BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Hněvotínská 3 779 00 Olomouc Czech Republic

Department of Immunology University Hospital Olomouc Zdravotníků 248 7 77900 Olomouc Czech Republic

Institute of Clinical and Molecular Pathology Faculty of Medicine and Dentistry Palacky University Olomouc Hněvotínská 3 779 00 Olomouc Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry and Czech Advanced Technology and Research Institute Palacky University Olomouc Hněvotínská 5 779 00 Olomouc Czech Republic

Laboratory of Ligand Engineering Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Research Center Průmyslová 595 252 50 Vestec Czech Republic

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Research Center Průmyslová 595 252 50 Vestec Czech Republic

Zobrazit více v PubMed

Wykes MN, Lewin SR. Immune checkpoint blockade in infectious diseases. Nat Rev Immunol. 2018;18(2):91–104. doi: 10.1038/nri.2017.112. PubMed DOI PMC

Wherry EJ. T cell exhaustion. Nat Immunol. 2011;12(6):492–499. doi: 10.1038/ni.2035. PubMed DOI

Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12(4):252–264. doi: 10.1038/nrc3239. PubMed DOI PMC

He X, Xu C. Immune checkpoint signaling and cancer immunotherapy. Cell Res. 2020;30(8):660–669. doi: 10.1038/s41422-020-0343-4. PubMed DOI PMC

Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of Pd-1, a novel member of the immunoglobulin gene superfamily. Upon Programmed Cell-Death Embo J. 1992;11(11):3887–3895. PubMed PMC

Sharpe AH, Pauken KE. The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol. 2018;18(3):153–167. doi: 10.1038/nri.2017.108. PubMed DOI

Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol. 2004;173(2):945–954. doi: 10.4049/jimmunol.173.2.945. PubMed DOI

Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 2002;99(19):12293–12297. doi: 10.1073/pnas.192461099. PubMed DOI PMC

Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439(7077):682–687. doi: 10.1038/nature04444. PubMed DOI

Antonia SJ, Kim SW, Spira AI, Ahn MJ, Ou SHI, Stjepanovic N, Fasolo A, Jagert D, Ottt PA, Wainberg ZA, et al. Safety and clinical activity of durvalumab (MEDI4736), an anti-PD-L1 antibody, in treatment-naive patients with advanced non small-cell lung cancer. J Clin Oncol. 2016;34(15):9029. doi: 10.1200/JCO.2016.34.15_suppl.9029. DOI

Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, Chow LQ, Vokes EE, Felip E, Holgado E, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373(17):1627–1639. doi: 10.1056/NEJMoa1507643. PubMed DOI PMC

Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, Daud A, Carlino MS, McNeil C, Lotem M. Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med. 2015;372(26):2521–2532. doi: 10.1056/NEJMoa1503093. PubMed DOI

Brahmer JR, Tykodi SS, Chow LQM, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366(26):2455–2465. doi: 10.1056/NEJMoa1200694. PubMed DOI PMC

Maute RL, Gordon SR, Mayer AT, McCracken MN, Natarajan A, Ring NG, Kimura R, Tsai JM, Manglik A, Kruse AC, et al. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proc Natl Acad Sci USA. 2015;112(47):E6506–6514. doi: 10.1073/pnas.1519623112. PubMed DOI PMC

Nishi W, Wakamatsu E, Machiyama H, Matsushima R, Saito K, Yoshida Y, Nishikawa T, Takehara T, Toyota H, Furuhata M, et al. Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters. Nat Commun. 2023;14(1):3157. doi: 10.1038/s41467-023-38512-7. PubMed DOI PMC

Ninomiya K, Hotta K. Pembrolizumab for the first-line treatment of non-small cell lung cancer. Expert Opin Biol Th. 2018;18(10):1015–1021. doi: 10.1080/14712598.2018.1522300. PubMed DOI

Antonia SJ, Villegas A, Daniel D, Vicente D, Murakami S, Hui R, Yokoi T, Chiappori A, Lee KH, de Wit M, et al. Durvalumab after chemoradiotherapy in stage III non-small-cell lung cancer. N Engl J Med. 2017;377(20):1919–1929. doi: 10.1056/NEJMoa1709937. PubMed DOI

Rittmeyer A, Barlesi F, Waterkamp D, Park K, Ciardiello F, von Pawel J, Gadgeel SM, Hida T, Kowalski DM, Dols MC, et al. Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial. Lancet. 2017;389(10066):255–265. doi: 10.1016/S0140-6736(16)32517-X. PubMed DOI PMC

Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E, Antonia S, Pluzanski A, Vokes EE, Holgado E, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med. 2015;373(2):123–135. doi: 10.1056/NEJMoa1504627. PubMed DOI PMC

