Publications by authors named "Josée Golay"

64 Publications

Third-party bone marrow-derived mesenchymal stromal cell infusion before liver transplantation: A randomized controlled trial.

Am J Transplant 2020 Dec 28. Epub 2020 Dec 28.

Aldo & Cele Daccò Clinical Research Center for Rare Diseases, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy.

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pretransplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary endpoint was the safety profile of MSC administration during the 1-year follow-up. In all, 19 patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56 NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pretransplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.
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http://dx.doi.org/10.1111/ajt.16468DOI Listing
December 2020

Tolerance to Bone Marrow Transplantation: Do Mesenchymal Stromal Cells Still Have a Future for Acute or Chronic GvHD?

Front Immunol 2020 11;11:609063. Epub 2020 Dec 11.

Center of Cellular Therapy "G. Lanzani", Division of Haematology, Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Mesenchymal Stromal Cells (MSCs) are fibroblast-like cells of mesodermal origin present in many tissues and which have the potential to differentiate to osteoblasts, adipocytes and chondroblasts. They also have a clear immunosuppressive and tissue regeneration potential. Indeed, the initial classification of MSCs as pluripotent stem cells, has turned into their identification as stromal progenitors. Due to the relatively simple procedures available to expand large numbers of GMP grade MSCs from a variety of different tissues, many clinical trials have tested their therapeutic potential . One pathological condition where MSCs have been quite extensively tested is steroid resistant (SR) graft versus host disease (GvHD), a devastating condition that may occur in acute or chronic form following allogeneic hematopoietic stem cell transplantation. The clinical and experimental results obtained have outlined a possible efficacy of MSCs, but unfortunately statistical significance in clinical studies has only rarely been reached and effects have been relatively limited in most cases. Nonetheless, the extremely complex pathogenetic mechanisms at the basis of GvHD, the fact that studies have been conducted often in patients who had been previously treated with multiple lines of therapy, the variable MSC doses and schedules administered in different trials, the lack of validated potency assays and clear biomarkers, the difference in MSC sources and production methods may have been major factors for this lack of clear efficacy . The heterogeneity of MSCs and their different stromal differentiation potential and biological activity may be better understood through more refined single cell sequencing and proteomic studies, where either an "anti-inflammatory" or a more "immunosuppressive" profile can be identified. We summarize the pathogenic mechanisms of acute and chronic GvHD and the role for MSCs. We suggest that systematic controlled clinical trials still need to be conducted in the most promising clinical settings, using better characterized cells and measuring efficacy with specific biomarkers, before strong conclusions can be drawn about the therapeutic potential of these cells in this context. The same analysis should be applied to other inflammatory, immune or degenerative diseases where MSCs may have a therapeutic potential.
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http://dx.doi.org/10.3389/fimmu.2020.609063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759493PMC
December 2020

The Role of Complement in the Mechanism of Action of Therapeutic Anti-Cancer mAbs.

Antibodies (Basel) 2020 Oct 28;9(4). Epub 2020 Oct 28.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

Unconjugated anti-cancer IgG1 monoclonal antibodies (mAbs) activate antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages, and these activities are thought to be important mechanisms of action for many of these mAbs in vivo. Several mAbs also activate the classical complement pathway and promote complement-dependent cytotoxicity (CDC), although with very different levels of efficacy, depending on the mAb, the target antigen, and the tumor type. Recent studies have unraveled the various structural factors that define why some IgG1 mAbs are strong mediators of CDC, whereas others are not. The role of complement activation and membrane inhibitors expressed by tumor cells, most notably CD55 and CD59, has also been quite extensively studied, but how much these affect the resistance of tumors in vivo to IgG1 therapeutic mAbs still remains incompletely understood. Recent studies have demonstrated that complement activation has multiple effects beyond target cell lysis, affecting both innate and adaptive immunity mediated by soluble complement fragments, such as C3a and C5a, and by stimulating complement receptors expressed by immune cells, including NK cells, neutrophils, macrophages, T cells, and dendritic cells. Complement activation can enhance ADCC and ADCP and may contribute to the vaccine effect of mAbs. These different aspects of complement are also briefly reviewed in the specific context of FDA-approved therapeutic anti-cancer IgG1 mAbs.
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http://dx.doi.org/10.3390/antib9040058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709112PMC
October 2020

Combined Anti-Cancer Strategies Based on Anti-Checkpoint Inhibitor Antibodies.

