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 Table of Contents  
SHORT COMMUNICATION
Year : 2017  |  Volume : 3  |  Issue : 4  |  Page : 139-145

Combination of Interleukin-11Rα chimeric antigen receptor T-cells and programmed death-1 blockade as an approach to targeting osteosarcoma cells In vitro


Department of Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA

Date of Submission14-Jan-2017
Date of Acceptance24-May-2017
Date of Web Publication14-Aug-2017

Correspondence Address:
Hatel Rana Moonat
Texas Children's Cancer and Hematology Centers, Texas Children's Hospital The Woodlands, 17580 I-45 S, Suite 450, The Woodlands, TX 77384
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ctm.ctm_3_17

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  Abstract 

Aim: To test whether combining interleukin (IL)-11Rα chimeric antigen receptor (CAR) T-cells with an anti-programmed death (PD-1) antibody (an immune checkpoint inhibitor) is an effective therapeutic approach in osteosarcoma (OS), allowing improved tumor eradication.
Methods: IL-11Rα-CAR T-cells were cocultured in vitro with human LM7 OS tumor cells (with and without anti-PD-1 antibody). Coculture of LM7 cells with purified T-cells served as the control. Cytotoxicity and surface PD-1 expression were analyzed in all groups.
Results: PD-1 expression increased during expansion of CAR T-cells. Exposure of immune cells to tumor cells in vitro subsequently decreased surface PD-1 expression on the CAR T-cells. Addition of an anti-PD-1 antibody (Clone J110) to further decrease surface PD-1 expression on CAR T-cells before coculture did not enhance cytotoxic effects of the CAR T-cells against LM7 cells.
Conclusion: This combination of IL-11Rα-CAR T-cells and an anti-PD-1 antibody did not provide any additional cytotoxic benefit over IL-11Rα-CAR T-cell therapy alone in this setting. Further studies are needed as simple interference with surface PD-1 expression alone may not be sufficient to inhibit this immune checkpoint pathway to then enhance IL-11Rα-CAR T-cell therapeutic effects.

Keywords: Chimeric antigen receptor T-cells, immune checkpoint inhibitors, osteosarcoma, programmed death-1


How to cite this article:
Moonat HR, Huang G, Dhupkar P, Schadler K, Gordon N, Kleinerman E. Combination of Interleukin-11Rα chimeric antigen receptor T-cells and programmed death-1 blockade as an approach to targeting osteosarcoma cells In vitro. Cancer Transl Med 2017;3:139-45

How to cite this URL:
Moonat HR, Huang G, Dhupkar P, Schadler K, Gordon N, Kleinerman E. Combination of Interleukin-11Rα chimeric antigen receptor T-cells and programmed death-1 blockade as an approach to targeting osteosarcoma cells In vitro. Cancer Transl Med [serial online] 2017 [cited 2017 Sep 24];3:139-45. Available from: http://www.cancertm.com/text.asp?2017/3/4/139/210635


  Introduction Top


Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents.[1] Current multimodal therapies for localized disease have improved the 5-year survival rate to 65%–70%,[2] but unfortunately a plateau in survival has been reached since the mid-1980s.[3] Patients with metastatic disease have even less promising outcomes, with their 5-year survival being only 31% or less.[4] New therapeutic approaches, therefore, are desperately needed.

Immunotherapy has recently demonstrated itself as a promising therapeutic strategy in oncology, particularly for the treatment of various heterogeneous tumors.[5],[6],[7],[8],[9],[10] Adoptive T-cell therapy is a specific type of immunotherapy that genetically modifies a patient's own primary T-cells by embedding a special surface receptor called a chimeric antigen receptor (CAR) to create antigen-targeted immune cells known as CAR T-cells. These surface receptors then serve as tumor-specific immunoreceptors that recognize and attack the patient's tumor. Adoptive transfer of these CAR T-cells is a way to deliver antigen-targeted cancer therapy.[11] This type of immunotherapy has shown promise against different hematologic malignancies.[12] As a consistent therapeutic option for solid tumors, however, CAR T-cell therapy is still considered to be in its infancy,[13] with further investigations still needed.

