Browsing by Author "Barth, Stefan"

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    Open Access
    Acquired immune responses to three malaria vaccine candidates and their relationship to invasion inhibition in two populations naturally exposed to malaria
    (BioMed Central, 2016-02-05) Addai-Mensah, Otchere; Seidel, Melanie; Amidu, Nafiu; Maskus, Dominika J; Kapelski, Stephanie; Breuer, Gudrun; Franken, Carmen; Owusu-Dabo, Ellis; Frempong, Margaret; Rakotozandrindrainy, Raphaël; Schinkel, Helga; Reimann, Andreas; Klockenbring, Torsten; Barth, Stefan; Fischer, Rainer; Fendel, Rolf
    Background: Malaria still represents a major cause of morbidity and mortality predominantly in several developing countries, and remains a priority in many public health programmes. Despite the enormous gains made in control and prevention the development of an effective vaccine represents a persisting challenge. Although several para site antigens including pre-erythrocytic antigens and blood stage antigens have been thoroughly investigated, the identification of solid immune correlates of protection against infection by Plasmodium falciparum or clinical malaria remains a major hurdle. In this study, an immuno-epidemiological survey was carried out between two populations naturally exposed to P. falciparum malaria to determine the immune correlates of protection. Methods: Plasma samples of immune adults from two countries (Ghana and Madagascar) were tested for their reactivity against the merozoite surface proteins MSP1-19, MSP3 and AMA1 by ELISA. The antigens had been selected on the basis of cumulative evidence of their role in anti-malarial immunity. Additionally, reactivity against crude P. falciparum lysate was investigated. Purified IgG from these samples were furthermore tested in an invasion inhibition assay for their antiparasitic activity. Results: Significant intra- and inter- population variation of the reactivity of the samples to the tested antigens were found, as well as a significant positive correlation between MSP1-19 reactivity and invasion inhibition (p < 0.05). Interestingly, male donors showed a significantly higher antibody response to all tested antigens than their female counterparts. In vitro invasion inhibition assays comparing the purified antibodies from the donors from Ghana and Madagascar did not show any statistically significant difference. Although in vitro invasion inhibition increased with breadth of antibody response, the increase was not statistically significant. Conclusions: The findings support the fact that the development of semi-immunity to malaria is probably con tingent on the development of antibodies to not only one, but a range of antigens and that invasion inhibition in immune adults may be a function of antibodies to various antigens. This supports strategies of vaccination including multicomponent vaccines as well as passive vaccination strategies with antibody cocktails.
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    Open Access
    Antibody engineering to evaluate binding, internalisation, and intracellular routing of tumour-targeting fusion proteins
    (2023) Karaan, Maryam; Barth, Stefan
    Breast cancer is a major global health crisis, particularly affecting women, and triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options. TNBC is challenging to treat due to the lack of specific therapeutic markers such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Current treatments primarily involve chemotherapy, radiotherapy, and surgery, as targeted therapies are limited. TNBC also exhibits significant heterogeneity among patients, emphasizing the need for precise diagnostic and therapeutic approaches. Immunotherapy, the manipulation of the immune system to target diseases, holds promise for precision medicine. Antibody-drug conjugates (ADCs) use antibodies to deliver drugs selectively. This study focused on a novel recombinant fusion protein format for ADCs using a single chain variable fragment (scFv) specific to chondroitin sulphate proteoglycan 4 (CSPG4), a tumour-associated antigen often overexpressed in TNBC. The scFv antibody derivative was genetically fused to a SNAP-tag, enabling stable and site-specific conjugation of the scFv to diagnostic and therapeutic substrates. To enhance antigen binding, internalisation, and therapeutic efficacy, a bivalent scFv fusion protein was created in tandem with the standard monovalent fusion protein. In vitro experiments using fluorophores and the cytotoxin, monomethyl auristatin F (AURIF), demonstrated that the bivalent fusion protein exhibited improved binding, faster uptake, and efficient release of the conjugate within target cells. Colocalization analysis revealed that the fusion proteins were routed to the lysosomal degradation pathway which is essential for the functionality of ADCs. The cell viability assays revealed that the enhanced binding and uptake of the bivalent fusion protein resulted in a more potent cytotoxic effect on antigen-positive TNBC cells. This study successfully compared mono- and bivalent αCSPG4-SNAP fusion proteins, revealing the superiority of the bivalent format in antigen binding and targeted drug delivery. It also, for the first time, explored the intracellular routing of scFv SNAP-tag fusion proteins upon uptake. The findings may influence the design of future scFv-based immunotherapeutics, possibly resulting in the incorporation of multiple scFv domains for increased efficacy. Furthermore, the diagnostic potential of these fusion proteins, aiding in prognosis prediction and patient responsiveness to targeted therapies have been highlighted. The versatility of SNAP-tag technology makes it relatively easy to transform immunodiagnostic fusion proteins into immunotherapeutic tools, potentially advancing TNBC management. In summary, this study contributes to the development of precision medicine tools for TNBC, addressing its complex nature and limited treatment options. The bivalent fusion protein format shows promise for improved TNBC therapy, offering a new avenue for research and potential clinical applications.
