Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 12840 Prague Czech Republic

Institute of Macromolecular Chemistry Czech Academy of Sciences Heyrovského nám 2 16206 Prague Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic

Zobrazit více v PubMed

Vesely M.D., Kershaw M.H., Schreiber R.D., Smyth M.J. Natural Innate and Adaptive Immunity to Cancer. Annu. Rev. Immunol. 2011;29:235–271. doi: 10.1146/annurev-immunol-031210-101324. PubMed DOI

Dunn G.P., Bruce A.T., Ikeda H., Old L.J., Schreiber R.D. Cancer immunoediting: From immunosurveillance to tumor escape. Nat. Immunol. 2002;3:991–998. doi: 10.1038/ni1102-991. PubMed DOI

Smyth M.J., Dunn G.P., Schreiber R.D. Cancer Immunosurveillance and Immunoediting: The Roles of Immunity in Sup-pressing Tumor Development and Shaping Tumor Immunogenicity. Adv. Immunol. 2006;90:1–50. doi: 10.1016/s0065-2776(06)90001-7. PubMed DOI

Aguilar O.A., Mesci A., Ma J., Chen P., Kirkham C.L., Hundrieser J., Voigt S., Allan D.S., Carlyle J.R. Modulation of Clr Ligand Expression and NKR-P1 Receptor Function during Murine Cytomegalovirus Infection. J. Innate Immun. 2015;7:584–600. doi: 10.1159/000382032. PubMed DOI PMC

Chen D.S., Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541:321–330. doi: 10.1038/nature21349. PubMed DOI

Zitvogel L., Tesniere A., Kroemer G. Cancer despite immunosurveillance: Immunoselection and immunosubversion. Nat. Rev. Immunol. 2006;6:715–727. doi: 10.1038/nri1936. PubMed DOI

Liu Y., Cao X. Immunosuppressive cells in tumor immune escape and metastasis. J. Mol. Med. 2015;94:509–522. doi: 10.1007/s00109-015-1376-x. PubMed DOI

Dunn G.P., Old L.J., Schreiber R.D. The Three Es of Cancer Immunoediting. Annu. Rev. Immunol. 2004;22:329–360. doi: 10.1146/annurev.immunol.22.012703.104803. PubMed DOI

Pascal V., Schleinitz N., Brunet C., Ravet S., Bonnet E., Lafarge X., Touinssi M., Reviron D., Viallard J.F., Moreau J.F., et al. Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions. Eur. J. Immunol. 2004;34:2930–2940. doi: 10.1002/eji.200425146. PubMed DOI

Bryceson Y.T., Björkström N.K., Mjösberg J., Ljunggren H. The Autoimmune Diseases. Elsevier; Amsterdam, The Netherlands: 2020. Natural Killer Cells; pp. 229–242.

Morvan M., Lanier L.L. NK cells and cancer: You can teach innate cells new tricks. Nat. Rev. Cancer. 2015;16:7–19. doi: 10.1038/nrc.2015.5. PubMed DOI

Shimasaki N., Jain A., Campana D. NK cells for cancer immunotherapy. Nat. Rev. Drug Discov. 2020;19:200–218. doi: 10.1038/s41573-019-0052-1. PubMed DOI

Hu W., Wang G., Huang D., Sui M., Xu Y. Cancer Immunotherapy Based on Natural Killer Cells: Current Progress and New Opportunities. Front. Immunol. 2019;10:1205. doi: 10.3389/fimmu.2019.01205. PubMed DOI PMC

Habif G., Crinier A., André P., Vivier E., Narni-Mancinelli E. Targeting natural killer cells in solid tumors. Cell. Mol. Immunol. 2019;16:415–422. doi: 10.1038/s41423-019-0224-2. PubMed DOI PMC

Farkona S., Diamandis E.P., Blasutig I.M. Cancer immunotherapy: The beginning of the end of cancer? BMC Med. 2016;14:73. doi: 10.1186/s12916-016-0623-5. PubMed DOI PMC

