Peptide Blocking CTLA-4 and B7-1 Interaction

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 sequence 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 with CTLA-4. (a) Total Docking Score of peptide in- interaction with the whole extracellular portion of CTLA-4 (S ); (b) Contribution to the Docking Score of peptide interaction teraction with the whole extracellular portion of CTLA-4 (Stottot); (b) Contribution to the Docking Score of peptide interaction 99 104 withwith 99MYPPPY104 looploop of of CTLA-4 CTLA-4 molecule molecule (S (Spp).).

Next, we analyzed the obtained 3D model of p344 peptide bound to the extracellular portion of CTLA-4. CTLA-4. Figure Figure 22 showsshows the the result resultss of of molecular molecular docking docking for for p344 p344 peptide peptide to theto the extracellular extracellular part part of the of theCTLA-4 CTLA-4 molecule. molecule. When When the B7-1 the ligand B7-1 ligand was embedded was embedded in the CTLA-4/p344in the CTLA-4/p344 docking docking complex complex in the orientation in the orientation adopted adoptedin the 1I8L in structure, the 1I8L structure, a notice- a noticeable superposition of B7-1 on the docked peptide was observed (Figure2a,b). able superposition of B7-1 on the docked peptide was observed (Figure 2a,b). According According to our calculations, 15 amino acid residues of B7-1 protein are located within 1 Å of the p344 peptide on the CTLA-4 molecule, so they are considered to be in clash with the Molecules 2021, 26, x FOR PEER REVIEW 3 of 7

Molecules 2021, 26, 253 3 of 7 to our calculations, 15 amino acid residues of B7-1 protein are located within 1 Å of the p344 peptide on the CTLA-4 molecule, so they are considered to be in clash with the pep- tidepeptide and therefore and therefore prevent prevent CTLA-4 CTLA-4 binding binding to B7-1 to (Figure B7-1 (Figure 2b). Thus,2b). Thus, it is conceivable it is conceivable that thethat p344 the peptide p344 peptide can cause can causea blockade a blockade of CTLA-4 of CTLA-4 and B7-1 and interaction. B7-1 interaction.

Figure 2. Model of p344 peptideFigure interaction 2. Model with of CTLA-4 p344 peptide and B7-1 interaction proteins. with Using CTLA-4 the CABS-dock and B7-1 proteins.server, p344 Using was the docked CABS-dock to CTLA-4 protein (blue ribbon).server, Next, p344 the wasB7-1 docked ligand (red to CTLA-4 ribbon) protein was embedded (blue ribbon). in the CTLA-4/p344 Next, the B7-1 complex ligand (redas in ribbon) PDB was 1I8L. p344 shown as sticks (a,embeddedb); the residues in the of CTLA-4/p344 B7-1 which are complex in clash aswi inth PDBthe docked 1I8L. p344 peptide shown are as shown sticks as (a ,spheresb); the residues (b). of B7-1 which are in clash with the docked peptide are shown as spheres (b). Further analysis of the modeled CTLA-4/p344 complex showed that the following Further analysis of the modeled CTLA-4/p344 complex showed that the following eleven residues of CTLA-4 are located within 3 Å from the docked peptide: Glu33, Val34, eleven residues of CTLA-4 are located within 3 Å from the docked peptide: Glu33, Val34, Arg35, Thr37, Glu48, Ala51, Thr53, Leu63, Glu97, Met99, and Tyr100. Among them, Glu33, Arg35, Thr37, Glu48, Ala51, Thr53, Leu63, Glu97, Met99, and Tyr100. Among them, Glu33, Thr37, and Thr53 form hydrogen bonds with residues Tyr7, Ser13, and Pro4 of the pep- Thr37, and Thr53 form hydrogen bonds with residues Tyr7, Ser13, and Pro4 of the peptide, tide, respectively. Residues Val34, Glu48, and Ala51 of CTLA-4 participate in attractive respectively. Residues Val34, Glu48, and Ala51 of CTLA-4 participate in attractive van van der Waals interactions with p344 peptide (evaluated by “Energy inspector” tool of der Waals interactions with p344 peptide (evaluated by “Energy inspector” tool of MVD MVD software). Measurements on CTLA-4/B7-1 complex (PDB 1I8L) gave the following software). Measurements on CTLA-4/B7-1 complex (PDB 1I8L) gave the following list list of thirteen CTLA-4 residues in the vicinity of 3 Å from B7-1 ligand: Glu33, Arg35, Thr53, of thirteen CTLA-4 residues in the vicinity of 3 Å from B7-1 ligand: Glu33, Arg35, Thr53, Leu63, Asp65, Lys95, Met99, Tyr100, Pro102, Pro103, Tyr104, Tyr105, and Leu106. It is seen Leu63, Asp65, Lys95, Met99, Tyr100, Pro102, Pro103, Tyr104, Tyr105, and Leu106. It is seen thatthat both both lists lists contain contain common common residues residues of of CT CTLA-4LA-4 (shown (shown in in italic), italic), indicating indicating that that the the formationformation of ofCTLA-4/p344 CTLA-4/p344 complex complex prevents prevents an effective an effective interaction interaction between between CTLA-4 CTLA-4 and B7-1.and It B7-1. should It should be noted be noted that thatMet99 Met99 and andTyr104 Tyr104 residues residues of CTLA-4 of CTLA-4 are are involved involved in inH- H- bondingbonding with with Tyr31 Tyr31 andand Lys36Lys36 of of the the B7-1 B7-1 ligand, ligand, respectively. respectively. Additionally, Additionally, Arg29, Arg29, Lys89, Lys89,and Asp90 and Asp90 residues residues of B7-1 of form B7-1 saltform bridges salt bridges with Glu33, with Glu33, Asp65, Asp65, and Arg35 and ofArg35 CTLA-4 of CTLA-4protein. protein. These anchoring These anchoring interactions interactions must be must significantly be significantly affected affected by the p344 by the peptide. p344 peptide.Next, we investigated by ELISA whether CTLA-4Fc can bind synthetic peptide p344. First, we confirm the ability of CTLA-4Fc fusion protein to bind B7-1 recombinant protein and mAb against human CTLA-4. The data presented in Figure3a shows that the recombinant CTLA-4Fc chimeric protein specifically interacts with the recombinant B7-1 protein and antibodies against human CTLA-4 protein. The higher level of interaction of the recombinant CTLA-4Fc protein with the anti-CTLA-4 mAb can be explained by the higher affinity of antibodies compared to the B7-1 protein. Molecules 2021, 26, x FOR PEER REVIEW 4 of 7