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    Comparison of the therapeutic efficacy of SNAP-tag fusion protein and the synergistic actions of chemotherapy and photodynamic therapy in killing resistant melanoma
    (2019) Biteghe, Fleury Augustin Nsole; Barth, Stefan
    Cutaneous melanoma is the deadliest form of skin cancer, which arises from epidermal pigment-producing cells called melanocytes. In melanoma, surgical excision of primary nonmetastatic tumor remains the gold standard of therapy worldwide. Upon metastases, melanoma becomes highly resistant to conventional radio-and chemotherapy. Chemotherapy, using dacarbazine (DTIC), remains the standard treatment option. In melanoma, chemotherapy failure has partly been attributed to a resistant population which is endowed with higher clonogenic potential, and aberrant expression of membrane proteins known as ABC transporters (ABCB5 and ABCG2), which mediate cellular resistance by extruding cytotoxic molecules from cells. To palliate these adverse effects, this study primarily aimed to investigate the efficacy of the synergistic actions of chemotherapy (Dacarbazine:DTIC) and hypericin-activated photodynamic therapy (HYP-PDT) in reducing chemoresistance in DTIC resistant (UCT Mel1DTICR2) and non-resistant melanoma cells (UCT Mel-1). To achieve these goals, the therapeutic efficacy of conventional therapies (DTIC, HYP-PDT and DTIC+HYP-PDT) was evaluated based on their ability to reduce cell viability; therapeutic resistant subpopulations; clonogenicity and ABC transporters (ABCB5 and ABCG2) in both melanoma cells. Additionally, the ability of the therapeutic treatments to efficiently halt cell division and activating cell death mechanisms, was assessed using cell cycle analysis and an Annexin-V assay. The results obtained showed that combination therapy was the most efficient therapy which was associated with a reduction in main populations (therapeutic resistant sub-population not expressing ABC transporters: MP), and clonogenic capacity in both melanoma cell types. Similarly, DTIC displayed a therapeutic efficacy which significantly reduced side populations (therapeutic resistant sub-population which were expressing ABC transporters: SP), and clonogenicity in UCT Mel-1 only. Interestingly, both ABCG2 and ABCB5 expressions were significantly increased in both melanoma cells, post combination therapy. Lastly, combination therapy and PDT were equally shown to induce a G1 cell cycle arrest, as opposed to DTIC which induced an S phase arrest. These cell cycle arrests were associated with efficient activation of apoptosis and necrosis, depending on the melanoma cell type, post HYP-PDT and combination therapy. Nevertheless, the efficacy of DTIC and HYP-PDT might respectively be limited by off target effects harming normal cells and low dosage in tumour cells which limiting their clinical utility. To address these challenges, this study aimed to develop a targeted tumor therapy, using the self-labelling activity of SNAP-tag fusion protein to conjugate synthetic small molecule lead substance toxin such as monomethyl Auristatin F (MMAF or AURIF). This cytotoxic payload was delivered to targeted cells, through genetic fusion of SNAP-tag to three different single chain fragments of an antibody (scFv1711, LsFv49 and scFv#34), which specifically treated tumor cells expressing epidermal growth factor (EGFR), melanotransferrin (p97) orfibroblast activation protein alpha (FAP-α) receptors, respectively. Achievement of this targeted therapy was performed through construction of three scFv-SNAP fusion proteins, which were expressed in HEK 293T cells. Thereafter, the purified scFv-SNAP fusion proteins were conjugated to BG-substrates to investigate their efficacy in specifically killing tumor cells. Binding and cytotoxic activities of the scFv-SNAP fusion proteins were performed using flow cytometry, and the XTT cell viability assay. All scFv-SNAP-fusion proteins were specifically bound to their target cells, indicating that AURIF conjugation did not compromise the binding activity of scFv-SNAP fusions. Finally, the cytotoxic assay confirmed that all scFv-SNAPAURIF conjugates induced a 50 percent reduction in cell viability at nanomolar concentrations in targeted cells which expressed their cognate antigens. To conclude, combination therapy was shown to be more efficient than monotherapies in killing chemoresistant melanoma cells, while SNAP-tag technology provided a superior, targeted therapeutic efficacy by sparing normal cells from unwanted toxic effects, and reducing the therapeutic requirement for high concentration of cytotoxic payloads.
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    Open Access
    Development and Characterization of recombinant immunotoxins for cervical carcinoma
    (2023) Henry, Marc; Barth, Stefan
    Cervical cancer is the second most frequent occurring cancer in South African women. Cervical cancer accounts for 41.7% of all cancer in black women and 37% in coloured women. If detected early the disease can be treated, however patients are only diagnosed once the cancer has reached its advanced stages. This poor outcome is due to personal reasons of the patient, lack of screening facilities or lack of education. Cervical cancer is a consequence of Human papillomavirus (HPV) infection. Whereby the virus infects epithelial cells of the cervix and integrates its viral DNA into the host's genome. The viral DNA encodes oncogenes and initiates various pathways within the cells to undergo oncogenesis. During oncogenesis the cells protrude various surface receptors to ensure growth and survival of the cancerous cells. Through many cycles of uncontrolled cell proliferation, the cancer will eventually form a tumour in the cervix, this usually occurs during the advanced stages of cervical cancer. Once the cancer has reached advanced stages current treatment has very limited therapeutic effect. This is due to the cancers' ability to develop resistance against current treatments i.e., chemoresistance which leads to a relapse. Therefore, the demand for novel therapeutics for treating advanced stage cancers are high. Immunotoxins are fusion proteins that consist of an antibody that binds specifically to the cancer cell connected to a protein toxin capable of killing the cell. Immunotoxins have proven to be a promising alternative to current treatment for cancer. Immunotoxins utilize the ability of antibodies to target cancerous cells without affecting the healthy cells due to certain surface receptors being overexpressed in cancerous cells and not in healthy cells. Studies have shown that cell surface receptors LGR5, EpCAM and CD90 are overexpressed in cervical carcinoma. Full length antibodies have been proven to be less effective when used against tumours whereas, various formats of antibody such as fragment antigen binding (Fab) and single chain variable fragment (scFv) demonstrated greater penetration properties. Pseudomonas exotoxin A (ETA) is a highly cytotoxic enzyme that modifies elongation factor 2 of the cell. This modification is irreversible and arrests protein synthesis thereby causing cell death. ETA is prone to causing a humoral response within patients. This unwanted effect can be prevented by introducing point mutations into the ETA gene. The point mutations are to reduce the chances of an immune response from occurring whilst maintaining the cytotoxic activity of the wildtype (wt) ETA. This study aims to produce scFv antibodies targeting the MSc Thesis - 13 - Marc Henry three overexpressed biomarkers found in cervical carcinoma connected to a wt ETA or a mutant variant generated from computer simulation by our collaborating partner Prof Paolo Carloni (Forschungszentrum Julich, Germany) described as de-immunised (dETA). The recombinant immunotoxins (rITs) were generated by designing a bacterial periplasmic expression plasmid containing ETA or dETA to be fused to a scFv antibody fragment targeting one of the three biomarkers (LGR5, CD90 and EpCAM). The corresponding scFv was cloned into this plasmid before being transformed into Escherichia coli (E. coli) BL21. The periplasm of E. coli BL21 was used to produce the rITs by performing bacterial expression under osmotic stress conditions. The rITs were purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified proteins were characterized by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blot analysis. Once expression of full-length proteins was confirmed the rITs were assessed on the cervical carcinoma cell line CaSki for binding and cytotoxicity studies. The results demonstrate that the periplasm of the E. coli BL21 can be utilized to generate complex fusion proteins, this being evidenced by the SDS PAGE and western blot results. Both ETA and dETA rITs exhibited strong binding towards CaSki cells. The cytotoxicity assays indicate that both ETA and dETA rITs are capable of targeted killing. However, ETA seems to demonstrate a more potent response compared to the dETA. This may be due to the computer simulated mutant R456T not being able to fully recover the enzymatic activity of B cell epitope depleted dETA to its original wt activity. The result of this study showcases that this form of treatment may potentially be effective treatment for patients experiencing advanced stages of cervical cancer. However, in future studies other cervical cancer lines should be assessed and eventually patient samples should be assessed in binding and cytotoxicity studies. Although this dETA variant seems to possess a lower potency compared to wildtype ETA immunogenicity analysis using mouse models to determine whether the dETA variant reduces the humoral response within the host would be essential.