Vivier E., Ugolini S., Blaise D., Chabannon C., Brossay L. Targeting natural killer cells and natural killer T cells in cancer. Nat. Rev. Immunol. 2012;12:239–252. doi: 10.1038/nri3174. PubMed DOI PMC

Chauhan S.K.S., Koehl U., Kloess S. Harnessing NK Cell Checkpoint-Modulating Immunotherapies. Cancers. 2020;12:1807. doi: 10.3390/cancers12071807. PubMed DOI PMC

Dahlberg C.I.M., Sarhan D., Chrobok M., Duru A., Alici E. Natural Killer Cell-Based Therapies Targeting Cancer: Possible Strategies to Gain and Sustain Anti-Tumor Activity. Front. Immunol. 2015;6:605. doi: 10.3389/fimmu.2015.00605. PubMed DOI PMC

Pfefferle A., Huntington N.D. You Have Got a Fast CAR: Chimeric Antigen Receptor NK Cells in Cancer Therapy. Cancers. 2020;12:706. doi: 10.3390/cancers12030706. PubMed DOI PMC

Liu E., Marin D., Banerjee P., Macapinlac H.A., Thompson P., Basar R., Kerbauy L.N., Overman B., Thall P., Kaplan M., et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N. Engl. J. Med. 2020;382:545–553. doi: 10.1056/NEJMoa1910607. PubMed DOI PMC

Nigro C.L., Macagno M., Sangiolo D., Bertolaccini L., Aglietta M., Merlano M.C. NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: Biological evidence and clinical perspectives. Ann. Transl. Med. 2019;7:105. doi: 10.21037/atm.2019.01.42. PubMed DOI PMC

Tay S.S., Carol H., Biro M. TriKEs and BiKEs join CARs on the cancer immunotherapy highway. Hum. Vaccines Immunother. 2016;12:2790–2796. doi: 10.1080/21645515.2016.1198455. PubMed DOI PMC

Gleason M.K., Ross J.A., Warlick E.D., Lund T.C., Verneris M.R., Wiernik A., Spellman S., Haagenson M.D., Lenvik A.J., Litzow M.R., et al. CD16xCD33 bispecific killer cell engager (BiKE) activates NK cells against primary MDS and MDSC CD33+ targets. Blood. 2014;123:3016–3026. doi: 10.1182/blood-2013-10-533398. PubMed DOI PMC

Felices M., Lenvik T.R., Davis Z.B., Miller J.S., Vallera D.A. Natural Killer Cells. Volume 1441. Humana Press; New York, NY, USA: 2016. Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells; pp. 333–346. PubMed DOI PMC

Sarhan D., Brandt L., Felices M., Guldevall K., Lenvik T., Hinderlie P., Curtsinger J., Warlick E., Spellman S.R., Blazar B.R., et al. 161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS. Blood Adv. 2018;2:1459–1469. doi: 10.1182/bloodadvances.2017012369. PubMed DOI PMC

Märklin M., Hagelstein I., Koerner S.P., Rothfelder K., Pfluegler M.S., Schumacher A., Grosse-Hovest L., Jung G., Salih H.R. Bispecific NKG2D-CD3 and NKG2D-CD16 fusion proteins for induction of NK and T cell reactivity against acute myeloid leukemia. J. Immunother. Cancer. 2019;7:143. doi: 10.1186/s40425-019-0606-0. PubMed DOI PMC

Han Y., Sun F., Zhang X., Wang T., Jiang J., Cai J., Gao Q., Hezam K., Liu Y., Xie J., et al. CD24 targeting bi-specific antibody that simultaneously stimulates NKG2D enhances the efficacy of cancer immunotherapy. J. Cancer Res. Clin. Oncol. 2019;145:1179–1190. doi: 10.1007/s00432-019-02865-8. PubMed DOI

