenolidamide is used in combination with cyclophosphamide and dexamethasone for patients with relapsed refractory disease. Multiply relapsed patients were given Revlimid 25mg po on days 1–21, dexamethasone 40mg po days 1–4 and days 12–15, and cyclophosphamide 500 mg po days 1, 8, 15 and 21 of a 28 day cycle for a maximum of 6 cycles of treatment. Prophylaxis with acyclovir, septrin and proton pump inhibitors was routinely used, 1 high risk patient received prophylactic anticoagulation. Toxicity profiles and response were assessed every 4 weeks. To date 18 patients have been included in the study with median age of 60.5 years (range 34–76). All were heavily pre-treated with a median of 4 previous lines of therapies (range 2–8). 12 patients had received high dose melphalan, 18 patients thalidomide and 16 patients bortezomib. One case had undergone an allogeneic BMT. The median time from diagnosis to treatment initiation was 55.5 months (range 11–122). To date 81 complete courses of therapy have been given to 18 patients with median number of 5 courses (range 1–6). 8 patients experienced neutropenia with the neutophil count falling below 0.5×109/L in a total number of 14 cycles, which resulted in a dose reduction or stopping of cyclophosphamide in 8 patients. 12 patients received GCSF to maintain their neutrophil count. 5 patients required dose reduction/omit of Revlimid. 4 patients required intravenous antibiotics for neutropenic fever. The side effect profile was manageable, importantly no patient experienced sedation, constipation or worsening of peripheral neuropathy. 2 patients with a heavy myeloma load suffered a DVT, but continued on therapy achieving a PR once anti-coagulation had been commenced. 14 of the 17 patients assessable for response achieved a response with 1 CR, 3 VGPR, 8 PR and 2 MR according to EBMT criteria. The median time to response was prompt at 32 days (range 21–68). To date only 2 patients have discontinued therapy because of a failure to respond to therapy and 1 stopped due to liver toxicity. 13 patients have completed treatment and 5 still remain on treatment. The combination of CRD is effective in heavily pretreated myeloma patients and has a manageable toxicity profile. In view of the neutropenia further studies using this combination are currently being investigated using cyclophosphamide on days 1 and 8 only.
Expanded Access Program (EAP) for Lenalidomide (Revlimid®) Plus Dexamethasone in over 1400 Subjects with Relapsed or Refractory Multiple Myeloma.
Authors: Chen Christine , Reece Donna E. , Siegel David , Niesvizky Ruben , Boccia Ralph Vincent , Stadtmauer Edward A. , Abonour Rafat , Irwin David , Polikoff Jonathan , Kumar Shaji , Gams Richard A. , Kenvin Laurie , McElveen Dale , Hu Angela , Pietronigro Dennis , Knight Robert D. , Zeldis Jerome B.
Published: October 2019

Background: Lenalidomide (Revlimid®) in combination with dexamethasone was approved in the US on June 29, 2006 for the treatment of subjects with multiple myeloma who had received at least one prior therapy. On February 28, 2005 based upon a positive interim analysis of two pivotal placebo-controlled Phase III studies, an independent Data Safety Monitoring Board recommended the studies be unblinded and all subjects in both studies be given access to lenalidomide. In April 2005, the FDA in association with myeloma patient advocacy groups requested Celgene establish an expanded access program to make lenalidomide plus dexamethasone available to subjects with relapsed or refractory multiple myeloma while the treatment was awaiting approval.

Aim: To provide lenalidomide to multiple myeloma subjects with a high likelihood of benefit and to obtain additional safety data.

Methods: Subjects with relapsed or refractory multiple myeloma that received at least 1 prior therapy were eligible. Subjects received 25 mg lenalidomide plus high-dose dexamethasone in 4-week cycles until disease progression was documented, study drug was discontinued, or lenalidomide became commercially available for this indication.

Results: Between September 8, 2005 and July 25, 2006, approximately 1400 subjects in the US and Canada were enrolled into the study. A data snapshot taken March 17, 2006 demonstrated that 746 subjects had been enrolled, median age was 63 years, 60% were male, and 66.5% had Stage III disease. Median time on study was 7.1 weeks (0.1–24.4) and median daily dose was 20.5 mg. At least one Grade 3 or 4 adverse event was reported in 261 (35%) of the 746 subjects. Most commonly reported Grade 3–4 events were neutropenia (7.9% of subjects), thrombocytopenia (6.0%), fatigue (3.6%), anemia (3.5%), pneumonia (3.1%) and hyperglycemia (2.0%). These most commonly reported Grade 3–4 adverse events were the same as those found in the previous pivotal studies, however, their frequencies of occurrence were lower in the current study probably due to ongoing data collection and differences in study maturity. Likewise, the most commonly reported adverse events (all grades) were the same as those reported in the two previous pivotal studies.

