The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tend to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutic is currently under exploration. As a comparative study, the hCD83ext was expressed as a glutathione-S-transferase (GST) fusion in two E. coli B strains, i.e. BL21 and Origami B having a reductive and oxidative cytoplasm. The final therapeutic products of hCD83ext produced by the two expression hosts exhibited significant differences in protein conformation and molecular properties, which presumably resulted from different disulfide patterns. The study highlights the importance of developing proper host/vector systems and biomanufacturing conditions for the production of recombinant therapeutic proteins with a consistent product quality. Cys27 in the hCD83ext was identified as a target for molecular manipulation. Two E. coli strains of BL21(DE3) and Origami B(DE3) were used as the expression host to produce the Cys27 mutants. It was observed that Cys27 was involved in the in vivo formation of intramolecular disulfide bonds when hCD83ext was expressed in Origami B(DE3). The Origami-derived protein products had a higher tendency than the BL21-derived counterparts for multimerization via the in vitro formation of intermolecular disulfide bonds. Various analyses were conducted to identify the structural differences among these mutant variants. Most importantly, molecular stability was enhanced by the Cys27 mutations since the Cys27 mutants derived from either BL21 or Origami were much less susceptible to degradation compared to wild-type hCD83ext. This study highlights the implications of aberrant disulfide bond formation on the production of therapeutic proteins. To address an inconsistent bioactivity issue that is primarily due to the aberrant formation of disulfide bonds associated with the presence of five cysteine residues, i.e. AA 27, 35, 100, 107, and 129, the molecular role that each cysteine plays upon the formation of intramolecular or intermolecular disulfide bonds was characterized, using various hCD83ext mutant variants derived by two E. coli expression hosts, i.e. BL21(DE3) and Origami B(DE3). Among the five cysteines, Cys100 and Cys129 can act as a bridging cysteine for in vitro multimerization via the formation of intermolecular disulfide bonds. The multimerization can be alleviated to some extent with less free Cys129 residues, associated with the possible formation of Cys27-Cys129 intramolecular disulfide bond. As a result, introducing the Cys27 mutation can increase the multimerization presumably via freeing more Cys129 residues. In addition, protein stability can be improved in the presence of the Cys27 mutation. The formation of the Cys27-Cys129 intramolecular disulfide bond appears to be more effective in the presence of the Cys100 mutation, resulting in the suppression of multimerization. The two conserved cysteine residues, i.e. Cys35 and Cys107, can be potentially linked to form an intramolecular disulfide bond, particularly when the protein is produced in Origami B(DE3).

The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tend to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutic is currently under exploration. As a comparative study, the hCD83ext was expressed as a glutathione-S-transferase (GST) fusion in two E. coli B strains, i.e. BL21 and Origami B having a reductive and oxidative cytoplasm. The final therapeutic products of hCD83ext produced by the two expression hosts exhibited significant differences in protein conformation and molecular properties, which presumably resulted from different disulfide patterns. The study highlights the importance of developing proper host/vector systems and biomanufacturing conditions for the production of recombinant therapeutic proteins with a consistent product quality. Cys27 in the hCD83ext was identified as a target for molecular manipulation. Two E. coli strains of BL21(DE3) and Origami B(DE3) were used as the expression host to produce the Cys27 mutants. It was observed that Cys27 was involved in the in vivo formation of intramolecular disulfide bonds when hCD83ext was expressed in Origami B(DE3). The Origami-derived protein products had a higher tendency than the BL21-derived counterparts for multimerization via the in vitro formation of intermolecular disulfide bonds. Various analyses were conducted to identify the structural differences among these mutant variants. Most importantly, molecular stability was enhanced by the Cys27 mutations since the Cys27 mutants derived from either BL21 or Origami were much less susceptible to degradation compared to wild-type hCD83ext. This study highlights the implications of aberrant disulfide bond formation on the production of therapeutic proteins. To address an inconsistent bioactivity issue that is primarily due to the aberrant formation of disulfide bonds associated with the presence of five cysteine residues, i.e. AA 27, 35, 100, 107, and 129, the molecular role that each cysteine plays upon the formation of intramolecular or intermolecular disulfide bonds was characterized, using various hCD83ext mutant variants derived by two E. coli expression hosts, i.e. BL21(DE3) and Origami B(DE3). Among the five cysteines, Cys100 and Cys129 can act as a bridging cysteine for in vitro multimerization via the formation of intermolecular disulfide bonds. The multimerization can be alleviated to some extent with less free Cys129 residues, associated with the possible formation of Cys27-Cys129 intramolecular disulfide bond. As a result, introducing the Cys27 mutation can increase the multimerization presumably via freeing more Cys129 residues. In addition, protein stability can be improved in the presence of the Cys27 mutation. The formation of the Cys27-Cys129 intramolecular disulfide bond appears to be more effective in the presence of the Cys100 mutation, resulting in the suppression of multimerization. The two conserved cysteine residues, i.e. Cys35 and Cys107, can be potentially linked to form an intramolecular disulfide bond, particularly when the protein is produced in Origami B(DE3).

