9 research outputs found
Selection and characterization of bispecific ADAPT molecules for enhanced biodistribution in cancer therapy
Established biopharmaceuticals such as antibodies and derivatives thereof are relatively large. In cancer therapy, this creates a steep drug concentration gradient within tumors, leaving cells far from blood vessels effectively untreated. Continuous pseudo treatments should foster the development of drug resistance and might lead to eventual disease relapse. Drug concentration gradients can be operationalized as tissue penetration efficiencies, which are functions of molecular size. However, small particles are also subject to potent renal clearance, collapsing the therapeutic window beyond clinical applications. In this masterâs thesis, spatial bispecificity was engineered into a single albumin binding domain (ABD). Resulting ABD derived affinity proteins (ADAPTs) are saved from urinary excretion by the grace of HSA, but in the more static microenvironment of tumors, following HSA dissociation, they are capable of tissue penetration efficiencies bestowed only upon smaller particles. To this end, phage display was used to raise ADAPTs against the cancer associated proteins human epidermal growth factor receptor 2 (HER2) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), but also the inflammation marker C-reactive protein (CRP). Via Sanger sequencing, 9 variants were picked for protein production and characterization, among which two spatially bispecific binders were found. ADAPTs were also evaluated for aggregation tendencies, structural conformity to library design, and thermal stability. One ADAPT, binding HER2, passed all tests of initial characterizations. Deep sequencing was used to analyze selection output, from which many more binders should be screened in future experiments.Etablerade biolĂ€kemedel liksom antikroppar och deras derivat Ă€r relativt stora protein. Som cancerterapeutiska skapar de sĂ„ledes branta koncentrationsgradienter utgĂ„ende frĂ„n tumörpenetrerande blodkĂ€rl. Detta riskerar att lĂ€mna vissa cancerceller utanför det terapeutiska fönstret. Det svaga selektionstryck som sĂ„ledes verkar i tumörperiferin fostrar cancerceller till att utveckla resistens mot detsamma. Koncentrationsgradienten beror pĂ„ proteinets vĂ€vnadspenetrarande förmĂ„ga, vilken Ă€r en funktion av proteinets storlek. Mindre proteiner borde dĂ€rmed lĂ€ttare ackumuleras i hela tumören och förebygga resistensutveckling. Problemet med smĂ„ proteiner Ă€r deras mycket korta halveringstid i serum, en följd av relativt obehindrad filtrering ut i urinen via njurarna. I det hĂ€r examensarbetet utvecklades rumsbispecifika bindare av cancerassocierade protein med hjĂ€lp av fagdisplayselektioner frĂ„n ett proteinbibliotek baserat pĂ„ en enda albuminbindande domĂ€n (ABD). Resulterande ABD deriverade affinitetsprotein (ADAPT) undkommer ovan nĂ€mnda filtrering tack vare sin naturligt starka interaktion med humant serumalbumin (HSA). I den mer lĂ„ngsamt flödande tumörmikromiljön tillĂ„ts ADAPTerna efter albumindissociation sedan utöva en bland biolĂ€kemedel överlĂ€gsen vĂ€vnadspenetration. Tre parallella selektionsspĂ„r utfördes mot de cancerassocierade mĂ„lproteinerna human epidermal growth factor receptor 2 (HER2) och carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) samt den utsöndrade inflammationsmarkören C-reaktivt protein (CRP). Via Sangersekvensering kunde flera kandidater identifieras. Bland 6 karakteriserade ADAPTer uppvisade samtliga hög HSA-affinitet, tre konstaterades interagera specifikt med sitt mĂ„lprotein, och tvĂ„ verkade binda Ă€ven rumsbispecifikt. ADAPTer utvĂ€rderades Ă€ven för sin benĂ€genhet att bilda aggregat, strukturell överensstĂ€mmelse med experimentell design, och vĂ€rmestabilitet. Endast en bindare, mot HER2, klarade sig genom alla prövningar som proteinkarakteriseringen innebar utan underkĂ€nt. Ăven en högparallel sekvensering utav selektionsresultat utfördes, men utanför de tidsramar som tillĂ€t ytterligare karakterisering
Selection and characterization of bispecific ADAPT molecules for enhanced biodistribution in cancer therapy
Established biopharmaceuticals such as antibodies and derivatives thereof are relatively large. In cancer therapy, this creates a steep drug concentration gradient within tumors, leaving cells far from blood vessels effectively untreated. Continuous pseudo treatments should foster the development of drug resistance and might lead to eventual disease relapse. Drug concentration gradients can be operationalized as tissue penetration efficiencies, which are functions of molecular size. However, small particles are also subject to potent renal clearance, collapsing the therapeutic window beyond clinical applications. In this masterâs thesis, spatial bispecificity was engineered into a single albumin binding domain (ABD). Resulting ABD derived affinity proteins (ADAPTs) are saved from urinary excretion by the grace of HSA, but in the more static microenvironment of tumors, following HSA dissociation, they are capable of tissue penetration efficiencies bestowed only upon smaller particles. To this end, phage display was used to raise ADAPTs against the cancer associated proteins human epidermal