Next, we investigated by ELISA whether CTLA-4Fc can bind synthetic peptide p344. First, we confirm the ability of CTLA-4Fc fusion protein to bind B7-1 recombinant protein and mAb against human CTLA-4. The data presented in Figure 3a shows that the recombinant CTLA-4Fc chimeric protein specifically interacts with the recombinant B7-1 protein and antibodies against human CTLA-4 protein. The higher level of interaction of the recombinant CTLA-4Fc protein with the anti-CTLA-4 mAb can be explained by the higher affinity of antibodies compared to the B7-1 protein. To verify the specificity of CTLA-4Fc interaction with synthetic peptides we incubated CTLA-4Fc and B7-1Fc with immobilized p344 or p333 peptides. Data presented in Figure 3b show that B7-1Fc did not bind p344 and p333 peptides. In contrast, CTLA-4Fc binds p344 in a dose-dependent manner (Figure 3b). No significant CTLA-4 interaction with control p333 peptide was observed. It appears that the Fc part of the CTLA-4Fc fusion protein did not participate in p344 peptide binding, since B7-1Fc protein containing the Molecules 2021, 26, 253 4 of 7 same amino acid sequence Fc did not bind p344. Therefore, the extracellular portion of the CTLA-4 molecule specifically binds the p344 peptide.

