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. Author manuscript; available in PMC: 2015 Sep 24.
Published in final edited form as: Leukemia. 2013 Jun 14;28(2):329–337. doi: 10.1038/leu.2013.177

Lenalidomide enhances the curative effect of a therapeutic vaccine and reverses immune suppression in mice bearing established lymphomas

Ippei Sakamaki 1,2, Larry W Kwak 1,2, Soung-chul Cha 1,2, Qing Yi 1,2, Beatrisa Lerman 1,2, Jian Chen 3, Sekhar Surapaneni 3, Scott Bateman 3, Hong Qin 1,2
PMCID: PMC4581849  NIHMSID: NIHMS674418  PMID: 23765229

Abstract

Immunomodulatory drugs (IMiDs) are effective therapeutic agents with direct inhibitory effects on malignant B- and plasma cells and immunomodulatory effects on the T cell activation. This dual function of IMiDs makes them appealing candidates for combination with an cancer vaccine. We investigated the immune stimulatory effects of lenalidomide, administrated to mice in doses which provided comparable pharmacokinetics to human patients, on the potency of a novel fusion DNA lymphoma vaccine. The combination was curative in the majority of mice with 8d pre-established syngeneic A20 lymphomas, compared with vaccine or lenalidomide alone and induced immune memory. In vivo depletion experiments established the requirement for effector CD8+ and CD4+ T cells in protective immunity. Unexpectedly, lenalidomide alone was also associated with reduced numbers of systemic MDSC and Treg in tumor-bearing, but not naïve mice, an effect that was independent of simple tumor burden reduction. These results confirm and extend results from other models describing the effect of lenalidomide on enhancing T-cell immunity highlight the potency of this effect, and provide a rational for clinical application. Independently, a novel mechanism of action reversing tumor-induced immune suppression by MDSC is suggested.

Keywords: Lenalidomide, DNAvaccine, adjuvant, T cell, lymphoma

Introduction

Lenalidomide, a derivative of thalidomide, is a FDA-approved drug for the treatment of multiple myeloma (MM). In addition MM, recent clinical studies have confirmed the therapeutic effectiveness of lenalidomide in a variety of B-cell malignancies, including lymphoma patients. For example, lenalidomide monotherapy induced an overall response rate (ORR) of 23% in patients with relapsed/refractory indolent lymphoma (n=43) and of 35% in patients with aggressive non-Hodgkin’s lymphoma (NHL) (n=217).1, 2 Combining lenalidomide with rituximab brought about an ORR of 84% in newly diagnosed indolent lymphoma patients (n=20).3 The combination therapy of lenalidomide with R-CHOP (rituximab-cyclophospamide, doxorubicin, vincristine and prednisone) also revealed a promising clinical outcome, resulting in 71% complete response in patients with aggressive NHL (n=21).4

Its antitumor properties include anti-angiogenesis and direct tumor cytotoxicity that involves multiple mechanisms of action, such as inhibition of cell proliferation, modulation of cytoskeleton, activation of apoptosis and suppression of oncogenes.5-12 In addition, thalidomide and its derivatives are classified as Immonomudulatory Drugs (IMiDs) due to the initial finding that thalidomide inhibits the production of tumor necrosis factor α.13 Subsequent studies found that thalidomide/IMiDs treatment resulted in an increase in interferon-γ and IL-2 secretion in MM patients, which was associated with priming NK cells for cytotoxicity.14 Mechanistic studies further revealed that IMiDs down-regulated interferon-γ and IL-2 suppressor of cytokine signaling (SOCS)1 expression in immune effector cells (T, NK and NKT), and thus enhanced the immune response in MM bone marrow microenvironment.15 In addition, the effect of IMiDs on T cell activation by stimulating the CD28 signaling pathway was shown 16, and this effect may result in polarization toward Th1 responses.17 A recent study suggests a potential role of lenalidomide in repairing T-cell immunologic synapse dysfunction induced by lymphoma cells.18 Taken together, these findings support the hypothesis that IMiDs may provide an adjuvant function in vaccines.

