Respiratory syncytial virus–approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus–approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy. Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus–approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy. Cytokine receptors are in a monomeric off-mode and execute signal transduction in a dimeric or multimeric on-mode after cytokine binding (1Croxford A.L. Mair F. Becher B. IL-23: one cytokine in control of autoimmunity.Eur. J. Immunol. 2012; 42: 2263-2273Crossref PubMed Scopus (139) Google Scholar). The on-mode can be interrupted and converted to the off-mode by depletion of the cytokine from the cytokine receptor, natural cytokine antagonists, or intracellular negative feedback mechanisms. Among others, antibodies as synthetic cytokine antagonists and agonists are a recent but promising development representing an own class of future therapeutic biomolecules (2Domachowske J. Madhi S. Simões E. Atanasova V. Cabañas F. Furuno K. et al.Safety of Nirsevimab for RSV in infants with heart or lung disease or prematurity.N. Engl. J. Med. 2022; 386: 892-894Crossref PubMed Scopus (43) Google Scholar, 3Spangler J. Moraga I. Jude K. Savvides C. Garcia K. A strategy for the selection of monovalent antibodies that span protein dimer interfaces.J. Biol. Chem. 2019; 294: 13876-13886Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Moreover, the approved chimeric antigen receptor (CAR) T-cell therapy for severe cases of acute lymphatic leukemia (4Si W. Li C. Wei P. Synthetic immunology: T-cell engineering and adoptive immunotherapy.Synth. Syst. Biotechnol. 2018; 3: 179-185Crossref PubMed Scopus (21) Google Scholar) is based on synthetic receptors (5Scheller J. 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Nanobodies are already used in diagnostic and therapeutic applications and are immunologically safe (13Pan S. Chia Y. Yee H. Fang Cheng A. Anjum C. Kenisi Y. et al.Immunomodulatory potential of anti-idiotypic antibodies for the treatment of autoimmune diseases.Future Sci. OA. 2020; 7FS0648PubMed Google Scholar). GFP-mCherry fusion proteins served as nanobody-cytokine receptor dimerizers (9Mossner S. Phan H. Triller S. Moll J. Conrad U. Scheller J. Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands.PLoS One. 2020; 15e0230804Google Scholar). GFP and mCherry as synthetic cytokine ligands and specific nanobodies as receptor entities enabled background-free and cell-type–specific activation of synthetic cytokine receptors due to the lack of existing human equivalents for these antigen–antibody interactions. However, the of a because therapeutic to the development of antibodies R. M. D. N. S. D. to fluorescent for PubMed Scopus Google which receptor binding of synthetic ligands. we an synthetic cytokine/cytokine receptor which be based on a nanobody as extracellular receptor This nanobody not to human and the synthetic ligand not be in We that an nanobody pair in which the antibody is not against a human protein be for Moreover, the antibody be for human therapeutic applications to are antibodies or nanobodies that specifically to the variable or of an antibody Y. S. antibodies: biological and J. 9: PubMed Scopus Google Scholar). we Palivizumab as for the development of anti-idiotypic Palivizumab is a antibody against an in the of the fusion protein of respiratory syncytial B. on the antibody for of respiratory syncytial PubMed Scopus Google Scholar). was approved in to infection and severe disease by RSV in infants Palivizumab the of RSV B. on the antibody for of respiratory syncytial PubMed Scopus Google Scholar). We a with by the selection of anti-idiotypic nanobodies using Palivizumab and dimeric Palivizumab single–chain variable fragment served as synthetic cytokine ligands to the synthetic anti-idiotypic Palivizumab nanobody (AIPVHH) cytokine receptor fusion A was with were for the VHH nanobody were by and by using E. K. for Biotechnol. PubMed Google Scholar). were with Palivizumab for were with to unspecific by a were with Palivizumab by an the were of were and anti-idiotypic nanobodies for Palivizumab were as proteins in and by the of to Palivizumab was by a of of of and of for The monomeric a with Palivizumab by a of and a of we used the anti-idiotypic