Correspondence
`
`To the editor:
`
`Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells,
`B cells, and HSCs because they lack the LDL receptor
`
`Vesicular stomatitis virus (VSV) G-protein pseudotyped lentiviral
`vectors (VSV-G-LVs) signify a major advancement in the gene and
`immunotherapy field as illustrated by successful clinical trials, for
`example, for Wiskott Aldrich Syndrome and leukodystrophies.1
`Although VSV-G-LVs allow efficient transduction of nondividing
`cells,2 they do not provide efficient transduction of quiescent T cells,
`
`B cells, and hematopoietic stem cells (HSCs), which hampers their
`application in gene and immune-therapy areas where conservation of
`cell phenotype is essential. Although these hurdles can be overcome
`in lymphocytes by LVs pseudotyped with measles virus envelope
`proteins (MV-LVs3-5), the reason as to why VSV-G-LVs were not
`efficient for gene transfer in these quiescent cells, and in particular in
`
`Downloaded from http://ashpublications.org/blood/article-pdf/123/9/1422/1378598/1422.pdf by guest on 08 October 2023
`
`Figure 1. Low expression of LDL receptor on
`1
`resting T cells, B cells, and CD34
`cells limits
`VSV-G-LV binding, fusion, and transduction of these
`gene-therapy targets. (A) Unstimulated human T cells,
`1
`B cells, and CD34
`cells (G0) or 24-hour prestimulated
`(stim) T cells (anti-CD3 1 anti-CD28 1 IL-2), B cells
`1
`(SAC 1 IL-2), and hCD34
`cells (TPO 1 SCF 1 Flk-3L)
`were transduced with a GFP-encoding VSV-G-LV
`vector at an MOI 5 50 (T and B cells) or MOI 5 100
`1
`1
`(CD34
`cells) and GFP
`cells were analyzed at day 3
`posttransduction by FACS (see supplemental Meth-
`ods, available on the Blood Web site); for LDL-R
`detection,
`freshly isolated or 24-hour prestimulated
`cells (see above) were incubated with the anti–LDL-R
`antibody (mouse mAb; R&D Systems) followed by
`staining with anti-mouse APC antibody (white open
`histograms), a control
`incubation with the latter
`antibody alone was performed (gray filled histogram);
`for
`fusion detection,
`freshly isolated or 24-hour
`prestimulated cells were incubated overnight with
`GFP gesicles7 at 4°C to allow only binding or at 37°C
`to allow binding followed by fusion. The cells were
`then treated with trypsin to remove the GFP gesicles
`at the cell surface that did not fuse with the cells.
`(B) Equivalent quantities of VSV-G-LV or LV particles
`without envelope (measured by p24 content) were
`incubated with freshly isolated or 24-hour prestimulated
`cells (2E5 cells) for 1 hour at 4°C and then washed
`4 times to remove unbound vector particles. The cells
`were pelleted and the cell-associated HIV capsid
`content (p24) was determined by ELISA (means 6 SD;
`n 5 3). The p24 signal for nonenveloped LVs was
`used as reference. (C) Entry through LDL-R was
`evaluated by blocking with a monoclonal antibody
`(C7, aLDL-R at 5 mg/mL; Santa Cruz Biotechnology)
`or by competition with soluble LDL receptor at 0.5 mg/mL
`(LDL-R 0.5; R&D Systems) or 5 mg/mL (LDL-R 5).
`A 1-hour preincubation of the prestimulated T cells,
`1
`B cells, and CD34
`cells with either blocking agent
`was performed before transduction with GFP-encoding
`VSV-G-LVs (MOI 50 for T and B cells; MOI 100 for
`1
`CD34
`cells) or MV-LVs (MOI of 10) for 48 hours,
`1
`followed by FACS analysis for detection of GFP
`cells
`(means 6 SD; n 5 3). aLDL-R, anti-low density lipid
`receptor antibody; ELISA, enzyme-linked immunosor-
`bent assay; FACS, fluorescence-activated cell sorter;
`IL, interleukin; mAb, monoclonal antibody; MOI, multi-
`plicity of infection; SAC, staphylococcus aureus Cowan;
`SCF, stem cell factor; TPO, trombopoietin. Blood sam-
`ples were obtained from healthy donors after informed
`consent and after local ethical committee approval
`in
`accordance with the Declaration of Helsinki.