Antibodies (Basel) 2020 May 20;9(2). Epub 2020 May 20.

Laboratoire de Biochimie et Thérapies Moléculaires, Faculté de Pharmacie, Université Saint Joseph de Beyrouth, Beirut 1100, Lebanon.

Therapeutic monoclonal antibodies for the treatment of cancer came of age in 1997, with the approval of anti-CD20 Rituximab. Since then, a wide variety of antibodies have been developed with many different formats and mechanisms of action. Among these, antibodies blocking immune checkpoint inhibitors (ICI) have revolutionized the field, based on the novelty of their concept and their demonstrated efficacy in several types of cancer otherwise lacking effective immunotherapy approaches. ICI are expressed by tumor, stromal or immune cells infiltrating the tumor microenvironment, and negatively regulate anti-tumor immunity. Antibodies against the first discovered ICI, CTLA-4, PD-1 and PD-L1, have shown significant activity in phase III studies against melanoma and other solid cancers, alone or in combination with chemotherapy or radiotherapy. However, not all cancers and not all patients respond to these drugs. Therefore, novel antibodies targeting additional ICI are currently being developed. In addition, CTLA-4, PD-1 and PD-L1 blocking antibodies are being combined with each other or with other antibodies targeting novel ICI, immunostimulatory molecules, tumor antigens, angiogenic factors, complement receptors, or with T cell engaging bispecific antibodies (BsAb), with the aim of obtaining synergistic effects with minimal toxicity. In this review, we summarize the biological aspects behind such combinations and review some of the most important clinical data on ICI-specific antibodies.
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http://dx.doi.org/10.3390/antib9020017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345008PMC
May 2020

Monoclonal Antibody Monitoring: Clinically Relevant Aspects, A Systematic Critical Review.

Ther Drug Monit 2020 02;42(1):45-56

Department of Diagnostic Medicine; Clinical and Experimental Pharmacokinetics Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

Monoclonal antibody (mAb) therapy does not usually lead to a clinical response in all patients and resistance may increase over time after repeated mAb administration. This lack or loss of response to the treatment may originate from different and little-known epigenetic, biomolecular, or pathophysiological mechanisms, although an inadequate serum concentration is perhaps the most likely cause, even if not widely recognized and investigated yet. Patient factors that influence the pharmacokinetics (PK) of a mAb should be taken into account. Multiple analyses of patient-derived PK data have identified various factors influencing the clearance of mAbs. These factors include the presence of antidrug antibodies, low serum albumin, high serum levels of C-reactive protein, high body weight, and gender differences among others. The same clearance processes involved in systemic clearance after intravenous administration are also involved in local first-pass catabolism after subcutaneous administration of mAbs. Therapeutic drug monitoring has been proposed as a way to understand and respond to the variability in clinical response and remission. For both classes of mAbs with anti-inflammatory and antitumor effects, dose-guided optimization based on the measurement of serum concentrations in individual patients could be the next step for a personalized and targeted mAb therapy.
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http://dx.doi.org/10.1097/FTD.0000000000000681DOI Listing
February 2020

Key Features Defining the Disposition of Bispecific Antibodies and Their Efficacy In Vivo.

Ther Drug Monit 2020 02;42(1):57-63

S.I.F.E.B.- Società Italiana di Farmacocinetica e Biofarmaceutica, Pavia, Italy.

