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ISSN (Print): 1381-6128
ISSN (Online): 1873-4286

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Research Article

PEGylation Strategy for Improving the Pharmacokinetic and Antitumoral Activity of the IL-2 No-alpha Mutein

Author(s): Marianniz Díaz-Hernández*, Janoi Chang-Calderón, Miguel Angel Álvarez, Ingrid Ruiz Ramírez, Olga Lidea Fernández Saez, Armando López Medinilla, Carlos Yordan González Castillo, Claudia Diaz Borges, Sum Lai Lozada Chang, Kalet León and Tania Carmenate*

Volume 29, Issue 44, 2023

Published on: 11 December, 2023

Page: [3579 - 3588] Pages: 10

DOI: 10.2174/0113816128279062231204110410

Price: $65

Abstract

Background: In a previous work, an IL-2Rβγ biased mutant derived from human IL-2 and called IL-2noα, was designed and developed. Greater antitumor effects and lower toxicity were observed compared to native IL-2. Nevertheless, mutein has some disadvantages, such as a very short half-life of about 9-12 min, propensity for aggregation, and solubility problems.

Objective: In this study, PEGylation was employed to improve the pharmacokinetic and antitumoral properties of the novel protein.

Methods: Pegylated IL-2noα was characterized by polyacrylamide gel electrophoresis, size exclusion chromatography, in vitro cell proliferation and in vivo cell expansion bioassays, and pharmacokinetic and antitumor studies.

Results: IL-2noα-conjugates with polyethylene glycol (PEG) of 1.2 kDa, 20 kDa, and 40 kDa were obtained by classical acylation. No significant changes in the secondary and tertiary structures of the modified protein were detected. A decrease in biological activity in vitro and a significant improvement in half-life were observed, especially for IL-2noα-PEG20K. PEGylation of IL-2noα with PEG20K did not affect the capacity of the mutant to induce preferential expansion of T effector cells over Treg cells. This pegylated IL-2noα exhibited a higher antimetastatic effect compared to unmodified IL-2noα in the B16F0 experimental metastases model, even when administered at lower doses and less frequently.

Conclusion: PEG20K was selected as the best modification strategy, to improve the blood circulation time of the IL-2noα with a superior antimetastatic effect achieved with lower doses.