FigureFigure 3.3. InteractionInteraction ofof CTLA-4FcCTLA-4Fc withwith ligand,ligand, antibodies,antibodies, andand syntheticsynthetic peptides.peptides. ((aa)) CTLA-4FcCTLA-4Fc fusionfusion proteinprotein 55µ μg/mLg/mL waswas addedadded toto thethe wellswells withwith immobilizedimmobilized recombinantrecombinant B7-1B7-1 proteinprotein (8(8µ μg/mL)g/mL) and anti-CTLA-4anti-CTLA-4 mAbmAb (8(8 µμg/mL).g/mL). Protein- freefree wellswells (PBS)(PBS) werewere usedused asas aa negativenegative control.control. ((bb)) CTLA-4FcCTLA-4Fc oror B7-1FcB7-1Fc atat indicatedindicated concentrationsconcentrations werewere addedadded toto thethe wellswells withwith immobilizedimmobilized syntheticsynthetic peptidespeptides p344p344 oror p333p333 (2(2µ μg/mL).g/mL). Binding of fusion proteins to the wells was determined using HRP conjugated mAb against the Fc part of human IgG. using HRP conjugated mAb against the Fc part of human IgG. ToThe verify next thestep specificity in this study of CTLA-4Fc was to evaluate interaction the withability synthetic of synthetic peptides peptides we incubated to block CTLA-4Fcthe binding and of B7-1FcCTLA-4 with to its immobilized natural ligand p344 B7-1 or p333 (Figure peptides. 4). In Data these presented experiments, in Figure CTLA-3b show4Fc was that preincubated B7-1Fc did not with bind either p344 p344 and p333or control peptides. p333 In peptide contrast, and CTLA-4Fc added to binds the wells p344 inwith a dose-dependent immobilized recombinant manner (Figure B7-1 protein3b). No (wit significanthout Fc CTLA-4fragment). interaction Data indicate with controlthat the p333addition peptide of CTLA-4Fc was observed. to immobilized It appears that B7-1 the increased Fc part of optical the CTLA-4Fc density fusion from protein0.02 (back- did notground) participate to 0.105, in similar p344 peptide data presented binding, in since Figure B7-1Fc 3а. This protein result containing indicates the that same CTLA-4Fc amino acidbind sequenceto B7-1. Preincubation Fc did not bind of CTLA-4Fc p344. Therefore, with p333 the did extracellular not notably portion change ofCTLA-4Fc the CTLA-4 and moleculeB7-1 binding. specifically However, binds incu thebation p344 peptide.of CTLA-4Fc in the presence of p344 decreased the bindingThe of next CTLA-4 step in to this B7-1 study by 42%. was toThus, evaluate these the results ability indicate of synthetic that the peptides p344 peptide, to block thewhich binding specifically of CTLA-4 interacts to its with natural CTLA-4, ligand can B7-1 partially (Figure 4block). In theseCTLA-4 experiments, and B7-1 interac- CTLA- 4Fctions. was preincubated with either p344 or control p333 peptide and added to the wells with immobilized recombinant B7-1 protein (without Fc fragment). Data indicate that the addition of CTLA-4Fc to immobilized B7-1 increased optical density from 0.02 (background) to 0.105, similar data presented in Figure3a. This result indicates that CTLA-4Fc bind to B7-1. Preincubation of CTLA-4Fc with p333 did not notably change CTLA-4Fc and B7-1 binding. However, incubation of CTLA-4Fc in the presence of p344 decreased the binding of CTLA-4 to B7-1 by 42%. Thus, these results indicate that the p344 peptide, which specifically interacts with CTLA-4, can partially block CTLA-4 and B7-1 interactions. Thus, during this work, a peptide interacting with the CTLA-4 molecule was identified and characterized. Using molecular modeling, we have shown that p344 peptide interacts with 99MYPPPY104 loop of CTLA-4, which is responsible for binding CTLA-4 to the B7-1 ligand. The experimental data obtained by ELISA confirmed modeling results regarding specificity CTLA-4 and p344 peptide interaction. Moreover, we have shown that p344 partially blocks the interaction of CTLA-4 with B7-1, which also confirms p344 binding near the MYPPPY motif. The p344 peptide presented in this work is a leading molecule for the development of immunostimulatory drugs that can regulate/prevent binding of ligands B7-1 (CD80) (and possibly B7-2 (CD86)) to the inhibitory CTLA-4 receptor and thereby may enhance the immune response. However, immunomodulatory properties of p344 peptide require validation in vitro and in vivo, which will be reported in upcoming publications. Molecules 26 Molecules 20212021,, 26,, 253x FOR PEER REVIEW 55 ofof 7

Figure 4. Blocking CTLA-4 and B7-1 interaction withwith p344p344 peptide.peptide. CTLA-4FcCTLA-4Fc (5(5 µμg/mL)g/mL) was incubated with p344 or p333 peptide (8(8 µμg/mL)g/mL) for 2 h h and and added added to the wells of a 96-well 96-well plate with immobilized recombinant B7-1 protein (8(8 µμg/mL).g/mL). The interactioninteraction ofof CTLA-4Fc CTLA-4Fc with with B7-1 B7-1 was was determined determined using usin HRPg HRP conjugated conjugated mAbs mAbs against against the Fcthe part Fc part of human of human IgG. IgG. One ofOne four of independentfour independent experiments experiments is shown. is shown.