Bispecific antibodies (BsAbs) are novel drugs, with only a few approved for clinical use. BsAbs are versatile molecules that come in many different forms and are designed and produced via genetic engineering. Although BsAbs share several pharmacokinetic (PK) and pharmacodynamic (PD) properties with monoclonal antibodies, they have their own unique characteristics based on their overall structure and specificities. BsAbs are generally more complex to investigate and develop than monoclonal antibodies, because they recognize at least 2 different antigens. Understanding their relative affinities to each target is crucial for determining their mechanism of action and efficacy. Moreover, the presence or absence of an Fc region determines, in part, their in vivo stability, distribution, and half-life. This study summarizes several PK and PD aspects that are specific for BsAbs and are important for the success of these new drugs. We emphasize previous PK/PD studies that have been fundamental for the correct prediction of appropriate dosages and schedules of these new drugs in clinical trials or for defining which drugs may take advantage of individualized and standardized drug monitoring for improved efficacy and safety.
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http://dx.doi.org/10.1097/FTD.0000000000000668DOI Listing
February 2020

Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation.

Blood 2019 03 17;133(13):1395-1405. Epub 2019 Jan 17.

Center of Cellular Therapy "G. Lanzani," Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab') antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.
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http://dx.doi.org/10.1182/blood-2018-07-864538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484458PMC
March 2019

Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19 tumors.

Cytotherapy 2018 08 7;20(8):1077-1088. Epub 2018 Aug 7.

Center of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy. Electronic address:

Background: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19 malignancies.

Methods: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs.

Results: CB-CIKs, like PB-CIKs, were mostly CD3 T cells with mean 45% CD3CD56 and expressing mostly TCR(T cell receptor)αβ with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4 cells, mostly CD56, as well as a greater proportion of naïve CCR7CD45RA and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8 cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19 tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph CD19 acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 10 CIK/kg.

Discussion: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.
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http://dx.doi.org/10.1016/j.jcyt.2018.06.003DOI Listing
August 2018

Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance.

Cytotherapy 2018 02 12;20(2):262-270. Epub 2017 Dec 12.

Centre of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Background: We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs).

Methods: For sterility tests 14-day cultures were performed in culture media detecting aerobic and anaerobic microorganisms.

Results: In this study, 22/1643 (1.3%) of apheretic products for autologous or allogeneic HSCT were contaminated, whereas 14/73 bone marrow (BM) harvests (17.8%) were positive. In 22 cases, the contaminated HSCs were infused to patients, but there was no evidence of any adverse impact of contamination on the hematologic engraftment or on infections. Indeed none of the five positive hemocultures detected in patients following infusion could be linked to the contaminated stem cell product. Our Cell Factory also generated 286 ATMPs in good manufacturing practice (GMP) conditions since 2007 and all final products were sterile. In three cases of mesenchymal stromal cell expansions, the starting BM harvests were contaminated, but the cell products at the end of expansion were sterile, presumably thanks to the presence of an antibiotic in the culture medium.

Discussion: The decreased rate of contamination of cell harvests observed with time suggests that routine sterility testing and communication of the results to the collecting centers may improve clinical practices. Furthermore, we recommend the use of antibiotics in the medium for ATMP expansion, to decrease the likelihood of expanding microorganisms within clean rooms. Finally we discuss the costs of sterility testing of ATMPs by GMP-approved external laboratories.
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http://dx.doi.org/10.1016/j.jcyt.2017.11.009DOI Listing
February 2018

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2017 Dec 13;23(12):2070-2078. Epub 2017 Jul 13.

USC Hematology and Bone Marrow Transplant Unit ASST Papa Giovanni XXIII Bergamo, Bergamo, Italy; Università degli Studi di Milano, Milan Italy.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.
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http://dx.doi.org/10.1016/j.bbmt.2017.07.005DOI Listing
December 2017

Direct targeting of cancer cells with antibodies: What can we learn from the successes and failure of unconjugated antibodies for lymphoid neoplasias?

Authors:
Josée Golay

J Autoimmun 2017 Dec 27;85:6-19. Epub 2017 Jun 27.