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[1]
Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 2012; 12(3): 180-90.
[http://dx.doi.org/10.1038/nri3156] [PMID: 22343569]
[2]
Malek TR, Castro I. Interleukin-2 receptor signaling: At the interface between tolerance and immunity. Immunity 2010; 33(2): 153-65.
[http://dx.doi.org/10.1016/j.immuni.2010.08.004] [PMID: 20732639]
[3]
Rosenberg SA. IL-2: The first effective immunotherapy for human cancer. J Immunol 2014; 192(12): 5451-8.
[http://dx.doi.org/10.4049/jimmunol.1490019] [PMID: 24907378]
[4]
Zhou Y, Quan G, Liu Y, et al. The application of interleukin-2 family cytokines in tumor immunotherapy research. Front Immunol 2023; 14: 1090311.
[http://dx.doi.org/10.3389/fimmu.2023.1090311] [PMID: 36936961]
[5]
Rosenberg SA. Progress in the development of immunotherapy for the treatment of patients with cancer. J Intern Med 2001; 250(6): 462-75.
[http://dx.doi.org/10.1046/j.1365-2796.2001.00911.x] [PMID: 11902815]
[6]
Klapper JA, Downey SG, Smith FO, Yang JC, Hughes MS, Kammula US. High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma: A retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006. Cancer 2008; 113(2): 293-301.
[7]
Marabondo S, Kaufman HL. High-dose interleukin-2 (IL-2) for the treatment of melanoma: Safety considerations and future directions. Expert Opin Drug Saf 2017; 16(12): 1347-57.
[http://dx.doi.org/10.1080/14740338.2017.1382472] [PMID: 28929820]
[8]
Siegel JP, Puri RK. Interleukin-2 toxicity. J Clin Oncol 1991; 9(4): 694-704.
[http://dx.doi.org/10.1200/JCO.1991.9.4.694] [PMID: 2066765]
[9]
Cesana GC, DeRaffele G, Cohen S, et al. Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol 2006; 24(7): 1169-77.
[http://dx.doi.org/10.1200/JCO.2005.03.6830] [PMID: 16505437]
[10]
MacDonald A, Wu TC, Hung CF. Interleukin 2-based fusion proteins for the treatment of cancer. J Immunol Res 2021; 2021: 1-11.
[http://dx.doi.org/10.1155/2021/7855808] [PMID: 34790830]
[11]
Zhang B, Sun J, Yuan Y, et al. Proximity-enabled covalent binding of IL-2 to IL-2Rα selectively activates regulatory T cells and suppresses autoimmunity. Signal Transduct Target Ther 2023; 8(1): 28.
[http://dx.doi.org/10.1038/s41392-022-01208-3] [PMID: 36588107]
[12]
Harris F, Berdugo YA, Tree T. IL-2-based approaches to Treg enhancement. Clin Exp Immunol 2023; 211(2): 149-63.
[http://dx.doi.org/10.1093/cei/uxac105] [PMID: 36399073]
[13]
Peterson LB, Bell CJM, Howlett SK, et al. A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease. J Autoimmun 2018; 95: 1-14.
[http://dx.doi.org/10.1016/j.jaut.2018.10.017] [PMID: 30446251]
[14]
Ghelani A, Bates D, Conner K, Wu M-Z, Lu J, Hu Y-L. Defining the threshold IL-2 signal required for induction of selective treg cell responses using engineered IL-2 muteins. Front Immunol 2020; 11: 1106.
[http://dx.doi.org/10.3389/fimmu.2020.01106]
[15]
Khoryati L, Pham MN, Sherve M, Kumari S, Cook K, Pearson J. Regulatory T cell expansion by a highly CD25-dependent IL-2 mutein arrests ongoing autoimmunity. bioRxiv 2019; 862789.
[http://dx.doi.org/10.1101/862789]
[16]
Carmenate T, Pacios A, Enamorado M, et al. Human IL-2 mutein with higher antitumor efficacy than wild type IL-2. J Immunol 2013; 190(12): 6230-8.
[http://dx.doi.org/10.4049/jimmunol.1201895] [PMID: 23677467]
[17]
Carmenate T, Montalvo G, Lozada SL, et al. The antitumor effect induced by an IL-2 ‘no-alpha’ mutein depends on changes in the CD8+ T lymphocyte/Treg cell balance. Front Immunol 2022; 13: 974188.