antibody to for anti-idiotypic binding of AIPVHH to because AIPVHH for binding with to Palivizumab of and but not of in a of with an of and and we that and have the or an anti-idiotypic binding to Palivizumab as a to with in or a that are anti-idiotypic to the anti-idiotypic of we the of and Palivizumab and and and the of and and Scopus Google Scholar, H. I. of the of proteins in from a on a Scopus Google Scholar, of and by PubMed Scopus Google Scholar, Franke D. of protein from 2018; PubMed Scopus Google Scholar, The Scholar). The of the and were used as and with the protein the We that and Palivizumab are and is a and Palivizumab a dimer in The of and Palivizumab in a of Palivizumab one the antigen–binding of Palivizumab and the on and We an and of and and of Palivizumab we of and of Palivizumab with of M. B. R. W. D. et of the Chem. PubMed Scopus Google Scholar). were fused to a for the transmembrane and intracellular domain of gp130 and and and The of AIPVHHgp130 proteins in was by against the N-terminal that of in was and was the the have an on the for and to the of the receptors on the are and due to of these to fusion protein of IL-6 and IL-6 receptor which and of signaling M. J. C. A. et al.A cytokine for human Biotechnol. PubMed Google Scholar). We that of the AIPVHHgp130 receptor by Palivizumab in synthetic cytokine receptor of signal and cellular of was the of Palivizumab induced of the for In not in to Palivizumab Palivizumab to in of the AIPVHHgp130 receptors We the the antigen-binding of Palivizumab and to be using We that be to synthetic gp130 receptors. a human mAb was to synthetic gp130 receptor we a of the Palivizumab to was for and but not for the of in with induced in the was for and the antibody in with the nor the and of the of was from to and on was already induced by a However, a was the efficient and Moreover, of the with from to in and Palivizumab efficiently activated synthetic receptor of Palivizumab binding to cellular is by a Palivizumab of with of Palivizumab in the of a of was to of with biological of is in with Palivizumab in the of a of or for of proteins were specific antibodies and one of of with of Palivizumab in the of a and of was to of with biological of is in with Palivizumab in the of a and of or for of proteins were specific antibodies and one of of with of Palivizumab in the of a and of was to of with biological of is in with Palivizumab in the of a of or for of proteins were specific antibodies and one of anti-idiotypic nanobodies for fragment human signal and of that Palivizumab efficiently activated the synthetic AIPVHHgp130 cytokine receptors Palivizumab is a of synthetic AIPVHHgp130 receptors, which be due to the and the of the antigen-binding we Palivizumab single-chain the variable domains of the and the were fused by a in the for and The and the of the variable domains of the or the In the variable domain of the is in the variable domain of the is and were fused to an of an antibody S. K. Conrad C. F. H. et interleukin by in with active 2021; Full Text Full Text PDF PubMed Scopus Google Scholar). The of or to the N-terminal of the was and the a of the variable domains of and as by A and and were in and and as from the A we the of and to by and Palivizumab with of and and of Palivizumab fragments. we on to and for induced cellular and of AIPVHHgp130 with an of for and for which is in with natural cytokine for receptor activation (6Engelowski E. Schneider A. Franke M. Xu H. Clemen R. Lang A. et al.Synthetic cytokine receptors transmit biological signals using artificial ligands.Nat. Commun. 2018; 9: 2034Crossref PubMed Scopus (29) Google Scholar). However, the of was the and to cellular were by was on by and were not activated by we the single-chain is active The activity of was for and with of and In cellular of was induced with with an of with induced in the was for by and We found that induced of with an of which was in the as for of and already induced of in was by the J. R. R. R. B. et of a a protein Med. Chem. 12: PubMed Scopus Google that activation was the activation of signaling was by we the to receptor activation of with we that the The of was in the but not in the of using the of from to not the of and not of and a neither Palivizumab nor induced cellular or of