Cancer vaccines are designed to train the immune system to evoke tumor-specific immunity against malignant cells. Recently, Sipuleucel-T, which targets prostatic acid phosphatase, was FDA-approved as a therapeutic vaccine for treatment of metastatic prostate cancer.19 Two other controlled, phase III clinical trials of cancer vaccines have reported positive results, including a melanoma peptide vaccine20 and a NHL idiotype protein vaccine.21 The latter showed that follicular lymphoma patients who were in minimal residual disease and received idiotype (Id)-KLH plus GM-CSF had prolonged disease-free survival, compared with those in the control cohort who were administered KLH plus GM-CSF control vaccine.21 Second generation idiotype DNA vaccines feature the single-chain variable fragment (scFv), which not only simplifies vaccine preparation, but also allows genetic fusion to enhance the immunogenicity of idiotype antigen. For example, preclinical data showed that genetic fusion of idiotype scFv with chemokine ligands facilitated antigen capture by dendritic cells (DC) that express corresponding chemokine receptors. The receptor-mediated endocytosis allows cross-presentation of the tumor antigen, leading to the development of cellular, rather than humoral immunity.22-24

Combination strategies including immune adjuvants may further enhance the therapeutic potency of cancer vaccines. In this study, we combined lenalidomide with a novel idiotype DNA vaccine in treatment of mice against A20 murine lymphoma. Our data showed that the combination was more effective than either agent as therapy against established tumor challenge in an effector T-cell-dependent manner. The results also suggest that lenalidomide ameliorates tumor-induced immune suppression by reducing the number of systemic myeloid-derived suppressor cells (MDSC) and regulatory T cells (Teg).

Materials and Methods

Animals and drug

Balb/c mice were purchased from National Cancer Institute (NCI). Female 6-8 weeks mice were used for individual experiments. Lenalidomide was provided by Celgene Corporation (Summit, New Jersey, USA). All mice were maintained in a pathogen-free mouse facility according to institutional guidelines. Animal studies were approved by the Institutional Animal Care and Use Committee at The University of Texas M. D. Anderson Cancer Center.

Vaccination and tumor challenge

In prophylactic setting, groups of 10 mice were immunized i.m. with 100 μg MCP3-sFv20 DNA vaccine 23 on days 0, 14, and 28 with or without lenalidomide treatment (5 mg/kg, i.p.) for 35 consecutive days. Two weeks after final vaccination, mice were challenged i.p. with 2 × 105 A20 lymphoma cells. In therapeutic setting, groups of 10 mice were challenged i.p. with 2 × 105 A20 cells on day 0 followed by 4 vaccinations on days 8, 10, 14, and 21, respectively. Lenalidomide (5 mg/kg) was given i.p. for 21 consecutive days starting on day 1. In all animal studies, data were statistically analyzed by using the Kaplan-Meier method with a log-rank P value.

Pharmacokinetic assay

A group of 15 Balb/c mice were treated i.p with a daily dose of 5 mg/kg lenalidomide for 35 consecutive days. Blood samples were collected 0.5, 1, 2, 4, 8 and 24 hours after the final dose of lenalidomide on Day 35. The samples were centrifuged at 4°C and 3,000 rpm for 5 minutes to harvest the plasma. A 150 μL aliquot of plasma was mixed well with an equal volume of Sorenson’s 25 mM Citrate Buffer at pH 1.5. The pharmacokinetic analysis was performed by Celgene Corporation (Summit, NJ). The samples were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a Sciex 5500 Qtrap Mass Spectrometer (AB Sciex, Concord, Canada) coupled to a Shimadzu HPLC System. Composite pharmacokinetic parameters were calculated using Watson LIMS™ (version 7.4, Thermo Fisher, Philadelphia, PA). All statistics were calculated using Watson LIMS.