En raison de leur haute spécificité d’interaction, les protéines thérapeutiques sont de plus en plus utilisées et représentent une part majoritaire du marché pharmaceutique. Néanmoins, ces molécules sont fragiles et leur stabilité est une problématique majeure pour l'industrie pharmaceutique. La dégradation des protéines thérapeutiques peut survenir à chaque étape de leur cycle de vie : production, stockage, transport et administration au patient. Les modifications chimiques, l'exposition à des forces de cisaillement (fort débit fluidique), la température, le pH et les interactions avec les matériaux et/ou les interfaces gazeuses sont autant de facteurs qui peuvent nuire à la stabilité de ces protéines. De plus, l'utilisation croissante de dispositifs médicaux automatisés pour la manipulation et l'injection de protéines thérapeutiques augmente drastiquement le risque de dégradation. Dans cette thèse, nous étudions l’effet et le rôle de la triple interface solide/liquide/air sur l'agrégation des protéines. Ce phénomène se produit fréquemment dans les procédés de manipulation d’une solution de protéines thérapeutiques (cavitation, agitation…). Lors d’un mouillage intermittent, les interfaces air/liquide et liquide/solide se confondent en une seule et même interface appelée triple interface ou ligne triple. La ligne triple est une zone favorisant fortement l'agrégation des protéines. Notre étude, basée sur l’insuline, montre que la ligne triple cause une accumulation progressive de protéines qui déclenche, après une période de nucléation, leur agrégation, précisément à l’endroit de cette ligne triple. Nos résultats démontrent aussi que les forces de cisaillement, seules, n’entrainent pas l’agrégation de l’insuline. De plus, nous observons que la diminution de la tension superficielle (induite par l’ajout de polysorbates) d'une solution de protéines réduit le risque de formation d’agrégats. En conclusion de ce travail, nous proposons des recommandations pour la conception des dispositifs médicaux de préparation et d’administration de protéines thérapeutiques. / Due to the high specificity of their interactions, proteins are increasingly used in therapy and represent a vast majority of the global pharmaceutical market. Nevertheless, these molecules are fragile and therapeutic protein stability is a major concern in pharmaceutical industry. Protein degradation and aggregation can occur at every step during production, storage, transport and delivery. In this thesis, we interrogate the possible role of intermittent wetting in protein aggregation. Intermittent wetting frequently occurs in protocols involving pumping (cavitation), agitation, and liquid handling. During intermittent wetting, the air/liquid and liquid/solid interfaces meet at a triple line or triple interface, which is a local trigger for protein aggregation because it concentrates the mechanical action of the recessing fluid on the surface adsorbed proteins. We study the effect of surface intermittent wetting on insulin aggregation. Our results demonstrate that the triple interface line, where an air/water interface meets a hydrophobic surface, allows progressive protein accumulation, and finally triggers local insulin aggregation. We also show that shear stress, alone, is not detrimental for protein stability. Additionally, Additives such as polysorbates were tested, showing that the modification of the surface tension of a protein solution impacts its ability to form aggregates. Based on this work, we propose recommendations for the design of drug delivery and preparation devices in order to limit the risk of protein aggregation at the triple interface.