[http://dx.doi.org/10.3389/fimmu.2022.974188] [PMID: 36059465]
[18]
Konrad MW, Hemstreet G, Hersh EM, et al. Pharmacokinetics of recombinant interleukin 2 in humans. Cancer Res 1990; 50(7): 2009-17.
[PMID: 2317789]
[19]
Zuma LK, Gasa NL, Makhoba XH, Pooe OJ. Protein PEGylation: Navigating recombinant protein stability, aggregation, and bioactivity. BioMed Res Int 2022; 2022: 1-7.
[http://dx.doi.org/10.1155/2022/8929715] [PMID: 35924267]
[20]
Ryan SM, Mantovani G, Wang X, Haddleton DM, Brayden DJ. Advances in PEGylation of important biotech molecules: Delivery aspects. Expert Opin Drug Deliv 2008; 5(4): 371-83.
[http://dx.doi.org/10.1517/17425247.5.4.371] [PMID: 18426380]
[21]
Abbina S, Parambath A. PEGylation and its alternatives: A summary. Engineering of Biomaterials for Drug Delivery Systems. Elsevier 2018; pp. 363-76.
[http://dx.doi.org/10.1016/B978-0-08-101750-0.00014-3]
[22]
Harris JM, Martin NE, Modi M. Pegylation. Clin Pharmacokinet 2001; 40(7): 539-51.
[http://dx.doi.org/10.2165/00003088-200140070-00005] [PMID: 11510630]
[23]
Łażewski D, Murias M, Wierzchowski M. Pegylation – In search of balance and enhanced bioavailability. J Med Sci 2022; 91(4): e761.
[http://dx.doi.org/10.20883/medical.e761]
[24]
Theyab A, Alsharif KF, Alzahrani KJ, et al. New insight into strategies used to develop long-acting G-CSF biologics for neutropenia therapy. Front Oncol 2023; 12: 1026377.
[http://dx.doi.org/10.3389/fonc.2022.1026377] [PMID: 36686781]
[25]
Diab A, Gogas H, Sandhu S, et al. Bempegaldesleukin plus nivolumab in untreated advanced melanoma: the open-label, phase III PIVOT IO 001 trial results. J Clin Oncol 2023; 41(30): 4756-67.
[http://dx.doi.org/10.1200/JCO.23.00172] [PMID: 37651676]
[26]
Raeber ME, Sahin D, Boyman O. Interleukin-2-based therapies in cancer. Sci Transl Med 2022; 14(670): eabo5409.
[http://dx.doi.org/10.1126/scitranslmed.abo5409] [PMID: 36350987]
[27]
Berkovits A, Yanez E, Rojas C, et al. Pegathor lymphoma, a phase 2 study of SAR444245 as a monotherapy or in combination with pembrolizumab for the treatment of adults and adolescents with relapsed or refractory B cell lymphoma. Blood 2022; 140(S1): 12020-1.
[http://dx.doi.org/10.1182/blood-2022-159980]
[28]
Kurfürst MM. Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 1992; 200(2): 244-8.
[http://dx.doi.org/10.1016/0003-2697(92)90460-O] [PMID: 1378701]
[29]
Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in microsoft excel. Comput Methods Programs Biomed 2010; 99(3): 306-14.
[http://dx.doi.org/10.1016/j.cmpb.2010.01.007] [PMID: 20176408]
[30]
Siekmann J, Turecek PL. PEGylation of human coagulation factor VIII and other plasma proteins. Polymer-Protein Conjugates. Elsevier 2020; pp. 155-74.
[31]
Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 2002; 54(4): 459-76.
[http://dx.doi.org/10.1016/S0169-409X(02)00022-4] [PMID: 12052709]
[32]
Wang X, Rickert M, Garcia KC. Structure of the quaternary complex of interleukin-2 with its α, ß, and γc receptors. Science 2005; 310(5751): 1159-63.
[http://dx.doi.org/10.1126/science.1117893] [PMID: 16293754]
[33]
Stauber DJ, Debler EW, Horton PA, Smith KA, Wilson IA. Crystal structure of the IL-2 signaling complex: Paradigm for a heterotrimeric cytokine receptor. Proc Natl Acad Sci 2006; 103(8): 2788-93.
[http://dx.doi.org/10.1073/pnas.0511161103] [PMID: 16477002]