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    Development of potential immunodiagnostic & therapeutic techniques using SNAP-fusion proteins as tools for the validation of Triple-negative Breast Cancer
    (2020) Magugu, Freddy-Junior Siybaulela; Barth, Stefan; Naran, Krupa
    Globally, breast cancer is the leading cause of death in the female population aged 45 and below with a breast cancer incidence reaching 18.1 million in the year 2018. Triple negative breast cancer (TNBC) is part of a group of cancers that lack the expression of Progesterone receptor (PR), Estrogen receptor (ER) and Human epidermal growth factor receptor 2 (HER2). TNBC is commonly associated with early stage metastasis with low survival rates as well as a high frequency of recurrence and proves to be problematic in both the young and elderly female populations. Conventional diagnostic methods for TNBCs include mammography, magnetic resonance imaging (MRI) and ultrasound while therapeutic methods include mastectomy and breast conserving surgery (coupled with radiation therapy). The lack of effective therapeutic options, poor prognostic value and high rates of metastasis, has made treatment of TNBC difficult. The major focus of this work was on the following tumour associated antigens (TAAs): CSPG4 (a transmembrane protein found in 50% of TNBC cases), EGFR (which is overexpressed in 13-76% of TNBCs), and MSLN (which is overexpressed in 67% of TNBCs) as potential targets for monospecific therapy. The evolution of antibody-based immunotherapy strategies has led to applications of single chain variable fragment (scFv) & single domain/nanobody (VHH) antibody formats for diagnostic and therapeutic purposes. In this work, these recombinant antibody fragments have been combined with SNAP-tag, a modified version of the human DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT), which autocatalytically binds benzyl-guanine modified substrates such as fluorophores or small molecule toxins covalently in a 1:1 stoichiometry. In this study, the primary aim was the comparison of different antibody formats fused to SNAPtag and the potential of these biopharmaceuticals towards immunodiagnosis and therapy of TNBCs. First functionalities of two scFv SNAP fusion proteins and one VHH SNAP fusion protein previously not having been described are provided through binding analyses on receptor positive tumour cell lines. This was achieved by in-silico design and molecular cloning of genetically fused antiCSPG4(scFv), -MSLN(scFv), -MSLN(VHH), -EGFR(scFv) & -EGFR(VHH) to SNAP-tag. The final constructs were confirmed by Sanger sequencing and subsequently transfected into a mammalian vector system (HEK293T) for transient expression of the engineered fusion proteins. Full length protein purified from cell culture supernatant was analysed for diagnostic/therapeutic activities dependant on the substrate attached in the form of a fluorophore or small molecule toxin resulting in recombinant antibody-drug conjugates (ADCs). The study shows promise in providing new immunodiagnostic and therapeutic agents that are specific and less harmful than the current state of the art procedure
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    Development Of Recombinant Immunotherapeutics for Triple-Negative Breast Cancer (TNBC)
    (2023) Ngwenya, Takunda; Barth, Stefan
    Breast cancer is the most common form of female malignancy globally. There are several subtypes of breast cancers including Triple-negative breast cancer (TNBC), which is mainly characterized by a lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is one of the most aggressive subtypes of breast cancer, with a 20% lower disease-free and overall survival rates than is observed for non-TNBC patients. It is characterized by higher rates of aggressive relapse, high metastatic potential, shorter survival rate and an overall aggressive and invasive clinical course. This subtype is most prevalent in young (≤50 years) female patients of African ancestry compared to other ethnicities. TNBC poses a significant challenge due to the lack of molecular targets. The current treatment of TNBC is achieved by chemotherapy, surgery and radiotherapy, collectively referred to as tri-modality therapy. However, tri-modality therapy results in disease relapse and adverse side effects. As a result, an ongoing need exists to develop a range of targeted therapeutics to effectively treat the majority of TNBC tumours. These targeted anti-cancer therapies aim to enable the selective killing of cancer cells while minimizing harm to healthy cells. This selective cytotoxicity would result in a significant improvement in TNBC treatment efficacy while reducing the side effects. The development of targeted anticancer therapies for TNBC is an active area of research and scientists are currently exploring various approaches to develop targeted anticancer therapies for TNBC and other cancer types. Antibody-Drug-Conjugates (ADCs) are proteins that combine an antibody and a toxin designed to selectively target and kill cancerous cells respectively. These are promising alternatives to conventional cancer treatment. This approach takes advantage of the specific cell surface receptors (CSRs) that are often present on cancerous cells and absent in healthy tissues. Among several CSRs that have been identified to be overexpressed in TNBC, CD90, LGR5 and EpCAM were considered for this study. Due to the limited effectiveness of full mAbs penetration into tumours, smaller antibody fragments such as scFv and Fabs have shown a greater ability to penetrate tumour tissues. These smaller antibody formats can be used to develop recombinant immunotoxins (rITs) using protein toxins as cell-killing moieties to achieve targeted destruction of cancer cells. Pseudomonas exotoxin A (ETA) is a potent enzyme that disrupts the function of eukaryotic elongation factor 2, causing inhibition of protein synthesis and leading to cell death. Due to the bacterial origin of ETA, scientists have discovered that it often triggers an unwanted humoral response in patients during clinical trials. To address this challenge, researchers have introduced mutations in the ETA gene to reduce the risk of causing an immunogenic response while preserving the cytotoxicity activity. This study aimed at developing single chain fragments of variability (scFvs) derived from publicly available sequences of antibodies that target three CSRs overexpressed in TNBC, genetically fused to wild-type ETA (wt ETA) or a mutant version of ETA that was generated using advanced computer simulation by our collaborating partner, Professor Paolo Carloni from Forschungszentrum Jülich, Germany. The mutated version is described as de-immunized ETA (dETA). The recombinant immunotoxins were generated by constructing a bacterial periplasmic expression plasmid, which included ETA or dETA fused to an scFv antibody fragment targeting each of the three biomarkers (CD90, LGR5 and EpCAM). The plasmid also contained the corresponding scFv that was inserted before the transformation of the plasmid into Escherichia coli (E. coli) BL21 for bacterial protein expression under osmotic stress conditions in the presence of compatible solutes. The rITs were then purified by immobilized metal ion chromatography (IMAC) and size exclusion chromatography (SEC). Confirmation of full-length rITs was done by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot analysis. Upon successful confirmation of full-length proteins, binding and cytotoxicity studies were conducted on TNBC cell line MDA-MB-468 using the rITs. In this study, all six designed plasmids were successfully cloned, and recombinant fusion proteins were expressed using the bacterial periplasm of E. coli BL21 as evidenced by SDS PAGE and western blot. The rITs displayed a strong binding affinity for MDA-MB-468 TNBC cells. Cytotoxicity results revealed that both ETA and dETA-rITs can both target and kill MDAMB-468 TNBC cells. ETA-based rITs exhibited more robust killing activity than dETA-based rITs, except for the rITs targeting CD90, in this case exceeding wt ETA enzymatic activity. This study showed potential for clinical application of the generated rITs' for TNBC immunodiagnostic use and the ability of ETA and dETA toxins to specifically kill TNBC antigenpositive tumour cells indicating targeted therapeutic potential. This work serves as proof of concept for investigating the use of rITs in TNBC tumour detection and patient stratification to offer personalized therapeutics.