Measurement of idiotype antibody

Mice were immunized according to the prophylactic setting schedule with or without lenalidomide treatment. One week after final vaccination mice were sacrificed to collect the blood samples. The antibody response was determined by measuring serum levels of anti-idiotype antibodies using ELISA with recombinant A20 idiotype protein (Favrille Biotech, San Diego, CA) as reported previously.25

In vivo T cell depletion assay

In vivo T cell depletion was achieved by intraperitoneal injection of mice with 200 μg anti-CD8 antibody (clone 2.43) and anti-CD4 antibody (clone GK1.5) as reported previously.23, 25 In brief, mice were immunized according to the prophylactic setting schedule with or without lenalidomide. Mice were then treated with anti-CD8 and/or anti-CD4 antibodies on days −7, −5, −3 before and day 14 after tumor challenge (day 0), respectively. The efficiency of T-cell depletion was assessed by staining peripheral blood mononuclear cells with CD3-FITC, CD8-PE, and CD4-APC (BD Biosciences).

Flow cytometric analysis

MDSC, Treg and NK cells were examined in immunized and lenalidomide-treated mice with or without being challenged with A20 tumor cells. A control group in tumor-bearing mice received 100 mg/kg cyclophospamide twice on days 13 and 14, respectively. Single cell suspension of splenocytes prepared from each individual mouse was labeled with Gr-1-FITC and CD11b-APC (BD Bioscience) for MDSC, CD4-FITC (BD Bioscience) and Foxp3-PE (eBioscience) for Treg, and CD49b-APC (BD Bioscience) for NK cells. The data were obtained on BD FACSCalibur flow cytomter and analyzed using FlowJo 7.2.5 software. Statistical analysis was performed by using a two-tailed Student’s t-test.

Results

Lenalidomide enhanced lymphoma vaccine-induced prophylactic and memory antitumor immunity

The immunomodulatory activity of lenalidomide provides a potential opportunity to use this anti-myeloma drug as a vaccine adjuvant. To test this hypothesis, we evaluated the antitumor effect of combination therapy with a previously descried lymphoma idiotype DNA fusion vaccine 23, 25 together with lenalidomide used in various doses and schedules. The most potent protective effect against lethal A20 murine lymphoma challenge was observed in the two groups treated with either a low dose (5 mg/kg) of lenalidomide for 35 consecutive days or a high dose (50 mg/kg) using an intermittent schedule of total 6 injections. The median tumor development time was 33 days for both groups compared with 26.5 days for vaccine alone controls. Interestingly, continuous administration of high dose lenalidomide failed to potentiate the vaccine-induced tumor protection (Supplementary Table 1). Lower doses of lenalidomide (0.5 mg/kg) were not effective (date not shown). Based on these preliminary results, the treatment schedule using 5 mg/kg for 35 consecutive days was chosen for further development of lenalidomide as a vaccine adjuvant. In addition, pharmacokinetic analysis showed that following the final dose on Day 35, lenalidomide was rapidly absorbed with Tmax of 0.5 hour, and the mean Cmax and AUCt values were 2520 ng/mL and 2940 ng*h/mL, respectively (Figure 1), which was comparable to plasma exposures achieved in human patients receiving a daily dose of 25 mg (AUCt: 2773 ng*h/mL).26 In the prophylactic experiment shown in Figure 2A, combing lenalidomide with the vaccine resulted in 80% long-term survival in tumor-challenged mice, compared with 30% in the vaccine alone group (P=0.046). Lenalidomide, when used alone, had no protective antitumor effect compared with PBS control. More than 75% of the mice protected from primary tumor challenge in the combination group were resistant to secondary challenge (p<0.01), suggesting the development of antitumor immune memory (Figure 2b).

Figure 1. Pharmacokinetics of plasma lenalidomide level in mice.

Figure 1

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Fifteen Balb/c mice were injected i.p with a daily dose of 5 mg/kg lenalidomide for 35 consecutive days. Blood samples were collected 0.5 (n=3), 1 (n=3), 2 (n=3), 4 (n=2), 8 (n=2) and 24 (n=2) hours after the final dose of lenalidomide on Day 35. Plasma was analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Composite pharmacokinetic parameters were calculated using Watson LIMS™ (version 7.4, Thermo Fisher, Philadelphia, PA).

Figure 2. Lenalidomide enhanced lymphoma vaccine-induced protective antitumor and memory immunity.