Conclusion: Preliminary data from this expanded access program in over 1400 subjects with multiple myeloma are consistent with results from two earlier Phase III pivotal studies. The EAP of lenalidomide plus dexamethasone in multiple myeloma represents a model of how government, advocacy groups, healthcare providers and industry can work together to quickly provide treatment to subjects in need while a clearly active treatment regimen is awaiting approval.
A Randomized Comparison of Dexamethasone + Thalidomide (Dex/Thal) vs Dex + Placebo (Dex/P) in Patients (pts) with Relapsing Multiple Myeloma (MM).
Authors: Fermand Jean-Paul , Jaccard Arnaud , Macro Margaret , Uzunhan Yurdagul , Allard Christian , Castaigne Sylvie , Divine Marine , Mariette Xavier , Leblond Veronique , Chevret Sylvie
Published: October 2019

We randomly compared a Dex/Thal and a Dex/placebo combination as treatment of MM pts in first or second relapse after high dose therapy (HDT) or conventional chemotherapy (CCT). 116 pts, aged 41 to 81 (med. 63 yrs), were enrolled between July 2001 and December 2004. Dex was given orally during 4 days at a dose of 40 mg/day every other week for 4 cycles and then monthly. Thal or placebo were given blindly at the initial dose of 100 mg, escalated up to 400 mg daily according to tolerance, and maintained for a maximum of 12 mths. In both arms, treatment initially included continuous infusions of Doxorubicin (9 mg/m2/d for 4 days 4–5 wk apart). In July 2002, because of a high incidence of thrombotic event (TE), the study was interrupted during a six-mth period, before restarting without Doxorubicin administration. Anticoagulation prophylaxis wasn’t systematically given at any time.

Main characteristics of pts included in both arms were similar, including age above 65 yrs (43% in the Dex/Thal arm, 44 % in the Dex/P arm), previous therapy (HDT 57% vs 56%), MM isotype (IgA: 26% vs 21%), serum β2m (≥3 mg/L, 48% vs 55%), serum creatinine (≤ 300 μmol/L in all pts) and albumin levels. Data were analyzed in an intent to treat basis, regarding EFS as the primary objective.

A partial response (defined by a 50% or more reduction of MM protein in serum) was achieved in 46% of all pts, in 65% of pts treated by Dex/Thal and in 28% of pts treated by Dex/P (p<.0001). At one year post randomization, 46.5% of pts in the Dex/Thal arm were alive and free of progression, compared to 31% in the Dex/P arm (p=.009). Death rates were 7% (n=4) and 5% (n=3), respectively.

Median dose of actually tolerated Thal was 150 mg daily. At least one ≥ grade II WHO adverse event was reported in 90% of pts who received Thal, as compared with 42% of pts who received the placebo (p<.0001). A TE was recorded in 12 (22%) of pts in the Dex/Thal arm, as compared to 4 (7%) of pts in the Dex/P arm (p= .02). Other frequent side effects were somnolence/mood changes (58% vs 21%), constipation/nausea (43% vs 11%), skin/mucosal dryness (26% vs 9%) and tremor (19% vs 4%). A symptomatic peripheral neuropathy was observed in 28% and 7% of pts, respectively. Besides the 25 pts who completed the trial (16 and 9 in both arms, respectively), toxicity was the main cause of treatment discontinuation in 21 (55%) pts of the Dex/Thal arm and in 3 (6%) pts of the Dex/P arm.

This randomized study demonstrates that the adjunction of Thal to Dex, although it increases toxicity, is a valuable therapeutic option for pts with relapsing MM since it produces higher response rate and longer remission duration than Dex alone.
The Registro Italiano Trombocitemie (GIMEMA Project): Preliminary Analysis of the First 801 Enrolled Patients.
Authors: Gugliotta Luigi , Tieghi Alessia , Gaidano Gianluca , Franceschetti Silvia , Specchia Giorgina , Carluccio Paola , Mazzucconi Maria Gabriella , Santoro Cristina , Dragani Alfredo , Ciancia Rosanna , Candoni Anna , Palmieri Fausto , Rumi Elisa , Antonioli Elisabetta , Villa Maria Rosaria , Cilloni Daniela , Federici Augusto , Vianelli Nicola , Radaelli Franca , Randi Maria Luigia , Scalzulli Potito , Melillo Lorella , Cacciola Emma , Cacciola Rossella , Gobbi Marco
Published: October 2019