Center of Cellular Therapy "G. Lanzani", USC Haematology, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Via Garibaldi 11-13, 24128, Bergamo, Italy. Electronic address:

Following approval in 1997 of the anti-CD20 antibody rituximab for the treatment of B-NHL and CLL, many other unconjugated IgG1 MAbs have been tested in pre-clinical and clinical trials for the treatment of lymphoid neoplasms. Relatively few have been approved however and these are directed against a limited number of target antigens (CD20, CD52, CCR4, CD38, CD319). We review here the known biological properties of these antibodies and discuss which factors may have led to their success or may, on the contrary, limit their clinical application. Common factors of the approved MAbs are that the target antigen is expressed at relatively high levels on the neoplastic targets and their mechanism of action is mostly immune-mediated. Indeed most of these MAbs induce ADCC and phagocytosis by macrophages, and many also activate complement, leading to target cell lysis. In contrast direct cell death induction is not a common feature but may enhance efficacy in some cases. Interestingly, a key factor for the success of several MAbs appears to be their capacity to skew immunity towards an anti-tumour mode, by inhibiting/depleting suppressor cells and/or activating immune cells within the microenvironment, independently of FcγRs. We also expose here some of the strategies employed by industry to expand the clinical use of these molecules beyond their original indication. Interestingly, due to the central role of lymphocytes in the control of the immune response, several of the antibodies are now successfully used to treat many different autoimmune diseases and have also been formally approved for some of these new indications. There is little doubt that this trend will continue and that the precise mechanisms of therapeutic MAbs will be further dissected and better understood in the context of both tumour immunology and autoimmunity.
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http://dx.doi.org/10.1016/j.jaut.2017.06.002DOI Listing
December 2017

The specific Bruton tyrosine kinase inhibitor acalabrutinib (ACP-196) shows favorable activity against chronic lymphocytic leukemia B cells with CD20 antibodies.

Haematologica 2017 10 22;102(10):e400-e403. Epub 2017 Jun 22.

Center of Cellular Therapy "G. Lanzani, USC Hematology, and Fondazione per la Ricerca Ospedale Maggiore, ASST Papa Giovanni XXIII, Bergamo, Italy.

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http://dx.doi.org/10.3324/haematol.2017.169334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622871PMC
October 2017

Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies.

Blood 2017 05 13;129(19):2636-2644. Epub 2017 Mar 13.

Center of Cellular Therapy G. Lanzani, Division of Hematology, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo.
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http://dx.doi.org/10.1182/blood-2016-08-735605DOI Listing
May 2017

Development of advanced therapies in Italy: Management models and sustainability in six Italian cell factories.

Cytotherapy 2016 Apr;18(4):481-6

Laboratory of Cell and Gene Therapy Stefano Verri, San Gerardo Hospital, Monza, Italy; Tettamanti Research Center, Pediatric Clinic Monza Brianza per il Bambino e la sua Mamma (MBBM) Foundation, Monza, Italy.

On November 10, 2014, the representatives of all six certified Good Manufacturing Practices (GMP) cell factories operating in the Lombardy Region of Italy convened a 1-day workshop in Milan titled "Management Models for the Development And Sustainability of Cell Factories: Public-Private Partnership?" The speakers and panelists addressed not only the many scientific, technological and cultural challenges faced by Lombardy Cell Factories, but also the potential impact of advanced therapy medicinal products (ATMPs) on public health and the role played by translational research in this process. Future perspectives for research and development (R&D) and manufacturing processes in the field of regenerative medicine were discussed as well. This report summarizes the most important issues raised by the workshop participants with particular emphasis on strengths and limitations of the R&D and manufacturing processes for innovative therapeutics in Lombardy and what can be improved in this context while maintaining GMP standards. The participants highlighted several strategies to translate patient-specific advanced therapeutics into scaled manufacturing products for clinical application. These included (i) the development of a synergistic interaction between public and private institutions, (ii) better integration with Italian regulatory agencies and (iii) the creation of a network among Lombardy cell factories and other Italian and European institutions.
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http://dx.doi.org/10.1016/j.jcyt.2016.01.002DOI Listing
April 2016

Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

J Immunol 2016 Apr 26;196(7):3199-211. Epub 2016 Feb 26.