Chen PL, Roh W, Reuben A, Cooper ZA, Spencer CN, Prieto PA, Miller JP, Bassett RL, Gopalakrishnan V, Wani K, et al. Analysis of immune signatures in longitudinal tumor samples yields insight into biomarkers of response and mechanisms of resistance to immune checkpoint blockade. Cancer Discov. 2016;6(8):827–837. doi: 10.1158/2159-8290.CD-15-1545. PubMed DOI PMC

Camidge DR, Doebele RC, Kerr KM. Comparing and contrasting predictive biomarkers for immunotherapy and targeted therapy of NSCLC. Nat Rev Clin Oncol. 2019;16(6):341–355. doi: 10.1038/s41571-019-0173-9. PubMed DOI

Hummelink K, van der Noort V, Muller M, Schouten RD, Lalezari F, Peters D, Theelen WSME, Koelzer VH, Mertz KD, Zippelius A, et al. PD-1T TILs as a predictive biomarker for clinical benefit to PD-1 blockade in patients with advanced NSCLC. Clin Cancer Res. 2022;28(22):4893–4906. doi: 10.1158/1078-0432.CCR-22-0992. PubMed DOI PMC

Uryvaev A, Passhak M, Hershkovits D, Sabo E, Bar-Sela G. The role of tumor-infiltrating lymphocytes (TILs) as a predictive biomarker of response to anti-PD1 therapy in patients with metastatic non-small cell lung cancer or metastatic melanoma. Med Oncol. 2018;35(3):25. doi: 10.1007/s12032-018-1080-0. PubMed DOI

Kaira K, Yamaguchi O, Kawasaki T, Hashimoto K, Miura Y, Shiono A, Mouri A, Imai H, Kobayashi K, Yasuda M, et al. Prognostic significance of tumor infiltrating lymphocytes on first-line pembrolizumab efficacy in advanced non-small cell lung cancer. Discov Oncol. 2023;14(1):6. doi: 10.1007/s12672-023-00615-4. PubMed DOI PMC

Niemeijer AN, Leung D, Huisman MC, Bahce I, Hoekstra OS, van Dongen G, Boellaard R, Du S, Hayes W, Smith R, et al. Whole body PD-1 and PD-L1 positron emission tomography in patients with non-small-cell lung cancer. Nat Commun. 2018;9(1):4664. doi: 10.1038/s41467-018-07131-y. PubMed DOI PMC

Ryman JT, Meibohm B. Pharmacokinetics of monoclonal antibodies. CPT Pharmacometrics Syst Pharmacol. 2017;6(9):576–588. doi: 10.1002/psp4.12224. PubMed DOI PMC

Lee CM, Tannock IF. The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors. BMC Cancer. 2010;10:255. doi: 10.1186/1471-2407-10-255. PubMed DOI PMC

Wu F, Fan J, He Y, Xiong A, Yu J, Li Y, Zhang Y, Zhao W, Zhou F, Li W, et al. Single-cell profiling of tumor heterogeneity and the microenvironment in advanced non-small cell lung cancer. Nat Commun. 2021;12(1):2540. doi: 10.1038/s41467-021-22801-0. PubMed DOI PMC

Karamitopoulou-Diamantis E, Andreou A, Perren A, Gloor B. Spatial immunoarchitectural intra-tumor heterogeneity is associated with early tumor recurrence in pancreatic cancer. Lab Invest. 2021;101(Suppl 1):1047–1048.

Francis DM, Thomas SN. Progress and opportunities for enhancing the delivery and efficacy of checkpoint inhibitors for cancer immunotherapy. Adv Drug Deliv Rev. 2017;114:33–42. doi: 10.1016/j.addr.2017.04.011. PubMed DOI PMC

Deng R, Bumbaca D, Pastuskovas CV, Boswell CA, West D, Cowan KJ, Chiu H, McBride J, Johnson C, Xin Y, et al. Preclinical pharmacokinetics, pharmacodynamics, tissue distribution, and tumor penetration of anti-PD-L1 monoclonal antibody, an immune checkpoint inhibitor. Mabs-Austin. 2016;8(3):593–603. doi: 10.1080/19420862.2015.1136043. PubMed DOI PMC

Pinotsis N, Chatziefthimiou SD, Berkemeier F, Beuron F, Mavridis IM, Konarev PV, Svergun DI, Morris E, Rief M, Wilmanns M. Superhelical architecture of the myosin filament-linking protein myomesin with unusual elastic properties. PLoS Biol. 2012;10(2):e1001261. doi: 10.1371/journal.pbio.1001261. PubMed DOI PMC

Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005;33(Web Server issue):W382–388. doi: 10.1093/nar/gki387. PubMed DOI PMC