and In summary, the dimeric are efficient anti-idiotypic synthetic cytokine ligands that signal transduction and not the activity of the synthetic gp130 cytokine receptors. dimeric Palivizumab and efficiently synthetic cytokine receptor in with a of and of Palivizumab that Palivizumab is an of receptor 2 was used to of of Palivizumab in of cellular The for of by Palivizumab was of Palivizumab was sufficient to For an of Palivizumab in of signal transduction in monomeric AIPVHH was for of of induced cellular with an of of and were and not as an because of to and The of these to and to The death receptor Fas and the trimeric D. functions in death and Biol. PubMed Scopus Google Scholar, V. A. receptor signal of in signaling Immunol. 2009; PubMed Scopus Google Scholar). trimeric binding D. the of 2020; 9: PubMed Scopus Google Scholar). as synthetic we that by the was induced by which efficient the trimeric or S. Floss D. Scheller J. Pro- and anti-apoptotic fate decisions induced by di- and trimeric synthetic cytokine receptors.iScience. 2021; 24102471Abstract Full Text Full Text PDF Scopus (3) Google Scholar). Here, the anti-idiotypic synthetic cytokine were to were fused to a for the transmembrane and intracellular domain of human with an N-terminal signal by a for against the N-terminal of synthetic receptors on were for activation of and of after in to gp130 The synthetic dimeric and ligands were to dimeric and receptor of was by of fused to the Fas was induced by of with and ligands induced activation after in and was not induced with of the synthetic ligands. to of was to in The of activated was and of ligand For activation was as as for and dimeric activated to to of was using after and of with synthetic cytokine ligands. treatment served as with was the control of in to and were synthetic cytokine found after dimeric receptor activation with not and receptor activation efficiently induced already after were after with and we have that activation of synthetic Fas efficiently induced that the nanobody-cytokine receptor pair is for synthetic cytokine receptor and anti-idiotypic antibody is against the of antibody, which the antigen-binding of the antibodies Y. S. antibodies: biological and J. 9: PubMed Scopus Google Scholar). In the fully synthetic cytokine/cytokine receptor system in in cellular activation gp130 or cellular which the of to synthetic cytokine receptors in one This system is because signal activation can be by nanobodies or which therapeutic nonphysiological targets in immunotherapy. death as and are in and the of T-cell therapy D. M. for T-cell Full Text Full Text PDF PubMed Scopus Google Scholar, M. B. E. B. F. H. et to the of chimeric antigen receptor N. Y. Sci. 2022; PubMed Scopus Google Scholar). We an approved therapeutic antibody the of a from the fully synthetic receptor system for the and activation of cytokine signaling in a the of activation of Recently, we developed a based on receptor fusion proteins activated by dimeric GFP and mCherry ligands (6Engelowski E. Schneider A. Franke M. Xu H. Clemen R. Lang A. et al.Synthetic cytokine receptors transmit biological signals using artificial ligands.Nat. Commun. 2018; 9: 2034Crossref PubMed Scopus (29) Google Scholar). Moreover, we that the of the in the synthetic dimeric ligand is for receptor activation (9Mossner S. Phan H. Triller S. Moll J. Conrad U. Scheller J. Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands.PLoS One. 2020; 15e0230804Google Scholar). This system was used to cytokine signaling for IL-6 and signaling, for IL-10 and signaling, and for the trimeric receptor class of tumor necrosis factor (6Engelowski E. Schneider A. Franke M. Xu H. Clemen R. Lang A. et al.Synthetic cytokine receptors transmit biological signals using artificial ligands.Nat. Commun. 2018; 9: 2034Crossref PubMed Scopus (29) Google Scholar, 7Mossner S. Floss D. Scheller J. Pro- and anti-apoptotic fate decisions induced by di- and trimeric synthetic cytokine receptors.iScience. 2021; 24102471Abstract Full Text Full Text PDF Scopus (3) Google Scholar, 8Mossner S. Kuchner M. Fazel Modares N. Knebel B. Al-Hasani H. Floss D. et al.Synthetic interleukin 22 (IL-22) signaling reveals biological activity of homodimeric IL-10 receptor 2 and functional cross-talk with the IL-6 receptor gp130.J. Biol. Chem. 2020; 295: 12378-12397Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, N. N. F. J. Xu H. E. et al.Synthetic a to in Microbiol. 