Peptide Blocking CTLA-4 and B7-1 Interaction
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molecules Communication Peptide Blocking CTLA-4 and B7-1 Interaction Stepan V. Podlesnykh 1, Kristina E. Abramova 1, Anastasia Gordeeva 1, Andrei I. Khlebnikov 2 and Andrei I. Chapoval 1,3,* 1 Russian-American Anti-Cancer Center, Altai State University, 61 Lenin St., 656049 Barnaul, Russia; [email protected] (S.V.P.); [email protected] (K.E.A.); [email protected] (A.G.) 2 Kizhner Research Center, National Research Tomsk Polytechnic University, 30 Lenin St., 634050 Tomsk, Russia; [email protected] 3 Center for Innovations in Medicine, Biodesign Institute, Arizona State University, 1001 S. McAllister Ave., Tempe, AZ 85281, USA * Correspondence: [email protected] Abstract: Discovery of the B7 family immune checkpoints such as CTLA-4 (CD152), PD-1 (CD279), as well as their ligands B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1, CD274), and B7-DC (PD-L2, CD273), has opened new possibilities for cancer immunotherapy using monoclonal antibodies (mAb). The blockade of inhibitory receptors (CTLA-4 and PD-1) with specific mAb results in the activation of cancer patients’ T lymphocytes and tumor rejection. However, the use of mAb in clinics has several limitations including side effects and cost of treatment. The development of new low-molecular compounds that block immune checkpoints’ functional activity can help to overcome some of these limitations. In this paper, we describe a synthetic peptide (p344) containing 14 amino acids that specifically interact with CTLA-4 protein. A 3D computer model suggests that this peptide binds to the 99MYPPPY104 loop of CTLA-4 protein and potentially blocks the contact of CTLA-4 receptor with B7-1 ligand. Experimental data confirm the peptide-specific interaction with CTLA-4 and its ability to partially block CTLA-4/B7-1 binding. The identified synthetic peptide can be used for the development of novel immune checkpoint inhibitors that can block CTLA-4 functional activity for cancer immunotherapy. Citation: Podlesnykh, S.V.; Abramova, K.E.; Gordeeva, A.; Keywords: peptides; immune checkpoints; peptide microarray; cancer; immunotherapy Khlebnikov, A.I.; Chapoval, A.I. Peptide Blocking CTLA-4 and B7-1 Interaction. Molecules 2021, 26, 253. https://doi.org/10.3390/molecules 1. Introduction 26020253 Receptors for the B7 family ligands expressed on the surface of T lymphocytes can Received: 18 December 2020 deliver both stimulating and inhibitory signals that regulate the immune response [1,2]. Accepted: 2 January 2021 Inhibitory receptor CTLA-4 (CD152) interact with ligands B7-1 (CD80) and B7-2 (CD86), Published: 6 January 2021 while PD-1 (CD279) receptor binds B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). These molecules are also termed as immunological checkpoints [1,2]. Injections of mon- Publisher’s Note: MDPI stays neu- oclonal antibodies (mAb) blocking immunological checkpoints, such as CTLA-4, PD-1, tral with regard to jurisdictional clai- and PD-L1, enhance antitumor immunity and lead to tumor rejection [3–6]. However, ms in published maps and institutio- the side effects and cost of manufacturing mAb for therapy limit their wider clinical use. nal affiliations. Low molecular weight agents that block immunological checkpoints can overcome some problems associated with the use of mAb for cancer therapy [7,8]. Several groups are developing low molecular weight agents such as aptamers, small molecules, binding parts of antibodies, and peptides for disruption of CTLA-4 binding to Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. its ligands [9–13]. Peptides for the therapy of various diseases are gaining more interest be- This article is an open access article cause of cell-free low-cost production, optimal half-life in circulation, ability to specifically distributed under the terms and con- bind to other proteins, and higher tissue penetration [14]. In this work, we introduce a pep- ditions of the Creative Commons At- tide (p344), which can specifically bind the CTLA-4 molecule. This peptide was identified tribution (CC BY) license (https:// using microarrays containing 330034 peptides with random amino acid sequences [15–17]. 