An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction
Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte anti...
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Veröffentlicht in: | ACS chemical biology 2020-02, Vol.15 (2), p.360-368 |
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creator | Ramanayake Mudiyanselage, Tharanga M.R Michigami, Masataka Ye, Zhengmao Uyeda, Atsuko Inoue, Norimitsu Sugiura, Kikuya Fujii, Ikuo Fujiwara, Daisuke |
description | Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4–B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a K D of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4–B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4–B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy. |
doi_str_mv | 10.1021/acschembio.9b00743 |
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We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4–B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a K D of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4–B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4–B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.</description><identifier>ISSN: 1554-8929</identifier><identifier>EISSN: 1554-8937</identifier><identifier>DOI: 10.1021/acschembio.9b00743</identifier><identifier>PMID: 31841301</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Sequence ; B7-1 Antigen - metabolism ; CTLA-4 Antigen - metabolism ; Dendritic Cells - drug effects ; Helix-Loop-Helix Motifs ; Humans ; Immunoglobulin Fc Fragments - metabolism ; Immunologic Factors - chemical synthesis ; Immunologic Factors - genetics ; Immunologic Factors - pharmacology ; Leukocytes, Mononuclear - drug effects ; Lymphocyte Activation - drug effects ; Peptide Library ; Peptides - chemical synthesis ; Peptides - genetics ; Peptides - pharmacology ; Protein Binding - drug effects ; Saccharomyces cerevisiae - genetics</subject><ispartof>ACS chemical biology, 2020-02, Vol.15 (2), p.360-368</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a452t-acb18484cdd6f0cd9dd36119f3c1ed8d9890ed4888fe7d680c9c78c252d5b4663</citedby><cites>FETCH-LOGICAL-a452t-acb18484cdd6f0cd9dd36119f3c1ed8d9890ed4888fe7d680c9c78c252d5b4663</cites><orcidid>0000-0002-6230-5534</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acschembio.9b00743$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acschembio.9b00743$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,776,780,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31841301$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ramanayake Mudiyanselage, Tharanga M.R</creatorcontrib><creatorcontrib>Michigami, Masataka</creatorcontrib><creatorcontrib>Ye, Zhengmao</creatorcontrib><creatorcontrib>Uyeda, Atsuko</creatorcontrib><creatorcontrib>Inoue, Norimitsu</creatorcontrib><creatorcontrib>Sugiura, Kikuya</creatorcontrib><creatorcontrib>Fujii, Ikuo</creatorcontrib><creatorcontrib>Fujiwara, Daisuke</creatorcontrib><title>An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction</title><title>ACS chemical biology</title><addtitle>ACS Chem. Biol</addtitle><description>Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4–B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a K D of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4–B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4–B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.</description><subject>Amino Acid Sequence</subject><subject>B7-1 Antigen - metabolism</subject><subject>CTLA-4 Antigen - metabolism</subject><subject>Dendritic Cells - drug effects</subject><subject>Helix-Loop-Helix Motifs</subject><subject>Humans</subject><subject>Immunoglobulin Fc Fragments - metabolism</subject><subject>Immunologic Factors - chemical synthesis</subject><subject>Immunologic Factors - genetics</subject><subject>Immunologic Factors - pharmacology</subject><subject>Leukocytes, Mononuclear - drug effects</subject><subject>Lymphocyte Activation - drug effects</subject><subject>Peptide Library</subject><subject>Peptides - chemical synthesis</subject><subject>Peptides - genetics</subject><subject>Peptides - pharmacology</subject><subject>Protein Binding - drug effects</subject><subject>Saccharomyces cerevisiae - genetics</subject><issn>1554-8929</issn><issn>1554-8937</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9UEtOwzAQtRCIlsIFWKBcIMWOncRmVyqglSKB1LKOEnuiukriKHEQ3XEHbshJcEkpO1ZvRu-jmYfQNcFTggNym8lObqDKtZmKHOOY0RM0JmHIfC5ofHqcAzFCF123xZjRiItzNKKEM0IxGSM5q71lVfU1-Curq77MrGl33gJK_f718ZkY0zj4Wb0XaKxWcOetoARp9Rt4y3qjc221qT1TePN1MvOZ09_HjrHQZnJPXaKzIis7uDrgBL0-PqznCz95flrOZ4mfsTCwfiZzdxZnUqmowFIJpWhEiCioJKC4ElxgUIxzXkCsIo6lkDGXQRioMGdRRCcoGHJla7quhSJtWl1l7S4lON03lv41lh4ac6abwdT0eQXqaPmtyAmmg8CZ063p29r98F_iN0-OfOY</recordid><startdate>20200221</startdate><enddate>20200221</enddate><creator>Ramanayake Mudiyanselage, Tharanga M.R</creator><creator>Michigami, Masataka</creator><creator>Ye, Zhengmao</creator><creator>Uyeda, Atsuko</creator><creator>Inoue, Norimitsu</creator><creator>Sugiura, Kikuya</creator><creator>Fujii, Ikuo</creator><creator>Fujiwara, Daisuke</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><orcidid>https://orcid.org/0000-0002-6230-5534</orcidid></search><sort><creationdate>20200221</creationdate><title>An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction</title><author>Ramanayake Mudiyanselage, Tharanga M.R ; Michigami, Masataka ; Ye, Zhengmao ; Uyeda, Atsuko ; Inoue, Norimitsu ; Sugiura, Kikuya ; Fujii, Ikuo ; Fujiwara, Daisuke</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a452t-acb18484cdd6f0cd9dd36119f3c1ed8d9890ed4888fe7d680c9c78c252d5b4663</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Amino Acid Sequence</topic><topic>B7-1 Antigen - metabolism</topic><topic>CTLA-4 Antigen - metabolism</topic><topic>Dendritic Cells - drug effects</topic><topic>Helix-Loop-Helix Motifs</topic><topic>Humans</topic><topic>Immunoglobulin Fc Fragments - metabolism</topic><topic>Immunologic Factors - chemical synthesis</topic><topic>Immunologic Factors - genetics</topic><topic>Immunologic Factors - pharmacology</topic><topic>Leukocytes, Mononuclear - drug effects</topic><topic>Lymphocyte Activation - drug effects</topic><topic>Peptide Library</topic><topic>Peptides - chemical synthesis</topic><topic>Peptides - genetics</topic><topic>Peptides - pharmacology</topic><topic>Protein Binding - drug effects</topic><topic>Saccharomyces cerevisiae - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ramanayake Mudiyanselage, Tharanga M.R</creatorcontrib><creatorcontrib>Michigami, Masataka</creatorcontrib><creatorcontrib>Ye, Zhengmao</creatorcontrib><creatorcontrib>Uyeda, Atsuko</creatorcontrib><creatorcontrib>Inoue, Norimitsu</creatorcontrib><creatorcontrib>Sugiura, Kikuya</creatorcontrib><creatorcontrib>Fujii, Ikuo</creatorcontrib><creatorcontrib>Fujiwara, Daisuke</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>ACS chemical biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ramanayake Mudiyanselage, Tharanga M.R</au><au>Michigami, Masataka</au><au>Ye, Zhengmao</au><au>Uyeda, Atsuko</au><au>Inoue, Norimitsu</au><au>Sugiura, Kikuya</au><au>Fujii, Ikuo</au><au>Fujiwara, Daisuke</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction</atitle><jtitle>ACS chemical biology</jtitle><addtitle>ACS Chem. Biol</addtitle><date>2020-02-21</date><risdate>2020</risdate><volume>15</volume><issue>2</issue><spage>360</spage><epage>368</epage><pages>360-368</pages><issn>1554-8929</issn><eissn>1554-8937</eissn><abstract>Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4–B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a K D of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4–B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4–B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>31841301</pmid><doi>10.1021/acschembio.9b00743</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0002-6230-5534</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Amino Acid Sequence B7-1 Antigen - metabolism CTLA-4 Antigen - metabolism Dendritic Cells - drug effects Helix-Loop-Helix Motifs Humans Immunoglobulin Fc Fragments - metabolism Immunologic Factors - chemical synthesis Immunologic Factors - genetics Immunologic Factors - pharmacology Leukocytes, Mononuclear - drug effects Lymphocyte Activation - drug effects Peptide Library Peptides - chemical synthesis Peptides - genetics Peptides - pharmacology Protein Binding - drug effects Saccharomyces cerevisiae - genetics |
title | An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction |
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