Laboratory of Ligand Engineering

Smal binding protein molecules with high specificity and affinity as an alternative to complex molecules of human antibodies.

Group leader: RNDr. Petr Malý, CSc.

RNDr. Petr Malý, CSc.Artificial binding proteins derived from small protein scaffolds represent a valuable alternative to commonly used antibodies. Novel binders with engineered affinity, high specificity or designed inhibitory function attract attention as key components for the development of novel biosensing devices, in vivo diagnostics and next-generation therapeutics. Small, robust, and soluble proteins with high thermal and hydrodynamic stability and without disulphide bonds are amenable to rational improvement and can be easily modified by gene-fusion approaches.

In our laboratory, which operates since 2008, we focus on engineering novel binders raised against human cytokines, their receptors or tumour markers. Recently we have established a model of three-helix bundle of albumin-binding domain (ABD) of streptococcal protein G as a master scaffold for the generation of high-complex combinatorial libraries. Using randomization of 11 pre-determined amino acid residues we engineered a library of a theoretical complexity of 1014 protein variants which was successfully used, in combination with ribosome display selection, for the generation of unique binders of human interferon gamma (hIFNg) with KD in the nanomolar range (Ahmad et al 2012). High-affinity binders of hIFNg were then used as key components for the construction of a novel SPR biosensor with the improved sensitivity for detection of hIFNg in diluted blood plasma (Šípová et al. 2012).

The assembled combinatorial library has been used in another project leading to the generation of inhibitory ABD variants targeting human IL-23 and its receptor (IL-23R), key elements of the Th17-mediated pro-inflammatory pathway. In this running project we have already identified a collection of ABD variants (REX binders) with sub- to nanomolar KD value for the binding of recombinant hIL-23R or IL-23R-Fc chimera. These so-called REX binders inhibit binding of p19 protein (a subunit of IL-23) to the IL-23 receptor in several arrangements of direct competition ELISA. We further demonstrate that REX variants bind to human cell lines K-562 and THP-1 and this binding correlates with IL-23R cell-surface expression. As binding of REX proteins to THP-1 cells can be substantially diminished by a high dose of the p19 protein, we conclude that we identified novel IL-23R-binding inhibitors that might be useful in designing novel anti-inflammatory tools. This is of special value as the molecular structure of the IL-23/IL-23R complex has not been described yet and, therefore, designing efficient inhibitors of IL-23 function with a promising therapeutic potential remains cumbersome. Currently, we have been working on the characterization of IL-23 binding ABD variants (ILP binders) and investigation of a possible immunomodulatory function of the most promising REX and ILP candidates.

In another part of our research activities, we closely collaborate with several Czech biotech companies in the project AFFIBINDER supported by the Ministry of Industry and Trade of the Czech Republic. Within this project, we develop a novel fluorescence-based screening method for large-scale identification of high-affinity binders raised against selected human oncomarkers. As a part of this project we develop novel high-affinity binders of human KLK2 and PSP94 prostate cancer biomarkers.

Bharadwaj Shiv
Daniel Lišková Veronika, Mgr., Ph.D.
Groza Yaroslava
Kadlčáková Petra
Kuchař Milan, Mgr., Ph.D.
Malá Linda, Mgr.
Malý Petr, RNDr., CSc.
Mierzwicka Joanna Maria
Panova Natalya
Petroková Hana, Ing., Ph.D.
Vaňková Lucie, Mgr., Ph.D.


  • Bharadwaj S, Groza Y, Mierzwicka JM, Malý P. Current understanding on TREM-2 molecular biology and physiopathological functions. Int Immunopharmacol. 2024 May 3;134:112042. doi: 10.1016/j.intimp.2024.112042. Epub ahead of print. PMID: 38703564.

  • Groza, Y., Lacina, L., Kuchař, M. et al. Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells. Cell Commun Signal 22, 261 (2024).

  • Mierzwicka, J.M., Petroková, H., Kafková, L.R. et al. "Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging." J Transl Med 22, 426 (2024).