99 104 creativecommons.org/licenses/by/ It appears that the identified peptide interacts with the MYPPPY amino acid sequence 4.0/). loop of the CTLA-4 molecule which is responsible for the binding of CTLA-4 with B7-1 [18]. Molecules 2021, 26, 253. https://doi.org/10.3390/molecules26020253 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 7 Molecules 2021, 26, 253 2 of 7 [15]. It appears that the identified peptide interacts with the 99MYPPPY104 amino acid se- quence loop of the CTLA-4 molecule which is responsible for the binding of CTLA-4 with B7-1 [16]. In this work, we confirmed the specificity of p344 peptide interaction with the CTLA-4In this work, protein we using confirmed ELISA. the p344 specificity peptide of presented p344 peptide in this interaction work is one with of thethe CTLA-4leading proteincandidates using for ELISA. the development p344 peptide of presented a low-molecular in this work agent is that one ofblocks the leading the interaction candidates of CTLA-4for the development with its ligands. of a This low-molecular peptide can agent be potentially that blocks used the interactionfor immunotherapy of CTLA-4 of withcan- cerits ligands.and other This immunologically peptide can be dependent potentially diseases. used for immunotherapy of cancer and other immunologically dependent diseases. 2. Results and Discussion 2. Results and Discussion Earlier, using peptide microarrays, we identified >350 peptides that specifically in- teractEarlier, with the using recombinant peptide microarrays, fusion proteins we B7-1Fc, identified B7-2Fc, >350 and peptides CTLA-4Fc that specifically[15]. Nineteen in- peptidesteract with out the of recombinant330034 peptides fusion presented proteins on B7-1Fc, microarrays, B7-2Fc, specifically and CTLA-4Fc interacted [15]. Nineteen with re- peptidescombinant out CTLA-4Fc of 330034 protein. peptides We presented synthesized on eight microarrays, peptides specificallythat showed interacted maximal inter- with actionrecombinant with CTLA-4Fc. CTLA-4Fc Among protein. synthesized We synthesized peptides, eight p344 peptides peptide that with showed amino maximal acid se- quenceinteraction ARHPSWYRPFEGCG with CTLA-4Fc. Among demonstrated synthesized reprod peptides,ucible p344 results peptide in witha series amino of acidexperi- se- quencements and ARHPSWYRPFEGCG was selected for further demonstrated analysis. reproducible This synthetic results peptide in a (Mw series = of1.66 experiments kDa) con- tainsand was 14 amino selected acid for residues, further analysis.the calculated This values synthetic of the peptide GRAVY (Mw index = 1.66 = − kDa)1.114, contains charac- 14 amino acid residues, the calculated values of the GRAVY index = −1.114, characterize terize this peptide as hydrophilic. Control peptide p333 (amino acid sequence this peptide as hydrophilic. Control peptide p333 (amino acid sequence EGLNRPSGGCG), EGLNRPSGGCG), which does not interact with CTLA-4Fc, has similar characteristics. which does not interact with CTLA-4Fc, has similar characteristics. We used a computational approach implemented on the CABS-dock server [17], We used a computational approach implemented on the CABS-dock server [19], which is intended for docking peptides without knowing the binding site on the protein which is intended for docking peptides without knowing the binding site on the protein macromolecule. In this case, the conformational mobility of the peptide and partial mo- macromolecule. In this case, the conformational mobility of the peptide and partial mobility bility of the protein are considered [17], which allows docking without special preparation of the protein are considered [19], which allows docking without special preparation (pre- (pre-optimization) of the crystal structure of the macromolecule. First, we found the opti- optimization) of the crystal structure of the macromolecule. First, we found the optimal mal docking poses and calculated interaction energy of CTLA-4 with peptide p344 and docking poses and calculated interaction energy of CTLA-4 with peptide p344 and control control p333. When we analyzed the calculated interaction energy of p344 and control p333. When we analyzed the calculated interaction energy of p344 and control p333 peptide p333 peptide with the whole CTLA-4 extracellular domain (Stot), both peptides showed with the whole CTLA-4 extracellular domain (Stot), both peptides showed similar values (Figuresimilar 1valuesa). However, (Figure when1a). However, we compared when the we interactioncompared energythe interaction of these energy peptides of withthese peptides99 with 104the 99MYPPPY104 loop, p344 peptides showed significantly higher values the MYPPPY loop, p344 peptides showed significantly higher values (Sp), suggesting (Sthatp), thesuggesting p344 peptide that the position p344 peptide on the CTLA-4 position molecule on the CTLA-4 is energetically molecule optimal is energetically near the 99 104 99 104 optimal99MYPPPY near104 theloop MYPPPY (Figure1b). loop It is(Figure known 1b). that It is the known99MYPPPY that the104 MYPPPYloop of CTLA-4 loop of is CTLA-4responsible is responsible for the binding for the of binding CTLA-4 of with CTLA-4 B7-1 [ 18with]. B7-1 [16]. Figure 1. Assessment calculated interaction energy of p344 peptide with CTLA-4. (a) Total Docking Score of peptide Figure 1. Assessment calculated interaction energy of p344 peptide