Epidemiological, diagnostic, prognostic and therapeutical data were retrospectively obtained by the Italian Registry in over 2000 Essential Thrombocythemia (ET) patients who mainly were diagnosed according to the PVSG criteria and were treated with potentially leukemogenic drugs. The Registro Italiano Trombocitemie (RIT), that is a GIMEMA project, has been activated in order to registry italian ET patients, to improve the diagnosis appropriateness (WHO criteria), to promote the acquisition of biological data, to evaluate the compliance to the therapeutical guidelines of the Italian Society of Hematology (SIE), to monitor in particular the ET patients receiving Interferons alpha and Anagrelide, to evaluate cases of pregnancy, pediatric age and familiarity, to define the prognostic value of the biological factors as JAK2 mutation, clonality, etc, to create a network for activation of new clinical and biological studies. The RIT, co-ordinated by the Hematology Unit of Reggio Emilia, is a web based registry that beside a public area comprehends a database of italian ET patients. The data, with respect of the privacy rules, are object of validation and analysis by various RIT expert subcommittees. Eighty hematological Institutions adhered to the RIT and 801 patients have been registered since June 2005. The ET diagnosis was done according to the PVSG (90%) and WHO (10%) criteria. The patients, 492 females and 309 males, had age <40 yr (19%), 40–60 yr (32%), 60–70 yr (19%), >70 yr (30%), and the median age was 59 years. At diagnosis the platelet count was >1000 ×109/L in 29% of cases (mean 932). Few patients had prior thrombosis (4%; major 2%) and prior hemorrhage (2.6%). Patients at high risk of thrombosis were 55% on considering age >60 yr and/or previous thrombosis and/or PLT count >1500 ×109/L, and 65% on considering the PLT count cut-off of 1000 ×109/L. The patients shown general thrombotic risk factors (70%), disease related symptoms (40%) and splenomegaly (25%). By applying the WHO diagnostic criteria, the picture of true ET was found in 27% of cases. Sixty pregnancies have been reported. Aspirin was administered in 72% of patients and cytoreduction was performed in 62% of them, with use of Hydroxyurea (65%), Anagrelide (10%), Interferons alpha (10%), Pipobroman (4%), Busulfan (2%). A separate analysis for patients treated with Anagrelide and Iterferons alpha is in progress. To improve the diagnostic approach, the RIT has promoted the bone marrow biopsy revision (WHO criteria) and the acquisition of the new biological parameters.
Generation of CD8+ Viral Specific T Cells from EBV and CMV Naive Individuals.
Authors: Bao Lei , Niyazova-Brewer Nargisa , Dunham Kimberly , Kenneth Kenneth , Sun Qi
Published: October 2019