`
`1422
`
`BLOOD, 27 FEBRUARY 2014 x VOLUME 123, NUMBER 9
`
`Page 1 of 3
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`KELONIA EXHIBIT 1017
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`

`

`BLOOD, 27 FEBRUARY 2014 x VOLUME 123, NUMBER 9
`
`CORRESPONDENCE
`
`1423
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`
`Caroline Costa
`Centre International de Recherche en Infectiologie,
`Enveloppe Virale et Ing ´enierie du Retrovirus Team,
`Institut National de la Sant ´e et de la Recherche M ´edicale U1111,
`Centre National de la Recherche Scientifique,
`Unit ´e Mixte de Recherche 5308, Universit ´e de Lyon-1,
`Ecole Normale Sup ´erieure de Lyon,
`Lyon, France
`
`Philippe-Emmanuel Mangeot
`Centre International de Recherche en Infectiologie,
`Cellular Biology of Viral Infections Team,
`The Scripps Research Institute,
`La Jolla, CA
`
`Bruce E. Torbett
`Department of Molecular and Experimental Medicine,
`The Scripps Research Institute,
`La Jolla, CA
`
`Cathy X. Wang
`Department of Molecular and Experimental Medicine,
`The Scripps Research Institute,
`La Jolla, CA
`
`Didier N `egre
`Centre International de Recherche en Infectiologie,
`Enveloppe Virale et Ing ´enierie du Retrovirus Team,
`Institut National de la Sant ´e et de la Recherche M ´edicale U1111,
`Centre National de la Recherche Scientifique,
`Unit ´e Mixte de Recherche 5308, Universit ´e de Lyon-1,
`Ecole Normale Sup ´erieure de Lyon,
`Lyon, France
`
`Franc¸ ois-Lo¨ıc Cosset
`Centre International de Recherche en Infectiologie,
`Enveloppe Virale et Ing ´enierie du Retrovirus Team,
`Institut National de la Sant ´e et de la Recherche M ´edicale U1111,
`Centre National de la Recherche Scientifique,
`Unit ´e Mixte de Recherche 5308, Universit ´e de Lyon-1,
`Ecole Normale Sup ´erieure de Lyon,
`Lyon, France
`
`Els Verhoeyen
`Centre International de Recherche en Infectiologie,
`Enveloppe Virale et Ing ´enierie du Retrovirus Team,
`Institut National de la Sant ´e et de la Recherche M ´edicale U1111,
`Centre National de la Recherche Scientifique,
`Unit ´e Mixte de Recherche 5308, Universit ´e de Lyon-1,
`Ecole Normale Sup ´erieure de Lyon,
`Lyon, France
`Institut National de la Sant ´e et de la Recherche M ´edicale U1065,
`Centre M ´editerran ´een de M ´edecine Mol ´eculaire,
`´E quipe “Contr ˆole M ´etabolique des Morts Cellulaires,”
`Nice, France
`
`F.A., C.L., and C.C. contributed equally.
`
`The online version of this article contains a data supplement.
`
`Contribution: F.A., C.L., C.C., D.N., and C.X.W. performed and designed
`experiments; B.E.T. and F.L.C. discussed results; and E.V. coordinated the
`project, designed and performed the experiments, analyzed the data, discussed
`results, and wrote the manuscript.
`
`Conflict-of-interest disclosure: The authors declare no competing financial
`interests.
`
`Correspondence: Els Verhoeyen, CIRI/EVIR, ENS de Lyon, 46 All ´ee d’Italie,
`69364 Lyon Cedex 07, France; e-mail: els.verhoeyen@ens-lyon.fr or els.
`verhoeyen@unice.fr; and Franc¸ois-Lo¨ıc Cosset, CIRI/EVIR, ENS de Lyon, 46
`All ´ee d’Italie, 69364 Lyon Cedex 07, France; e-mail: flcosset@ens-lyon.fr.
`
`References
`
`1. Verma IM. Medicine. Gene therapy that works. Science. 2013;341(6148):853-855.
`
`2. Naldini L, Bl ¨omer U, Gallay P, et al. In vivo gene delivery and stable
`transduction of nondividing cells by a lentiviral vector. Science. 1996;
`272(5259):263-267.