Kuchar M, Kosztyu P, Liskova VD, Cerny J, Petrokova H, Vroblova E, Maly M, Vankova L, Krupka M, Kafkova LR, et al. Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice. Virulence. 2021;12(1):1271–1287. doi: 10.1080/21505594.2021.1920251. PubMed DOI PMC

Sali A, Blundell TL. Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol. 1993;234(3):779–815. doi: 10.1006/jmbi.1993.1626. PubMed DOI

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Kozakov D, Beglov D, Bohnuud T, Mottarella SE, Xia B, Hall DR, Vajda S. How good is automated protein docking? Proteins. 2013;81(12):2159–2166. doi: 10.1002/prot.24403. PubMed DOI PMC

Kozakov D, Brenke R, Comeau SR, Vajda S. PIPER: an FFT-based protein docking program with pairwise potentials. Proteins. 2006;65(2):392–406. doi: 10.1002/prot.21117. PubMed DOI

Daniel Liskova V, Kosztyu P, Kuchar M, Cerny J, Bharadwaj S, Petrokova H, Vroblova E, Krupka M, Maly M, Zosincukova T, et al. Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies. Front Immunol. 2022;13:1066361. doi: 10.3389/fimmu.2022.1066361. PubMed DOI PMC

UniProt C. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019;47(D1):D506–D515. doi: 10.1093/nar/gky1049. PubMed DOI PMC

Islam S, Vick E, Huber B, Morales C, Spier C, Cooke L, Weterings E, Mahadevan D. Co-targeting aurora kinase with PD-L1 and PI3K abrogates immune checkpoint mediated proliferation in peripheral T-cell lymphoma: a novel therapeutic strategy. Oncotarget. 2017;8(59):100326–100338. doi: 10.18632/oncotarget.22222. PubMed DOI PMC

Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, Rosenberg SA. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009;114(8):1537–1544. doi: 10.1182/blood-2008-12-195792. PubMed DOI PMC

Han Y, Liu D, Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res. 2020;10(3):727–742. PubMed PMC

Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12(4):298–306. doi: 10.1038/nrc3245. PubMed DOI

Bruni D, Angell HK, Galon J. The immune contexture and Immunoscore in cancer prognosis and therapeutic efficacy. Nat Rev Cancer. 2020;20(11):662–680. doi: 10.1038/s41568-020-0285-7. PubMed DOI

Pages F, Mlecnik B, Marliot F, Bindea G, Ou FS, Bifulco C, Lugli A, Zlobec I, Rau TT, Berger MD, et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. Lancet. 2018;391(10135):2128–2139. doi: 10.1016/S0140-6736(18)30789-X. PubMed DOI

Havel JJ, Chowell D, Chan TA. The evolving landscape of biomarkers for checkpoint inhibitor immunotherapy. Nat Rev Cancer. 2019;19(3):133–150. doi: 10.1038/s41568-019-0116-x. PubMed DOI PMC

Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJM, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568. doi: 10.1038/nature13954. PubMed DOI PMC

Richards DA. Exploring alternative antibody scaffolds: antibody fragments and antibody mimics for targeted drug delivery. Drug Discov Today Technol. 2018;30:35–46. doi: 10.1016/j.ddtec.2018.10.005. PubMed DOI

Vazquez-Lombardi R, Phan TG, Zimmermann C, Lowe D, Jermutus L, Christ D. Challenges and opportunities for non-antibody scaffold drugs. Drug Discov Today. 2015;20(10):1271–1283. doi: 10.1016/j.drudis.2015.09.004. PubMed DOI

Son S, Park J, Seo H, Lee HT, Heo YS, Kim HS. A small-sized protein binder specific for human PD-1 effectively suppresses the tumour growth in tumour mouse model. J Drug Target. 2020;28(4):419–427. doi: 10.1080/1061186X.2019.1669042. PubMed DOI

Bryan CM, Rocklin GJ, Bick MJ, Ford A, Majri-Morrison S, Kroll AV, Miller CJ, Carter L, Goreshnik I, Kang A, et al. Computational design of a synthetic PD-1 agonist. Proc Natl Acad Sci U S A. 2021;118(29):e2102164118. doi: 10.1073/pnas.2102164118. PubMed DOI PMC

Liang Z, Tian Y, Cai W, Weng Z, Li Y, Zhang H, Bao Y, Li Y. High-affinity human PD-L1 variants attenuate the suppression of T cell activation. Oncotarget. 2017;8(51):88360–88375. doi: 10.18632/oncotarget.21729. PubMed DOI PMC

Lin X, Lu X, Luo G, Xiang H. Progress in PD-1/PD-L1 pathway inhibitors: from biomacromolecules to small molecules. Eur J Med Chem. 2020;186:111876. doi: 10.1016/j.ejmech.2019.111876. PubMed DOI