  • Raskova-Kafkova, L., Mierzwicka, J.M., Chakraborty, P., Jakubec, P., Fischer, O., Skarda, J., Maly, P., Raska, M. (2024) "NSCLC: from tumorigenesis, immune checkpoint misuse to current and future targeted therapy" Front. Immunol. 15:1342086. doi: 10.3389/fimmu.2024.1342086


  • Bharadwaj S, Sahoo AK and Yadava U (2023), Editorial: Advances in the therapeutic targeting of human matrix metalloproteinases in health and disease. Front. Mol. Biosci. 10:1150474. doi:10.3389/fmolb.2023.1150474

  • Plavec T.V., Klemenčič K., Kuchař M., Malý P., Berlec A. (2023) “Secretion and surface display of binders of IL-23/IL-17 cytokines and their receptors in Lactococcus lactis as a therapeutic approach against inflammation”. European Journal of Pharmaceutical Sciences, 190, 106568.doi:10.1016/j.ejps.2023.106568

  • Song X., Kirtipal N., Lee S., Malý P., Bharadwaj S. (2023) “Current therapeutic targets and multifaceted physiological impacts of caffeine”.Phytotherapy Research. 1–41. DOI: 10.1002/ptr.8000


  • Daniel Lišková, V.,Kosztyu, P., Kuchar, M., Cerny, J., Bharadwaj, S., Petrokova, H., Vroblova, E., Krupka, M., Maly, M., Zosincukova, T., Sulc, J., Raskova Kafkova, L., Raska, M., Maly, P. (2022) "Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodiesas non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies" Front. Immunol. 13:1066361. doi:10.3389/fimmu.2022.1066361

  • Groza, Y., Jemelková, J., Raskova Kafkova, L., Maly, P., Raska, M. (2022) "IL-6 and its role in IgA nephrophaty development" Cytokine and Growth FactRev 66 (1-14)

  • Kirtipal, N., Kumar, S., Dubay, S. K., Dwivedi, V. D., Babu, K. G., Malý, P., Bharadway, S. (2022)"Understanding on the possible routes for SARS CoV-2 invasion via ACE2 in the host linked with multiple organs damage", Inf., Gen. and Evo.99 / 105254.
  • Kumar, S., El-Kafrawy, S.A. , Bharadwaj, S., Maitra, S. S. , Alandijany, T.A., Faizo, A.A., Khateb, A.M., Dwivedi, V.D., Azhar, E.I. (2022) "Discovery of Bispecific Lead Compounds from Azadirachta indica against ZIKA NS2B-NS3 Protease and NS5 RNA Dependent RNA Polymerase Using Molecular Simulations" Molecules, 27, 2562.

  • Kumar, S., Kumar, S.K., Maitra, S. S., Malý, P., Bharadway, S., Sharma, P., Dwivedi, V.D. (2022) "Viral informatics: bioinformatics-based solution for managing viral infections" Bioinformatics 1-36, //
  • Kumar, G.S., Moustafa, M., Sahoo, A.K., Malý, P., Bharadwaj, S. (2022) „Computational Investigations on the Natural Small Molecule as an Inhibitor of Programmed Death Ligand 1 for Cancer Immunotherapy“ Life, 12 (659).


  • Dostál, J., Brynda, J., Vaňková, L.,  Rehana Zia, S., Pichová, I., Heidingsfeld, O., Lepšík, M. (2021) Structural determinants for subnanomolar inhibition of the secreted aspartic protease Sapp1p from Candida parapsilosis. Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 914-921
  • Kuchař, M., Kosztyu, P., Daniel Lišková, V., Čern,ý J., Petroková, H., Vróblová, H., Malý, M., Vaňková,L., Křupka, M.,Rašková Kafková, L., Turánek Knotigová, P., Dušková, J., Dohnálek, J., Mašek, J., Turánek, J., Raška, M., Malý, P. (2021) Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice. Virulence, 12:1,1271-1287
  • Lee, K. E., Bharadwaj, S., Sahoo, A. K., Yadava, U., & Kang, S. G. (2021). "Determination of tyrosinase-cyanidin-3-O-glucoside and (-/+)-catechin binding modes reveal mechanistic differences in tyrosinase inhibition." Sci Rep, 11(1), 24494. (ASEP)


  • Smejkal, J., P. Maly, M. Kuchar, N. Panova, A. Semeradtova, P. Aubrecht, M. Stofik, J. Maly (2020). “Cell immunocapture microfluidic chip based on high-affinity recombinant protein binders.” Biosens. Bioelectron 172: 112784.
  • Viteckova Wunschova, A., Novobilsky, A., Hlozkova, J., Scheer, P., Petrokova, H., Jirik, R., Kulich, P., Bartheldyova, E., Hubatka, F., Jonas, V., Mikulik, R., Maly, P., Turanek, J., & Masek, J. (2020). "Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies." Pharmaceutics, 12(12).