A terapia gênica consiste em introduzir uma sequência de nucleotídeos, codificante ou não-codificante, que tenha a capacidade de interferir na progressão de uma doença por ativação, inativação ou modulação da expressão de um gene alvo. Uma das rotas de transferência gênica é a ex vivo. Essa rota consiste em realizar a modificação gênica ex vivo de um tipo celular do organismo afetado, seguida do transplante celular no indivíduo para a correção do fenótipo. Dentre os vários vetores virais utilizados para transferência gênica, o sistema lentiviral é considerado um dos mais eficazes, visto que é capaz de manter altos níveis de expressão a longo prazo. Além disso, dentre os vetores virais que se integram no genoma, esse é considerado o mais seguro, tendo apresentado apenas um caso com relatado de efeito colateral sem reação adversa. Uma nova tecnologia utilizada para padronizar sistemas biológicos é a biologia sintética. Essa tecnologia pode ser utilizada como ferramenta para produzir e/ou otimizar sistemas de expressão para produção de proteínas de interesse terapêutico. O objetivo desse trabalho é gerar um vetor lentiviral sintético, para terapia gênica ex vivo, para reposição enzimática. Foram geradas quatro linhagens celulares humanas com produção transiente das proteínas terapêuticas Prot_Ctr e Prot_Mut sob controle dos promotores CMV e HeF1a. Para padronização do ensaio de transfecção, foram geradas duas linhagens celulares humanas que expressão da proteína fluorescente verde (GFP) sob controle dos mesmos promotores citados acima. Como controle negativo das linhagens produtoras da proteína terapêutica, foi gerada uma linhagem como vetor plasmidial vazio pLV_GTW_Mock. O nível de expressão do mRNA relativo às proteínas terapêuticas variou de 3.581,95 ± 1.322 a 10.377,18 ± 2.562 URE, não havendo diferenças significativas entre as linhagens produzidas (p > 0,05). O nível de produção da proteínas terapêuticas variou de 30,98 UI/mL a 241,1 UI/mL. A linhagem com maior produção dessa proteína foi a 293T/HeF1a_Prot_Mut, e essa diferença é significativa (p < 0,05). A partir da transfecção dos plasmídeos auxiliares e plasmídeo portador do cDNA que codifica a Prot-Mut na linhagem celular 293-T foi obtido o vetor lentiviral com título de 1,68x107 pLV/mL. Em conclusão, é possível gerar um vetor lentiviral funcional portador da Prot_Mut para utilização em terapia gênica ex vivo. Espera-se que o produto desse projeto de pesquisa gere uma patente. Para evitar a quebra de novidade, atividade inventiva e suficiência descritiva, requisitos mínimos de patenteabilidade, os resultados foram apresentados sem mencionar a doença e o gene em estudo / Gene therapy involves introducing a nucleotide sequence, coding or non-coding that can to interfere with the progression of a disease by activating, inactivating or modulating the expression of a target gene. One of the gene transfer routes is the ex vivo. This route consists in producing a ex vivo gene modification of a cellular kind of the affected organismo, following the transplant of those cells to correct the fenotype. Among the various viral vectors used for gene transfer, the lentiviral system is considered one of the most effective, since it is able to maintain high levels of long-term expression. Between the viral vectors that integrate into the genome, the lentiviral system is considered the safest, and It has only filed a case with reported side effect without adverse reaction. A new technology used to standardize biological systems is the synthetic biology. This technology can be used as a tool to produce and/or optimize expression systems for production of proteins of therapeutic interest. The aim of this study is to generate a synthetic lentiviral vector for ex vivo gene therapy, for enzyme replacement therapy. It were generated four human cell lines with transient production of therapeutic proteins Prot_Ctr and Prot_Mut under the control of CMV and HeF1a promoters. To standardize the transfection assay were generated two human cell lines expressing green fluorescent protein (GFP) under control of the same promoters cited above. As a negative control of the producing cell lines, It was generated a human cell line with an empty plasmid vector pLV_GTW_Mock. The level of mRNA expression relative to the therapeutic proteins ranged from 3581,95 ± 1322 to 10377,18 ± 2562 REU, there were no significant differences among the produced cell lines ( p > 0.05 ). The production level of therapeutic proteins ranged from 30.98 IU/mL to 241.1 IU/mL. The cell line with the highest production of the therapeutic protein was 93T/HeF1a_Prot_Mut+, and this difference is significant (p < 0.05 ). From the transfection of the plasmid containing the cDNA encoding the Prot_Mut and packing plasmid It was obtained lentiviral vector with the titer 1,68x107 pLV/mL. In conclusion, it is possible to generate a functional lentiviral vector carrying the Prot_Mut for use in ex vivo gene therapy. It is expected that the product of this research project generates a patent. To avoid breaking novelty, inventive activity and descriptive sufficiency, minimum requirements for patentability, the results were presented without mentioning the disease and the gene under study