Wu Q, Jiang L, Li SC, He QJ, Yang B, Cao J. Small molecule inhibitors targeting the PD-1/PD-L1 signaling pathway. Acta Pharmacol Sin. 2021;42(1):1–9. doi: 10.1038/s41401-020-0366-x. PubMed DOI PMC

Cheng X, Veverka V, Radhakrishnan A, Waters LC, Muskett FW, Morgan SH, Huo J, Yu C, Evans EJ, Leslie AJ, et al. Structure and interactions of the human programmed cell death 1 receptor. J Biol Chem. 2013;288(17):11771–11785. doi: 10.1074/jbc.M112.448126. PubMed DOI PMC

Tang S, Kim PS. A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery. Proc Natl Acad Sci U S A. 2019;116(49):24500–24506. doi: 10.1073/pnas.1916916116. PubMed DOI PMC

Lin DY, Tanaka Y, Iwasaki M, Gittis AG, Su HP, Mikami B, Okazaki T, Honjo T, Minato N, Garboczi DN. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors. Proc Natl Acad Sci USA. 2008;105(8):3011–3016. doi: 10.1073/pnas.0712278105. PubMed DOI PMC

Latchman Y, Wood C, Chemova T, Iwai Y, Malenkovich N, Long A, Bourque K, Boussiotis V, Nishimura H, Honjo T, et al. PD-L2, a novel B7 homologue, is a second ligand for PD-1 and inhibits T cell activation. Faseb J. 2001;15(4):A345–A345.

Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, Fitz LJ, Malenkovich N, Okazaki T, Byrne MC, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192(7):1027–1034. doi: 10.1084/jem.192.7.1027. PubMed DOI PMC

Zhang X, Schwartz JC, Guo X, Bhatia S, Cao E, Lorenz M, Cammer M, Chen L, Zhang ZY, Edidin MA, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity. 2004;20(3):337–347. doi: 10.1016/S1074-7613(04)00051-2. PubMed DOI

Said EA, Dupuy FP, Trautmann L, Zhang Y, Shi Y, El-Far M, Hill BJ, Noto A, Ancuta P, Peretz Y, et al. Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection. Nat Med. 2010;16(4):452–459. doi: 10.1038/nm.2106. PubMed DOI PMC

Bally AP, Lu P, Tang Y, Austin JW, Scharer CD, Ahmed R, Boss JM. NF-κB regulates PD-1 expression in macrophages. J Immunol. 2015;194(9):4545–4554. doi: 10.4049/jimmunol.1402550. PubMed DOI PMC

Wen BY, Zhao L, Wang YC, Qiu CN, Xu ZM, Huang KL, Zhu H, Li ZM, Li HJ. Nanobodies targeting the interaction interface of programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-1/PD-L1) Prep Biochem Biotech. 2020;50(3):252–259. doi: 10.1080/10826068.2019.1692217. PubMed DOI

Miyazaki T, Aoki W, Koike N, Sato T, Ueda M. Application of peptide barcoding to obtain high-affinity anti-PD-1 nanobodies. J Biosci Bioeng. 2023;136(3):173–181. doi: 10.1016/j.jbiosc.2023.07.002. PubMed DOI

Zhang Y, Yang S, Jiang D, Li Y, Ma S, Wang L, Li G, Wang H, Zhang A, Xu G. Screening and identification of an anti-PD-1 nanobody with antitumor activity. Biosci Rep. 2022;43(1):BSR20221546. doi: 10.1042/BSR20221546. PubMed DOI PMC

Ding ZQ, Sun SY, Wang X, Yang XM, Shi W, Huang XN, Xie SX, Mo FZ, Hou XQ, Liu AQ, et al. Nanobody-based trispecific T cell engager (Nb-TriTE) enhances therapeutic efficacy by overcoming tumor-mediated immunosuppression. J Hematol Oncol. 2023;16(1):115. doi: 10.1186/s13045-023-01507-4. PubMed DOI PMC

Kosztyu P, Kuchar M, Cerny J, Barkocziova L, Maly M, Petrokova H, Czernekova L, Liskova V, Raskova Kafkova L, Knotigova P, et al. Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice. EBioMedicine. 2019;47:247–256. doi: 10.1016/j.ebiom.2019.07.015. PubMed DOI PMC

Smejkal J, Maly P, Kuchar M, Panova N, Semeradtova A, Aubrecht P, Stofik M, Maly J. Cell immunocapture microfluidic chip based on high-affinity recombinant protein binders. Biosens Bioelectron. 2021;172:112784. doi: 10.1016/j.bios.2020.112784. PubMed DOI

Frejd FY, Kim KT. Affibody molecules as engineered protein drugs. Exp Mol Med. 2017;49:e306. doi: 10.1038/emm.2017.35. PubMed DOI PMC