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Exploitation of interactions with the neonatal Fc receptor to manipulate biological half-lives for therapeutic applications
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering. (Gräslund group)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein engineering provides powerful tools to create useful proteins with desired properties. In this thesis, rational design principles have been used for development of fusion proteins that can interact with the neonatal Fc receptor (FcRn) for potential medical applications. 

FcRn is widely expressed in the human body. The natural ligands of FcRn are immunoglobulin G (IgG) and serum albumin (SA). FcRn can bind to both proteins in a pH dependent manner and endow them with an unusually long half-life in vivo. Protein building blocks interacting directly or indirectly with FcRn may potentially be used to either piggy-back on the FcRn-system for extension of the in vivo half-life or to saturate the system to decrease the in vivo half-life of the natural ligands. In this thesis, I have explored an FcRn binding affibody molecule (ZFcRn) and/or an albumin binding domain (ABD) for these purposes. 

In study I and II, the prolactin receptor was found to often be expressed in glioblastoma multiforme tumors from patients as well as in glioblastoma multiforme cells lines. We investigated a novel antagonist of the prolactin receptor in vitro and found that it could block signaling through the receptor as well as cellular invasiveness. An antagonist of prolactin receptor could thus potentially become a drug for treatment of glioblastoma multiforme. However, the antagonist will likely have a short plasma half-life due to its small molecular size, which limits its usability. Therefore, it was expressed as a fusion to ABD, which interacts indirectly with FcRn. The produced fusion protein was found to be able to block signaling through the prolactin receptor in vitro and also had a prolonged plasma half-life in vivo

The goal of study III was to investigate the properties of human growth hormone (hGH) when it was expressed as a protein fusion with ZFcRn, interacting directly with FcRn, and/or ABD. The fusion proteins, ZFcRn-hGH, ABD-hGH, and ZFcRn-ABD-hGH could be recombinantly expressed and successfully purified to homogeneity. They had the expected binding abilities to FcRn, SA and hGH receptor. They were all found to be able to induce signaling over the plasma membrane in a model cell line. 

Patients suffering from many autoimmune diseases produce particular IgG molecules, which are responsible for the disease symptoms. A potential treatment could be to increase the catabolism of these IgGs to relieve disease symptoms. In study IV, an FcRn interacting affibody molecule was investigated for IgG depletion by blocking the IgG/FcRn interaction. In vitro, we first found that the affibody molecule shares a common binding site with IgG on FcRn, which indicates that the affibody should be able to block IgG from binding to FcRn. In vivo, we injected large amounts of the affibody molecules in different formats in mice and found up to 39% reduction of total endogenous IgG. In a clinical setting, reduction of total IgG level would also reduce the disease causing IgGs, and potentially ameliorate the symptoms of IgG-driven autoimmune diseases. 

Taken together, I have in this thesis explored application of FcRn interacting molecules for extension of biological half-lives of therapeutically relevant proteins and reduction of total IgG level by FcRn blocking. 

Abstract [sv]

Proteinteknik tillhandahåller kraftfulla verktyg för att skapa användbara proteiner med önskade egenskaper. I denna avhandling har rationella designprinciper använts för utveckling av fusionsproteiner som kan interagera med neonatal Fc-receptorn (FcRn) för potentiella medicinska tillämpningar. 

FcRn uttrycks allmänt i människokroppen. De naturliga liganderna aför FcRn är immunglobulin G (IgG) och serumalbumin (SA). FcRn kan binda till båda proteinerna på ett pH-beroende sätt och förse dem med en ovanligt lång halveringstid in vivo. Proteinbyggstenar som interagerar direkt eller indirekt med FcRn kan potentiellt användas för att antingen åka snålskjuts på FcRn-systemet för förlängning av halveringstiden in vivo eller för att mätta systemet för att minska halveringstiden in vivo för de naturliga liganderna. I den här avhandlingen har jag utforskat en FcRn-bindande affibody molekyl (ZFcRn) och en albuminbindande domän (ABD) för dessa ändamål. 