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    Development of SNAP-tag based fusion proteins as novel auristatin F-containing immunoconjugates and photoimmunotheranostics in the detection and treatment of triple-negative breast cancer
    (2022) Mungra, Neelakshi; Barth, Stefan
    Breast cancer represents one of the most common forms of female malignancy of the 21st century. Among the various breast cancer subtypes, triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). As an idiosyncratic disease, TNBC displays a conspicuously aggressive and invasive clinical course, with an unexplained partiality towards women of African ancestry. Its acute heterogeneity and complexity behave as mutually reinforcing negative factors, which further complicate prognosis, thereby increasing the burden of breast cancer-related mortality. With the absence of well-defined molecular targets in TNBC, there is a heightened reliance on tri-modality therapy (surgery, radiotherapy and chemotherapy), albeit with an increasing incidence of adverse effects and disease relapse. To this end, there is an urgent need to develop an arsenal of targeted diagnostics and therapeutics, which can be synergized to cover the vast majority of triple-negative breast tumors, paving the way towards the development of personalized regimens suitable for the particular needs and disease of each patient. As such, achieving selective cytotoxicity, with minimal or no collateral damage to healthy tissues, embodies the holy grail of targeted anti-cancer therapies. For instance, the high affinity and specificity of monoclonal antibodies (mAbs) and derivatives thereof, have cemented their application as revolutionary tools in the selective delivery of drugs to malignant cells. These therapeutic proteins, also known as antibody-drug conjugates (ADCs), might exhibit several advantages compared to their small-molecule counterparts, but their widespread clinical use is hampered by various developmental considerations. Traditional conjugation strategies employed to arm mAbs with cytotoxic warheads, usually give rise to heterogeneous mixtures of ADC species, bearing non-uniform drug-to-antibody ratios (DARs), pharmacologic characteristics and safety profiles. Fortunately, the implementation of self-labeling tags (such as SNAP-tag, CLIP-tag and Halo-tag) are providing renewed impetus to homogeneous recombinant immunotherapeutics development. More precisely, SNAP-tag is an engineered mutant of the human O(6)-alkylguanine-DNA alkyltransferase, endowed with the ability to specifically and irreversibly react with benzylguanine (BG) derivatives, forming a stable product. Based on the above premises, this research aims to use SNAP-tag technology as a cutting-edge site-specific conjugation method to: (1) develop a comprehensive antibody platform, consisting of single-chain antibody fragments (scFvs) genetically fused to SNAP-tag, to specifically screen and evaluate their predictive potential for chondroitin sulfate proteoglycan 4 (CSPG4), CD44 and aspartate (aspartyl/asparaginyl) β-hydroxylase (ASPH)-positive TNBC cells, (2) generate functional recombinant ADC formulations as robust delivery systems carrying the antimitotic drug monomethyl auristatin F (MMAF/AURIF), concurrently overcoming production constraints to yield therapeutically viable and homogeneous combination products, and (3) provide a fail-safe system that overcomes the lack of specificity of photodynamic therapy (PDT), by coupling scFv-SNAP-tag to the potent near-infrared (NIR) photosensitizer (PS) IRDye700DX (IR700) and to demonstrate its selective dose-dependent cytotoxic activities in vitro. Following in silico design of the open reading frames (ORFs) coding for each construct, standardized molecular cloning techniques were implemented to generate recombinant mammalian expression plasmids, encompassing Ig-Kappa leader as an efficient protein secretory system. After confirmation of the DNA integrity, protein expression was achieved through transient transfection into HEK293T cells. Thereafter, the resulting histidine-tagged fusion proteins (αCSPG4(scFv)-SNAP, αCD44(scFv)-SNAPf and αASPH(scFv)-SNAP) were harvested from the cell culture supernatant and subjected to immobilized metal affinity chromatography (IMAC). In order to evaluate the outcome of this protein expression and purification step, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were used to confirm the presence of full-length recombinant SNAP-tag based fusion proteins based on their molecular weights. Integration of the fluorescent dye Alexa Fluor 488 into the fusion proteins was carried out to investigate the self-labeling activity of the SNAP-tag moiety, as well as to provide qualitative and quantitative insights into the binding potential of the antibody fragments towards their cognate antigens. Subsequently, the AURIF and IR700-based immunoconjugates were generated by conjugating scFv-SNAP with their respective BG-modified substrates, in a defined 1:1 stoichiometric reaction. The specific and dose-dependent biological activities of the resulting bifunctional therapeutic proteins were then assessed on TNBC cells. In this study, pCB-αCD44(scFv)-SNAPf was successfully cloned and all 3 fusion proteins were effectively expressed, although with low yields and purity, yet adequate for downstream in vitro characterization. After showcasing the self-labeling potential of the SNAP-tag component, surface binding of the fluorescently labeled product was demonstrated on antigen-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell killing ability of the novel AURIF-based recombinant ADCs and IR700 photoimmunoconjugates, was illustrated by the induction of a 50% reduction in cell viability (IC50 value) at nanomolar to micromolar concentrations on target cell lines. This observable selective cytotoxicity revealed that conjugation of BG derivatives to SNAP-tag, did not affect the binding potential of the antibody fragment, nor abrogated the cytocidal activity of the payload. As a proof of concept, this research builds on existing work that promulgates the use of SNAP-tag as a state-of-the-art conjugation strategy that can circumvent the challenges associated with the use of antibodies as effective delivery systems for therapeutic molecules. By harnessing the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically acceptable immunoconjugates, the results herein presented, also highlight the prospects of such agents in disease-specific tumor suppression. While various architectural modifications could further improve cytotoxic activities of future combination products, this research underscores the duality of SNAP-tag in the development of immunodiagnostics and therapeutics, that could potentially be instrumental in instilling a shift towards a personalized medicine stratagem. In conclusion, the combination of such immunoconjugates with a robust companion diagnostic panel provided by SNAP-tag, represents a first step towards the effective management of TNBC, with potential impact on the economic, social and clinical settings.