Disposable technology has gained increasing acceptance in the biopharmaceutical industry over the last decade, and provides many advantages over conventional stainless steel equipment. Disposable rocking bioreactors (RBs) are widely employed for cultivation of recombinant mammalian and insect cell lines, although the perception of inadequate mass transfer has prevented their application to bioprocesses based on microbial platforms. In an effort to thoroughly evaluate the suitability of disposable RBs for cultivation of aerobic microorganisms, a comparative study of one-dimensional (1D) and two-dimensional (2D) disposable RBs, and the conventional stirred tank reactor (STR) was performed. The comparison involved: 1) physical characterization of oxygen mass transfer efficiency and mixing intensity, 2) batch cultivation of Escherichia coli BL21 for comparison of growth characteristics, and 3) batch cultivation of recombinant E. coli BL21 expressing a clinical therapeutic, hCD83ext (the extracytoplasmic domain of human CD83). Oxygen mass transfer (evaluated as the mass transfer coefficient, kLa) was comparable between the 1D RB and STR (approximately 150 h-1) at low working volume (WV), declining linearly with increasing WV, while kLa was highest in the 2D RB for all tested WVs, providing the maximum kLa (394 h-1) at 3 L WV. Fast mixing (t95 of 8-20 s) was observed in all three systems for water and aqueous carboxymethylcellulose (CMC) solutions. Batch growth characteristics of E. coli BL21 were similar in each system, although acetate accumulation was significant in the 1D RB. Batch production of GST-hCD83ext (glutathione S-transferase-hCD83ext fusion protein) resulted in similar soluble protein yields and inclusion body formation between bioreactors. Although cell growth and protein expression were comparable between all bioreactors, the 1D RB is not considered a suitable cultivation system for E. coli under experimental conditions given the significant acetate accumulation observed and high supplemental oxygen requirement for low cell density cultures. On the other hand, considering its formidable mass transfer capacity and overall performance in batch cultivations, the CELL-tainer® is an attractive alternative to the STR for cultivation of recombinant E. coli expressing high value therapeutic proteins.

Un inconvénient majeur de l’utilisation des protéines thérapeutiques est leur immunogénicité,c'est-à-dire le déclenchement chez les patients d’une réponse immunitaire, avec production d’anticorps (anti-drug antibodies, ADA). Parmi les facteurs contributifs, les agrégats de protéines dans les spécialités administrées pourraient jouer un rôle majeur dans l’immunogénicité. Par ailleurs, la présence d’ADA de haute affinité et de divers isotypes suggère la mise en place d’une réponse immunitaire classique, faisant intervenir les cellules présentatrices d’antigènes et plus particulièrement les cellules dendritiques.Nous avons développé un modèle d’étude de l’impact d’agrégats de protéines sur la maturation de cellules dendritiques, dérivées de monocytes isolés du sang(moDC). Dans ce but, des agrégats d’hormone de croissance (GH) et d’anticorps (Rituximab) ou d’IgG1 polyclonale ont été produits et caractérisés.Nous avons montré que ces agrégats induisent la maturation des moDC, objectivée par une augmentation de l’expression de marqueurs d’activation et de co-stimulation (CD40, CD80,CD83, CD86 et HLA-DR), et par l’augmentation dela production de cytokines et chimiokines proinflammatoires (IL-6, IL-8,IL-12p40, CCL2, CCL3,CCL4 et CXCL10).En utilisant un modèle de co-culture allogénique,nous avons montré que les moDC stimulées par les agrégats induisent la prolifération de lymphocytes TCD4+, dont la polarisation dépendait de la nature de la protéine. Ainsi les agrégats de GH conduisent à une production majoritaire d’IFNγ, signe d’une réponse de type Th1, tandis que les agrégats d’anticorps induisent une réponse mixte, Th1, Th2,Th17 (production d’IFNγ, IL-5, IL-13 et IL-17.Enfin, nous avons commencé l’étude des mécanismes intra cellulaires impliqués dans l’activation des moDC, en montrant que les agrégats de GH induisent la