[34]
Eftink MR. Intrinsic fluorescence of proteins. Lakowicz JR. Topics in Fluorescence Spectroscopy: Volume 6: Protein Fluorescence. Boston, MA: Springer US 2000; pp. 1-15.
[35]
Sun C, Jiang X, Li B, Li S, Kong XZ. Fluorescence behavior and mechanisms of poly(ethylene glycol) and their applications in Fe3+ and Cr6+ detections, data encryption, and cell imaging. ACS Sustain Chem Eng 2021; 9(14): 5166-78.
[http://dx.doi.org/10.1021/acssuschemeng.1c00250]
[36]
Elliott VL, Edge GT, Phelan MM, et al. Evidence for metabolic cleavage of a PEGylated protein in vivo using multiple analytical methodologies. Mol Pharm 2012; 9(5): 1291-301.
[http://dx.doi.org/10.1021/mp200587m] [PMID: 22480236]
[37]
Moosmann A, Blath J, Lindner R, Müller E, Böttinger H. Aldehyde PEGylation kinetics: A standard protein versus a pharmaceutically relevant single chain variable fragment. Bioconjug Chem 2011; 22(8): 1545-58.
[http://dx.doi.org/10.1021/bc200090x] [PMID: 21780828]
[38]
Pasut G, Veronese FM. State of the art in PEGylation: The great versatility achieved after forty years of research. J Control Release 2012; 161(2): 461-72.
[http://dx.doi.org/10.1016/j.jconrel.2011.10.037] [PMID: 22094104]
[39]
Ekladious I, Colson YL, Grinstaff MW. Polymer–drug conjugate therapeutics: Advances, insights and prospects. Nat Rev Drug Discov 2019; 18(4): 273-94.
[http://dx.doi.org/10.1038/s41573-018-0005-0] [PMID: 30542076]
[40]
Cheng TL, Chuang KH, Chen BM, Roffler SR. Analytical measurement of PEGylated molecules. Bioconjug Chem 2012; 23(5): 881-99.
[http://dx.doi.org/10.1021/bc200478w] [PMID: 22242549]
[41]
Charych DH, Hoch U, Langowski JL, et al. NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models. Clin Cancer Res 2016; 22(3): 680-90.
[http://dx.doi.org/10.1158/1078-0432.CCR-15-1631] [PMID: 26832745]
[42]
Saha-Shah A, Sun S, Kong J, Zhong W, Mann BF. Design and study of PEG linkers that enable robust characterization of PEGylated proteins. ACS Pharmacol Transl Sci 2021; 4(4): 1280-6.
[http://dx.doi.org/10.1021/acsptsci.1c00112] [PMID: 34423265]
[43]
Ranjbar B, Gill P. Circular dichroism techniques: Biomolecular and nanostructural analyses - A review. Chem Biol Drug Des 2009; 74(2): 101-20.
[http://dx.doi.org/10.1111/j.1747-0285.2009.00847.x] [PMID: 19566697]
[44]
Alva A, Daniels GA, Wong MKK, et al. Contemporary experience with high-dose interleukin-2 therapy and impact on survival in patients with metastatic melanoma and metastatic renal cell carcinoma. Cancer Immunol Immunother 2016; 65(12): 1533-44.
[http://dx.doi.org/10.1007/s00262-016-1910-x] [PMID: 27714434]
[45]
Dhupkar P, Gordon N. Interleukin-2: Old and new approaches to enhance immune-therapeutic efficacy. Adv Exp Med Biol 2017; 995: 33-51.
[http://dx.doi.org/10.1007/978-3-319-53156-4_2] [PMID: 28321811]
[46]
Lykhopiy V, Malviya V, Humblet-Baron S, Schlenner SM. “IL-2 immunotherapy for targeting regulatory T cells in autoimmunity”. Genes Immun 2023; 24(5): 248-62.
[http://dx.doi.org/10.1038/s41435-023-00221-y] [PMID: 37741949]
[47]
Mayolo K, Rito M. Proteínas PEGiladas: Producción, purificación y aplicaciones. Rev Mex Ing Quim 2010; 9(1): 17-27.
[48]
Kang JS, DeLuca PP, Lee KC. Emerging PEGylated drugs. Expert Opin Emerg Drugs 2009; 14(2): 363-80.
[http://dx.doi.org/10.1517/14728210902907847] [PMID: 19453284]
[49]