Figure 2

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(a) Ten Balb/c mice per group were immunized intramuscularly with 100 μg plasmid DNA encoding MCP3 chemokine-fused A20 lymphoma-derived idiotype antigen (MCP3-sFv) on days 0, 14 and 28. Lenalidomide was given i.p at a dose 5 mg/kg/day for 35 consecutive days starting on Day 0. Control mice were injected with plasmid DNA without lenalidomide (Vac), PBS, or lenalidomide alone (Len). Two weeks after final vaccination, all mice were challenged with a lethal dose of 2×105 A20 lymphoma cells by intraperitoneal injection and followed for survival. Survival differences between groups were analyzed by log-rank test. The data represent 3 independent experiments. (b) Tumor-free mice from the Vac+Len group (n=17) were re-challenged with 2×105 A20 tumor cells intraperitoneally along with normal Balb/c mice (n=20) and followed for survival. The data showed combined results from 3 independent experiments.

Antigen-specific antibody responses are not enhanced by lenalidomide

Antibodies against the antigen (idiotype) were easily detected in 4 out of 5 mice immunized with the vaccine alone (Figure 3a). However, adding lenalidomide to the vaccination did not potentiate antigen-specific humoral immunity. The serum titers of antibodies in mice immunized with the combination vaccine + lenalidomide were similar to those found in the mice receiving vaccine alone (Figure 2b). The specificity of antibody response was confirmed by showing that the antibodies found in mice receiving vaccine, either alone or combining with lenalidomide, did not bind to an isotype-matched Ig of irrelevant idiotype (not shown) and by the observation that neither lenalidomide nor PBS treated mice developed idiotype-specific antibody responses (Figure 3c-d). These data suggest that the adjuvant effect of lenalidomide is not due to the enhancement of antibody responses.

Figure 3. Lenalidomide had no effect on antigen-specific antibody responses.

Figure 3

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Five mice per group were vaccinated as in Figure 1. Serum samples were obtained 7 days after final vaccination and were examined for anti-idiotype antibodies by enzyme-linked immunoabsorbent assay using plates coated with recombinant A20 idiotype protein. Bound antibodies were detected by HRP-conjugated anti-mouse IgG1. The data are representative of 2 identical experiments.

T cell-mediated immunity is involved in the vaccine-potentiating effect of lenalidomide

We attempted to detect A20 idiotype peptide-specific T cell responses to a reported MHC Class I binding-epitope in the vaccinated mice, but failed to observe splenic CD8+ T cells that could specifically recognize the candidate peptide by ELISPOT (data not shown). Alternatively, we performed in vivo T-cell depletion to determine the role of cellular immunity in the protective antitumor effect of the combination therapy. T-cell depletion was achieved by i.p injection of anti-CD8 (clone 2.43) and /or anti-CD4 (clone GK 1.5) monoclonal antibodies at the effector phase. The results showed that tumor protection elicited by the combination of vaccine + lenalidomide was abrogated partially by CD4+ or CD8+ T cell depletion alone, but completely abrogated by CD8 T-cell depletion in combination with CD4 T-cell depletion. Specifically, without T-cell depletion, 60% of vaccinated mice were alive on Day 60 after tumor challenge, compared with 30% with the treatment of anti-CD4 antibodies, and 10% for CD8 or zero for CD8/CD4 depletion (Figure 4). Taken together, the results suggest that T cells, especially CD8 effector cells are required for the vaccine-potentiating effect of lenalidomide.

Figure 4. Effector T cells were required in vivo for the enhanced tumor protection induced by the combination of Vaccine + Lenalidomide.

Figure 4

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In vivo T-cell depletion was achieved by intraperitoneal injection of 200 μg monoclonal antibodies against CD8 (clone 2.43) and/or CD4 (clone GK1.5) using a protocol described previously. 25 Ten mice per group were vaccinated with MCP3-sFv DNA vaccine plus lenalidomide as in Figure 1 and then were treated with depleting mAb starting 1 week after final vaccination. Control mice received vaccine without depleting antibodies, or PBS alone. All mice were challenged with A20 tumor cells as in Figure 1 and followed for survival.