phosphorylation de p38MAPK, ERK, JNK et NF-κB (p65). Ces mêmes voies de signalisation sont impliquées dans l’expression de CXCL10,chimiokine connue pour induire la polarisation Th1.Au final, ces résultats confirment l’effet immunomodulateur des agrégats de protéines sur les cellules dendritiques et précisent leur rôle de signal de danger conduisant à la mise en place d’une réponse immunitaire contre les protéines thérapeutiques. / A major drawback in therapeutic biological products (BP) use is the development of anti-drug antibodies (ADA) in patients. Among other factors, BP aggregates seems to play a major role in immunogenicity. Moreover, the presence of ADA with high affinity and different isotypes suggest a CD4 T-cell dependent immune response and therefore a pivotal role for antigen presenting cells, such as dendritic dells (DC).In order to determine if BP aggregates participate to DC activation, aggregates form human growth hormone (GH) and antibodies (Rituximab and polyclonal IgG1) were produced and characterized.Their impact was tested on a model of monocytederived dendritic cells (mo-DC).We have shown aggregates were able to induce moDC maturation, as observed with increase of key co-stimulatory and maturation markers (CD40, CD80, CD83, CD86 and HLA-DR), and by increase of pro-inflammatory cytokines and chemokines (IL-6, IL-8, IL-12p40, CCL2, CCL3, CCL4, CXCL10).Using an allogenic model of co-culture, we have shown that moDC stimulated with aggregates were able to induce CD4+ T cells proliferation.Polarization was different following the nature of the protein. GH aggregates were able to induceIFNγ, sign of Th1 response, whereas antibody aggregates induced Th1, Th2, Th17 mix response (with production of IFNγ, IL-5, IL-13 and IL-17).Finally, we started to study intracellular mechanisms involved in moDC activation, by showing that GH aggregates were able to induce p38MAPK, ERK, JNK and NF-κB (p65)phosphorylation. These pathways are involved in CXCL10 expression, which is implicated in Th1 polarization. These results confirmed the immunomodulary effect of protein aggregates on DC and their role as danger signal, inducing an immune response against therapeutic proteins.

Affinity proteins constitute an important category of cancer therapeutics. Owing to properties such as high target affinity and selectivity, therapeutic proteins offer more targeted therapy than small molecule drugs. The target molecules are typically proteins that are overexpressed on the surface of tumour cells, such as membrane-bound receptors. However, these surface proteins are usually expressed in normal tissues as well, resulting in on-target off-tumour toxicity. Proteins with a higher tissue selectivity are thus needed. Here, this has been addressed by developing prodrug proteins dependent on cancer-specific proteases for activation. The prodrugs were composed of a target-binding affibody (active domain) connected to a masking affibody (masking domain) by a peptide linker including a protease substrate. The target of the prodrugs developed in this project was the HER2 receptor, which is overexpressed in several cancer types. Three prodrug candidates were developed, produced and characterised based on their ability to be activated by their respective protease. The hypothesis that the prodrugs could be activated and thus bind to HER2 in cancer cells was tested using biosensor assays, as well as preliminary cancer cell assays. One of the three candidates showed strong potential to be used as a targeted therapy for cancer treatment in the future. / Affinitetsproteiner utgör en viktig kategori av cancerläkemedel. Jämfört med småmolekylära läkemedel är affinitetsproteiner mer riktade, då de har högre affinitet och selektivitet än små molekyler. Oftast utgörs det molekylära målet av ett protein som överuttrycks på ytan av cancerceller, så som membranbundna receptorer. Dessvärre uttrycks de flesta cancerspecifika proteiner i mindre mängd även i normal vävnad. Detta leder till oönskade effekter som kan ge upphov till biverkningar. I syfte att utveckla mer vävnadsspecifika läkemedel har här affibody-baserade “prodrugs”, beroende av cancerspecifika proteaser för aktivering, tagits fram. Prodrug-proteinerna i detta projekt är riktade mot HER2-receptorn, som är överuttryckt i flera typer av cancer. Tre kandidater togs fram och utvärderades med avseende på deras förmåga att aktiveras av sina respektive proteaser. För att testa hypotesen att kandidaterna kunde binda till HER2 på cancerceller efter proteasaktivering användes biosensoranalys samt experiment med cancerceller. En av kandidaterna visade stark potential att kunna användas som ett riktat läkemedel mot cancer i framtiden.