Moosmann A, Müller E, Böttinger H. Purification of PEGylated proteins, with the example of PEGylated lysozyme and PEGylated scFv. Protein Downstream Processing. Springer 2014; pp. 527-38.
[50]
Na DH, Park EJ, Youn YS, et al. Sodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alpha. Electrophoresis 2004; 25(3): 476-9.
[http://dx.doi.org/10.1002/elps.200305684] [PMID: 14760640]
[51]
Shang X. Purification and synthesis of PEGylated protein. The Pennsylvania State University 2014.
[52]
Pfister D, Morbidelli M. Process for protein PEGylation. J Control Release 2014; 180: 134-49.
[http://dx.doi.org/10.1016/j.jconrel.2014.02.002] [PMID: 24531008]
[53]
Belén LH, Rangel-Yagui CO, Beltrán Lissabet JF, et al. From synthesis to characterization of site-selective PEGylated proteins. Front Pharmacol 2019; 10: 1450.
[http://dx.doi.org/10.3389/fphar.2019.01450] [PMID: 31920645]
[54]
Ramos-de-la-Peña AM. Progress and challenges in PEGylated proteins downstream processing: A review of the last 8 years. Int J Pept Res Ther 2020; 26(1): 333-48.
[55]
Shadish JA, DeForest CA. Site-selective protein modification: From functionalized proteins to functional biomaterials. J Mater 2020; 2(1): 50-77.
[56]
Zhang B, Sun J, Wang Y, Ji D, Yuan Y, Li S. Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells. Nat Biomed Eng 2021; 5(11): 1288-305.
[http://dx.doi.org/10.1038/s41551-021-00797-8]
[57]
Moro-Pérez L, Lozada-Chang SL, Rivas-García G, et al. Purification and conformational characterization of a novel interleukin-2 mutein. Protein J 2021; 40(6): 917-28.
[http://dx.doi.org/10.1007/s10930-021-10025-6] [PMID: 34643845]
[58]
Turecek PL, Bossard MJ, Schoetens F, Ivens IA. PEGylation of biopharmaceuticals: A review of chemistry and nonclinical safety information of approved drugs. J Pharm Sci 2016; 105(2): 460-75.
[http://dx.doi.org/10.1016/j.xphs.2015.11.015] [PMID: 26869412]
[59]
Grace M, Youngster S, Gitlin G, et al. Structural and biologic characterization of pegylated recombinant IFN-α2b. J Interferon Cytokine Res 2001; 21(12): 1103-15.
[http://dx.doi.org/10.1089/107999001317205240] [PMID: 11798469]
[60]
Meng F, Manjula BN, Smith PK, Acharya SA. PEGylation of human serum albumin: Reaction of PEG-phenyl-isothiocyanate with protein. Bioconjug Chem 2008; 19(7): 1352-60.
[http://dx.doi.org/10.1021/bc7003878] [PMID: 18572961]
[61]
Giuseppe D, Luca B, Chiara B, et al. NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1–29)-NH2. J Am Chem Soc 2003; 125(12): 3458-70.
[62]
Liu X, Kouassi KGW, Vanbever R, Dumoulin M. Impact of the PEG length and PEGylation site on the structural, thermodynamic, thermal, and proteolytic stability of mono-PEGylated alpha-1 antitrypsin. Protein Sci 2022; 31(9): e4392.
[http://dx.doi.org/10.1002/pro.4392] [PMID: 36040264]
[63]
Santos JHPM, Torres-Obreque KM, Meneguetti GP, Amaro BP, Rangel-Yagui CO. Protein PEGylation for the design of biobetters: From reaction to purification processes. Braz J Pharm Sci 2018; 54(spe): 54.
[http://dx.doi.org/10.1590/s2175-97902018000001009]
[64]
Gupta V, Bhavanasi S, Quadir M, Singh K, Ghosh G, Vasamreddy K. Protein PEGylation for cancer therapy: Bench to bedside. J Cell Commun Signal 2019; 13(3): 319-30.
[http://dx.doi.org/10.1007/s12079-018-0492-0]
[65]
Foser S, Schacher A, Weyer KA, et al. Isolation, structural characterization, and antiviral activity of positional isomers of monopegylated interferon α-2a (PEGASYS). Protein Expr Purif 2003; 30(1): 78-87.