I studie I och II befanns prolaktinreceptorn ofta uttryckt i glioblastoma multiforme tumörer från patienter såväl som i glioblastoma multiforme cellinjer. Vi undersökte en ny antagonist av prolaktinreceptorn in vitro och fann att den kunde blockera signalering genom receptorn såväl som cellulär invasivitet. En prolaktinreceptorantagonist kan således potentiellt bli ett läkemedel för behandling av glioblastoma multiforme. Men antagonisten kommer troligen att ha en kort halveringstid i blodet på grund av dess lilla storlek, vilket begränsar användbarheten. Därför uttrycktes det som en fusion till ABD, som interagerar indirekt med FcRn. Det producerade fusionsproteinet visade sig kunna blockera signalering genom prolaktinreceptorn in vitro och hade också en förlängd halveringstid i blodet in vivo

Målet med studie III var att undersöka egenskaperna hos humant tillväxthormon (hGH) när det uttrycktes som en proteinfusion med ZFcRn som interagerar direkt med FcRn och/eller ABD. Fusionsproteinerna, ZFcRn-hGH, ABD-hGH och ZFcRn-ABD-hGH kunde uttryckas rekombinant och kunde framgångsrikt renas till homogenitet. De hade de förväntade bindningsförmågorna till FcRn, SA och hGH-receptorn. De visade sig alla kunna inducera signalering över plasmamembranet i en modellcellinje. 

Patienter som lider av många autoimmuna sjukdomar producerar specifika IgG-molekyler som är ansvariga för sjukdomens symtom. En potentiell behandling kan vara att öka katabolismen av dessa IgG för att lindra sjukdomssymtomen. I studie IV undersöktes en FcRn-interagerande affibodymolekyl för IgG-minskning, detta genom att blockera IgG/FcRn-interaktionen. In vitro fann vi först att affibodymolekylerna delar ett gemensamt bindningsställe med IgG på FcRn, vilket indikerade att affibodymolekylen borde kunna blockera IgG från bindning till FcRn. In vivo injicerade vi stora mängder av affibodymolekylen i olika format i möss och fann upp till 39% reduktion av den totala mängden endogent IgG. I en klinisk kontext skulle reduktion av total IgG också minska nivån av de IgG som driver sjukdomen och potentiellt minska symtomen hos patienter som lider av IgG-drivna autoimmuna sjukdomar. 

Sammantaget har jag i den här avhandlingen undersökt tillämpningar av FcRn-interagerande molekyler för förlängning av biologiska halveringstider av terapeutiskt relevanta proteiner och dessutom reduktion av totalt IgG genom FcRn-blockering. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. , p. 87
Series
TRITA-CBH-FOU ; 2019:41
Keywords [en]
Glioblastoma Multiforme (GBM), Prolacin (Prl), Prolacin receptor (PrlR), Prolactin antagonist (PrlRA), Albumin binding domain (ABD), serum albumin (SA), half-life extension, FcRn, Affibody, human growth hormone (hGH), immunoglobulin G (IgG), blocking, autoimmune disease
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-256536ISBN: 978-91-7873-268-5 (print)OAI: oai:DiVA.org:kth-256536DiVA, id: diva2:1346897
Public defence
2019-09-20, Oskar Kleins Auditorium, Roslagstullsbacken 21, Albanova University Center, Stockholm, 10:30 (English)
Opponent
Supervisors
Note

QC 2019-08-29

Available from: 2019-08-29 Created: 2019-08-29 Last updated: 2019-08-29Bibliographically approved
List of papers
1. Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme
Open this publication in new window or tab >>Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme
Show others...
2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 48, p. 79558-79569Article in journal (Refereed) Published
Abstract [en]

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

Place, publisher, year, edition, pages
Impact Journals LLC, 2016
Keywords
prolactin, prolactin receptor, prolactin receptor antagonist, STAT5, GBM
National Category
Cancer and Oncology Cell Biology
Identifiers
urn:nbn:se:kth:diva-199535 (URN)10.18632/oncotarget.12840 (DOI)000389636000109 ()2-s2.0-84998678038 (Scopus ID)
Note

QC 20170116

Available from: 2017-01-16 Created: 2017-01-09 Last updated: 2019-08-29Bibliographically approved
2. An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation
Open this publication in new window or tab >>An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation
2019 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 5, article id e0215831Article in journal (Refereed) Published
Abstract [en]

Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (K-D = 2.3 +/- 0.2 nM) and mouse serum albumin (K-D = 0.38 +/- 0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21 +/- 3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2019
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-252381 (URN)10.1371/journal.pone.0215831 (DOI)000467148400008 ()31063493 (PubMedID)2-s2.0-85065793714 (Scopus ID)
Note

QC 20190718

Available from: 2019-07-18 Created: 2019-07-18 Last updated: 2019-08-29Bibliographically approved
3. Human growth hormone can transduce signals to phosphorylate STAT5 as a fusion with an affibody molecule binding the neonatal Fc receptor and an albumin binding domain
Open this publication in new window or tab >>Human growth hormone can transduce signals to phosphorylate STAT5 as a fusion with an affibody molecule binding the neonatal Fc receptor and an albumin binding domain
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Growth hormone replacement therapy has been used to treat children and adults with

growth hormone deficiency for more than three decades. Growth hormone has a short

biological half-life, requiring daily subcutaneous injections, and long acting versions are

therefore desirable. In this study we have analyzed three fusion proteins, ZFcRn-hGH,

ABD-hGH and ZFcRn-ABD-hGH, consisting of the human growth hormone (hGH) and an

affibody molecule binding the neonatal Fc receptor (ZFcRn) and/or an albumin binding

domain (ABD). Both ZFcRn and the ABD may have the ability to endow hGH with an

extended plasma half-life. The fusion proteins could be recombinantly expressed in the

periplasmic space of Escherichia coli and easily purified to homogeneity. All three fusion

proteins appeared to have a strong interaction with the growth hormone receptor. ABDhGH

and ZFcRn-ABD-hGH had a strong affinity for HSA (KD 0.006 and 0.02 nM,

respectively). ZFcRn-hGH and ZFcRn-ABD-hGH had moderately strong affinity for mouse

neonatal Fc receptor at pH 6.0 (KD 200 and 100 nM, respectively). The fusion proteins

thus retained the expected binding abilities of the individual domains. Further

characterization showed that the fusion proteins could induce phosphorylation of signal

transducer and activator of transcription 5 (STAT5) in the model cell line U251-MG,

further showing that the hGH-part of the fusion proteins was functional.

Keywords
ABD, affibody molecule, albumin binding domain, human growth hormone, neonatal Fc receptor, prolactin receptor, STAT5
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-257350 (URN)
Note

QC 20190906

Available from: 2019-08-29 Created: 2019-08-29 Last updated: 2019-09-06Bibliographically approved
4. In vivo depletion of serum IgG by an affibody molecule binding the neonatal Fc receptor
Open this publication in new window or tab >>In vivo depletion of serum IgG by an affibody molecule binding the neonatal Fc receptor
Show others...
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5141Article in journal (Refereed) Published
Abstract [en]

Lowering the total level of Immunoglobulin G (IgG) in circulation is a promising general treatment option for many autoimmune diseases driven by pathogenic autoantibodies. The half-life of IgG in circulation is unusually long as a consequence of its interaction with the neonatal Fc receptor (FcRn), which protects it from lysosomal degradation by cells in contact with blood. Blocking the IgG/FcRn interaction prevents FcRn-mediated rescue, which may lead to increased catabolism and a lowering of the total IgG level. Here, we find that an engineered alternative scaffold protein, an affibody molecule, interacting specifically with FcRn, is able to block the IgG/FcRn interaction in vitro. The affibody molecule (Z(FcRn)) was expressed alone or as a fusion to an albumin binding domain (ABD), to extend its half-life in circulation, in both cases with retained affinity and blocking potential. Repeated i.v. injections in mice of Z(FcRn) and Z(FcRn)-ABD were found to result in an up to 40% reduction of the IgG serum-level after 5 days. Potential applications of Z(FcRn) as a general treatment modality for autoimmune diseases are discussed.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-225708 (URN)10.1038/s41598-018-23481-5 (DOI)000428163600012 ()29572538 (PubMedID)2-s2.0-85044363795 (Scopus ID)
Note

QC 20180411

Available from: 2018-04-11 Created: 2018-04-11 Last updated: 2019-08-29Bibliographically approved

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