The interleukin (IL)-11Rα chain has been identified as a molecular target overexpressed in several different types of cancers, including OS.[14] This target was utilized as the tumor-specific immunoreceptor in a previous study of CAR T-cells by our group. The engineered IL-11Rα-CAR T-cells showed greater cytotoxicity against various OS cell lines in vitro compared to unmodified control T-cells.[11] In addition, in vivo studies using a KRIB mouse lung metastasis model of OS demonstrated that mice treated with IL-11Rα-CAR T-cells had fewer pulmonary metastases than those treated with unmodified control T-cells alone.[11] Although that study successfully showed a significant tumor response with CAR T-cell therapy, there was evidence of residual tumor cells and tumor burden after the therapy, clearly indicating that CAR T-cells as monotherapy was not sufficient to completely eradicate disease.

A potential mechanism underlying tumor cells' evasion of the immune system is frequent exploitation of immunosuppressive mechanisms already in place, ultimately leading to immune tolerance and escape. One such mechanism is the programmed death-1/programmed death-ligand-1 (PD-1/PD-L1) pathway. PD-1 is a cell-surface receptor induced on immune cells upon activation, including B- and T-lymphocytes. Interaction of PD-1 with one of its ligands PD-L1 (normally found on resting immune cells, macrophages, and vascular endothelial cells) negatively suppresses the immune response under physiologic circumstances to allow for self-tolerance, T-cell exhaustion, and protection against autoimmunity.[15],[16],[17] Not surprisingly then, PD-L1 is also found on tumor cells to be constitutively overexpressed in several malignancies such as melanoma, lung cancer, renal cell carcinoma, and colorectal cancers as a way to diminish optimal immune potential.[17],[18],[19],[20],[21],[22] The PD-L1 status in OS has recently become clearer, with several OS cell lines and patient tissue samples showing a variable range of surface PD-L1 expression [23] that could be targeted.

In our study presented here, we examined the potential benefit of combining IL-11Rα-CAR T-cells with an anti-PD-1 antibody against OS cells in vitro. We hypothesized that this combination could effectively maximize the therapeutic potential of IL-11Rα-CAR T-cell therapy against OS by eliminating the immune escape pathway that is utilized by the tumor cells. We further analyzed surface PD-1 expression on the immune cells under various conditions during treatment to increase our understanding of this unique pathway. Our data indicate that disruption of the PD-1/PD-L1 pathway through an anti-PD-1 antibody does not increase in vitro cytotoxic activity of IL-11Rα-CAR T-cells against OS cells.


  Methods Top


Generation of interleukin-11Rα-chimeric antigen receptor T-cells

The cDNA encoding IL-11Rα-CAR was constructed and cloned using a DNA sleeping beauty expression plasmid (pSBSO) to create the transposon plasmid pSBSO-IL-11-CAR as previously described.[24],[25] To express the SB11 transposase, the DNA plasmid pCMV-SB11 was used as previously described.[11] Human peripheral blood mononuclear cells (PBMCs) were harvested from buffy coats isolated from blood samples donated by healthy adult volunteers (Gulf Coast Regional Blood Center, Houston, TX, USA).[26] PBMCs were either immediately electroporated to generate CAR T-cells or cryopreserved in liquid nitrogen for electroporation at a later time. Genetically unmodified T-cells used as control cells were purified from the same buffy coats using the EasySEP Human T Cell Isolation Kit (Stem Cell Technologies, Cambridge, MA, USA).

Nucleofector solution (Human CD34 Cell Nucleofector Kit; Amaxa) was mixed with 15 μg pSBSO-IL-11-CAR plasmid and 5 μg pCMV-SB11 plasmid, transferred to a cuvette, and electroporated into PBMCs using Amaxa Nucleofector II (Program U-14). Transfected cells were then transferred to 6-well plates with 4 mL/well of prewarmed phenol-free RPMI medium (supplemented with Glutamax [Thermo Fisher Scientific, Grand Island, NY, USA] and fetal bovine serum [FBS]). Cells were incubated overnight; 24 h after electroporation (day 1), CAR T-cells were stimulated with gamma-irradiated artificial antigen-presenting cells (aAPCs; preloaded with anti-CD3 antibody OKT3) at a ratio of 1:1 of CAR T-cells to aAPCs, 50 U/mL recombinant human IL-2, and 30 ng/mL IL-21. aAPCs were K562 clone 4 cells modified to express certain surface molecules (including IL-11Rα). CAR T-cells were kept at a concentration of 0.5–1 × 106 cells/mL in complete culture medium in which IL-2/IL-21 was replenished and half of the medium changed every 48 h. Control T-cells were propagated identically with gamma-irradiated aAPCs, IL-2, and IL-21. Both cell types underwent one cycle of ex vivo expansion.

