Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation
Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-express...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-04-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1341013/full |
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| author | H.A. Daniel Lagassé Jiayi Ou Zuben E. Sauna Basil Golding |
| author_facet | H.A. Daniel Lagassé Jiayi Ou Zuben E. Sauna Basil Golding |
| author_sort | H.A. Daniel Lagassé |
| collection | DOAJ |
| description | Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains. |
| first_indexed | 2024-04-24T11:55:13Z |
| format | Article |
| id | doaj.art-2ae01388a1334f9cb319d59268a22ae9 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-24T11:55:13Z |
| publishDate | 2024-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-2ae01388a1334f9cb319d59268a22ae92024-04-09T04:45:58ZengFrontiers Media S.A.Frontiers in Immunology1664-32242024-04-011510.3389/fimmu.2024.13410131341013Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activationH.A. Daniel Lagassé0Jiayi Ou1Zuben E. Sauna2Basil Golding3Division of Hemostasis, Office of Plasma Protein Therapeutics CMC, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesDivision of Hemostasis, Office of Plasma Protein Therapeutics CMC, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesDivision of Hemostasis, Office of Plasma Protein Therapeutics CMC, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesOffice of Plasma Protein Therapeutics CMC, Office of Therapeutic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesRecombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.https://www.frontiersin.org/articles/10.3389/fimmu.2024.1341013/fullFc-fusionFc gamma receptorsnatural killer cellsFc gamma receptor IIIaFactor VIII light chaindiscoidin domain |
| spellingShingle | H.A. Daniel Lagassé Jiayi Ou Zuben E. Sauna Basil Golding Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation Frontiers in Immunology Fc-fusion Fc gamma receptors natural killer cells Fc gamma receptor IIIa Factor VIII light chain discoidin domain |
| title | Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation |
| title_full | Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation |
| title_fullStr | Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation |
| title_full_unstemmed | Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation |
| title_short | Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation |
| title_sort | factor viii moiety of recombinant factor viii fc fusion protein impacts fc effector function and cd16 nk cell activation |
| topic | Fc-fusion Fc gamma receptors natural killer cells Fc gamma receptor IIIa Factor VIII light chain discoidin domain |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1341013/full |
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