`
`HSCs, remains unclear. Recently, Finkelstein et al revealed a long-
`kept secret of VSV by identifying its receptor, the low-density lipid
`receptor (LDL-R), explaining its broad tropism.6 This finding promp-
`ted us to evaluate LDL-R levels on unstimulated T, B, and CD341
`cells. Strikingly, we confirmed a very low expression of LDL-R,
`coinciding with VSV-G-LV–mediated poor transduction in these 3 cell
`lineages (Figure 1A). Stimulation of T cells through the T-cell receptor
`or of human CD341 (hCD341) cells with “early-acting cytokines”
`remarkably upregulated the LDL-R surface expression and permitted
`efficient VSV-G-LV transduction. In contrast, B-cell receptor stim-
`ulation augmented LDL-R expression only marginally, in agreement
`with poor VSV-G-LV transduction levels (Figure 1A and Frecha
`et al4). Binding of the different cell lineages with VSV-G-LVs was
`detected by incubation with the VSV-G-LVs followed by HIV capsid
`(p24) detection. VSV-G-LVs bound efficiently to stimulated T and
`hCD341 cells but not B cells and barely attached to their resting
`counterparts (Figure 1B). In contrast, MV-LVs efficiently attached to
`both stimulated and unstimulated cells (Figure 1B). Next, we used
`particles formed by VSV-G protein (gesicles7) incorporating high
`levels of green fluorescent protein (GFP) through a farnesylation
`tag to verify fusion of VSV-G protein with 3 cell
`lineages
`(Figure 1A, right panels). Resting T, B, and CD341 cells showed
`a poor GFP signal upon contact with GFP-loaded gesicles, while
`the GFP signal was evident for prestimulated cells, except for
`B cells (Figure 1A), confirming the presence of VSV and thus the
`VSV-G-LV receptor, LDL-R. Accordingly, VSV-G-LV trans-
`duction of resting T and B cells also resulted in very low levels of
`reverse-transcribed viral DNA.4
`Finally, we confirmed the requirement for VSVG-LV entry and
`transduction through the LDL-R and its family members using an
`anti–LDL-R antibody or by competition with soluble LDL-R, resulting
`in reduction or almost complete inhibition of transduction, respectively
`(Figure 1C). In contrast, MV-LVs were not sensitive to these LDL-
`blocking or -competing agents. Interestingly, IL-7–stimulated T-cell
`VSV-G-LV transduction8 coincided with LDL-R upregulation and was
`inhibited upon LDL-R blocking. Additionally, low-level transduction
`in resting cells was lost upon LDL-R blocking (data not shown).
`In conclusion, although cellular postentry blocks may still play a
`role in VSV-G-LV transduction of resting T cells, B cells, and
`HSCs, we confirmed here that VSV-G-LV entry is compromised
`by the low expression of the VSV receptor LDL-R and its family
`members. Therefore, other LV pseudotypes (eg, MV-LVs) are
`more adapted for gene transfer in these invaluable resting gene-
`therapy targets.9
`
`Fouzia Amirache
`Centre International de Recherche en Infectiologie,
`Enveloppe Virale et Ing ´enierie du Retrovirus Team,
`Institut National de la Sant ´e et de la Recherche M ´edicale U1111,
`Centre National de la Recherche Scientifique,
`Unit ´e Mixte de Recherche 5308, Universit ´e de Lyon-1,
`Ecole Normale Sup ´erieure de Lyon,
`Lyon, France
`
`Camille L ´evy
`Centre International de Recherche en Infectiologie,
`Enveloppe Virale et Ing ´enierie du Retrovirus Team,
`Institut National de la Sant ´e et de la Recherche M ´edicale U1111,
`Centre National de la Recherche Scientifique,
`Unit ´e Mixte de Recherche 5308, Universit ´e de Lyon-1,
`Ecole Normale Sup ´erieure de Lyon,
`Lyon, France
`
`Page 2 of 3
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`
`1424
`
`CORRESPONDENCE
`
`BLOOD, 27 FEBRUARY 2014 x VOLUME 123, NUMBER 9
`
`3. Frecha C, Costa C, N `egre D, et al. Stable transduction of quiescent T cells without
`induction of cycle progression by a novel lentiviral vector pseudotyped with measles
`virus glycoproteins. Blood. 2008;112(13):4843-4852.
`
`4. Frecha C, Costa C, L ´evy C, et al. Efficient and stable transduction of resting
`B lymphocytes and primary chronic lymphocyte leukemia cells using measles
`virus gp displaying lentiviral vectors. Blood. 2009;114(15):3173-3180.
`
`5. Frecha C, L ´evy C, Costa C, et al. Measles virus glycoprotein-pseudotyped lentiviral
`vector-mediated gene transfer into quiescent lymphocytes requires binding to both
`SLAM and CD46 entry receptors. J Virol. 2011;85(12):5975-5985.
`
`6. Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein M. LDL receptor and
`its family members serve as the cellular receptors for vesicular stomatitis virus.
`Proc Natl Acad Sci U S A. 2013;110(18):7306-7311.
`
`7. Mangeot PE, Dollet S, Girard M, et al. Protein transfer into human cells by
`VSV-G-induced nanovesicles. Mol Ther. 2011;19(9):1656-1666.
`
`8. Verhoeyen E, Dardalhon V, Ducrey-Rundquist O, Trono D, Taylor N,
`Cosset FL. IL-7 surface-engineered lentiviral vectors promote survival and
`efficient gene transfer in resting primary T lymphocytes. Blood. 2003;101(6):
`2167-2174.