Flow cytometry

The phenotypes of IL-11Rα-CAR T-cells and control T-cells were analyzed through flow cytometry to obtain baseline profiles before expansion (on day 1) and before coculture with LM7 OS cells (on either day 5 or day 6). The number of cells used per flow sample for analysis was approximately 0.1–0.2 × 106 cells/sample. Cell surface antigen expression to characterize both immune cell types was done utilizing fluorochrome-conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies. Phycoerythrin-conjugated goat anti-human Fcγ was used to detect cell surface expression of the Fc portion of IL-11Rα-CAR (not present on control T-cells). From this, percentage of CAR T-cells present per sample was estimated by the number of Fc+ cells for that point in time. Surface PD-1 expression on both CAR and control T-cells was also analyzed before expansion (on day 1), on day 6 before coculture with LM7 cells, and after coculture with LM7 cells at different effector:target (E:T) ratios, with or without anti-PD-1 antibody. The number of cells used per flow sample for analysis was again approximately 0.1–0.2 × 106 cells/flow sample. Unstained cell samples were used as negative controls. Percentage of CAR T-cells expressing PD-1 was estimated by the number of Fc+/PD-1+ cells for that particular condition. Data acquisition was conducted on a BD FACSCalibur, with the percentage of cells in an analyzed region calculated using FlowJo V10 software (FlowJo LLC., Ashland, OR, USA).

Treating with anti-human PD-1 (Clone J110) before cytotoxicity assay

To test the impact of blocking PD-1/PD-L1 pathway in vitro before the immune cell-tumor cell coculture setup, we pretreated immune cells in the following manner. Immune cells were first treated with different doses of the anti-PD-1 antibody (5, 10, 20, and 25 μg/mL) to determine a specific nontoxic dose using the 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cytotoxicity assay. All the doses tested had no toxicity effects against OS cells. We selected the highest dose tested, 25 μg/mL per 1 × 106 immune cells. An appropriate number of immune cells were harvested during each experiment and resuspended at 1 × 106 cells/mL in complete culture media and seeded in a standard 6-well plate. Thereafter, 25 μg/mL of anti-PD-1 antibody was added to each well and left at 37°C for 15 h.

In vitro coculture of interleukin-11Rα-chimeric antigen receptor T-cells with LM7 human osteosarcoma cells

The human LM7 OS cell line was cultured in Eagle's modified essential medium supplemented with glutamine, sodium pyruvate, nonessential amino acids, penicillin, and FBS. To determine the cytotoxic effect of coculture with IL-11Rα-CAR T-cells on the LM7 cells, the calcein release assay was used. The LM7 cells were harvested and loaded with calcein AM (2 mL calcein/1 × 106 cells) by incubating for 1 h at 37°C. The cells were then washed twice with phosphate-buffered saline solution to remove excess calcein. Effector immune cells (with or without anti-PD-1 antibody) were first plated in a 96-well plate, and the target LM7 cells (now loaded with calcein) were then plated in the wells at E:T ratios of 5:1 and 1:1. The plate was subjected to gentle centrifugation to establish contact between effector and target cells and was then incubated for 15 h at 37°C. After the coculture period was completed, cells were again subjected to gentle centrifugation, and 100 μL of supernatant from each well was transferred to a fluorescence plate reader for analysis of excitation/emission values. Percentage of cell lysis was calculated. Lysis induced by 2% Triton-X was used as the “maximum” (100%) lysis value.