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    Development of SNAP-tag-based fusion proteins targeting HIV-1 viral reservoirs
    (2020) Cingo, Siphelele Sanele; Barth, Stefan
    Background Globally, the HIV/AIDS epidemic has cost over 35 million lives and approximately a further 37 million people are currently infected with HIV. In South Africa alone, more than 7 million people are HIV positive. Since the initiation of combination antiretroviral therapy (cART), viral replication can be supressed below the limit of detection by conventional testing. There is, however, no approved therapy for the cure of HIV. This is because HIV establishes viral reservoirs in memory CD4+ T-cells, where replication is low or arrested, allowing prolonged survival. Since there is little or no replication, a therapeutic strategy which targets the viral production and replication becomes ineffective and upon cessation of antiretroviral therapy a dramatic viral relapse occurs. The eradication of HIV, therefore, requires the targeted killing of the reservoir cells, or latency reversal followed by the prevention of further infection using cART. Targeting of cell-surface antigens for therapeutic purposes is the basis of immunotherapy. FDA-approved monoclonal antibodies such as Trastuzumab have been used to treat breast cancer via the human epidermal growth factor 2 (HER2) receptor. Immunotoxins (ITs) composed of an antibody fragment fused to apoptosis-inducing protein toxins targeting cellsurface antigens have been used for therapy of refractory leukaemia. The anti-CD22 recombinant IT Moxetumomab pasudotox based on Pseudomonas aeruginosa exotoxin A (ETA) has been FDA approved to treat hairy cell leukaemia. Moxetumomab pasudotox targets the antigen CD22 found on the surface of tumour cells. The HIV neutralizing VHH-nanobody J3, isolated from an immunised Llama has demonstrated anti-HIV properties against more than 95 % of HIV strains in vitro. As part of an ongoing project to develop a J3-ETA IT, this work sought to produce a J3-SNAP fusion protein by osmotic stress expression in the presence of compatible solutes in the periplasmic space of E. coli. SNAP-tag is a self-labelling protein that covalently binds benzylguanine (BG)-modified substrates in a 1:1 stoichiometric ratio. When recombinantly fused to any protein of interest, SNAP-tag allows the stable labelling of the protein of interest of in vitro and in vivo imaging. The periplasmic space of bacteria has been reported as a dedicated compartment to express functional proteins of interest. Furthermore, osmotic stress expression in the presence of compatible solutes has been reported to result in up to a thousand-fold increase in protein yield for difficult to express proteins. This study ultimately aimed to understand whether a functional J3-SNAP or J3-ETA can be expressed under osmotic stress in the presence of compatible solutes, in the periplasmic space of E. coli. 11 Experimental work In this study, a SNAP-tag-based fusion protein and an ETA-based IT were designed using J3, an anti-HIV-1 Env VHH-nanobody isolated from an immunised llama. Using the SnapGene® software (v.5.0.8, GSL Biotech LLC, USA), in silico design and cloning of an ETA-based IT J3-ETA and SNAP-tag-based fusion protein J3-SNAP was performed. Molecular cloning of designed open reading frames (ORFs) was performed into appropriate bacterial expression plasmid vectors. Plasmid vectors confirmed to contain the required ORFs by Sanger sequencing were transformed into E. coli BL21-DE3. Histidine-tagged J3-SNAP was expressed by osmotic stress in the presence of compatible solutes. J3-SNAP was purified by IMAC and assessed by SDS-PAGE and Western blot analysis. To ascertain the binding of J3- SNAP to cells expressing HIV-1 Env in vitro, recombinant Env protein was transiently transfected into HEK293T-cells to generate an Env expressing cell line. Cell-surface binding of SNAP-Surface® Alexa Fluor® 488 -conjugated J3-SNAP on Env expressing HEK293Tcells was assessed by confocal microscopy analysis. Results Successful expression of J3-SNAP in E. coli BL21-DE3 was confirmed by SDS-PAGE and Western blot analysis. The J3-SNAP fusion protein was subsequently purified by IMAC. Purified J3-SNAP was conjugated to the benzyl guanine-modified fluorophore SNAPSurface® Alexa Fluor® 488 and full-length conjugated protein was confirmed by combinations of SDS-PAGE and Western blot analysis. Cell-surface binding of J3-SNAP to HIV-1 Env-expressing HEK293T-cells was demonstrated in vitro by confocal microscopy analysis. These results prompted the generation of the IT, J3-ETA, by replacing SNAP-tag with ETA. Conclusion Successful binding studies suggest using J3 to target HIV-1 Env. Accessing patient probes would allow for the confirmation of these results for future human applications. Future in vitro studies would need to confirm the selective elimination of Env expressing T-cells by J3-ETA and thereafter confirmed on Env-positive patient probes.
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    Establishment of recombinant antibody technologies allowing for the generation of SNAP-tag fusion proteins
    (2023) Nundalall, Trishana; Barth, Stefan; Naran Krupa
    Triple negative breast cancer (TNBC's) is a highly aggressive and invasive subtype of breast cancer, typically characterised by the lack of estrogen receptor (ER), progesterone receptor (PR) and Human epidermal growth factor receptor 2 (HER2) with an inexplicable partiality towards African women. The acute heterogenicity and complexity of TNBC tumours, together with a lack of well-defined molecular targets, complicates prognosis of the diseases resulting in patient reliance on traditional therapies, like chemotherapy, radiotherapy, and surgery, which are associated with elevated incidence of adverse effects and relapse. A major contributor to the heterogenicity of TNBCs is the tumour microenvironment which is composed of tumour infiltrating lymphocytes (TILs), tumour cells, healthy cells, and tumour vasculature. TILs have commonly been used as a prognostic marker and show robust predictive value for TNBC. In-depth analysis of the TIL composition within TNBC tumours may provide greatly beneficial information for the development of newer tumour microenvironment changing therapies and could assist doctors in understanding what therapies a particular patient maybe susceptible to. Thus, the diagnosis and therapy of this disease may greatly benefit from improved molecular profiling and patient stratification. Precision medicine seeks to provide such a solution, by dividing patients into subpopulations based on disease-specific profiles. The identification of new molecular targets would provide the basis for development of novel therapies. To this end, one of the major aims of this thesis was to develop a phage display based screening technique which could be utilised to isolate novel TNBC specific cancer antibodies. Once selected these antibodies could be used to generate TNBC specific therapies. Specific monoclonal antibodies (mAbs) and derivatives thereof, have already been established as a revolutionary tool for drug delivery to cancerous cells. Such antibodies have been conjugated to cytotoxic drugs to form antibody-drug conjugates, which may exhibit multiple advantages over their unconjugated counterparts, but their general use in clinical application has been restricted due to developmental deliberations. Historical conjugation strategies used for the generation of ADCs commonly resulted in heterogeneous mixtures of ADC species, with varying drug-to-antibody ratios resulting in unpredictable pharmacologic characteristics and safety profiles. In more recent time, self-labelling tags such as Snaptag have provided a means of developing homogenised recombinant