Engineering Bispecificity into a Single Albumin-Binding Domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed

ADHD medication and sexuality : A qualitative interview study about lived experiences of the impact of stimulant medication on sexuality and how the health care is perceived to respond to this

Bakgrund: När individer diagnostiseras med ADHD är det inte ovanligt att individen påbörjar en centralstimulerande läkemedelsbehandling. Men forskningen kring läkemedelsbehandlingens påverkan på sexualiteten är skral både nationellt och internationellt. Det finns ett behov av att få bättre förståelse de konsekvenser som centralstimulerande läkemedelsbehandling kan ha för individers sexuella hälsa och välbefinnande. Syfte: För att fördjupa kunskapen inom detta område syftade denna studie att undersöka hur vuxna personer med ADHD upplever att centralstimulerande läkemedelsbehandling påverkar deras sexuella hälsa och välbefinnande samt vilka upplevelser de har av hälso- och sjukvårdens bemötande i relation till frågor som rör sexualitet och ADHD-läkemedel.Metod: Studien utgår ifrån en kvalitativ forskningsdesign bestående av nio semistrukturerade intervjuer. Empirin har analyserats utifrån en reflexiv tematisk analys.Resultat: Resultatdelen bygger på tre huvudteman: Medicinska biverkningars påverkan på sexualitet, Intervjupersoners upplevelser av sitt medicinerande sexuella jag – individuellt och relationellt och Sexualitet i en vårdkontext. Dessa har analyserats med hjälp av begreppen compulsory sexuality och compulsory able-bodiedness för att förstå hur normer kring sexualitet och kroppslig funktion samverkar och påverkar individers upplevelse av sin sexualitet. Slutsats: Resultaten visar att centralstimulerande läkemedel påverkar sexualiteten både positivt och negativt i relation till kroppsliga, psykologiska och sociala faktorer. I studien uppkommer även behov och önskemål om att sexologiska frågeställningar och information om sexuella biverkningar lyfts i hälso- och sjukvården. Resultaten är av vikt för den kliniska sexologin för att ämnet ska synliggöras och uppmärksammas i praktiken för att möjliggöra sexuellt välbefinnande hos vuxna personer med ADHD. Background: When individuals are diagnosed with ADHD it is not uncommon that they begin a treatment with stimulant medication. However, research about the impact of said treatment is sparse, both nationally as well as internationally. There lies a need to make better understanding of what consequences central stimulants can have for the individual's sexual health and well-being. Aim: To deepen the knowledge within this field, the study aims to examine how adults diagnosed with ADHD experience how being treated with central stimulants affects their sexual health and well-being, as well as what experiences these individuals have regarding how they are being approached by health care professionals with reference to questions about sexuality and medication for ADHD. Method: A qualitative research method has been used in this study, consisting of nine semi-structured interviews. The observations have been analyzed with a reflexive thematic analysis.Results: The results are divided into three major themes: The influence that medical side effects have on sexuality, How the interviewees view their medicating sexual self - individually and with others and Sexuality in a health care context. The themes have been analyzed together with the concepts of compulsory sexuality and compulsory able-bodiedness in order to understand how social norms regarding sexuality and bodily function intertwine with - and affect – individuals’ view on their own sexuality. Conclusion: The results show that central stimulants affect sexuality in both positive and negative ways regarding bodily, psychological and social factors. The study also shows that there is a need for questions of sexological matter to be brought up by health care professionals, as well as giving information about potential side effects that have an impact on sexual health. The results are important for the clinical sexology field and practition, in order to raise awareness for this subject and to enable sexual well-being for adults with ADHD.

Protein engineering to explore and improve affinity ligands

In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering. Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface ofStreptococcus. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix. Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue. The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region. In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein. Keywords:binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.NR 2014080

ADHD medication and sexuality : A qualitative interview study about lived experiences of the impact of stimulant medication on sexuality and how the health care is perceived to respond to this

Bakgrund: När individer diagnostiseras med ADHD är det inte ovanligt att individen påbörjar en centralstimulerande läkemedelsbehandling. Men forskningen kring läkemedelsbehandlingens påverkan på sexualiteten är skral både nationellt och internationellt. Det finns ett behov av att få bättre förståelse de konsekvenser som centralstimulerande läkemedelsbehandling kan ha för individers sexuella hälsa och välbefinnande. Syfte: För att fördjupa kunskapen inom detta område syftade denna studie att undersöka hur vuxna personer med ADHD upplever att centralstimulerande läkemedelsbehandling påverkar deras sexuella hälsa och välbefinnande samt vilka upplevelser de har av hälso- och sjukvårdens bemötande i relation till frågor som rör sexualitet och ADHD-läkemedel.Metod: Studien utgår ifrån en kvalitativ forskningsdesign bestående av nio semistrukturerade intervjuer. Empirin har analyserats utifrån en reflexiv tematisk analys.Resultat: Resultatdelen bygger på tre huvudteman: Medicinska biverkningars påverkan på sexualitet, Intervjupersoners upplevelser av sitt medicinerande sexuella jag – individuellt och relationellt och Sexualitet i en vårdkontext. Dessa har analyserats med hjälp av begreppen compulsory sexuality och compulsory able-bodiedness för att förstå hur normer kring sexualitet och kroppslig funktion samverkar och påverkar individers upplevelse av sin sexualitet. Slutsats: Resultaten visar att centralstimulerande läkemedel påverkar sexualiteten både positivt och negativt i relation till kroppsliga, psykologiska och sociala faktorer. I studien uppkommer även behov och önskemål om att sexologiska frågeställningar och information om sexuella biverkningar lyfts i hälso- och sjukvården. Resultaten är av vikt för den kliniska sexologin för att ämnet ska synliggöras och uppmärksammas i praktiken för att möjliggöra sexuellt välbefinnande hos vuxna personer med ADHD. Background: When individuals are diagnosed with ADHD it is not uncommon that they begin a treatment with stimulant medication. However, research about the impact of said treatment is sparse, both nationally as well as internationally. There lies a need to make better understanding of what consequences central stimulants can have for the individual's sexual health and well-being. Aim: To deepen the knowledge within this field, the study aims to examine how adults diagnosed with ADHD experience how being treated with central stimulants affects their sexual health and well-being, as well as what experiences these individuals have regarding how they are being approached by health care professionals with reference to questions about sexuality and medication for ADHD. Method: A qualitative research method has been used in this study, consisting of nine semi-structured interviews. The observations have been analyzed with a reflexive thematic analysis.Results: The results are divided into three major themes: The influence that medical side effects have on sexuality, How the interviewees view their medicating sexual self - individually and with others and Sexuality in a health care context. The themes have been analyzed together with the concepts of compulsory sexuality and compulsory able-bodiedness in order to understand how social norms regarding sexuality and bodily function intertwine with - and affect – individuals’ view on their own sexuality. Conclusion: The results show that central stimulants affect sexuality in both positive and negative ways regarding bodily, psychological and social factors. The study also shows that there is a need for questions of sexological matter to be brought up by health care professionals, as well as giving information about potential side effects that have an impact on sexual health. The results are important for the clinical sexology field and practition, in order to raise awareness for this subject and to enable sexual well-being for adults with ADHD.

Protein A chromatography for antibody purification

Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.</p