growth factor receptor 2 (HER2) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), but also the inflammation marker C-reactive protein (CRP). Via Sanger sequencing, 9 variants were picked for protein production and characterization, among which two spatially bispecific binders were found. ADAPTs were also evaluated for aggregation tendencies, structural conformity to library design, and thermal stability. One ADAPT, binding HER2, passed all tests of initial characterizations. Deep sequencing was used to analyze selection output, from which many more binders should be screened in future experiments.Etablerade biolĂ€kemedel liksom antikroppar och deras derivat Ă€r relativt stora protein. Som cancerterapeutiska skapar de sĂ„ledes branta koncentrationsgradienter utgĂ„ende frĂ„n tumörpenetrerande blodkĂ€rl. Detta riskerar att lĂ€mna vissa cancerceller utanför det terapeutiska fönstret. Det svaga selektionstryck som sĂ„ledes verkar i tumörperiferin fostrar cancerceller till att utveckla resistens mot detsamma. Koncentrationsgradienten beror pĂ„ proteinets vĂ€vnadspenetrarande förmĂ„ga, vilken Ă€r en funktion av proteinets storlek. Mindre proteiner borde dĂ€rmed lĂ€ttare ackumuleras i hela tumören och förebygga resistensutveckling. Problemet med smĂ„ proteiner Ă€r deras mycket korta halveringstid i serum, en följd av relativt obehindrad filtrering ut i urinen via njurarna. I det hĂ€r examensarbetet utvecklades rumsbispecifika bindare av cancerassocierade protein med hjĂ€lp av fagdisplayselektioner frĂ„n ett proteinbibliotek baserat pĂ„ en enda albuminbindande domĂ€n (ABD). Resulterande ABD deriverade affinitetsprotein (ADAPT) undkommer ovan nĂ€mnda filtrering tack vare sin naturligt starka interaktion med humant serumalbumin (HSA). I den mer lĂ„ngsamt flödande tumörmikromiljön tillĂ„ts ADAPTerna efter albumindissociation sedan utöva en bland biolĂ€kemedel överlĂ€gsen vĂ€vnadspenetration. Tre parallella selektionsspĂ„r utfördes mot de cancerassocierade mĂ„lproteinerna human epidermal growth factor receptor 2 (HER2) och carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) samt den utsöndrade inflammationsmarkören C-reaktivt protein (CRP). Via Sangersekvensering kunde flera kandidater identifieras. Bland 6 karakteriserade ADAPTer uppvisade samtliga hög HSA-affinitet, tre konstaterades interagera specifikt med sitt mĂ„lprotein, och tvĂ„ verkade binda Ă€ven rumsbispecifikt. ADAPTer utvĂ€rderades Ă€ven för sin benĂ€genhet att bilda aggregat, strukturell överensstĂ€mmelse med experimentell design, och vĂ€rmestabilitet. Endast en bindare, mot HER2, klarade sig genom alla prövningar som proteinkarakteriseringen innebar utan underkĂ€nt. Ăven en högparallel sekvensering utav selektionsresultat utfördes, men utanför de tidsramar som tillĂ€t ytterligare karakterisering
Selection and characterization of bispecific ADAPT molecules for enhanced biodistribution in cancer therapy
Established biopharmaceuticals such as antibodies and derivatives thereof are relatively large. In cancer therapy, this creates a steep drug concentration gradient within tumors, leaving cells far from blood vessels effectively untreated. Continuous pseudo treatments should foster the development of drug resistance and might lead to eventual disease relapse. Drug concentration gradients can be operationalized as tissue penetration efficiencies, which are functions of molecular size. However, small particles are also subject to potent renal clearance, collapsing the therapeutic window beyond clinical applications. In this masterâs thesis, spatial bispecificity was engineered into a single albumin binding domain (ABD). Resulting ABD derived affinity proteins (ADAPTs) are saved from urinary excretion by the grace of HSA, but in the more static microenvironment of tumors, following HSA dissociation, they are capable of tissue penetration efficiencies bestowed only upon smaller particles. To this end, phage display was used to raise ADAPTs against the cancer associated proteins human epidermal growth factor receptor 2 (HER2) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), but also the inflammation marker C-reactive protein (CRP). Via Sanger sequencing, 9 variants were picked for protein production and characterization, among which two spatially bispecific binders were found. ADAPTs were also evaluated for aggregation tendencies, structural conformity to library design, and thermal stability. One ADAPT, binding HER2, passed all tests of initial characterizations. Deep sequencing was used to analyze selection output, from which many more binders should be screened in future experiments.Etablerade biolĂ€kemedel liksom antikroppar och deras derivat Ă€r relativt stora protein. Som cancerterapeutiska skapar de sĂ„ledes branta koncentrationsgradienter utgĂ„ende frĂ„n tumörpenetrerande blodkĂ€rl. Detta riskerar att lĂ€mna vissa cancerceller utanför det terapeutiska fönstret. Det svaga selektionstryck som sĂ„ledes verkar i tumörperiferin fostrar cancerceller till att utveckla resistens mot detsamma. Koncentrationsgradienten beror pĂ„ proteinets vĂ€vnadspenetrarande förmĂ„ga, vilken Ă€r en funktion av proteinets storlek. Mindre proteiner borde dĂ€rmed lĂ€ttare ackumuleras i hela tumören och förebygga