immunotherapeutics. Snaptag is a modified version of a human DNA repair enzyme, O6 - alkylguanine-DNA-alkyltransferase (AGT) which naturally removes alkyl residues from damaged DNA. The enzyme reacts specifically with benzylguanine (BG) derivatives via irreversible transfer of alkyl groups to cysteine residues forming stable end products. In this thesis, Snaptag technology, together with other antibody discovery and manipulation tools was used to develop a methodology allowing for the generation of disease specific fusion proteins. Specifically, these fusion proteins consist of single-chain antibody fragments genetically fused to snaptag, allowing for the generation of recombinant ADCs that could be used as a drug delivery system carrying any BG-modified drug to a disease specific targets. In addition, snaptag interacts with BG in a 1:1 stoichiometry giving rise to homogenised combination products which when fused to a scFv provides a fail-safe target-specific therapeutic option. In addition to antibody conjugates, one of the most promising of all mAb based therapies currently used, are checkpoint inhibitors. In a balanced immune response, immune activation is counteracted with immunoregulatory pathways such as checkpoint inhibition. These negative regulatory pathways are necessary for maintaining tolerance and preventing hyperactivation, and are governed by cell surface, inhibitory receptors known as ‘'checkpoint inhibitors''. Blocking of checkpoint pathways during chronic infections and cancer has been shown to improve T-cell functions leading to reduced viral load and tumour burden. These findings have been translated into clinical application where checkpoint inhibitors, which are monoclonal antibodies targeting CTLA-4, PD1, PD-L1 or other inhibitory ligands, have been used to block these inhibitory interactions. The main intention of this research was to develop a methodology which could be used to generate snaptag based recombinant fusion proteins with potential diagnostic and therapeutic applications. Several snaptag based fusion proteins were developed using the recommended methodology these included fusion proteins targeting breast cancer specific antigen BCK1, checkpoint inhibitors PDL1, B7.1/CD80 (interacts with CTLA-4),and TIL characterising markers CD3, CD4, CD8, CD19 and CD20. In addition, to demonstrate the versatility and robustness of this methodology we sought to develop a snaptag based fusion protein not targeting breast cancer related antigens. Zika virus, an emerging infectious disease, currently lacking specific therapies was chosen for this purpose. An scFv derived from antibodies targeting the the Zika-DIII envelop protein, which is essential to the viral infection cycle was used in the snap fusion protein. The resulting ZIKA-DII-snap fusion protein demonstrated specific binding to zika virus membrane fractions. This research demonstrates the feasibility of using snaptag technology as a state-of-the-art conjugation strategy capable of bypassing the challenges previously associated with using antibodies as an effective delivery system for therapeutic drugs. By combining the applicability of snaptag technology with other antibody isolation and manipulation tools we were able to generate several functional snaptag based recombinant fusion proteins. Establishment of this methodology represents an important first step in generating medically necessary, pharmaceutically acceptable immunoconjugates that is instrumental in shifting general therapy towards a more personalized precision medicine approach.
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    Evaluation of tumour-associated antigens to optically label cutaneous basal cell carcinoma for surgical excision
    (2022) Madheswaran, Suresh; Barth, Stefan
    Basal cell carcinoma (BCC) is the most common skin cancer worldwide, with South Africa having the highest incidence rate only after Australia. The most effective treatment modality for BCC is tumor excision via Mohs surgery (pioneered by Dr. Frederic Mohs of the University of Wisconsin in 1930), a microscopically controlled surgery that removes a tumor piecemeal in layers until each layer is free of any neoplastic tissue. The major drawback of Mohs excision is that the surgeon might miss any neoplastic tissue as the tumor margin is not always well defined, and the tumor often could extend beyond the superficial layers of skin. Moreover, it's a time-consuming, expensive procedure that takes generally 3-4 h, at times even more, if several rounds of excisions are warranted. In South Africa, at the time of writing, therapy using the surgery cost around R45,000. The status quo thus necessitates identifying BCC cells both in the superficial layers and beyond the layers of the skin in individual patients. Our aim was to identify BCC-specific cell surface proteins and design, engineer, and test a range of SNAPtag–based antibody fusion proteins that would specifically bind to and detect such BCC cell surface receptors. The SNAP-tag antibody technology is based on the genetic fusion of a disease-specific ligand to a protein tag derived from O6-alkylguanine-DNA alkyltransferase, which would allow for covalent auto-labeling of the corresponding antibody based fusion proteins with benzylguanine-modified (BG) substrates (e.g., fluorophores) under physiological conditions with high efficiency at 1:1 stoichiometry. This would allow to develop a unique immunological screening modality which should allow to visually label BCC lesions for a more precise surgical excision. The best-performing SNAP-tag–based diagnostic antibodies resulting from these studies would be further evaluated in the future in suitable mouse models, thus aiming to reduce the time needed for surgical removal of BCC lesions and complete removal of the tumor from both superficial and deep layers of the skin by a single-excision procedure. We used an integrated computational tool to re-analyze publicly available cDNA microarray data in combination with theoretical search to identify BCC-associated antigens. Accordingly, six different antigens were selected and single-chain variable fragments (scFv) targeting these antigens were cloned in fusion with SNAP-tag encoding gene into a custom expression vector for production in a secretory mammalian system. scFv-SNAP-tag protein was isolated from the cell culture supernatant by immobilized metal affinity chromatography and eluted protein samples were analyzed by gel electrophoresis and immunoblotting. The absolute amount of the full-length protein was quantified by densitometry. Purified scFv-SNAP-tag proteins were validated for specific binding to corresponding antigen-positive cells by flow cytometry and confocal microscopy. Of the six different scFv-SNAP-tag fusion proteins cloned, four were successfully expressed in HEK293T cells. The specific binding to EpCAM, EMA, CSPG4, and CD138 antigenexpressing cell lines was observed on incubation with scFvUBS54-SNAP-tag, scFvID405- SNAP-tag, 9.2.27scFv-SNAP-tag, and scFvh-STL002-SNAP-tag, respectively. In addition, we showed the selective cell killing effect of scFvUBS54-SNAP after conjugating it with the cytotoxic drug BG-modified auristatin-F (BG-AF). In conclusion, we identified various cell surface antigens along with one possibly novel antigen for BCC detection and therapy. Further, we successfully designed and synthesized SNAP tag based antibody fusion proteins and showed their functional activity by selective binding to the corresponding antigens on the surface of tumor cells. Based on these findings, we presume that these antibodies can effectively bind to BCC and can confirm EpCAM as one of the target antigens, which has already been reported to be a standard immunophenotypic marker for differential BCC diagnosis.