Lenalidomide reduced immune suppressor cells in tumor-bearing mice

To further explore potential cellular mechanisms of the adjuvant effect of lenalidomide, we investigated its effects on other immune cells including myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and natural killer cells (NK). Treatment of 5 mg/kg lenalidomide did not change the numbers of these immune cells in naïve mice (Supplementary Figures 1 and 2); However, in tumor-bearing mice, lenalidomide treatment was associated with a reduction in splenic MDSC (1.39% ± 0.05), comparable to non-tumor-bearing naïve mice, as well as tumor regression in 60% of mice (not shown). To determine whether the effect of lenalidomide on MDSC numbers could be dissociated from tumor burden reduction, we treated mice with cyclophosphamide, which also induced tumor regression. Although cyclophosphamide induced complete tumor regression in all treated mice (not shown), its effect on MDSC was insignificant (8.26% ± 1.35) (Figure 5a and Supplementary Table 2). Unlike their differential roles on MDSC, lenalidomide and cyclophosphamide treatments both were associated with reduction in splenic Treg in tumor-bearing mice (Figure 5b and Supplementary Table 2).

Figure 5. Lenalidomide reverses systemic tumor-induced immune suppression.

Figure 5

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Five Balb/c mice per groups were injected with 2×105 A20 lymphoma cells intraperitoneally on day 0. Mice were then treated either with 5 mg/kg lenalidomide for 21 consecutive days starting on day 1, or with 100 mg/kg cyclophosphamide on days 13 and 14 or nothing (controls). All mice were sacrificed three weeks after tumor challenge. Splenocyte single cell suspensions were prepared and individual mice were analyzed by specific immunostaining for MDSC (a), Treg (b) and NK cells (c). Plots are shown with percentage of cell subpopulations analyzed for representative mice and composite results are shown from each respective group. Differences between groups were analyzed by a two-tailed Student’s t-test. ** indicates P < 0.01 compared with tumor-bearing mice (no treatment). The data represent 2 identical experiments.

A slight but statistically significant reduction of splenic NK cell numbers was observed in tumor-bearing compared with naïve mice (6.6% ± 0.51 vs. 8.53% ± 0.4). However, treatment with cyclophosphamide was associated with further reduction in NK cells (4.21% ± 0.58). Lenalidomide treatment restored NK cell numbers (8.54% ± 0.29) (Figure 5C and Supplementary Table 2). Altogether, our data suggest that lenalidomide has the potential to reverse systemic tumor-induced immune suppression by reducing MDSC, Treg and possibly rescuing NK cells, and this effect is independent of its effects on simple tumor burden reduction.

Combining lenalidomide with lymphoma vaccine demonstrates additive therapeutic antitumor effect against established tumor burdens

A therapeutic study against established tumors (Figure 6a) was carried out based on the findings above that lenalidomide both potentiates adaptive T-cell immunity and ameliorates systemic tumor-induced immune suppression. Compared with 15% and 25% survival in lenalidomide (p=0.0052) and vaccine treated mice (p=0.035), respectively, on day 70, the combination was associated with 50% long term survival. Vaccine + lenalidomide combination, lenalidomide alone, and vaccine alone groups were all superior to PBS controls (P<0.0001, 0.0043 and 0.0008, respectively, vs. PBS) (Figure 6b).

Figure 6. The additive therapeutic effects of vaccine plus lenalidomide cured established tumors.

Figure 6

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Ten Balb/c mice per group were first challenged with 2×105 A20 lymphoma cells intraperitoneally on day 0. The mice were then vaccinated i.m. with plasmid DNA encoding MCP3-sFv on day 8, 10, 14 and 21, along with 5 mg/kg i.p. lenalidomide for 21 consecutive days starting on day 1 (a) and followed for survival (b). Control mice received DNA vaccine alone, PBS only or lenalidomide alone. Data represent combined results from 2 independent experiments.

Discussion

Given the inherent weak immunogenicity of cancer vaccines, there is a need for clinical-grade immune adjuvants which can potentiate cancer-specific immune responses. Currently, clinically approved vaccine adjuvants are limited. Several vaccine adjuvants have shown promise but are still in development including Toll-like receptor agonists and cardiotoxin.25, 27, 28 Pomalidomide, another IMiDs family member, was reported to boost the anti-tumor effect of an irradiated tumor cell vaccination in mouse colon cancer and melanoma models.29 However, our current study is among the first reports of lenalidomide as an immune adjuvant to enhance the efficacy of cancer vaccine. The fact that lenalidomide is FDA-approved for its antitumor activity in multiple myeloma makes it particularly attractive for consideration as an adjuvant in combination with novel vaccine therapies 30 in clinical trials in this and other B-cell malignancies.

