Browsing by Subject "EGFR"

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    Open Access
    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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    Open Access
    Host somatic variation between women living with HIV with cervical intraepitheial lesions (CIN3) and their HIV negative counterparts
    (2024) Mabizela, Nosipho; Dandara, Collet. Soko, Nyarai; Naidoo, Richard
    Despite the use of antiretroviral therapy, cervical cancer remains a leading malignancy in women with HIV, who face a six-fold increased risk. Infection with HIV and HPV has been linked to accelerated cervical cancer development. However, there are limited studies on the role of host somatic variations in HIV-positive and HIV-negative women on cervical cancer. Understanding these variations may help identify potential genetic factors that contribute to accelerated cervical cancer development and differential response to treatment. This knowledge is important in targeting interventions and improving outcomes for women with HIV and cervical cancer. Therefore, this study aims to investigate host somatic genetic variation between cervical biopsies obtained from HIV-positive or HIV-negative women with histologically confirmed CIN3 to determine potential differences in genomic landscapes and HPV infection between HIV-positive and HIV-negative women. The matched case-control study utilized archived cervical biopsies from 88 women (44 HIV positive, 44 HIV-negative) attending Groote Schuur Hospital Cancer Clinic between 2020 and 2022. The cases and controls were carefully age matched. HPV infection and type were confirmed using the Anyplex™ II HPV28 Detection kit. In cervical cancer, six hotspot regions in the four commonly mutated genes (TP53, PIK3CA, PTEN, and EGFR) were genotyped using Polymerase Chain Reaction and validated using Sanger Sequencing. Missense variant pathogenicity was assessed using SIFT, Polyphen-2, and ClinVar tools. The median age was [37 years (IQR:34-41)] for HIV-positive women and [35 years (IQR:32- 43)] for HIV-negative women. In the HIV-negative cohort the women reported tobacco smoking (p<0.0001), menstruation irregularities (p=0.005), and contraception usage (p=0.019). These parameters were statistically significant when compared to HIV-positive cohort. Common HPV types identified were HPV 16 (43/88, 49%), 35 (12/88, 14%), and 58 (10/88, 11%). A total of 232 genetic variants were identified, with HIV-positive women having a significantly higher burden of pathogenic variants (31%) compared to 15% among the HIV-negative (p=0.0406). Identified mutations included stop-gain, missense, synonymous, and intron variants. The genes TP53 and PIK3CA had more stop-gain variants among HIV-positive women (4/5) compared to HIV-negative women with 1/5 of the 5 mutations. These damaging variants were more prevalent in women under 50 in both cohorts. In conclusion, younger women (<50 years) showed predominantly damaging variants, indicating more aggressive cancer, and a possible reason for early onset in the younger cohort. HIV-positive women displayed a higher mutation burden in PIK3CA and pathogenic variants in TP53, emphasizing the need to further explore these genes in gene expression studies.