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    Generation of Glioblastoma specific SNAP based antibody fusion proteins for future radiolabelling application
    (2023) Mayuni, Grace; Barth, Stefan
    Each year, Central Nervous System (CNS) cancers affect about 300,000 individuals globally, accounting for 1-2% of all adult cancer cases. Despite being rare, CNS cancers reportedly have disproportionately high morbidity and mortality rates compared to their incidence. Globally, Glioblastoma Multiforme (GBM) is the most aggressive CNS malignancy, accounting for 45% of brain tumours, with less than 5% of patients surviving for more than 5 years. To date, complete surgical resection followed by adjuvant chemotherapy and/or radiotherapy has been the standard of care treatment. This has been shown to not be curative, as it can only slightly improve prognosis and increase patient survival from 3–4 months to 14–16 months after diagnosis. However, it has been noted that early diagnosis increases the chance of patients going into complete remission following early treatment. Unfortunately, current methods of diagnosing GBM are very invasive and imprecise, necessitating more targeted, and more reliable diagnostic techniques. The use of antibody-based targeted agents in GBM treatment is being advanced due to their ability to specifically differentiate tumour masses from healthy tissue. The success of antibody-based targeted approaches lies in the selection of biomarkers that are selectively found in tumour cell populations. One type of biomarker is known as tumour-associated antigens (TAAs), which are antigenic substances present at a much relatively lower incidence on normal cells but highly correlated with certain tumour cells. The focus of this study was on two TAAs that have been established as being overexpressed in GBM: (i) the transmembrane protein epidermal growth factor receptor (EGFR), which is associated with tumour cell proliferation, and (ii) Fibroblast Activation Protein alpha (FAPα), an active protease in the stromal tissue of the GBM tumour microenvironment. SNAP-tag® fusion antibodies against the abovementioned TAAs were employed in this study. The fusion antibody is a recombinant antibody-based fusion protein created by fusing the SNAPtag®, a modified version of the DNA repair enzyme O6 -alkylguanine DNA alkyltransferase (hAGT), to a single chain variable fragment (scFv) region of an antibody, thereby providing a tool that can detect and label TAAs. The SNAP-tag® element is engineered to irreversibly react with any benzylguanine (BG) substrates, including labelling dyes or toxins, with a controlled 1:1 Masters Thesis 14 Grace Mayuni stoichiometry. According to previous studies, SNAP-tag fusion proteins have exhibited strong cytotoxic profiles as an immunotherapeutic tool as well as good imaging profiles as an immunodiagnostic tool. In this study, the recombinant SNAP fusion proteins, anti-FAPα (scFv)-SNAP and anti-EGFR (scFv)-SNAP were transiently expressed in mammalian cell culture. The recombinant protein was purified from harvested cell culture supernatant using immobilized metal ion affinity chromatography (IMAC). The enzymatic activity of the SNAP-tag element was tested using BGmodified fluorophores. To characterize highly specific in vitro labelling, the SNAP-tag-based fusion proteins were conjugated to BG-Alexa Fluor 647 to generate an immunofluorescent protein that can label TAAs in live tumour cells. To characterize the immunotherapeutic activity, the SNAP-tag fusion proteins were conjugated to the small molecule toxin, BG-Auristatin F, to generate an ADC that can kill TAA-expressing cells in vitro. Anti-FAPα (scFv)-SNAP and anti-EGFR (scFv)-SNAP fusion proteins were successfully expressed in this study with sufficient yields for small-scale purification. Through a series of selflabelling experiments with the BG-modified fluorophores, BG-Alexa Fluor 488 and BG-Alexa Fluor 647, the enzymatic activity of the SNAP-tag element of each construct was confirmed. The optimal binding ratio of fusion protein: BG-substrate conjugate for downstream experiments was determined to be 1:1. Using live cell imaging, the biological functionality of the fusion protein construct through site-specific labelling on antigen-expressing cell lines was demonstrated. Finally, this study investigated the cytotoxicity of SNAP-fusion proteins conjugated to toxic payloads. Using a cell proliferation assay, the next-generation ADC, anti-EGFR (scFv) SNAPAURIF, was tested and validated for its cell-killing activity. This ADC was able to elicit high rates of killing, exhibiting IC50 values in nanomolar ranges in three different antigen-positive tumour cell lines, with no cytotoxicity observed in the antigen-negative tumour cell lines. The targeted activity of this recombinant SNAP fusion protein format validates its biological function, making it suitable for further preclinical investigation. This study aimed to characterize SNAP fusion proteins that can target GBM-specific TAAs. It successfully demonstrated the optimal conjugation efficiencies and validated the site-specific targeted activity of the scFv SNAP fusion antibody format. This work serves as a proof-of-concept Masters Thesis 15 Grace Mayuni towards investigating the use of radio-labelled SNAP-tag-based antibody fusion proteins in GBM tumour detection and patient stratification to offer personalized therapeutics.
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    Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity
    (2021-01-11) Seidel-Greven, Melanie; Addai-Mensah, Otchere; Spiegel, Holger; Chiegoua Dipah, Gwladys N; Schmitz, Stefan; Breuer, Gudrun; Frempong, Margaret; Reimann, Andreas; Klockenbring, Torsten; Fischer, Rainer; Barth, Stefan; Fendel, Rolf
    Abstract Background Plasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirixâ„¢ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates. Material and methods Using antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1–3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays. Results The selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2. Conclusion We have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.
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    Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    (BioMed Central, 2016-02-17) Klose, Diana; Saunders, Ute; Barth, Stefan; Fischer, Rainer; Jacobi, Annett M; Nachreiner, Thomas
    Background: In an earlier study we developed a unique strategy allowing us to specifically eliminate antigenspecific murine B cells via their distinct B cell receptors using a new class of fusion proteins. In the present work we elaborated our idea to demonstrate the feasibility of specifically addressing and eliminating human memory B cells. Results: The present study reveals efficient adaptation of the general approach to selectively target and eradicate human memory B cells. In order to demonstrate the feasibility we engineered a fusion protein following the principle of recombinant immunotoxins by combining a model antigen (tetanus toxoid fragment C, TTC) for B cell receptor targeting and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’) to induce apoptosis after cellular uptake. The TTC-ETA’ fusion protein not only selectively bound to a TTC-reactive murine B cell hybridoma cell line in vitro but also to freshly isolated human memory B cells from immunized donors ex vivo. Specific toxicity was confirmed on an antigen-specific population of human CD27+ memory B cells. Conclusions: This protein engineering strategy can be used as a generalized platform approach for the construction of therapeutic fusion proteins with disease-relevant antigens as B cell receptor-binding domains, offering a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.