Chester C., Fritsch K., Kohrt H.E. Natural Killer Cell Immunomodulation: Targeting Activating, Inhibitory, and Co-stimulatory Receptor Signaling for Cancer Immunotherapy. Front. Immunol. 2015;6:601. doi: 10.3389/fimmu.2015.00601. PubMed DOI PMC

Koch J., Steinle A., Watzl C., Mandelboim O. Activating natural cytotoxicity receptors of natural killer cells in cancer and infection. Trends Immunol. 2013;34:182–191. doi: 10.1016/j.it.2013.01.003. PubMed DOI

Memmer S., Weil S., Beyer S., Zöller T., Peters E., Hartmann J., Steinle A., Koch J. The Stalk Domain of NKp30 Contributes to Ligand Binding and Signaling of a Preassembled NKp30-CD3ζ Complex. J. Biol. Chem. 2016;291:25427–25438. doi: 10.1074/jbc.M116.742981. PubMed DOI PMC

Brandt C.S., Baratin M., Yi E.C., Kennedy J., Gao Z., Fox B., Haldeman B., Ostrander C.D., Kaifu T., Chabannon C., et al. The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans. J. Exp. Med. 2009;206:1495–1503. doi: 10.1084/jem.20090681. PubMed DOI PMC

Binici J., Koch J. BAG-6, a jack of all trades in health and disease. Cell. Mol. Life Sci. 2013;71:1829–1837. doi: 10.1007/s00018-013-1522-y. PubMed DOI PMC

Kruse P.H., Matta J., Ugolini S., Vivier E. Natural cytotoxicity receptors and their ligands. Immunol. Cell Biol. 2013;92:221–229. doi: 10.1038/icb.2013.98. PubMed DOI

Wang W., Guo H., Geng J., Zheng X., Wei H., Sun R., Tian Z. Tumor-released Galectin-3, a Soluble Inhibitory Ligand of Human NKp30, Plays an Important Role in Tumor Escape from NK Cell Attack. J. Biol. Chem. 2014;289:33311–33319. doi: 10.1074/jbc.M114.603464. PubMed DOI PMC

Kaifu T., Escalière B., Gastinel L.N., Vivier E., Baratin M. B7-H6/NKp30 interaction: A mechanism of alerting NK cells against tumors. Cell. Mol. Life Sci. 2011;68:3531–3539. doi: 10.1007/s00018-011-0802-7. PubMed DOI PMC

Li Y., Wang Q., Mariuzza R.A. Structure of the human activating natural cytotoxicity receptor NKp30 bound to its tumor cell ligand B7-H6. J. Exp. Med. 2011;208:703–714. doi: 10.1084/jem.20102548. PubMed DOI PMC

Skořepa O., Pazicky S., Kalousková B., Bláha J., Abreu C., Ječmen T., Rosůlek M., Fish A., Sedivy A., Harlos K., et al. Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation. Cancers. 2020;12:1998. doi: 10.3390/cancers12071998. PubMed DOI PMC

Xu X., Narni-Mancinelli E., Cantoni C., Li Y., Guia S., Gauthier L., Chen Q., Moretta A., Vély F., Eisenstein E., et al. Structural Insights into the Inhibitory Mechanism of an Antibody against B7-H6, a Stress-Induced Cellular Ligand for the Natural Killer Cell Receptor NKp30. J. Mol. Biol. 2016;428:4457–4466. doi: 10.1016/j.jmb.2016.09.011. PubMed DOI

Arnon T.I., Markel G., Bar-Ilan A., Hanna J., Fima E., Benchetrit F., Galili R., Cerwenka A., Benharroch D., Sion-Vardy N., et al. Harnessing Soluble NK Cell Killer Receptors for the Generation of Novel Cancer Immune Therapy. PLoS ONE. 2008;3:e2150. doi: 10.1371/annotation/eaf9794a-9325-4977-952d-51285c3f6c6a. PubMed DOI PMC