2022; PubMed Scopus Google Scholar). The and ligands and However, GFP and mCherry are to the which in the of these synthetic ligands R. M. D. N. S. D. to fluorescent for PubMed Scopus Google Scholar). we that approved antibodies as synthetic ligands for anti-idiotypic nanobody-cytokine receptors. was that antibodies not have the human targets or clinically and antibodies with or as E. F. M. M. IL-6 2020; PubMed Scopus Google F. of in the from to J. 2009; PubMed Scopus Google or D. The of in 2012; 12: PubMed Scopus Google Scholar) were because these human we antibodies that and Palivizumab B. on the antibody for of respiratory syncytial PubMed Scopus Google Scholar) and the human Nirsevimab (2Domachowske J. Madhi S. Simões E. Atanasova V. Cabañas F. Furuno K. et al.Safety of Nirsevimab for RSV in infants with heart or lung disease or prematurity.N. Engl. J. Med. 2022; 386: 892-894Crossref PubMed Scopus (43) Google human A. Y. Juarez E. M. J. D. et treatment for with antibody Engl. J. Med. 2021; PubMed Scopus Google C. D. J. Y. J. Y. et al.A therapeutic antibody receptor binding domain of Commun. 2021; 12: PubMed Scopus Google and in to with a 2022; Full Text Full Text PDF PubMed Scopus Google Scholar) or and in to with a 2022; Full Text Full Text PDF PubMed Scopus Google human S. P. during an of disease in the of Engl. J. Med. 2022; 386: PubMed Scopus Google Scholar) and the antibody S. P. during an of disease in the of Engl. J. Med. 2022; 386: PubMed Scopus Google and human J. A. M. et for the treatment of Engl. J. Med. 2009; PubMed Scopus Google Scholar). We Palivizumab to the anti-idiotypic nanobodies because Palivizumab the in of on of for infants and of for respiratory syncytial 2014; PubMed Scopus Google Scholar). Palivizumab an in the A of the fusion protein of RSV and the of RSV B. on the antibody for of respiratory syncytial PubMed Scopus Google Scholar). nanobody-cytokine receptor signaling was efficiently activated after Palivizumab by However, of antibodies in we Palivizumab in dimeric and fusion which were highly We that the of the variable was for an efficient of nanobody-cytokine receptors, which be a for antibody The of antibodies can be the fragment antigen–binding and the The is the the variable by one domain of the or in the and antibody In and domains in In the fusion which were the active synthetic cytokine the domains of the were in synthetic antibodies with variable domains the of were and as A. A. E. A. W. S. et antibodies in with human in Biotechnol. 2009; PubMed Scopus Google Scholar). to be Palivizumab antibody as or synthetic anti-idiotypic cytokine signaling one domain the we that these antibody not synthetic cytokine receptors. the due to the with antigen-binding Palivizumab an antibody the and domains be of in future for production have been and the antibodies have B. R. A. S. M. and therapeutic of 2020; 9: Scopus Google Scholar). The of nanobody of the antibodies the of receptor For receptor be with GFP or mCherry fused to Palivizumab We have not because to as synthetic ligands. The fusion proteins the of or synthetic receptor was in that Fas signaling activated based on ligands. AIPVHH and to in with studies on receptor signaling C. H. S. C. J. et as a strategy to antibody PubMed Scopus Google Scholar, M. S. C. C. M. et al.A of Fas receptor antibodies that an and 2012; PubMed Scopus Google Scholar). can be with or using the for J. C. Li Y. E. Y. K. et fusion proteins based on domain in an for or and PubMed Scopus Google Scholar, R. strategies with 2012; PubMed Scopus Google Scholar). the of be In that antibody as efficient synthetic these antibody be used to antibodies as synthetic ligands. The antibodies in the and are in development A. M. J. P. antibodies: a of the 2021; Scopus Google Scholar). used in can be nonimmunogenic is of T-cell Nanobodies have a of to to the human in applications Y. F. S. F. A nanobodies and variable 2021; 9: PubMed Scopus Google one or of the in be the system been to in (6Engelowski E. Schneider A. Franke M. Xu H. Clemen R. Lang A. et al.Synthetic cytokine receptors transmit biological signals using artificial ligands.Nat. Commun. 