Adoptive T cell immunotherapy (ATCI) with viral specific T cells, as exemplified by ATCI against Epstein-Bar virus (EBV) and Cytomegalovirus (CMV) with viral specific T cells generated from virus-experienced individuals, is efficacious against viral reactivation in immuno-compromised hosts. EBV-seronegative solid organ transplant recipients and CMV-seropositive stem cell transplant patients receiving CMV-seronegative grafts are at high risk of EBV-driven lymphoproliferation and CMV reactivation, respectively. However, due to the absence of virus-specific memory T cells, ex vivo techniques for generating virus-specific CD8+ CTL from virus-naive individuals remain to be developed for reproducibility and efficiency. To extend ATCI to the above patients, we are developing novel ex vivo systems to expand virus specific CD8+ CTL from seronegative individuals. We designed a two step stimulation for the naive T cells to develop into specific T cells. The first step, “de-naiviation”, involves non-specific but high-affinity stimulation of CD45RO/CD25/CD56/CD14 depleted peripheral blood mononuclear cells with anti-CD3 and -CD28 antibodies. The “de-naiviated” T cells were then antigen-specifically stimulated by antigen presenting cells expressing both EBV and CMV antigens. Peripheral blood mononuclear cells from an EBV/CMV seronegative individual were depleted for two rounds with micro-beads conjugated with antibodies against CD45RO, CD25, CD14 and CD56. The resultant cells were a homogenous population of cells mostly CD45RO−/CD45RA+/CD3+/CD25−, the phenotype for naïve T cells. After a period of expansion stimulated by a cocktail containing anti-CD3 (OKT3) and -CD28, 90% of the RO-T cells became RO+/RA−, the phenotype of memory T cells. Nearly all the CD4+ cells and most the CD8 cells became CD25+, suggesting recent activation. Then the “de-naiviated” T cells were stimulated with autologous EBV immortalized B lymphoblastoid cells transduced and expressing the CMV pp65 (CMVpp65) antigen. After three rounds of stimulation, the T cells were screened for specific production of interferon-gamma (IFNg) by ELISA. Clones were isolated from the primed T cells, and FACS analysis showed that the T cell clones produced IFNg in response to EBV BLCL expressing CMVpp65. These T cells also antigen-specificly expressed IL2 and GMCSF. Interestingly, while the cells were predominantly CD8+/CD3+, some of the cells were also positive for CD56, suggesting newly differentiated effector T cells. Work is ongoing to further characterize the T cell clones for antigen specificity, functionality and differentiation status. The novel approach we are developing has the potential to generate EBV and CMV specific CD8+ CTL from virus naive individuals for adoptive T cell immunotherapy against viral infections.
Sensitive In Vivo Detection of Primary T Cells Expressing Membrane-Anchored Gaussia Luciferase for the Study of Adoptive T Cell Immunotherapy in Murine Models of Malignancy.
Authors: Brentjens Renier J. , Santos Elmer , Yeh Raymond , La Perle Krista , Toledo-Crow Ricardo , Nikhamin Yan , Punzalan Blesida , Entenberg David , Aranda Iana , Punzalan Bleserene , Larson Steven , Sadelain Michel
Published: October 2019

Current approaches to in vivo bioluminescent imaging (BLI) of T cells utilizing luciferase enzymes may be compromised by poor transduction efficiencies in primary T cells, and low photon emissions. Here we describe a novel and widely applicable approach to in vivo BLI of primary T cells utilizing a membrane-anchored form of the naturally secreted Guassia luciferase (GLuc) enzyme, termed exGLuc. We constructed exGLuc by fusion of the GLuc gene to the coding region of the CD8 transmembrane domain. The resulting exGLuc enzyme is anchored to the cell surface of retrovirally transduced cells. In vitro, cells which expressed the exGLuc enzyme demonstrated a markedly (>9 fold) increased bioluminescent signal when compared to cells which expressed the native GLuc (GLuc), the related Rhenilla luciferase (RLuc), a membrane-anchored form of RLuc (exRLuc), or a green fluorescent protein (GFP)-fire fly luciferase fusion protein. Following injection into SCID-Beige mice, MHC-mismatched C57BL6 T cells transduced to express exGLuc were detected by BLI generating graft versus host disease at an earlier time point and demonstrated a >10 fold increased bioluminescent signal when compared to infused C57BL6 T cells expressing GLuc, RLuc, or exRLuc. We further demonstrate homing of OVA-specific DO11.10 exGLuc+ T cells to A20(OVA) but not A20(GFP) subcutaneous tumors in both SCID-Beige and Balb/c mice. We further successfully applied this strategy to the study of in vivo human T cell homing using a xenogeneic SCID-Beige tumor model. Specifically, we demonstrate that human T cells, retrovirally co-transduced to express both a CD19-specific chimeric antigen receptor (CAR), 19z1, and exGLuc, could be monitored over time, and that these T cells quite rapidly home to subcutaneous CD19+ acute lymphoblastic leukemia (NALM-6) tumors when compared to T cells co-expressing an irrelevant CAR with exGLuc. Furthermore, we were able to demonstrate for the first time specific homing of 19z1/exGLuc+ human T cells to systemic NALM-6 tumor, detecting exGLuc+ T cells in deep tissues including the bone marrow, spleen, and liver. We conclude that the exGLuc enzyme emits a superior bioluminescent signal when compared to other commonly utilized luciferase enzymes; that primary T cells are readily transduced to express the exGLuc enzyme; and that exGLuc+ T cells may be accurately monitored in vivo over time by BLI. Based on these data, we believe that this novel approach to primary T cell in vivo imaging will significantly enhance the study of adoptive T cell immunotherapy in murine models of leukemia and other malignancies.
Redirecting Human T Lymphocytes toward Tumor-Associated Antigens by T Cell Receptor (TCR) Replacement.
Authors: Neudorfer Julia , Sommermeyer Daniel , Peschel Christian , Blankenstein Thomas , Uckert Wolfgang , Bernhard Helga
Published: October 2019