Statistical analysis

Flow cytometry and cell culture experiments with calcein release assay were done at least in triplicates and repeated at least two times. Cytotoxicity differences in IL-11Rα-CAR T-cells (both treated and untreated) and control T-cells (treated and untreated) were analyzed using the Student's paired t-test. All the results were considered to be statistically significant at values of P < 0.05.


  Results Top


Baseline expression of surface antigens on interleukin- 11Rα-chimeric antigen receptor T-cells and unmodified control T-cells

The surface antigen expression profiles of IL-11Rα-CAR T-cells (performed 24 h after electroporation) and of unmodified control T-cells (performed 24 h after isolation) were assessed through flow cytometry and included analysis for surface expression of CD3, CD4, CD8 antigens. CAR T-cells were identified by surface expression of the Fc domain, which was not present on our unmodified pure T-cells that we utilized as our control. Twenty-four hours after generation of IL-11Rα-CAR T-cells through electroporation, we had approximately 63% of total cells which demonstrated Fc CAR surface expression, with 42% of cells also co-expressing CD4+ antigen and 17% co-expressing CD8+ antigen [Figure 1]. For our unmodified T-cells, approximately 80% of cells were profiled as co-expressing CD3+/CD4+ while 20% showed CD3+/CD8+ co-expression (data not shown).
Figure 1: Characterization of interleukin-11Rα-chimeric antigen receptor T-cells through flow cytometry, 24 h after electroporation (day 1). Representative dot plots shown. Number of cells per analysis = 0.1–0.2 × 106 cells/sample. (a) Unstained cells (negative control). Cells co-expressing (b) CD3/Fc; (c) CD8/Fc; (d) CD4/Fc

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Surface PD-1 expression of interleukin- 11Rα-chimeric antigen receptor T-cells and unmodified T-cells increased during expansion

Surface PD-1 expression of both IL-11Rα-CAR T-cells and purified T-cells was analyzed through flow cytometry during the 1st week of expansion to document and follow expression trends. Baseline surface PD-1 expression on day 1 of expansion (defined as 24 h following electroporation or isolation) was < 1% on both of our immune cell types [Figure 2]a. By day 6 of expansion, surface PD-1 expression increased to 60% for CAR T-cells [Figure 2]b and to 50% for purified control T-cells (data not shown).
Figure 2: Surface programmed death-1 trends of interleukin-11Rα-chimeric antigen receptor T-cells (effector cells), ±anti-programmed death-1. (a) Day 1; (b) day 6 before coculture with LM7 (target cells), (c) after coculture (5:1) effector:target (−Ab); (d) after (1:1) effector:target (−Ab); (e) After (5:1) effector:target (+Ab); (f) after (1:1) effector:target (+Ab)

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Coculture with LM7 osteosarcoma cells decreased surface PD-1 expression of immune cells (with or without PD-1 blockade)

Surface PD-1 expression of IL-11Rα-CAR T-cells and purified T-cells was analyzed after in vitro coculture with LM7 OS cells. Immune cells were either pretreated with the anti-human PD-1 antibody (25 μg/mL) for 15 h before being cocultured or not pretreated with antibody before coculture. Effector immune cells and target LM7 OS cells were cocultured at different E:T ratios to determine whether different ratios were influential on cytotoxic activity of the T-cells. For IL-11Rα-CAR T-cells cocultured with LM7 cells at E:T ratios of (5:1) and (1:1) (no anti-PD-1 antibody), surface PD-1 expression decreased from 60% at baseline to 32% and 28%, respectively [Figure 2]c and [Figure 2]d. A similar decrease in PD-1 expression on control T-cells was observed when they were cocultured with no antibody (data not shown). When IL-11Rα-CAR T-cells and unmodified T-cells were both pretreated with anti-PD-1 antibody, surface PD-1 expression was observed to be < 1% across both (5:1) and (1:1) E:T ratios for both immune cell types [Figure 2]e and [Figure 2]f. Therefore, while pretreatment with an anti-PD-1 antibody almost completely abrogated surface PD-1 expression as expected, exposure to LM7 OS cells alone also suppressed PD-1 expression in both of our immune cell types.