  • Kosztyu, P., M. Kuchar, J. Cerny, L. Barkocziova, M. Maly, H. Petrokova, L. Czernekova, V. Liskova, L. Raskova Kafkova, P. Knotigova, J. Masek, J. Turanek, P. Maly and M. Raska (2019). "Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice." EBioMedicine 47: 247-256.
  • Petrokova, H., J. Masek, M. Kuchar, A. Viteckova Wunschova, J. Stikarova, E. Bartheldyova, P. Kulich, F. Hubatka, J. Kotoucek, P. T. Knotigova, E. Vohlidalova, R. Hezova, E. Maskova, S. Macaulay, J. E. Dyr, M. Raska, R. Mikulik, P. Maly and J. Turanek (2019). "Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders." Pharmaceutics 11(12).
  • Plavec, T. V., M. Kuchar, A. Benko, V. Liskova, J. Cerny, A. Berlec and P. Maly (2019). "Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation." Microorganisms 7(5).


  • Hlavnickova, M., M. Kuchar, R. Osicka, L. Vankova, H. Petrokova, M. Maly, J. Cerny, P. Arenberger and P. Maly (2018). "ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis." Int J Mol Sci 19(10).
  • Semeradtova, A., M. Stofik, L. Vankova, P. Maly, O. Stanek and J. Maly (2018). "Optical microchips based on high-affinity recombinant protein binders-Human serum albumin detection in urine." Sensors and Actuators B-Chemical 272: 441-447.
  • Skrlec, K., P. Zadravec, M. Hlavnickova, M. Kuchar, L. Vankova, H. Petrokova, L. Krizova, J. Cerny, A. Berlec and P. Maly (2018). "p19-Targeting ILP Protein Blockers of IL-23/Th-17 Pro-Inflammatory Axis Displayed on Engineered Bacteria of Food Origin." Int J Mol Sci 19(7).


  • Krizova, L., M. Kuchar, H. Petrokova, R. Osicka, M. Hlavnickova, O. Pelak, J. Cerny, T. Kalina and P. Maly (2017). "p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells." Autoimmunity 50(2): 102-113.


  • Maly, J., O. Stanek, J. Frolik, M. Maly, F. Ennen, D. Appelhans, A. Semeradtova, D. Wrobel, M. Stofik, T. Knapova, M. Kuchar, L. C. Stastna, J. Cermak, P. Sebo and P. Maly (2016). "Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications." Macromol Biosci 16(4): 553-566.
  • Zadravec, P., L. Mareckova, H. Petrokova, V. Hodnik, M. P. Nanut, G. Anderluh, B. Strukelj, P. Maly and A. Berlec (2016). "Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit." Plos One 11(9).


  • Mareckova, L., H. Petrokova, R. Osicka, M. Kuchar and P. Maly (2015). "Novel binders derived from an albumin-binding domain scaffold targeting human prostate secretory protein 94 (PSP94)." Protein Cell 6(10): 774-779.


  • Kuchar, M., L. Vankova, H. Petrokova, J. Cerny, R. Osicka, O. Pelak, H. Sipova, B. Schneider, J. Homola, P. Sebo, T. Kalina and P. Maly (2014). "Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells." Proteins 82(6): 975-989.


  • Mikulecky, P., J. Cerny, L. Biedermannova, H. Petrokova, M. Kuchar, J. Vondrasek, P. Maly, P. Sebo and B. Schneider (2013). "Increasing affinity of interferon-gamma receptor 1 to interferon-gamma by computer-aided design." Biomed Research International 2013: 752514.


  • Ahmad, J. N., J. Li, L. Biedermannova, M. Kuchar, H. Sipova, A. Semeradtova, J. Cerny, H. Petrokova, P. Mikulecky, J. Polinek, O. Stanek, J. Vondrasek, J. Homola, J. Maly, R. Osicka, P. Sebo and P. Maly (2012). "Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G." Proteins 80(3): 774-789.
  • Bibova, I., I. Linhartova, O. Stanek, V. Rusnakova, M. Kubista, M. Suchanek, M. Vasakova and P. Sebo (2012). "Detection of immune cell response to M. tuberculosis-specific antigens by quantitative polymerase chain reaction." Diagnostic Microbiology and Infectious Disease 72(1): 68-78.
  • Sipova, H., V. Sevcu, M. Kuchar, J. N. Ahmad, P. Mikulecky, R. Osicka, P. Maly and J. Homola (2012). "Surface plasmon resonance biosensor based on engineered proteins for direct detection of interferon-gamma in diluted blood plasma." Sensors and Actuators B-Chemical 174: 306-311.


