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.

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.



Daniel Lišková Veronika, Mgr., Ph.D.
Groza Yaroslava
Hlavničková Marie, Mgr.
Kadlčáková Petra
Kuchař Milan, Mgr., Ph.D.
Malá Linda, Mgr.
Malý Petr, RNDr., CSc.
Panova Natalya
Petroková Hana, Ing., Ph.D.
Šulc Josef
Vaňková Lucie, Mgr., Ph.D.
Zosinčuková Tereza, Bc.


  • 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


  • 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: 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 protein blockers of IL-23/IL-17 axis as intestinal inflammation inhibitors secreted by probiotic bacteria. 2021-2024

TA ČR (program TREND): FW01010350, Malý: Léčebný efekt bakteriofágů a jejich endolyzinů na oportunní bakteriální infekce vytvářející biofilm. 2020-2024.

MŠMT (program OP VVV): CZ.02.1.01/0.0/0.0/16_025/0007397, Malý: Centrum pro rekombinantní biotechnologie a imunoterapeutika (CEREBIT). 2018-2022.

AZV: 16-29738A, Malý: Detekce cirkulujících nádorových buněk (CTC) u pacientů s adenokarcinomem plic pomocí mikrofluidního čipu. 2016-2019.

AZV: 16-30299A, Malý: Nanoliposomální systémy pro rychlou diagnostiku trombu pomocí MRI. 2016-2019.

AZV: 16-27676A, Malý: Imunomodulační ligandy cílené na prozánětlivou dráhu IL-23/Th17 jako nový nástroj k vývoji zevních antipsoriatických léků. 2016-2019.

AVČR: SAZU-16-01, Malý: Využití bakterií mléčného kvašení k produkci nových terapeutických proteinů odvozených od struktury albumin vážící domény a k jejich uvolňování ve střevě. 2016-2018. (Partnerské pracoviště: Jozef Stefan Institute, SAZU, Slovinsko).

AZV: 15-32198A, Malý: Příprava rekombinantních mimotopů indukujících neutralizační protilátky proti HIV-1 gp120 glykoproteinu pomocí technologie vysokoafinitních ligandů. 2015-2018.

TA ČR (program EPSILON): TH01010837, Malý: Vývoj unikátních vakcín proti závažným onemocněním zvířat. 2015-2016.

MPO (program TIP): FR-TI4/667, Vývoj nových vazebných biomolekul pro in vitro diagnostické použití v onkologii. 2012-2014.

GAČR: GAP303/10/1849, Malý: Imunomodulační ligandy potlačující funkci lidského IL-23. 2010-2013.