Centre National de la Recherche Scientifique UPS3044 "Baculovirus et Thérapie," F-30380 Saint-Christol-Lèz Alès, France;

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.
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http://dx.doi.org/10.4049/jimmunol.1501592DOI Listing
April 2016

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy.

Haematologica 2015 Jan 24;100(1):77-86. Epub 2014 Oct 24.

Center of Cellular Therapy "G. Lanzani", Division of Hematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.
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http://dx.doi.org/10.3324/haematol.2014.107011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281316PMC
January 2015

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

J Immunol 2014 Nov 29;193(9):4739-47. Epub 2014 Sep 29.

Centro Trapianto Midollo Osseo, USC Ematologia, Azienda Ospedaliera Papa Giovanni XXIII, 24127 Bergamo, Italy;

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1401550DOI Listing
November 2014

Direct involvement of CD56 in cytokine-induced killer-mediated lysis of CD56+ hematopoietic target cells.

Exp Hematol 2014 Dec 6;42(12):1013-21.e1. Epub 2014 Sep 6.

USS Centre of Cellular Therapy "G.Lanzani", USC Hematology and Bone Marrow Transplantation Unit, A.O. Papa Giovanni XXIII, Bergamo, Italy. Electronic address:

Cytokine-induced killer (CIK) cells are in-vitro-expanded T lymphocytes that represent a heterogeneous population. A large majority of CIK cells are CD3(+)CD56(+), and this population has been shown to confer a cytotoxic effect against tumor targets. The scope of this work was to study whether CD56 has a direct role in CIK-mediated cytotoxicity. Blocking of CD56 with the anti-CD56 monoclonal antibody GPR165 significantly reduced CIK-mediated lysis of three CD56(+) hematopoietic tumor cell lines (AML-NS8, NB4, and KCL22), whereas no effect was observed on three CD56(-) hematopoietic tumor cell lines (K562, REH, and MOLT-4). Knockdown of CD56 in CIK cells by short interfering RNA made the cells less cytotoxic against a CD56(+) target, and knockdown of CD56 in target cells with lentiviral short hairpin RNA significantly altered their susceptibility to CIK-mediated lysis. Our data suggest that homophilic interaction between CD56 molecules may occur in tumor-cell recognition, leading to CIK-mediated cell death.
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http://dx.doi.org/10.1016/j.exphem.2014.08.005DOI Listing
December 2014

Frequent occurrence of non-malignant genetic alterations in clinical grade mesenchymal stromal cells expanded for cell therapy protocols.

Haematologica 2014 Jun 28;99(6):e94-7. Epub 2014 Mar 28.

USS Center of Cellular Therapy "G. Lanzani", A.O. Papa Giovanni XXIII, Bergamo, Italy USSD Laboratorio di Genetica Medica, A.O. Papa Giovanni XXIII, Bergamo, Italy USC Hematology, A.O. Papa Giovanni XXIII, Bergamo, Italy

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http://dx.doi.org/10.3324/haematol.2014.104711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040903PMC
June 2014

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

Biol Blood Marrow Transplant 2014 Mar 7;20(3):375-81. Epub 2013 Dec 7.

HSCT Pediatric Unit and Laboratory of Cell Therapy "S. Verri", San Gerardo Hospital, Monza, Italy.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.
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http://dx.doi.org/10.1016/j.bbmt.2013.11.033DOI Listing
March 2014

Comment on "Reduced T-dependent humoral immunity in CD20-deficient mice".

Authors:
Josée Golay

J Immunol 2013 Dec;191(12):5783

Center of Cellular Therapy, "G. Lanzani" Division of Hematology, Hospital Papa Giovanni XXIII, 24128 Bergamo, Italy.

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http://dx.doi.org/10.4049/jimmunol.1390065DOI Listing
December 2013

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab.

Blood 2013 Nov 8;122(20):3482-91. Epub 2013 Oct 8.

Center of Cellular Therapy "G. Lanzani," Division of Haematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy;

Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcγR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab')2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B.
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http://dx.doi.org/10.1182/blood-2013-05-504043DOI Listing
November 2013

Lessons for the clinic from rituximab pharmacokinetics and pharmacodynamics.