Kellner C., Maurer T., Hallack D., Repp R., Van De Winkel J.G.J., Parren P.W.H.I., Valerius T., Humpe A., Gramatzki M., Peipp M., et al. Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity. J. Immunol. 2012;189:5037–5046. doi: 10.4049/jimmunol.1201321. PubMed DOI

Kellner C., Günther A., Humpe A., Repp R., Klausz K., Derer S., Valerius T., Ritgen M., Brüggemann M., Van De Winkel J.G., et al. Enhancing natural killer cell-mediated lysis of lymphoma cells by combining therapeutic antibodies with CD20-specific immunoligands engaging NKG2D or NKp30. OncoImmunology. 2015;5:e1058459. doi: 10.1080/2162402X.2015.1058459. PubMed DOI PMC

Peipp M., Derer S., Lohse S., Staudinger M., Klausz K., Valerius T., Gramatzki M., Kellner C. HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell-mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity. Oncotarget. 2015;6:32075–32088. doi: 10.18632/oncotarget.5135. PubMed DOI PMC

Zhang T., Wu M.-R., Sentman C.L. An NKp30-Based Chimeric Antigen Receptor Promotes T Cell Effector Functions and Antitumor Efficacy In Vivo. J. Immunol. 2012;189:2290–2299. doi: 10.4049/jimmunol.1103495. PubMed DOI PMC

Wu M.-R., Zhang T., Demars L.R., Sentman C.L. B7H6-specific chimeric antigen receptors lead to tumor elimination and host antitumor immunity. Gene Ther. 2015;22:675–684. doi: 10.1038/gt.2015.29. PubMed DOI PMC

Hua C.K., Gacerez A.T., Sentman C.L., Ackerman M.E. Development of unique cytotoxic chimeric antigen receptors based on human scFv targeting B7H6. Protein Eng. Des. Sel. 2017;30:713–721. doi: 10.1093/protein/gzx051. PubMed DOI PMC

Vaněk O., Nálezková M., Kavan D., Borovičková I., Pompach P., Novák P., Kumar V., Vannucci L., Hudeček J., Hofbauerová K., et al. Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice. FEBS J. 2008;275:5589–5606. doi: 10.1111/j.1742-4658.2008.06683.x. PubMed DOI

Kolenko P., Skálová T., Vaněk O., Štěpánková A., Dušková J., Hašek J., Bezouška K., Dohnálek J. The high-resolution structure of the extracellular domain of human CD69 using a novel polymer. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2009;65:1258–1260. doi: 10.1107/S1744309109043152. PubMed DOI PMC

Deming T.J., Klok H.-A., Armes S.P., Becker M.L., Champion J.A., Chen E.Y.-X., Heilshorn S.C., van Hest J.C.M., Irvine D.J., Johnson J.A., et al. Polymers at the Interface with Biology. Biomacromolecules. 2018;19:3151–3162. doi: 10.1021/acs.biomac.8b01029. PubMed DOI

Jo S.D., Nam G.-H., Kwak G., Yang Y., Kwon I.C. Harnessing designed nanoparticles: Current strategies and future perspectives in cancer immunotherapy. Nano Today. 2017;17:23–37. doi: 10.1016/j.nantod.2017.10.008. DOI

Aikins M.E., Xu C., Moon J.J. Engineered Nanoparticles for Cancer Vaccination and Immunotherapy. Accounts Chem. Res. 2020;53:2094–2105. doi: 10.1021/acs.accounts.0c00456. PubMed DOI PMC

Thangam R., Patel K.D., Kang H., Paulmurugan R. Advances in Engineered Polymer Nanoparticle Tracking Platforms towards Cancer Immunotherapy—Current Status and Future Perspectives. Vaccines. 2021;9:935. doi: 10.3390/vaccines9080935. PubMed DOI PMC