Our pharmacokinetic data revealed that intraperitoneal injection of 5 mg/kg lenalidomide in mice resulted in an AUC value comparable to that achieved after oral administration of 25 mg lenalidomide in MM patients.26 This finding suggests that the dosing of drug used in mice results in exposure levels comparable to those observed in human patients receiving lenalidomide, highlighting the feasibility of its use as a potential vaccine adjuvant in humans. For example, it is tempting to speculate that an active vaccine immunotherapy could enhance the effectiveness of lenalidomide maintenance therapy to eradicate residual disease.

Our data suggest that one of the immune mechanisms of lenalidomide-induced adjuvant effect is to facilitate the development specific T-cell immunity against a model B-cell tumor antigen. Depletion of effector T cells abrogated the antitumor effect, highlighting the crucial role of T-cell immunity in lenalidomide-combined vaccine therapy. The effect of IMiDs on T cell immune response has been documented previously. For example, Dredge et al. reported that pomalidomide enhanced Th1 immune response.29 The similar finding was observed on thalidomide and lenalidomide, showing increased production of Th1 cytokines including IL-2 and IFNγ upon IMiDs treatment.31 Mechanistically, lenalidomide directly induces tyrosine phosphorylation of CD28 on T cells, leading to activation of downstream signaling targets including PI13K and NF-kB.16 In combination with the idiotype vaccine, lenalidomide showed a clear additive antitumor effect against A20 lymphoma in the therapeutic setting (Figure 6). Given both its vaccine adjuvant and direct antitumor effects, combining lenalidomide with cancer vaccines in maintenance therapy may provide an effective strategy for eradicating minimal residue disease and preventing relapse. Indeed, that lenalidomide potentiated vaccine responses was recently reported in a clinical study where lenalidomide was used in combination with pneumococcal vaccines in patients with multiple myeloma.32 Taken together, these studies confirm and extend the hypothesis that that lenalidomide could serve as an ideal adjuvant in combination with cancer vaccines designed to activate T-cell immunity.

An independent finding was that lenalidomide-associated reduction of numbers of Treg and MDSC and rescue of NK cells were observed only in tumor-bearing but not naïve mice, suggesting a direct role of lenalidomide on reversing systemic tumor-induced immune suppression. Our findings on Treg and NK cells are consistent with limited previous reports, which showed that lenalidomide inhibited the proliferation and function of Treg17, 33 and improved the number of NK cells.34 However, our data are the first to reveal that lenalidomide dramatically reduced numbers of MDSC in tumor-bearing mice. This effect did not likely result from simple reduction of tumor burden, as cyclophosphamide treatment alone failed to reduce the number of MDSC. Experiments outside of the scope of this study are in progress to confirm the effect of lenalidomide on regulation of MDSC function. It is also not clear whether lenalidomide has a direct cytotoxic effect on MDSC. A recent publication showed that cereblon, the major target of thalidomide teratogenicity,35 is essential to lenalidomide-induced cytotoxicity on multiple myeloma cells.9 Thus, assaying for direct cytotoxicity of lenalidomide on MDSC and comparing cereblon expression on MDSC between tumor-bearing and naïve mice may provide additional insight into the mechanism of MDSC reduction by lenalidomide. Finally, additional experiments will be planned characterizing lenalidomide-induced changes in function of MDSC and dendritic cells isolated directly from the tumor bed.

Supplementary Material

Suppl Fig Legends
Suppl Figs

ACKNOWLEDGEMENTS

Work was supported by a contract from Celgene Corporation and by grants from the Leukemia and Lymphoma Society (TRP 6003-07 and SCOR 7262-08), Cancer Prevention and Research Institute of Texas (RP 100457) and Department of Defense (W81XWH-07) to LWK.

Footnotes

Conflict of interest

Larry W. Kwak: research funding by Celgene

Jian Chen, Sekhar Surapaneni, Scott Bateman: employment by Celgene

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