MAbs 2013 Nov-Dec;5(6):826-37. Epub 2013 Aug 8.

IRCCS Policlinico San Matteo; Pavia, Italy.

The anti-CD20 antibody rituximab (RTX; Rituxan®, MabThera®) was the first anti-cancer antibody approved by the US Food and Drug Administration in 1997 and it is now the most-studied unconjugated therapeutic antibody. The knowledge gained over the past 15 y on the pharmacodynamics (PD) of this antibody has led to the development of a new generation of anti-CD20 antibodies with enhanced efficacy in vitro. Studies on the pharmacokinetics (PK) properties and the effect of factors such as tumor load and localization, antibody concentration in the circulation and gender on both PK and clinical response has allowed the design of optimized schedules and novel routes of RTX administration. Although clinical results using newer anti-CD20 antibodies, such as ofatumumab and obinutuzumab, and novel administration schedules for RTX are still being evaluated, the knowledge gained so far on RTX PK and PD should also be relevant for other unconjugated monoclonal antibody therapeutics, and will be critically reviewed here.
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http://dx.doi.org/10.4161/mabs.26008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896596PMC
January 2015

The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia.

PLoS One 2013 8;8(3):e58424. Epub 2013 Mar 8.

Oncology, Nerviano Medical Sciences, Nerviano, Milano, Italy.

CD56 is expressed in 15-20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56(+) monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56(+) AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058424PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592825PMC
September 2013

Ofatumumab is more efficient than rituximab in lysing B chronic lymphocytic leukemia cells in whole blood and in combination with chemotherapy.

J Immunol 2013 Jan 5;190(1):231-9. Epub 2012 Dec 5.

Laboratory of Cellular Therapy G. Lanzani, Division of Hematology, Ospedali Riuniti di Bergamo, 24128 Bergamo, Italy.

Ofatumumab (OFA) is a human anti-CD20 Ab approved for treatment of fludarabine-refractory B chronic lymphocytic leukemia (B-CLL). The efficacy of different immunotherapeutic strategies is best investigated in conditions that are as physiologic as possible. We have therefore compared the activity OFA and rituximab (RTX), alone or in combination with chemotherapeutic agents in unmanipulated whole blood assays, using flow cytometry. OFA (10-100 μg/ml) lysed B-CLL targets in whole blood more efficiently and with faster kinetics than RTX, with a mean 56% lysis at 24 h compared with 16%. This activity of OFA was fully complement dependent, as shown by >99% inhibition by anti-C5 Ab eculizumab and a lack of NK cell activation in whole blood. OFA-mediated NK cell activation was blocked by complement. OFA-mediated lysis could be increased an additional 15% by blocking CD55 and CD59 complement inhibitors. Interestingly, OFA-mediated lysis correlated significantly with CD20 expression levels (r(2) = 0.79). OFA showed overlapping dose response curves similar to those for RTX in phagocytosis assays using either human macrophages or neutrophils. However, phagocytosis was inhibited in the presence of serum or whole blood. Finally, combined treatment with mafosfamide and fludarabine showed that these therapeutic drugs are synergistic in B-CLL whole blood assays and show superior activity when combined with OFA compared with RTX. These results confirm in B-CLL samples and in physiologic conditions the superior complement mediated cytotoxicity induced by OFA alone compared with RTX, the lack of NK cell activation, and phagocytosis in these conditions and suggest effective chemoimmunotherapy strategies using this new generation anti-CD20 Ab.
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http://dx.doi.org/10.4049/jimmunol.1202645DOI Listing
January 2013

Givinostat and hydroxyurea synergize in vitro to induce apoptosis of cells from JAK2(V617F) myeloproliferative neoplasm patients.

Exp Hematol 2013 Mar 27;41(3):253-60.e2. Epub 2012 Oct 27.

Division of Hematology, Ospedali Riuniti, Bergamo, Italy.