Janisova L., Gruzinov A., Zaborova O.V., Velychkivska N., Vaněk O., Chytil P., Etrych T., Janoušková O., Zhang X., Blanchet C., et al. Molecular Mechanisms of the Interactions of N-(2-Hydroxypropyl)methacrylamide Copolymers Designed for Cancer Therapy with Blood Plasma Proteins. Pharmaceutics. 2020;12:106. doi: 10.3390/pharmaceutics12020106. PubMed DOI PMC

Apostolovic B., Danial M., Klok H.-A. Coiled coils: Attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials. Chem. Soc. Rev. 2010;39:3541–3575. doi: 10.1039/b914339b. PubMed DOI

Utterström J., Naeimipour S., Selegård R., Aili D. Coiled coil-based therapeutics and drug delivery systems. Adv. Drug Deliv. Rev. 2020;170:26–43. doi: 10.1016/j.addr.2020.12.012. PubMed DOI

Pola R., Laga R., Ulbrich K., Sieglová I., Král V., Fábry M., Kabešová M., Kovář M., Pechar M. Polymer Therapeutics with a Coiled Coil Motif Targeted against Murine BCL1 Leukemia. Biomacromolecules. 2013;14:881–889. doi: 10.1021/bm3019592. PubMed DOI

Wu K., Liu J., Johnson R.N., Yang J., Kopeček J. Drug-Free Macromolecular Therapeutics: Induction of Apoptosis by Coiled-Coil-Mediated Cross-Linking of Antigens on the Cell Surface. Angew. Chem. Int. Ed. 2010;49:1451–1455. doi: 10.1002/anie.200906232. PubMed DOI PMC

Wu K., Yang J., Liu J., Kopeček J. Coiled-coil based drug-free macromolecular therapeutics: In vivo efficacy. J. Control. Release. 2012;157:126–131. doi: 10.1016/j.jconrel.2011.08.002. PubMed DOI PMC

Chu T.-W., Yang J., Zhang R., Sima M., Kopeček J. Cell Surface Self-Assembly of Hybrid Nanoconjugates via Oligonucleotide Hybridization Induces Apoptosis. ACS Nano. 2014;8:719–730. doi: 10.1021/nn4053827. PubMed DOI PMC

Chu T.-W., Zhang R., Yang J., Chao M.P., Shami P.J., Kopeček J. A Two-Step Pretargeted Nanotherapy for CD20 Cross-linking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab. Theranostics. 2015;5:834–846. doi: 10.7150/thno.12040. PubMed DOI PMC

Zhang L., Fang Y., Yang J., Kopeček J. Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells. J. Control. Release. 2017;263:139–150. doi: 10.1016/j.jconrel.2016.12.025. PubMed DOI PMC

Zhang L., Fang Y., Li L., Yang J., Radford D.C., Kopeček J. Human Serum Albumin-Based Drug-Free Macromolecular Therapeutics: Apoptosis Induction by Coiled-Coil-Mediated Cross-Linking of CD20 Antigens on Lymphoma B Cell Surface. Macromol. Biosci. 2018;18:e1800224. doi: 10.1002/mabi.201800224. PubMed DOI PMC

Gambles M.T., Li J., Wang J., Sborov D., Yang J., Kopeček J. Crosslinking of CD38 Receptors Triggers Apoptosis of Malignant B Cells. Molecules. 2021;26:4658. doi: 10.3390/molecules26154658. PubMed DOI PMC

Pastorekova S., Gillies R.J. The role of carbonic anhydrase IX in cancer development: Links to hypoxia, acidosis, and beyond. Cancer Metastasis Rev. 2019;38:65–77. doi: 10.1007/s10555-019-09799-0. PubMed DOI PMC

Pechar M., Pola R., Laga R., Ulbrich K., Bednárová L., Maloň P., Sieglová I., Král V., Fábry M., Vaněk O. Coiled Coil Peptides as Universal Linkers for the Attachment of Recombinant Proteins to Polymer Therapeutics. Biomacromolecules. 2011;12:3645–3655. doi: 10.1021/bm200897b. PubMed DOI