The gene transfer of alpha and beta chains derived from a defined TCR has been successfully applied to endow T cells with specificities directed against tumor-associated antigens. However, it is still unclear if the transfer of TCR genes into T cells that already express an endogenous TCRalpha and beta chain leads to engineered T cells expressing four different TCR complexes on their cell surface. Mixed TCR heterodimers composed of endogenous and exogenous TCR chains may acquire new specificities, which may cause unwanted reactions in patients following adoptive T cell transfer. We examined the possibility of mixed TCR heterodimer formation using defined conditions of single TCR chain transfer into human cytotoxic T cell (CTL) clones specific for CMV and Melan-A, respectively. After stimulation for three days CTLs were retrovirally transduced with the beta chain derived from a gp100-specific TCR. The expression of the exogenous (transduced) and the endogenous beta chain was distinguished by flow cytometry using antibodies against the different Vbeta motives. Indeed, CTLs that had been transduced with the single beta chain expressed this chain on the cell surface indicating the formation of mixed TCRs, because the expression of the exogenous TCRbeta chain requires the pairing with the endogenous TCRalpha chain. Furthermore, we transduced the CTL clones with both the TCRalpha and beta chain derived from a gp100-specific TCR. The transduced T cells were positively stained with an A2/gp100 multimer documenting the correct formation of the exogenous TCR chains. Functionality of transduced CTL clones was tested by antigen-specific IFN-gamma release and cytolytic activity. The TCR-transduced T cells were sorted with the A2/gp100 multimer and expanded for two weeks. Double staining with HLA multimers for the endogenous and the transduced TCRs showed the downregulation of the endogenous TCRs in three different CTL clones. In one CTL clone, the endogenous TCR was even replaced by the exogenous TCR as documented by flowcytometry and antigen-specific T cell function. In conclusion, transfer of single TCR chains in CTL clones can result in the formation of TCR heterodimers. However, our results also show that complete TCRs are predominantly expressed or can even replace other TCRs following transfer. The development of dominant TCRs will facilitate the therapeutical approaches of adoptive transfer regimens based on TCR-transduced T cells, because dominant TCRs can be selected or TCRs can be modified to be more dominant.
Alloreactivity as a Source of High Avidity T Cell Clones Specific for Cancer Testis-Antigens Associated with Multiple Myeloma: Prospects for Adoptive Immunotherapy with Primary T Cells Redirected toward Multiple Myeloma by T Cell Receptor Gene Transfer.
Authors: Kronig Holger , Hofer Kathrin , Neudorfer Julia , Peschel Christian , Bernhard Helga
Published: October 2019

Cancer testis (CT)-antigens belong to a class of tumor antigens that are aberrantly expressed in a variety of hematological malignancies including multiple myeloma. Owing to their restricted gene expression, CT-antigens represent potential target antigens for immunotherapeutical approaches such as vaccination and adoptive T cell transfer. As the CT-antigens are self antigens, the majority of CT-antigen-specific autologous T cells display a low avidity T cell receptor (TCR), which often results in a weak tumor recognition efficiency.

Our group has been focusing on the isolation of highly avid T cells against CT-antigens that are expressed in multiple myeloma, in particular MAGE-C1, MAGE-C2, and NY-ESO-1. The experimental approach was based on the stimulation of allo-restricted cytotoxic T cells, because highly avid T cells recognizing peptide epitopes in context with foreign HLA-alleles are not depleted in the thymus. HLA-A2-negative T cells were stimulated with HLA-A2-positive allogeneic dendritic cells that had been exogenously loaded with HLA-A2-binding peptides derived from NY-ESO-1, MAGE-C1 or MAGE-C2. Using this technique we were able to isolate allo-HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones with peptide-dominant binding against known and novel peptide epitopes derived from NY-ESO-1, MAGE-C1 and MAGE-C2. The expanded peptide-specific CTL clones lysed HLA-A2-positive myeloma cell lines expressing NY-ESO-1, MAGE-C1 and MAGE-C2, respectively. Of note, the MAGE-C1-specific T cells crossreacted with the corresponding MAGE-C2 peptide due to the existing sequence homology between MAGE-C1 and MAGE-C2. Current experiments focus on redirecting primary T cells toward myeloma cells by retroviral gene transfer of CT-antigen-specific TCRs.

The establishment of a set of high avidity TCRs specific for CT-antigens facilitates the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of multiple myeloma.