2018; 9: 2034Crossref PubMed Scopus (29) Google the nanobody-cytokine receptor pair not been in vivo and have to be in nanobodies and are used in the or of the used in been Moreover, strategies to that is to to human heavy–chain variable domain C. R. D. S. Muyldermans S. K. strategy to a antibody and of a nanobody Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). a nanobody against factor to was the clinically approved in and the M. S. F. P. P. J. et treatment for Engl. J. Med. 2019; PubMed Scopus Google Scholar). as are tools be or the activity of was with Palivizumab experimental and were in with and In for of Palivizumab in a of were with or were A of were the of and was and was the from of the was to utilizing the as J. D. D. J. B. et of antibodies from using and J. 2020; Scholar, J. D. S. A. et in with the rapid of antibody from J. 2019; PubMed Scopus Google Scholar). For of were using in the to the signal of and in the the was by and an to VHH was using the and and used for a with pair and The was of as D. M. J. C. C. et against therapeutic antibodies anti-idiotypic 7: PubMed Scopus Google Scholar). were with Palivizumab for unspecific were with a human by with were with Palivizumab by an the were of were The for the of Palivizumab was by The of Palivizumab was from The variable and variable were using The variable and were by a The were the for an N-terminal signal and and a by a human in and In the a was the and The for AIPVHH an N-terminal signal and by and AIPVHH were by and and The were for synthetic cytokine receptors were by fusion of for the signal a by the anti-idiotypic nanobody of the extracellular domain the transmembrane domain and intracellular domain of the cytokine receptors gp130 and Fas. The to from human gp130 were representing of the the and the The to from the human Fas were representing of the the and were by and and in (6Engelowski E. Schneider A. Franke M. Xu H. Clemen R. Lang A. et al.Synthetic cytokine receptors transmit biological signals using artificial ligands.Nat. Commun. 2018; 9: 2034Crossref PubMed Scopus (29) Google Scholar). gp130 was by Fas using and in For transduction of with the for the synthetic receptors, the were S. Floss D. Scheller J. Pro- and anti-apoptotic fate decisions induced by di- and trimeric synthetic cytokine receptors.iScience. 2021; 24102471Abstract Full Text Full Text PDF Scopus (3) Google Scholar). The of was A. B. K. H. et is a natural of and to the proinflammatory of PubMed Scopus Google Scholar). The was from were in with and with of was in the of human M. J. C. A. et al.A cytokine for human Biotechnol. PubMed Google Scholar). were in a of to in a with on an and antibodies were from was from and were from was from Palivizumab antibody was from The antibody was from were with the for and as in S. Floss D. Scheller J. Pro- and anti-apoptotic fate decisions induced by di- and trimeric synthetic cytokine receptors.iScience. 2021; 24102471Abstract Full Text Full Text PDF Scopus (3) Google Scholar). were in as with of was with for 2 the were for synthetic receptor and were using to the were to a of in of the were used for to the the was by for by of the for The of the was and by proteins a were using A a of The was with of were to using a the protein were and the was to with proteins a were using to the proteins were to using were by and were in was by and functional For the and A were Palivizumab and were to a a of or were ligand and by from to with an with were a of for and the was for were in of and The were and to a binding using the We the on the A. F. 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M. protein to Methods. 2022; PubMed Scopus Google Scholar, Franke D. M. et in the for 2012; PubMed Scopus Google Scholar) to The the and the were used as for the This is a as in B. M. K. J. et binding to the protein with therapeutic potential in 2021; PubMed Scopus Google Scholar, D. V. B. R. of the antibody 2022; PubMed Scopus Google Scholar). The nanobody was created with the nanobody domain was used as a and the and were with M. K. Y. S. M. protein to Methods. 2022; PubMed Scopus Google Scholar, Franke D. M. et in the for 2012; PubMed Scopus Google Scholar). The and from Palivizumab were used as a for the with