[http://dx.doi.org/10.1016/S1046-5928(03)00055-X] [PMID: 12821324]
[66]
Hou Y, Lu H. Protein PEPylation: A new paradigm of protein-polymer conjugation. Bioconjug Chem 2019; 30(6): 1604-16.
[http://dx.doi.org/10.1021/acs.bioconjchem.9b00236] [PMID: 31045353]
[67]
Ibrahim M, Ramadan E, Elsadek NE, Emam SE, Shimizu T, Ando H. Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products. J Control Release 2022; 351: 215-30.
[68]
Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003; 2(3): 214-21.
[http://dx.doi.org/10.1038/nrd1033] [PMID: 12612647]
[69]
Caliceti P, Veronese FM. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)–protein conjugates. Adv Drug Deliv Rev 2003; 55(10): 1261-77.
[http://dx.doi.org/10.1016/S0169-409X(03)00108-X] [PMID: 14499706]
[70]
Wu K, Ma J, Bai W, et al. Short-term intratracheal use of PEG- modified IL-2 and glucocorticoid persistently alleviates asthma in a mouse model. Sci Rep 2016; 6(1): 31562.
[http://dx.doi.org/10.1038/srep31562] [PMID: 27527926]
[71]
Xiao Q, Ashton DS, Jones ZB, Thompson KP. Long-range PEG stapling: Macrocyclization for increased protein conformational stability and resistance to proteolysis. RSC Chem Biol 2020; 1(4): 273-80.
[72]
Yang JC, Topalian SL, Schwartzentruber DJ, et al. The use of polyethylene glycol-modified interleukin-2 (PEG-IL-2) in the treatment of patients with metastatic renal cell carcinoma and melanoma. Cancer 1995; 76(4): 687-94.
[http://dx.doi.org/10.1002/1097-0142(19950815)76:4<687::AID-CNCR2820760424>3.0.CO;2-M] [PMID: 8625167]
[73]
Ptacin JL, Caffaro CE, Ma L, et al. An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism. Nat Commun 2021; 12(1): 4785.
[http://dx.doi.org/10.1038/s41467-021-24987-9] [PMID: 34373459]
[74]
Milla ME, Ptacin JL, Ma L, et al. THOR-707, a novel not-alpha IL-2, promotes all key immune system anti-tumoral actions of IL-2 without eliciting vascular leak syndrome (VLS). Ann Oncol 2019; 30: v501.
[http://dx.doi.org/10.1093/annonc/mdz253.051]
[75]
Janku F, Abdul-Karim R, Azad A, Bendell J, Falchook G, Gan HK. Abstract LB041: THOR-707 (SAR444245), a novel not-alpha IL-2 as monotherapy and in combination with pembrolizumab in advanced/metastatic solid tumors: Interim results from hammer, an open-label, multicenter phase 1/2 study. Cancer Res 2021; 81(S13): LB041-LB.
[76]
Falchook G, Gan H, Fu S, McKean M, Azad A, Sommerhalder D. 481 Phase 1/2 study of THOR-707 (SAR444245), a pegylated recombinant non-alpha IL-2, as monotherapy and in combination with pembrolizumab or cetuximab in patients (pts) with advanced solid tumors. JITC 2021; 9(S2).
[http://dx.doi.org/10.1136/jitc-2021-SITC2021.481]
[77]
Janku F, Abdul-Karim R, Azad A, et al. Preliminary results from an open-label, multicenter phase 1/2 dose escalation and expansion study of THOR-707, a novel not-Alpha IL-2, as a single agent in adult subjects with advanced or metastatic solid tumors. Eur J Cancer 2020; 138: S11.
[http://dx.doi.org/10.1016/S0959-8049(20)31094-7]
[78]
Charych D, Khalili S, Dixit V, et al. Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. PLoS One 2017; 12(7): e0179431.
[http://dx.doi.org/10.1371/journal.pone.0179431] [PMID: 28678791]
[79]
Diab A, Tykodi SS, Daniels GA, et al. Bempegaldesleukin plus nivolumab in first-line metastatic melanoma. J Clin Oncol 2021; 39(26): 2914-25.
[http://dx.doi.org/10.1200/JCO.21.00675] [PMID: 34255535]

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