Effect on cytotoxic activity of interleukin-11Rα-chimeric antigen receptor T-cells after decreasing surface PD-1 expression

Cytotoxicity against LM7 cells when cocultured with either IL-11Rα-CAR T-cells or unmodified T-cells (with or without antibody pretreatment) was measured using a calcein release assay. Exposure of LM7 tumor cells to IL-11Rα-CAR T-cells alone (without antibody) at (1:1) E:T ratio demonstrated cell lysis at approximately16%–56%; at (5:1) E:T ratio, cell lysis was approximated at 49%–69%. Decreasing surface PD-1 expression with antibody pretreatment before coculture did not appear to enhance the cytotoxic activity of the CAR T-cells in our investigation as cell lysis percentages with pretreated IL-11Rα-CAR T-cells were found to be 8%–49% at (1:1) E:T ratio and 40%–54% at (5:1) E:T ratio [Figure 3]. Similar results were observed for our control T-cell population with or without antibody pretreatment [Figure 3]. Thus, the impact of decreasing surface PD-1 expression on both immune cell types was not further translated into an enhanced cytotoxic effect in our study.
Figure 3: Cytotoxicity of immune cells against LM7 osteosarcoma cells was not enhanced after disruption of programmed death-1/programmed death ligand 1 pathway through anti-programmed death-1 antibody. Different effector:target ratios are shown with or without antibody. Averaged data for two experiments are shown

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  Discussion Top


Adoptive cellular therapy with CAR T-cells allows for delivery of targeted therapy through the identification of tumor-specific antigens. Recent studies have shown promise with CAR T-cells in the setting of OS both in vitro and in vivo, including HER2 CAR T-cells and IGF1R-/ROR1-specific CAR T-cells when used as monotherapy.[27],[28] Although Huang et al.[11] specifically showed therapeutic benefit of IL-11Rα-CAR T-cells against OS both in vivo and in vitro, it was without complete tumor eradication. Even though PD-1/PD-L1 blockade is relatively new to the OS context, its clinical success in a variety of other tumor types [9],[10],[29],[30] encouraged us to further investigate its potential role in this setting. Addition of an anti-PD-1 antibody to HER2-CAR T-cells targeting sarcoma and breast tumor cells has shown improved functionality of the CAR T-cells following PD-1 blockade.[31] Given this possibility of synergism between CAR T-cells and PD-1/PD-L1 blockade, our study investigated the antitumor efficacy of a novel combination against OS with IL-11Rα-CAR T-cells and an anti-PD-1 antibody.

Indeed, the time-dependent increase in surface PD-1 expression of both genetically modified and unmodified T-cells following one cycle of expansion in our study is consistent with what is reported in the literature for PD-1 expression trends following T-cell activation.[32],[33] Our data also provide additional description of surface PD-1 trends during expansion on a particular type of CAR T-cells that has previously not been reported. As both immune cell types underwent activation with an anti-CD3 antibody and aAPCs, PD-1 expression increased during expansion due to these persistent activating agents that were part of our culture system. IL-2 that was also present in our culture is another known inducer of PD-1 expression in vitro,[34] likely contributing to our findings.

Analysis of the influence on immune cell surface PD-1 expression upon exposure to tumor cells was crucial to improve our understanding of this pathway, particularly for this tumor context. We speculate that the decreased surface PD-1 expression we observed on both anti-PD-1 treated and untreated CAR T-cells after tumor cell exposure could be explained by a possible receptor internalization, rather than true downstream inhibition and complete blockade of the PD-1/PD-L1 pathway, as published data have shown that T-cell exhaustion upon exposure to stimuli leads to increase PD-1 expression rather than a decrease.[35] This mechanism if it exists could serve as a primary “checkpoint” through which immune cells are able to self-preserve since constitutive engagement with PD-L1 leads to T-cell apoptosis and exhaustion.[32] This particular trend for PD-1 expression on immune cells following tumor cell exposure in vitro has not consistently been reported in the context of OS. Furthermore, although surface PD-L1 expression on tumor cells was not determined in our study, one has to consider what influential role its expression could have played, as it is a known highly inducible ligand that has been qualified as a response predictor for both anti-PD-1 and anti-PD-L1 therapies in a variety of other cancer settings already.[36],[37],[38],[39],[40]

