Affinity maturation, a key strategy of the vertebrates’ adaptive immunity, is the process whereby more efficient antibodies are generated following initial antigen encounter. Increasing affinity of the antibody molecule is chaperoned by somatic mutations in the genetic fragments for the variable region of the heavy and light chains, resulting in a more efficient antigen-binding of the mature antibody molecule. This thesis is part of a wider project, which aims at studying the structural basis of the phenomenon. Particularly, the work presented here focuses on two scFvs (single-chain Fragment variable), NQ10/2.2.5 and NQ19/22.21, elicited during the early stages of the antibody response to the hapten 2-phenyl-1,3-oxazol-5-one. Although the two recombinant scFv antibody fragments share the same VHOx1/VkOx1 germline combination of heavy and light chains, NQ10/2.2.5 exhibits low levels of binding affinity and moderate production levels, in contrast to NQ19/22.21, which is instead associated to significant affinity for its antigen and high expression levels. Furthermore, considering the sequences of NQ10/2.2.5 as a reference, NQ19/22.21 has eleven mutations located in the variable region of the heavy chain (VHOx1) and five mutations in the variable portion of the light chain (VkOx1). Preliminary data obtained in our laboratory demonstrate that if the variable region of NQ10/2.2.5 light chain is swapped with that of NQ19/22.21, the expression levels of the soluble NQ10/2.2.5 scFv remain low. Instead, if the variable region of NQ10/2.2.5 heavy chain is exchanged with that of NQ19/22.21, the production levels of soluble NQ10/2.2.5 scFv increase significantly. Such results suggest that, differently from its VL, the mutations introduced in the NQ19/22.21 heavy chain upon maturation significantly affect the production levels of NQ10/2.2.5. In a previous work, computational modelling tools based on sequence homology were exploited for the construction of structural models of both NQ10/2.2.5 and NQ19/22.21 scFv. The work proposed in this thesis constitutes the essential in vitro counterpart of the prior in silico analysis. The two anti-phOx scFvs were dissected in their “top” and “bottom” structural portions and six constructs were submitted to gene synthesis or generated by site-directed mutagenesis: • NQ10topNQ19bottom VH-NQ10 VL, providing five mutations, out of eleven, from NQ19/22.21 to the NQ10/22.21 scaffold; • NQ10bottomNQ19top VH-NQ10 VL, providing six mutations, out of eleven, from NQ19/22.21 to the NQ10/22.21 scaffold; The “top” portion of NQ19/22.21 containing six mutations was further dissected in two distinct groups of 4 and 2 mutations and, finally, the single mutations of the pair were added to the group of 4 mutations: • NQ10bottomNQ19top4+H99G VH-NQ10 VL; • NQ10bottomNQ19top4+S101D VH-NQ10 VL. These constructs were expressed in E. coli Origami(DE3) bacterial cells and their production levels were analysed by western blot, with the aim to discriminate the favourable mutations, able to improve the sequence of NQ10/2.2.5 scFv, towards an increase in the scFv solubility and, possibly, in its stability and affinity.
Analisi dei livelli di produzione dei frammenti anticorpali scFv anti-fenilossazolone NQ10/2.2.5 e NQ19/22.21. Questa tesi si inserisce in un progetto più ampio, che ha lo scopo di studiare le basi strutturali della maturazione anticorpale in vitro. In questo lavoro si propone l’analisi del formato scFv (single-chain Fragment variable) di due anticorpi, NQ10/2.2.5 e NQ19/22.21, che rappresentano due momenti della maturazione dell’affinità nel sistema sperimentale dell’aptene 2-fenil-1,3-ossazol-5-one. Nonostante, infatti, i due scFvs condividano la medesima sequenza germinale (VHOx1/VkOx1) codificante per le catene pesanti e leggere, NQ10/2.2.5 presenta bassa affinità di legame per l’antigene e bassi livelli di produzione, mentre NQ19/22.21 mostra alta affinità e livelli di produzione piuttosto significativi. NQ19/22.21 differisce da NQ10/2.2.5 per undici mutazioni puntiformi nella regione variabile della catena pesante (VH) e cinque nella regione variabile della catena leggera (VL), intervenute durante il processo di maturazione. Esperimenti precedenti condotti nel nostro laboratorio allo scopo di individuare la causa di tale differenza nei livelli di produzione hanno dimostrato che la sostituzione della VL di NQ10/2.2.5 con quella di NQ19/22.21 è irrilevante; la sostituzione della VH, invece, influenza significativamente i livelli di produzione. Da quanto emerso, se ne deduce che, se le mutazioni che cadono a livello della VL sono per lo più ininfluenti, non è così per le mutazioni che cadono nella VH, suggerendo che i fattori che influenzano la produzione dei scFv NQ10/2.2.5 e NQ19/22.21 risiedono a livello delle undici mutazioni che si accumulano progressivamente nella regione variabile della catena pesante di NQ10/2.2.5. Seguendo questa ipotesi, in un lavoro precedente sono stati usati metodi computazionali per l’elaborazione di