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    Photoimmunotheranostic targeting of CSPG4-positive melanoma cells using SNAP-tag technology
    (2018) Malindi, Zaria; Barth, Stefan; Padayachee, Eden; Biteghe, Fleury Augustin Nsole
    Melanoma is one of the most aggressive and inherently resistant cancers and the most dangerous skin cancer. While it accounts for fewer than 5% of skin cancer cases, 80% of skin cancer related deaths are attributed to melanoma. While resection remains the gold standard for melanoma treatment, surgery is only effective in providing local control of the disease if the cancer is detected in the early stages. Once melanoma enters the later stages, and particularly in the metastatic phase, recurrence is probable, and no adequate treatment exists. Previous work in this group has shown that photodynamic therapy (PDT) presents an opportunity to induce cell death in melanoma cells through the production of ROS and singlet oxygen at doses high enough to overwhelm the resistance mechanisms of the cancer. In this study, we investigated the use of recombinant SNAP-tag-based antibody fusion proteins as a means of delivering phototoxic molecules directly to cancer cells expressing the CSPG4 and PD-L1 cell surface receptors. SNAP-tag is an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase. It reacts autocatalytically and in a strictly 1:1 coupling chemistry with substrates that have been modified with benzylguanine (BG). Through genetic fusion of this self-labelling protein with a tumour targeting antibody, we developed a recombinant immunoconjugate able to carry BG-modified photosensitizers to selectively target and eliminate malignant melanoma cells. Conjugation of the SNAP-tag fusion protein with the fluorescent dye Alex Fluor 488 showed that anti-CSPG4-SNAP binds specifically to melanoma cells expressing the CSPG4 surface antigen. Binding was tested across a range of cell lines presenting melanoma in its radial and vertical growth phases, in the metastatic growth phase, in its chemoresistant form, and in both its pigmented and unpigmented forms. This binding data thus confirms CSPG4 as a suitable targeted for this treatment strategy. Conjugation of the fusion protein with the BGmodified photosensitizer IRDye 700DX (IR700) has produced no phototoxicity as of yet. In light of the convincing binding analysis, it is concluded that inefficient solubilization of the lyophilized product resulted in inadequate conjugation of BG-IR700 with SNAP-tag. Nonetheless, steps have been planned to resolve the problem in future ongoing work on this project, and we remain confident in the applicability of this technology. The results for the PD-L1 fusion protein were inconclusive. In summary, SNAP-tag technology offers a simple and efficient method for immunofluorescent detection of cancerous cells. These fusions proteins are versatile as they 1) can contain any antibody targeting a tumour-associated or tumour-specific antigen of choice and 2) can be endowed with a wide variety of substrates, as long as the latter contains the BG moiety.
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    Synthesis and biological evaluation of auristatin-F recombinant antibody-drug conjugates for cancer therapy
    (2020) Huysamen, Allan Martin; Hunter, Roger; Barth, Stefan
    This study serves as a contribution to the emerging field of antibody-drug conjugate (ADC) therapeutics, in which novel ADCs were generated and biologically evaluated for therapeutic potential and specificity towards cancer cells. SNAP-tag site-directed conjugation was employed in order to overcome the current challenges of heterogeneous ADC combination products resulting from standard conjugation methods. The SNAP-tag fusion proteins were generated using recombinant DNA technology, and incorporated antibody single-chain variable fragments (scFvs) exhibiting specificity towards the epidermal growth factor receptor (EGFR), chondroitin sulfate proteoglycan receptor (CSPG4) and CD44 receptors more highly expressed in various cancer types. The design and successful synthesis of two BG-linkers was followed by coupling to auristatin-F, a powerful microtubule poison, using both Cu(I)-catalysed cycloaddition and amide coupling chemistry. The BG-linkers each incorporated a noncleavable polyethylene glycol spacer, one of which included a dansyl fluorescent tag in order to aid in purification and characterisation. Ultimately, this study succeeded in producing two novel BG-modified ADC-precursors and various ADCs by virtue of SNAP-tag conjugation, three of which exhibited therapeutic potential following in vitro cytotoxicity assays. Cytotoxicity assays were undertaken by treating A431, SK-Mel-28, MDA-MB468, Hs578T and A2058 cell lines with ADCs generated from various scFv-SNAP fusion proteins, in which selectivity towards target receptor positive cell lines was demonstrated. Quantification of the efficacy of generated ADCs was afforded by IC50 values, resulting from dose-response curves, which ranged from 44 nM – 180 nM. Collectively, insights were gained into the subtleties governing the interface of molecular and macromolecular chemistry that will facilitate further research into the development of ADC cancer therapeutics.
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    Validation of an aASPH-SNAP-tag fusion protein for immunodiagnosis of triple-negative breast cancer
    (2023) Friedberg, Chardae; Barth, Stefan
    Breast cancer is a serious health concern for women from as young as 20 years old and to this day is well known to be the leading cause of cancer incidences and mortalities in females. This disease is known as a heterogeneous disease that requires different systemic therapies, therefore early diagnosis and proper therapeutics are of utmost importance. The triple-negative breast cancer (TNBC) type is the most critical breast cancer subtype due to little to no expression present of the more commonly known breast cancer markers such as human epidermal growth factor receptor 2 (HER2), estrogen(ER) and progesterone (PR). This subtype was also found to have a 2-fold higher increased risk in individuals with African ancestry, intensifying the African breast cancer burden. Currently available diagnostics and therapeutics for TNBC are still a concern due to this subtype being associated with poor prognosis, a high metastasis rate and the ability to become easily resistant to available therapeutics due to an immunosuppressive tumour micro-environment and low immunogenicity. Therefore, a need to improve TNBC diagnosis and therapy is an urgent matter. Antibody technologies have been quite successfully applied to improve diagnostics and therapeutics for different diseases which include cancer. Limitations associated with the use of full-length antibodies have led to the development of antibody derivatives which includes single chain fragments of variability (scFv) where a heavy chain variable region of a full-length antibody would have been recombinantly fused to its light chain variable region. The SNAP-tag is commonly known as the engineered version of the human DNA repair enzyme O6 -alkylguanineDNA-alkyltransferase (AGT) that has the ability to recognize and bind to any benzyl-guanine modified substrates in a 1:1 stoichiometry. This makes SNAP-tag technology a favourable tool to use for studying the different properties of antibody derivatives. As part of this research, recombinant single chain fragment variables were recombinantly fused to SNAP-tag to generate a unique antibody format allowing to full exploit the specific binding activity of an antibody with the self-labelling ability of SNAP and create a versatile tool to develop recombinant immunodiagnostics and -therapeutics.