An interesting feature of our current study was the lack of improved cytotoxic activity following decreased surface PD-1 expression in our anti-PD-1-treated groups. Engagement of PD-L1 ligand on tumor cells with PD-1 receptor on T-cells plays an influential role in preventing tumor-infiltrating lymphocytes from physically reaching tumor cells in vivo.[41],[42] Blockade of this trail then should lead to improved T-cell function in theory for OS, similarly to what has been noted in a variety of other oncologic settings already.[9],[10],[29],[30] As with most complex pathways, there were likely other parallel immune checkpoints not blocked in our study that likely continued to exhibit inhibitory effects on T-cell function and thus resulting in the lack of improved cytotoxic activity that we noted. Recently, it was shown that cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), another protein receptor that functions as an immune checkpoint, was upregulated in tumor-infiltrating T-cells in a metastatic OS mouse model once they became resistant to PD-1.[41] In our tumor cells, we did not assess simultaneous CTLA-4 expression to see if similar trends occurred when PD-1 expression decreased. Similarly, we did not investigate the impact of surface PD-1 blockade on other downstream signaling proteins such as PI3K/Akt signaling. Under normal physiologic conditions, PD-1/PD-L1 engagement results in inhibition of PI3K/Akt pathway, leading to blockade of T-cell proliferation and survival.[43],[44] It is possible that simple surface blockade of PD-1 signaling with a neutralizing antibody was not enough to overcome these downstream inhibitory effects that continued to block T-cell survival.[44] Similarly, myeloid-derived suppressor cells (MDSCs) have been known to play a role in T-cell exhaustion [45] and are known to be present in normal healthy donors at low levels. It is likely that a small percentage of MDSCs were present in the buffy coats from which our CAR T-cells were generated, thus contributing to additional T-cell suppression distinctive of the PD-1/PD-L1 pathway.

Our experimental design did have the following limitations that need to be considered when interpreting our results. First, we only utilized flow cytometry as our preferred method of detection for PD-1 expression, thus determining and analyzing only surface expression trends with this modality. Surface protein expression is not always reflective of further downstream pathway effects, mRNA levels, or ultimate protein expression. If we would have quantified total PD-1 expression through Western blot or immunohistochemistry, for example, we may have found additional data in regard to our PD-1 trends, particularly after tumor cell exposure, which appeared to be counter-intuitive. Furthermore, as mentioned previously, we did not quantify correlating PD-L1 expression on our tumor cells at the time of our PD-1 measurements, which could have allowed for direct comparisons and correlations. Future experiments measuring PD-L1 expression under the same conditions and time points are likely going to be crucial to our understanding of this pathway. Furthermore, even though our results demonstrated a significant decrease in surface PD-1 expression on our immune cells with our dose of anti-PD-1 antibody that we used, it is possible that higher antibody doses are needed to truly impact cytotoxicity on tumor cells. Finally, additional cell lines should be tested before broad conclusions are made for OS tumor cells with this particular combination. We further recommend future in vivo studies that naturally will recapitulate the true tumor microenvironment which is crucial to our understanding and potentially influencing cancer behavior.

In conclusion, a combination approach to obtain more favorable responses is not an uncommon occurrence in oncology, especially in the setting of challenging scenarios such as OS. Previous demonstration of antitumor effect against OS with IL-11Rα-CAR T-cells prompted us to investigate whether cytotoxic activity and tumor cell killing could be enhanced by combining these CAR T-cells with an anti-PD-1 antibody. Our results showed the combination offered no additional benefit compared to IL-11Rα- CAR T-cells alone. Further studies are warranted, however, to validate whether this combination is truly optimal and to better understand the downstream impact following decreased surface PD-1expression, particularly in regard to OS.

Acknowledgment

We thank the Scientific and Department Publications at MD Anderson Cancer Center for their assistance in editing of the manuscript.

Financial support and sponsorship

The study was supported in part by the Mary V and John A Reilly Distinguish Chair to E S Kleinerman and the Reliant Energy Fund.

Conflicts of interest

There are no conflicts of interest.

 
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