modelli strutturali di entrambi i scFvs. Le mutazioni identificate nella NQ19/22.21-VH sono state dissezionate in diverse combinazioni e localizzate sul modello strutturale di NQ10/2.2.5. Questa tesi si concentra su una fase di lavoro di validazione delle predizioni in vitro e di ulteriore analisi, durante la quale sono stati disegnati e sintetizzati alcuni costrutti genici con metodi di gene-synthesis e di mutagenesi sito-diretta. Inizialmente, i frammenti anticorpali scFv NQ10/2.2.5 e NQ19/22.21 sono stati concettualmente suddivisi in una componente strutturale “top” (alta) e una “bottom” (bassa): NQ10topNQ19bottom VH-NQ10 VL, fornendo allo scaffold strutturale della regione variabile della catena pesante di NQ10/2.2.5 cinque mutazioni, su undici, di NQ19/22.21, localizzate lontano dal sito di legame; NQ10bottomNQ19top VH-NQ10 VL, fornendo allo scaffold strutturale della regione variabile della catena pesante di NQ10/2.2.5 sei mutazioni, su undici, di NQ19/22.21, tutte localizzate in prossimità del sito di legame dell’aptene; In base ai risultati ottenuti, la parte alta “top” di NQ19/22.21, contenente sei mutazioni, è stata ulteriormente dissezionata in due gruppi, contenenti 2 e 4 mutazioni ciascuno e, successivamente. Al gruppo di 4 mutazioni è stata poi aggiunta una sola mutazione alla volta: NQ10bottomNQ19top4 + H99G VH-NQ10 VL; NQ10bottomNQ19top4 + S101D VH-NQ10 VL. Tali costrutti sono stati espressi in cellule E. coli Origami(DE3). L’analisi dei livelli di espressione associati a ciascun costrutto, eseguita in questa tesi, si propone di identificare le mutazioni driver, che primariamente influenzano la stabilità di NQ10/2.2.5.
Dissecting the basis of production levels of NQ10/2.2.5 and NQ19/22.21 anti-phenyloxazolone scFv antibody fragments.
PALMISANO, KATIA
2021/2022
Abstract
Affinity maturation, a key strategy of the vertebrates’ adaptive immunity, is the process whereby more efficient antibodies are generated following initial antigen encounter. Increasing affinity of the antibody molecule is chaperoned by somatic mutations in the genetic fragments for the variable region of the heavy and light chains, resulting in a more efficient antigen-binding of the mature antibody molecule. This thesis is part of a wider project, which aims at studying the structural basis of the phenomenon. Particularly, the work presented here focuses on two scFvs (single-chain Fragment variable), NQ10/2.2.5 and NQ19/22.21, elicited during the early stages of the antibody response to the hapten 2-phenyl-1,3-oxazol-5-one. Although the two recombinant scFv antibody fragments share the same VHOx1/VkOx1 germline combination of heavy and light chains, NQ10/2.2.5 exhibits low levels of binding affinity and moderate production levels, in contrast to NQ19/22.21, which is instead associated to significant affinity for its antigen and high expression levels. Furthermore, considering the sequences of NQ10/2.2.5 as a reference, NQ19/22.21 has eleven mutations located in the variable region of the heavy chain (VHOx1) and five mutations in the variable portion of the light chain (VkOx1). Preliminary data obtained in our laboratory demonstrate that if the variable region of NQ10/2.2.5 light chain is swapped with that of NQ19/22.21, the expression levels of the soluble NQ10/2.2.5 scFv remain low. Instead, if the variable region of NQ10/2.2.5 heavy chain is exchanged with that of NQ19/22.21, the production levels of soluble NQ10/2.2.5 scFv increase significantly. Such results suggest that, differently from its VL, the mutations introduced in the NQ19/22.21 heavy chain upon maturation significantly affect the production levels of NQ10/2.2.5. In a previous work, computational modelling tools based on sequence homology were exploited for the construction of structural models of both NQ10/2.2.5 and NQ19/22.21 scFv. The work proposed in this thesis constitutes the essential in vitro counterpart of the prior in silico analysis. The two anti-phOx scFvs were dissected in their “top” and “bottom” structural portions and six constructs were submitted to gene synthesis or generated by site-directed mutagenesis: • NQ10topNQ19bottom VH-NQ10 VL, providing five mutations, out of eleven, from NQ19/22.21 to the NQ10/22.21 scaffold; • NQ10bottomNQ19top VH-NQ10 VL, providing six mutations, out of eleven, from NQ19/22.21 to the NQ10/22.21 scaffold; The “top” portion of NQ19/22.21 containing six mutations was further dissected in two distinct groups of 4 and 2 mutations and, finally, the single mutations of the pair were added to the group of 4 mutations: • NQ10bottomNQ19top4+H99G VH-NQ10 VL; • NQ10bottomNQ19top4+S101D VH-NQ10 VL. These constructs were expressed in E. coli Origami(DE3) bacterial cells and their production levels were analysed by western blot, with the aim to discriminate the favourable mutations, able to improve the sequence of NQ10/2.2.5 scFv, towards an increase in the scFv solubility and, possibly, in its stability and affinity.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15165