Thus,Synthetic during peptides this work, capable a peptide of blocking interacting immunological with the CTLA-4 checkpoint molecule can was be used identi- as fieda substitute and characterized. of mAb for Using immunotherapy molecular modeling, of autoimmune we have diseases shown andthat cancer.p344 peptide Peptides in- teractscompare with to 99 mAb,MYPPPY have104 several loop of advantages; CTLA-4, which including is responsible small size, for binding which provides CTLA-4 to more the B7-1efficient ligand. penetration The experimental into tissues ordata tumors; obtain lowed immunogenicity,by ELISA confirmed which modeling allows an results unlimited re- gardingnumber specificity of injections; CTLA-4 relatively and simplep344 peptide chemical interaction. synthesis, Moreover, which reduces we have the shown cost of that the p344drug. partially The strategy blocks described the interaction in this of paper CTLA-4 can with be used B7-1, to which search also for peptidesconfirms interactingp344 bind- withing near other the molecules MYPPPY thatmotif. regulate the immune response, for example, PD-1, PD-L1, and PD-L2,The B7-H3 p344 (CD276),peptide presented B7-H4, B7-H5 in this (VISTA) work is other. a leading molecule for the development of immunostimulatory drugs that can regulate/prevent binding of ligands B7-1 (CD80) (and3. Materials possibly and B7-2 Methods (CD86)) to the inhibitory CTLA-4 receptor and thereby may enhance the immunePeptides response. used in thisHowever, work immunomodula were identifiedtory using properties a microarray of p344 containing peptide require 330034 peptidesvalidation with in vitro random and in amino vivo, acidwhich sequences, will be reported according in upcoming to the protocol publications. described earlier [15Synthetic]. Briefly, peptides recombinant capable fusion of blocking protein immunological CTLA-4Fc, containing checkpoint Fc portion can be ofused human as a substituteIgG, was applied of mAb on for peptide immuno microarrays,therapy of andautoimmune interaction diseases with peptides and cancer. was detectedPeptides usingcom- parefluorescence-labeled to mAb, have several mAb advantages; against human including IgG. For small this size, work, which we used provides peptide more p344 efficient with penetrationamino acid sequenceinto tissues ARHPSWYRPFEGCG or tumors; low immu whichnogenicity, interacted which with allows the CTLA-4Fc an unlimited molecule. num- Anotherber of injections; peptide p333relatively (amino simple acid sequencechemical synthesis, EGLNRPSGGCG) which reduces did not the bind cost CTLA-4 of the indrug. the Theexperiments strategy withdescribed microarrays in this andpaper was can used be used in this to work search as for a negative peptides control. interacting with otherFor molecules 3D modeling, that regulate the structure the immune of the B7-1/CTLA-4response, for example, complex wasPD-1, downloaded PD-L1, and from PD- L2,the B7-H3 Protein (CD276), Data Bank B7-H4, (code B7-H5 1I8L). (VISTA) The substructure other. containing an extracellular portion of CTLA-4 protein was exported from the downloaded file and sent to the CABS-dock server (protein-peptide3. Materials and Methods docking software http://biocomp.chem.uw.edu.pl/CABSdock/)[19]. When performing the simulation, the strategy of “blind” docking was used without speci- Peptides used in this work were identified using a microarray containing 330034 pep- fying the binding site on the CTLA-4 molecule: only the protein structure and amino acid tides with random amino acid sequences, according to the protocol described earlier [15]. sequence of the peptide were uploaded to the server; the optional search settings were not Briefly, recombinant fusion protein CTLA-4Fc, containing Fc portion of human IgG, was applied. The best conformation of the p344 peptide in complex with the CTLA-4 molecule, applied on peptide microarrays, and interaction with peptides was detected using fluo- obtained as an output from the CABS-dock server, was imported into the Molegro Virtual rescence-labeled mAb against human IgG. For this work, we used peptide p344 with Docker (MVD) program (Molegro ApS, Denmark) for further analysis and visualization. amino acid sequence ARHPSWYRPFEGCG which interacted with the CTLA-4Fc mole- The Docking Score value reflecting the degree of interaction of peptides with an extracellu- cule. Another peptide p333 (amino acid sequence EGLNRPSGGCG) did not bind CTLA- lar part of CTLA-4 molecule (Stot), as well as the contribution to this value made only by 4 in 99the experiments104 with microarrays and was used in this work as a negative control. the MYPPPY loop amino acid sequence (Sp), was estimated using the Ligand Energy For 3D modeling, the structure of the B7-1/CTLA-4 complex was downloaded from Inspector tool included in the MVD program. When calculating Stot and Sp, the MolDock thescore Protein function Data was Bank used (code [20]. 1I8L). The substructure containing an extracellular portion of CTLA-4In the protein experimental was exported work, from we used the download recombinanted fusionfile and proteins sent to the CTLA-4Fc CABS-dock and B7-1Fc, server (protein-peptidecontaining Fc portion docking of human software IgG, http://b purchasediocomp.chem.uw.edu.pl/CABSdock/) from R&D Systems (USA). Phosphate- [17]. Molecules 2021, 26, 253 6 of 7

