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Monoklonska protitelesa (mAb) izotipa IgG uporabljamo kot biološke učinkovine za zdravljenje rakavih obolenj, , avtoimunih in kroničnih vnetnih, infekcijskih in številnih drugih bolezni. mAb široko uporabljamo tudi v diagnostiki v različnih formatih predvsem zaradi njihove izjemne specifičnosti in visoke afinitete do raznolikih antigenov. Ligandi protiteles predstavljajo pomemben element v proizvodnji mAb. Večinoma temeljijo na bakterijskih immunoglobulin-vezavnih proteinih - stafilokoknem proteinu A in streptokoknem proteinu G, a so taki ligandi navadno dragi in podvrženi denaturaciji in odpuščanju iz afinitetnih kolon. Peptidi so preprostejši in cenovno dostopnejši od proteinskih ligandov, s kemijsko sintezo pa jih lahko proizvedemo v velikih količinah. Imajo ugodnejši toksikološki in imunogeni profil, in navadno vežejo tarčne proteine z zmerno afiniteto, zato lahko elucijo izvedemo pri relativno milih pogojih. V okviru preteklih raziskav smo identificirali peptidni ligand min19Fc-Q6D (GSYWYDVWF) in na njem osnovali usmerjeno peptidno knjižnico, predstavljeno na bakteriofagu. Izbrane položaje nonapeptida, ki niso esencialni za vezavo, smo delno ali popolnoma randomizirali do teoretične diverzitete 64000 klonov na aminokislinskem nivoju. Fagmid pIT2 smo priredili tako, da smo skrajšali distančnik, ki povezuje predstavljen polipeptid s fagnim proteinom p3, saj je znano, da dolgi fleksibilni distančniki negativno vplivajo na navidezne afinitete peptidnih ligandov. V prirejen fagmid subklonirali knjižnični insert DNA in generirali bakteriofagno knjižnico. V eni sami selekcijski stopnji smo knjižnico izpostavili tarči (zmesi človeških imunoglobulinov G), vezalce pa eluirali v več korakih s pufri s padajočimi vrednostmi pH. Izhodiščno knjižnico (pred selekcijo) in elucijske frakcije smo sekvencirali s tehnologijo sekvenciranja naslednje generacije (NGS) in v testu fagna ELISA primerjali relativno afiniteto najbolj obogatenih klonov. Najvišjo afiniteto je izkazoval peptid A (GSYWYNVWF), katerega smo kasneje uporabili za konstrukcijo afinitetnih matric z distančnimi ročicami različnih dolžin in rigidnosti. Matrica z razvejanim distančnikom tris(2-aminoetil)amin je izkazovala največjo statično vezavno kapaciteto, kar smo pripisali gostoti liganda oz. avidnostnemu učinku in učinkoviti izpostavitvi liganda topilu. Končni cilj doktorske naloge je bila konstrukcija afinitetne kromatografske kolone z najbolj ugodno distančno ročico (tris(2-aminoetil)amin) in peptidom A z izboljšanimi lastnostmi (v primerjavi z vodnico min19Fc-Q6D). Kolona je izkazovala primerljivo dinamično vezavno kapaciteto (~43 mg protiteles IgG1 /mL nosilca) in specifičnost kot komercialna kolona na osnovi proteina A, izkazovala pa je večjo stabilnostjo (tj. odpornost proti 0,5 M NaOH). Peptid min19Fc-Q6D smo poskušali kokristalizirati s fragmentom Fc (pridobljenim s fragmentacijo monoklonskega IgG1 s papainom). Difrakcijski vzorec pridobljenih monokristalov (ki so se formirali pri zelo različnih pogojih) smo posneli z visokoresolucijsko rentgensko difrakcijo na sinhrotronu (Elettra, Trst). Pridobili smo podatke v resoluciji 2,2 Å in razrešili strukturo z umeščanjem modela v elektronsko gostoto. Pri eni izmed domen CH2 je bila elektronska gostota razpršena, elektronske gostote peptida pa ni bilo zaznati. Tako nismo uspeli dobiti vpogleda v vezavno mesto za peptidni ligand na fragmentu Fc in naravo interakcije. Predpostavljamo, da nejasno definirana elektronska gostota v območju domen CH2 nakazuje konformacijsko spremembo v določenem deležu molekul Fc v kristalu, ki jo sproži interakcija s peptidom. Monoclonal antibodies (mAbs) of IgG isotype are widely used as therapeutics for treatments for cancer, autoimmune and chronic inflammatory, infectious, and many other diseases. They also make invaluable biorecognition agents due to remarkable specificity and affinity towards diverse antigens. Affinity chromatography based on bacterial immunoglobulin-binding proteins (i.e., staphylococcal protein A (spA) or streptococcal protein G (spG)) represents the cornerstone of antibody isolation and purification. Such protein affinity ligands suffer from ligand instability and leaching. Peptides represent a viable alternative to complex protein ligands. As affinity ligands, they bind target proteins with high specificity, but moderate affinity, thus requiring mild elution conditions. They are also small and simple, and thus available through cost-efficient chemical synthesis with consistent quality even on a large scale. Furthermore, the potential leaching of peptides from affinity matrices is not a major issue regarding the augmented product immunogenicity. We have previously identified a peptide ligand (min19FC-Q6D GSYWYDVWF) that binds to human IgGs. We designed a focused phage display peptide library based on randomization of selected non-essential residues of the parent peptide. Non-essential residues were either fully or partially randomized to produce a library of 64000 peptide members. Phagemid pIT2 was modified to remove the encoded long peptide linker anchoring the displayed peptide to the p3 phage coat protein, as we have observed that long flexible linkers negatively influence binding. We have subcloned the library insert into the modified pIT2-SL phagemid and generated the phage library. A single selection round was performed using a stepwise elution with buffers of progressively descending pH values. The initial library (before the panning session) and eluted fractions were subjected to deep sequencing to identify highly enriched clones. These were then tested for affinity in phage display ELISA assay. Peptide A (GSYWYNVWF) exhibited the highest affinity compared to other enriched binders and the parent peptide. Peptide A was used to construct several affinity matrices with linker molecules of different length and rigidity. The most effective resin for antibody capture utilized the brached linker tris(2-aminoethyl)amine. We suspect that the high capture capacity was due to the increased ligand density (compared to other linker molecules)/avidity effects and forced ligand orientation towards the solute. The goal of this doctoral thesis was the construction of an affinity column for IgG capture. The column constructed with the selected branched linker and peptide A demonstrated a dynamic binding capacity (~43 mg IgG1 /mL resin) and specificity on par with commercially employed protein A-based resin, but with higher stability (i.e., resistance to 0,5 M NaOH). We have attempted to obtain peptide:Fc region interaction information via X-ray crystallography. Fc fragment was obtained by papain digestion of monoclonal IgG1 and co-crystallized with the parent peptide min19Fc-Q6D. Several crystals were obtained under various conditions. Using the high-resolution diffraction data (resolution approx. 2,2 Å collected at Elettra Sincrotrone Trieste) we solved the structure employing phasing by molecular replacement. However, there was electron density scattering in one of the CH2 region, and no bound peptide was observed. We assume that the lack of defined electron density in the CH2 domain region indicates a conformational change of a fraction of Fc molecules in the crystal, triggered by interaction with the peptide. |