resistensutveckling. Problemet med smĂ„ proteiner Ă€r deras mycket korta halveringstid i serum, en följd av relativt obehindrad filtrering ut i urinen via njurarna. I det hĂ€r examensarbetet utvecklades rumsbispecifika bindare av cancerassocierade protein med hjĂ€lp av fagdisplayselektioner frĂ„n ett proteinbibliotek baserat pĂ„ en enda albuminbindande domĂ€n (ABD). Resulterande ABD deriverade affinitetsprotein (ADAPT) undkommer ovan nĂ€mnda filtrering tack vare sin naturligt starka interaktion med humant serumalbumin (HSA). I den mer lĂ„ngsamt flödande tumörmikromiljön tillĂ„ts ADAPTerna efter albumindissociation sedan utöva en bland biolĂ€kemedel överlĂ€gsen vĂ€vnadspenetration. Tre parallella selektionsspĂ„r utfördes mot de cancerassocierade mĂ„lproteinerna human epidermal growth factor receptor 2 (HER2) och carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) samt den utsöndrade inflammationsmarkören C-reaktivt protein (CRP). Via Sangersekvensering kunde flera kandidater identifieras. Bland 6 karakteriserade ADAPTer uppvisade samtliga hög HSA-affinitet, tre konstaterades interagera specifikt med sitt mĂ„lprotein, och tvĂ„ verkade binda Ă€ven rumsbispecifikt. ADAPTer utvĂ€rderades Ă€ven för sin benĂ€genhet att bilda aggregat, strukturell överensstĂ€mmelse med experimentell design, och vĂ€rmestabilitet. Endast en bindare, mot HER2, klarade sig genom alla prövningar som proteinkarakteriseringen innebar utan underkĂ€nt. Ăven en högparallel sekvensering utav selektionsresultat utfördes, men utanför de tidsramar som tillĂ€t ytterligare karakterisering
The GRPR Antagonist [<sup>99m</sup>Tc]Tc-maSSS-PEG<sub>2</sub>-RM26 towards Phase I Clinical Trial: Kit Preparation, Characterization and Toxicity
Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [99mTc]Tc-maSSS-PEG2-RM26 (based on [D-Phe6, Sta13, Leu14-NH2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 °C was monitored for 18 months. The biological properties of [99mTc]Tc-maSSS-PEG2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG2-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16â24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics
Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours
ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)(2)- (DiADAPT6L2), and -(SSSG)(3)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In-111 and I-125, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization
Radionuclide therapy using ABD-fused ADAPT scaffold protein : Proof of Principle
Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.De tvÄ första författarna delar förstaförfattarskapet</p
Swe-Clarin : Language Resources and Technology for Digital Humanities
CLARIN is a European Research Infrastructure Consortium (ERIC), which aims at (a) making extensive language-based materials available as primary research data to the humanities and social sciences (HSS); and (b) offering state-of-the-art language technology (LT) as an eresearch tool for this purpose, positioning CLARIN centrally in what is often referred to as the digital humanities (DH). The Swedish CLARIN node Swe-Clarin was established in 2015 with funding from the Swedish Research Council. In this paper, we describe the composition and activities of Swe-Clarin, aiming at meeting the requirements of all HSS and other researchers whose research involves using text and speech as primary research data, and spreading the awareness of what Swe-Clarin can offer these research communities. We focus on one of the central means for doing this: pilot projects conducted in collaboration between HSS researchers and Swe-Clarin, together formulating a research question, the addressing of which requires working with large language-based materials. Four such pilot projects are described in more detail, illustrating research on rhetorical history, second-language acquisition, literature, and political science. A common thread to these projects is an aspiration to meet the challenge of conducting research on the basis of very large amounts of textual data in a consistent way without losing sight of the individual cases making up the mass of data, i.e., to be able to move between Morettiâs âdistantâ and âclose readingâ modes. While the pilot projects clearly make substantial contributions to DH, they also reveal some needs for more development, and in particular a need for document-level access to the text materials. As a consequence of this, work has now been initiated in Swe-Clarin to meet this need, so that Swe-Clarin together with HSS scholars investigating intricate research questions can take on the methodological challenges of big-data language-based digital humanities
Modern quantum chemistry with [Open]Molcas
MOLCAS/OpenMolcas is an ab initio electronic structure program providing a large set of computational methods from HartreeâFock and density functional theory to various implementations of multiconfigurational theory. This article provides a comprehensive overview of the main features of the code, specifically reviewing the use of the code in previously reported chemical applications as well as more recent applications including the calculation of magnetic properties from optimized density matrix renormalization group wave functions