buffered saline (PBS) (Biolot, Russia), Tween-20 (Helicon, Russia), and bovine serum albumin (BSA) (Amresco, USA) were used for the preparation of basic working solutions. High-purity pyrogen-free deionized water was obtained using the UVOI-MF-1812 water treatment system (Akvalab, Russia). The peptides p344 (ARHPSWYRPFEGCG) and p333 (EGLNRPSGGCG) were synthesized at OOO “Alfa-Organika” (Russia) using the methods of classical peptide synthesis in solution. The resulting lyophilized peptides were dissolved in PBS (pH 7.3) and stored at 4 ◦C. The binding speciﬁcity of peptides to recombinant protein molecules was determined by ELISA. Before peptide coating, 96-well plates (Corning, USA) were treated with glu- taraldehyde (0.125%) 200 µL/well for 2 h at room temperature. After washing with PBST (PBS + 0.05% Tween-20) and drying the plates synthetic peptides 100 µL/well at 2 µg/mL in PBS (pH 9.5) were added to the wells and incubated for 3 h at 37 ◦C in a shaker. After peptide coating 200 µL of blocking solution containing 2% BSA and 0.05% Tween-20 in PBS was added to each well and plates were incubated for 2 h at room temperature. After washing different concentrations of CTLA-4Fc and B7-1Fc were added into the wells in triplicates 100 µL/well and plates were incubated for 1 h at 37 ◦C. After washing 100 µL of anti-human IgG antibody conjugated with HRP (1:10.000 dilution) was added to the wells and incubated for 1 h at room temperature. Plates were washed six times with PBST solution and TMB substrate was added (50 µL/well). Plates were kept in the dark at room temperature for 1–5 min. The reaction was stopped by adding 50 µL of 1 M Н2SO4 to each well. The optical density of the reaction was recorded at wavelength 450 nm using an iMark photometer (Bio-Rad, Hercules, CA, USA). The binding of CTLA-4Fc to B7-1 and mouse mAb against human CTLA-4 was also assessed by ELISA. Recombinant protein B7-1 without Fc or mouse mAb against human CTLA-4 (R&D Systems) were coated on the plates at 8 µg/mL wells. CTLA-4Fc was added to the wells at 5 µg/mL. Binding of CTLA-4Fc was detected with anti-human IgG HRP. The blocking activity of peptides was analyzed in similar experiments. CTLA-4Fc (5 µg/mL) was incubated with p344 or p333 (8 µg/mL) peptides and added to the wells coated with 8 µg/mL B7-1 without Fc. Binding of CTLA-4Fc was detected with anti-human IgG HRP, as described above.

Author Contributions: Methodology, S.V.P., K.E.A., and A.G.; formal analysis, molecular modeling, A.I.K. and S.V.P.; visualization S.V.P.; writing—original draft preparation, S.V.P.; writing—review and editing, A.I.C., A.I.K., S.V.P.; project administration, A.I.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Russian Foundation for Basic Research (17-54-33003); Ministry of Science and Higher Education of the Russian Federation (FZMW-2020-0007); and by the Tomsk Polytechnic University Competitiveness Enhancement Program. Institutional Review Board Statement: This article does not contain any research involving humans or the use of animals as objects. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conﬂicts of Interest: The authors declare no conﬂict of interest. Sample Availability: Samples of selected compounds are available from the authors.

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