<p> Cell encapsulation aims at the delivery of a therapeutic protein to a patient from transplanted cells. Conventional approaches involve immune-isolating cell lines that have been genetically modified to express a therapeutic protein, in alginate-based microcapsules. The long-term success of this approach hinges on the structural stability of the microcapsules, as well as their ability to maintain an environment suitable for the long-term survival of encapsulated cells. The most commonly studied type of microcapsule is the alginate-poly-Llysine-alginate (APA) microcapsule. However, the main concern with AP A microcapsules is the Joss of structural integrity during long-term implantation due to the exchange of calcium ions with other physiological ions, as well as the loss of the polyelectrolyte overcoats. </p> <p> In order to increase the structural stability of the microcapsules, we developed and characterized a number of synthetic polyelectrolytes that undergo phase separation upon complexation, and which are capable of forming covalent cross-links. These reactive polyelectrolytes are designed to take the place of poly-L-lysine and the outer alginate layer. We also explored combining cross-linkable synthetic polyanions with sodium alginate to strengthen the Ca Alginate core, by forming a core cross-linked network extending throughout the microcapsules. The polyelectrolyte complexes, encapsulation processes and microcapsule properties were studied in detail using extensive characterization techniques, including collaborative work on cell viability and host-immune response. </p> <p> Overall, this thesis describes a novel approach and prom1smg materials for cell encapsulations that offer enhanced microcapsule resistance to chemical and mechanical stresses, while preserving the desired biocompatibility. These materials may ultimately be useful for clinical immunosuppressive therapies. </p> / Thesis / Doctor of Philosophy (PhD)

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

Cocaine is a plant-based illicit drug commonly involved in substance use disorder. Although cocaine overdose and cocaine use disorders cause adverse health consequences to individuals and the economic burden on their family and society, there are no FDA (Food and Drug Administration) approved medications for treatment. Recently, it has been recognized that delivery of cocaine hydrolase (CocH) is a promising therapeutic strategy. Human butyrylcholinesterase (hBChE), the primary enzyme involved in cocaine metabolism in human, have advantages over other candidates for the development of CocH. Previous studies in our laboratory have designed and characterized hBChE mutants that have ~4,000-fold improved catalytic efficiency against naturally occurring (-)-cocaine as compared to the wild-type hBChE. Besides the catalytic efficiency, the biological half-life is another essential factor that influences the desired therapeutic value in the long-term treatment of cocaine use disorder. In order to provide prolonged effects to reduce administration frequency in clinical use, efforts have been made to increase the retention time of CocHs in blood circulation by fusing CocHs with other thermostable proteins or their mutants, including human serum albumin (Albu) or the Fc region of the human IgG (Fc). In this dissertation, we demonstrated the clinical potential and the benefits of long-lasting CocHs for cocaine overdose treatment. We used rodent models to show the ability of AlbuCocH1 to block or reverse manifestations of toxic effects of cocaine. In addition, a concomitant LC-MS/MS-based analysis was conducted to investigate the pharmacokinetic profile of a lethal dose of cocaine with the presence of AlbuCocH1. These experimental data demonstrated AlbuCocH1 as an effective cocaine detoxification agent by accelerating the metabolism of cocaine. In order to examine the potential therapeutic value of Fc-fused CocHs in the treatment of cocaine use disorder, we conducted a series of behavioral experiments in rats to evaluate the effectiveness and duration of Fc-fused CocHs in blocking or attenuating cocaine-induced psychostimulant and discriminative stimulus effects. In addition, the intravenous self-administration model was used to investigate the long-term effectiveness of Fc-fused CocHs in blocking or attenuating the reinforcing effects of cocaine. It has been shown that a single dose of E30-6-Fc (3 mg/kg) was able to effectively alter the cocaine dose-response curve and attenuate the reinforcing efficacy of cocaine for at least a month in both male and female rats. In summary, AlbuCocH1 (TV-1380), which failed to meet the primary efficacy endpoint in clinical trials for facilitating abstinence in cocaine-dependent subjects with a weekly dosing schedule (due to the short biological half-life), is more suitable to be developed as a cocaine detoxification agent. On the contrary, the newly designed Fc-fused CocH (e.g. CocH3-Fc, E30-6-Fc) with higher catalytic efficiency and longer biological half-life will be beneficial for long-term abstinence management in cocaine-dependent individuals.