We investigated whether clinically achievable concentrations of the histone deacetylase (HDAC) inhibitors givinostat and hydroxyurea induce synergistic cytotoxicity in Jak2(V617F) cells in vitro and through which possible mechanism. Givinostat and hydroxyurea at low doses potentiated the pro-apoptotic effects of each other in the Jak2(V617F) HEL and UKE1 cell lines. Givinostat induced 6.8%-20.8% and hydroxyurea (HU) 20.4%-42.4% cell death alone and 35.8%-75.3% in combination. The effect was statistically significant using the median effect Chou-Talalay method, resulting in a combination index less than 1, indicating synergy. Givinostat alone induced cell cycle arrest of the cell lines in G0/G1 and hydroxyurea in S phase, whereas both drugs together led to a G1 block. At the molecular level, hydroxyurea counteracted the induction of p21CDKN1A by Givinostat and potentiated caspase 3 activation, explaining at least in part the increased apoptosis observed in presence of both compounds. We also verified the effect of the same drugs in colony assays of freshly isolated Jak2(V617F) polycythemia vera cells. In this case, low doses of the compounds were additive to each other. These results suggest that combined treatment with givinostat and hydroxyurea is a potential strategy for the management of Jak2(V617F) myeloproliferative neoplasms.
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http://dx.doi.org/10.1016/j.exphem.2012.10.013DOI Listing
March 2013

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2(V617F) myeloproliferative neoplasm cells.

Exp Hematol 2012 Aug 8;40(8):634-45.e10. Epub 2012 May 8.

USC Hematology, Ospedali Riuniti, Bergamo, Italy.

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.
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http://dx.doi.org/10.1016/j.exphem.2012.04.007DOI Listing
August 2012

Lepidopteran cells, an alternative for the production of recombinant antibodies?

MAbs 2012 May-Jun;4(3):294-309. Epub 2012 Apr 26.

CNRS UPS3044 Baculovirus et Thérapie, CNRS GDR3260, ACCITH Anticorps et Ciblage Thérapeutique and LabEx MabImprove, Saint Christol Lèz Alès, France.

Monoclonal antibodies are used with great success in many different therapeutic domains. In order to satisfy the growing demand and to lower the production cost of these molecules, many alternative systems have been explored. Among them, the baculovirus/insect cells system is a good candidate. This system is very safe, given that the baculoviruses have a highly restricted host range and they are not pathogenic to vertebrates or plants. But the major asset is the speed with which it is possible to obtain very stable recombinant viruses capable of producing fully active proteins whose glycosylation pattern can be modulated to make it similar to the human one. These features could ultimately make the difference by enabling the production of antibodies with very low costs. However, efforts are still needed, in particular to increase production rates and thus make this system commercially viable for the production of these therapeutic agents.
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http://dx.doi.org/10.4161/mabs.19942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355492PMC
February 2013

Mechanism of action of therapeutic monoclonal antibodies: promises and pitfalls of in vitro and in vivo assays.

Arch Biochem Biophys 2012 Oct 25;526(2):146-53. Epub 2012 Feb 25.

Laboratory of Cellular Therapy G. Lanzani, USC Haematology, Ospedali Riuniti, Bergamo, Italy.

Therapeutic monoclonal antibodies (mAbs) are mostly used in cancer, as anti-infectious agents and as immunomodulatory drugs, and are amongst the most active area of research and development in the pharmaceutical industry. This class of drugs comprises unconjugated antibodies or antibody fragments, antibody-drug conjugates, radio-immunoconjugates and bispecific/trispecific molecules. A better understanding of the mechanism of action of successful mAbs is fundamental for the selection of more active and less toxic mAbs of new generation. Furthermore reliable screening of new compounds at an early stage of preclinical development, for both efficacy and toxicity, should allow the selection of the best molecules at an early stage, and improve the rate of success of this class of drugs. Here we review the major methods that are employed for testing the activity of therapeutic mAbs in vitro and in vivo in small animal models and point out to some of the pitfalls in these assays.
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http://dx.doi.org/10.1016/j.abb.2012.02.011DOI Listing
October 2012