Kissel M., Peschke P., Šubr V., Ulbrich K., Strunz A.M., Kühnlein R., Debus J., Friedrich E. Detection and cellular localisation of the synthetic soluble macromolecular drug carrier pHPMA. Eur. J. Nucl. Med. Mol. Imaging. 2002;29:1055–1062. doi: 10.1007/s00259-002-0835-0. PubMed DOI

Aricescu A.R., Lu W., Jones E.Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006;62:1243–1250. doi: 10.1107/S0907444906029799. PubMed DOI

Vaněk O., Celadova P., Skořepa O., Bláha J., Kalousková B., Dvorská A., Poláchová E., Pucholtová H., Kavan D., Pompach P., et al. Production of recombinant soluble dimeric C-type lectin-like receptors of rat natural killer cells. Sci. Rep. 2019;9:17836. doi: 10.1038/s41598-019-52114-8. PubMed DOI PMC

Csaderova L., Debreova M., Radvak P., Stano M., Vrestiakova M., Kopacek J., Pastorekova S., Svastova E. The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration. Front. Physiol. 2013;4:271. doi: 10.3389/fphys.2013.00271. PubMed DOI PMC

Bláha J., Pachl P., Novák P., Vaněk O. Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-density transfection of suspension adapted HEK293S GnTI— cells. Protein Expr. Purif. 2015;109:7–13. doi: 10.1016/j.pep.2015.01.006. PubMed DOI

Durocher Y. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002;30:E9. doi: 10.1093/nar/30.2.e9. PubMed DOI PMC

Pekar L., Klausz K., Busch M., Valldorf B., Kolmar H., Wesch D., Oberg H.-H., Krohn S., Boje A.S., Gehlert C.L., et al. Affinity Maturation of B7-H6 Translates into Enhanced NK Cell–Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement. J. Immunol. 2020;206:225–236. doi: 10.4049/jimmunol.2001004. PubMed DOI PMC

Scheuermann T.H., Brautigam C.A. High-precision, automated integration of multiple isothermal titration calorimetric thermograms: New features of NITPIC. Methods. 2014;76:87–98. doi: 10.1016/j.ymeth.2014.11.024. PubMed DOI PMC

Zhao H., Piszczek G., Schuck P. SEDPHAT—A platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods. 2015;76:137–148. doi: 10.1016/j.ymeth.2014.11.012. PubMed DOI PMC

Brautigam C.A. Calculations and Publication-Quality Illustrations for Analytical Ultracentrifugation Data. Methods Enzym. 2015;562:109–133. doi: 10.1016/bs.mie.2015.05.001. PubMed DOI

Rozbeský D., Kavan D., Chmelík J., Novák P., Vaněk O., Bezouška K. High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli. Protein Expr. Purif. 2011;77:178–184. doi: 10.1016/j.pep.2011.01.013. PubMed DOI

Schuck P. Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophys. J. 2000;78:1606–1619. doi: 10.1016/S0006-3495(00)76713-0. PubMed DOI PMC

Skálová T., Kotýnková K., Dušková J., Hašek J., Koval’ T., Kolenko P., Novák P., Man P., Hanč P., Vaněk O., et al. Mouse Clr-g, a Ligand for NK Cell Activation Receptor NKR-P1F: Crystal Structure and Biophysical Properties. J. Immunol. 2012;189:4881–4889. doi: 10.4049/jimmunol.1200880. PubMed DOI

Skálová T., Bláha J., Harlos K., Dušková J., Koval’ T., Stránský J., Hašek J., Vaněk O., Dohnálek J. Four crystal structures of human LLT1, a ligand of human NKR-P1, in varied glycosylation and oligomerization states. Acta Crystallogr. Sect. D Biol. Crystallogr. 2015;71:578–591. doi: 10.1107/S1399004714027928. PubMed DOI PMC