  • Krejcirikova, V., P. Pachl, M. Fabry, P. Maly, P. Rezacova and J. Brynda (2011). "Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition." Acta Crystallographica Section D-Structural Biology 67: 204-211.



Patenty a patentové přihlášky

  • Maly, P.; Raska, M.; Kuchar, M.; Kosztyu, P.; Cerny, J; Daniel Liskova, V.; Petrokova, H. Polypeptides mimicking MPER and V3-loop HIV-1 Env glycoprotein epitopes.  2021. Evropská patentová přihláška. Ref. No.
  • Maly, P.; Raska, M.; Kuchar, M.; Kosztyu, P.; Turanek, J.;. Petrokova, H. PCT/CZ2020/050066. Polypeptides mimicking epitope of broadly neutralizing antibody VRC01 as antigens for a vaccine preventing HIV-1 infection.

  • Maly, P.; Raska, M.; Kuchar, M.; Kosztyu, P.; Turanek, J.;. Petrokova, H. CZ308617. Polypeptidy mimikující epitop široce neutralizující protilátky VRC01 jako antigeny pro vakcíny proti infekci virem HIV-1.

  • Maly, P., L.; Kuchar, M.; Petrokova, H. ( 2019). CZ307849. Polypeptides for the treatment of autoimmune diseases based on blocking the p19 subunit of the IL-23 human cytokine., Biotechnologický ústav AVCR, v.v.i.

  • Maly, P., P.; Kuchar, M.; Vankova, L.; Petrokova, H. ( 2017). EP2922560. Polypeptide Antagonists of Human IL-23 Receptor for Treatment of Autoimmune Diseases.

  • Maly, P., P.; Kuchar, M.; Vankova, L.; Petrokova, H. ( 2014). CZ304514. Polypeptides for treatment of autoimmune diseases based on human IL-23 receptor blockade., Biotechnologický ústav AVCR, v.v.i.

AZV ČR: NU23-05-00203 High-affinity protein binders of hepatitis C virus broadly-neutralizing antibodies as a base for preventative vaccine. 2023-2026. Co-principal investigator.

TAČR - National Competence Centres: TN02000122 „REcombinant TEchnologies for MEDicine (RETEMED)“. 2023-2028. Co-principal investigator

TAČR - National Competence Centres: TN02000017 „National Centre for Biotechnology in Veterinary Medicine (NaCeBiVet)“. 2023-2028. Co-principal investigator. Principal Investigator of the Subproject.

AZV: NU21-03-00372, Malý: Small binding proteins targeting PD-1/PD-L1 immune checkpoint proteins as new diagnostic tools. 2021-2024.

GAČR: 21-16423K, Malý: Small binding proteins targeting PD-1/PD-L1 immune checkpoint proteins as new diagnostic tools. 2021-2024

TA ČR (program TREND): FW01010350, Malý: The therapeutic effect of bacteriophages and their endolysins on opportunistic bacterial infections forming biofilm. 2020-2024.

MŠMT (program OP VVV): CZ.02.1.01/0.0/0.0/16_025/0007397, Malý: Recombinant Biotechnology and Immunotherapy Centre (CEREBIT). 2018-2022.

AZV: 16-29738A, Malý: Detection and evaluation of circulating tumor cells (CTCs) in patients with lung adenocarcinoma by microfluidic chip technology. 2016-2019.

AZV: 16-30299A, Malý: Nanoliposomal systems for rapid diagnosis of thrombi by MRI. 2016-2019.

AZV: 16-27676A, Malý: Immunomodulatory ligands targeted to IL-23/Th17 pro-inflammatory axis as novel tools for development of topical drugs for treatment of psoriasis. 2016-2019.

AVČR: SAZU-16-01, Malý: Lactic acid bacteria-mediated intestinal delivery of novel therapeutic protein binders derived from scaffold of albumin-binding domain. 2016-2018. (Partners Institute: Jozef Stefan Institute, SAZU, Slovinsko).

AZV: 15-32198A, Malý: Construction of recombinant mimotopes for induction of neutralizing antibodies against HIV-1 gp120 glycoprotein using high-affinity binders approach. 2015-2018.

TA ČR (program EPSILON): TH01010837, Malý: Development of unique vaccines against serious diseases of animals. 2015-2016.

MPO (program TIP): FR-TI4/667, Novel binding biomolecule development for tumor in vitro diagnostics. 2012-2014.

GAČR: GAP303/10/1849, Malý: Immunomodulatory ligands suppressing function of human IL-23. 2010-2013.