`
`9. Frecha C, L ´evy C, Cosset FL, Verhoeyen E. Advances in the field of lentivector-
`based transduction of T and B lymphocytes for gene therapy. Mol Ther. 2010;
`18(10):1748-1757.
`
`© 2014 by The American Society of Hematology
`
`To the editor:
`
`Statin and aspirin use is associated with improved outcome of FCR therapy in
`relapsed/refractory chronic lymphocytic leukemia
`
`Statins and aspirin are widely prescribed medications that have long
`been associated with improved survival outcome in patients with
`various types of cancers.1,2 Both statins and aspirin were found to
`induce apoptosis of chronic lymphocytic leukemia (CLL) cells.3,4
`The intake of statins and aspirin was associated with reduced inci-
`dence of CLL.5,6 However, statin intake did not affect treatment-free
`survival in patients with early CLL.7,8 Whether statin or aspirin use
`will benefit patients with advanced CLL is unknown.
`Therefore, we retrospectively investigated the clinical outcome of
`patients with relapsed/refractory CLL treated with salvage fludar-
`abine, cyclophosphamide, and rituximab (FCR)9 with or without
`concomitant statins, aspirin, or both. We analyzed 280 patients who
`received salvage FCR between 1999 and 2012. The patients’ median
`age was 59 years (range: 31-84). The median progression-free
`survival (PFS) of all patients was 1.7 years, and the median overall
`survival (OS) was 4.0 years. Of the 280 patients, 58 patients received
`statins, aspirin, or both; 21 (8%) were taking aspirin only; 17 (6%)
`statins only; and 20 (7%) used both for at least 1 month prior to,
`during, and 1 month after salvage therapy. Among statin users, 15
`patients (41%) were using atorvastatin, 12 patients (32%) were using
`simvastatin, 7 patients (19%) were using pravastatin, 2 patients (5%)
`were using rosuvastatin, and 1 patient (3%) was using lovastatin.
`Clinical characteristics of statin and/or aspirin users were similar to
`those of nonusers except for age. Patients on both statin and aspirin
`were 6 years older than nonusers (P , .01).
`The overall response rate of patients receiving statins and aspirin
`concomitantly was superior (100%; 40% complete response, 60%
`partial response) to that of other patients (81% for aspirin-only users,
`82% for statin-only users, and 72% for those who took neither drug;
`P , .01). Early death (during chemotherapy and up to 6 weeks
`afterward) was not observed in patients receiving aspirin, statins, or
`both but occurred in 6% of nonusers. Patients receiving both statins
`and aspirin had median PFS and OS of 6.1 and 9.2 years, respectively,
`compared with 1.6 years and 3.7 years in nonusers (PFS P 5 .003; OS
`P 5 .05; Figure 1). Compared with nonusers, patients who took both
`statins and aspirin had a 66% reduced risk of disease progression and
`a 60% reduced risk of death (PFS hazard ratio [HR] 5 0.34, 95%
`confidence interval [CI] 5 0.18-0.65, P , .001; OS HR 5 0.40, 95%
`CI 5 0.21-0.79, P 5 .008).
`In a fitted multivariate model controlling for clinicopatholog-
`ical characteristics found to be statistically significant from uni-
`variate analyses including Rai stage, cytogenetic abnormalities, the
`number of previous treatments, refractoriness to fludarabine, IgVH
`mutation status, b2-microglobulin, hemoglobin, platelet, lactate
`
`dehydrogenase, and creatinine level, use of both medications was
`also associated with a much more favorable outcome (PFS ad-
`justed HR 5 0.27, 95% CI 5 0.14-0.53, P # .001; OS adjusted
`HR 5 0.29, 95% CI 5 0.15-0.58, P , .001), whereas single-agent
`use of aspirin or statins did not affect PFS or OS.
`Our findings demonstrate for the first time that concurrent
`administration of statins and aspirin to CLL patients with relapsed/
`refractory disease receiving salvage FCR significantly improve both
`response rate and survival. This is consistent with previous pre-
`clinical studies suggesting the possible synergistic effect between
`statins and chemotherapy.10 Therefore, a prospective study aimed at
`evaluating the effects of statins and aspirin in CLL patients receiving
`chemoimmunotherapy is warranted.
`
`Young Kwang Chae
`Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Long Trinh
`Department of Leukemia, Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Preetesh Jain
`Department of Leukemia, Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Xuemei Wang
`Department of Biostatistics, Division of Quantitative Sciences,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Uri Rozovski
`Department of Leukemia, Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`William G. Wierda
`Department of Leukemia, Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Michael J. Keating
`Department of Leukemia, Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Zeev Estrov
`Department of Leukemia, Division of Cancer Medicine,
`The University of Texas MD Anderson Cancer Center,
`Houston, TX
`
`Page 3 of 3
`
`