Joyce M.G., Tran P., Zhuravleva M.A., Jaw J., Colonna M., Sun P.D. Crystal structure of human natural cytotoxicity receptor NKp30 and identification of its ligand binding site. Proc. Natl. Acad. Sci. USA. 2011;108:6223–6228. doi: 10.1073/pnas.1100622108. PubMed DOI PMC

Bláha J., Kalousková B., Skořepa O., Pažický S., Novák P., Vaněk O. High-level expression and purification of soluble form of human natural killer cell receptor NKR-P1 in HEK293S GnTI— Cells. Protein Expr. Purif. 2017;140:36–43. doi: 10.1016/j.pep.2017.07.016. PubMed DOI

Herrmann J., Berberich H., Hartmann J., Beyer S., Davies K., Koch J. Homo-oligomerization of the Activating Natural Killer Cell Receptor NKp30 Ectodomain Increases Its Binding Affinity for Cellular Ligands. J. Biol. Chem. 2014;289:765–777. doi: 10.1074/jbc.M113.514786. PubMed DOI PMC

Pechar M., Pola R., Laga R., Braunova A., Filippov S.K., Bogomolova A., Bednarova L., Vanek O., Ulbrich K. Coiled coil peptides and polymer-peptide conjugates: Synthesis, self-assembly, characterization and potential in drug delivery systems. Biomacromolecules. 2014;15:2590–2599. doi: 10.1021/bm500436p. PubMed DOI

Matta J., Baratin M., Chiche L., Forel J.M., Cognet C., Thomas G., Farnarier C., Piperoglou C., Papazian L., Chaussabel D., et al. Induction of B7-H6, a ligand for the natural killer cell-activating receptor NKp30, in inflammatory conditions. Blood. 2013;122:394–404. doi: 10.1182/blood-2013-01-481705. PubMed DOI

Wykoff C.C., Beasley N.J., Watson P., Turner K.J., Pastorek J., Sibtain A., Wilson G., Turley H., Talks K.L., Maxwell P., et al. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res. 2000;60:7075–7083. PubMed

Sowa T., Menju T., Chen-Yoshikawa T.F., Takahashi K., Nishikawa S., Nakanishi T., Shikuma K., Motoyama H., Hijiya K., Aoyama A., et al. Hypoxia-inducible factor 1 promotes chemoresistance of lung cancer by inducing carbonic anhydrase IX expression. Cancer Med. 2016;6:288–297. doi: 10.1002/cam4.991. PubMed DOI PMC

Ulbrich K., Šubr V., Strohalm J., Plocová D., Jelínková M., Říhová B. Polymeric Drugs Based on Conjugates of Synthetic and Natural Macromolecules. I. Synthesis and Physico-Chemical Characterisation. J. Control. Release. 2000;64:63–79. doi: 10.1016/S0168-3659(99)00141-8. PubMed DOI

Pola R., Král V., Filippov S.K., Kaberov L., Etrych T., Sieglová I., Sedláček J., Fábry M., Pechar M. Polymer Cancerostatics Targeted by Recombinant Antibody Fragments to Gd2-Positive Tumor Cells. Biomacromolecules. 2019;20:412–421. doi: 10.1021/acs.biomac.8b01616. PubMed DOI

Perrier S., Takolpuckdee P., Westwood J., Lewis D.M. Versatile Chain Transfer Agents for Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerization to Synthesize Functional Polymeric Architectures. Macromolecules. 2004;37:2709–2717. doi: 10.1021/ma035468b. DOI

Green N.M. A Spectrophotometric Assay for Avidin and Biotin Based on Binding of Dyes by Avidin. Biochem. J. 1965;94:23C–24C. doi: 10.1042/bj0940023C. PubMed DOI

Wood C.W., Woolfson D.N. CCBuilder 2.0: Powerful and accessible coiled-coil modeling. Protein Sci. 2018;27:103–111. doi: 10.1002/pro.3279. PubMed DOI PMC