ComparativeIn VitroCytotoxicity Studies of177Lu-CHX-A″-DTPA-Trastuzumab and177Lu-CHX-A″-DTPA-F(ab′)2-Trastuzumab in HER2-Positive Cancer Cell Lines

Sharma, Rohit; Kameswaran, Mythili; Dash, Ashutosh

doi:10.1089/cbr.2019.2882

Cited by 6 publications

(3 citation statements)

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“…Since their discovery in the late 1980s, the ITGB superfamily members are demonstrated to be involved in the regulation of cancer development and progression ( Cooper and Giancotti, 2019 ). The role of ITGBs in the tumorigenesis and pathogenesis of various cancer types has increasingly received attention ( Sharma et al, 2020 ; Fujita et al, 2021 ; Nurzat et al, 2021 ; Paindelli et al, 2021 ). Here, in this paper, we evaluate the utility of ITGBs as biomarkers in GC, and we utilize bioinformatics tools for investigating the underlying mechanisms by ITGB5 affected GC.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Analysis of the Expression and Prognosis for ITGBs: Identification of ITGB5 as a Biomarker of Poor Prognosis and Correlated with Immune Infiltrates in Gastric Cancer

Liu

Fang

et al. 2022

Front. Cell Dev. Biol.

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Background: Integrin β superfamily members (ITGBs) are documented to play important roles in various biological processes, and accumulating evidence suggests that ITGBs are associated with carcinogenic effects in several malignancies. Gastric cancer (GC) is a complicated and highly heterogeneous disease; however, the expression and prognostic values of eight ITGBs and potential mechanism in GC remain largely unclear.Methods: The expression and prognostic significance of ITGBs in GC were systematically analyzed through Gene Expression Profiling Interactive Analysis, Human Protein Atlas, Kaplan–Meier Plotter, and cBioPortal databases. Then, the mRNA transcription data and corresponding clinical data of GC were downloaded from the Gene Expression Omnibus database as a testing cohort, and differentially expressed and prognostic genes were identified. The correlation between ITGB5 expression and overall survival and various clinical parameters were found by using univariate/multivariable Cox regression and Kaplan–Meier survival analysis. Additionally, differential analysis of gene expression profiles in low- and high-ITGB5 expression groups and pathway enrichment analysis was performed. Finally, the correlation of ITGB5 expression with immune infiltrates in GC was clarified.Results: Compared with adjacent normal tissue, the results reveal that the mRNA levels of ITGB1-2 and ITGB4-8 are significantly higher in GC, and immunohistochemistry results show the consistency between RNA and protein expression levels. Cox regression and Kaplan–Meier survival analysis indicate that high ITGB5 expression contributes to a poor prognosis and could be an independent prognostic factor in GC patients. Besides this, gene functional enrichment analysis indicates that ITGB5 expression is significantly associated with extracellular matrix organization, cell-substrate adhesion, and ossification. The KEGG pathway analysis of ITGB5 shows a close association between ITGB5 and focal adhesion, ECM-receptor interaction, phagosome, and PI3K-Akt signaling pathway. Last, the infiltrating level of CD4+ T cells, macrophages, and dendritic cells are positively related to the expression of ITGB5, especially macrophages, and lower levels of macrophages predict a better prognosis in GC in our study.Conclusion: Our findings investigate that ITGB5 may function as a valid biomarker of prognosis, and high expression of ITGB5 predicts poor prognosis for patients with GC. Besides this, it might be a potential target of precision therapy against GC.

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Section: Discussionmentioning

confidence: 99%

Comprehensive Analysis of the Expression and Prognosis for ITGBs: Identification of ITGB5 as a Biomarker of Poor Prognosis and Correlated with Immune Infiltrates in Gastric Cancer

Liu

Fang

et al. 2022

Front. Cell Dev. Biol.

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“…The concentrated antibody was added to 0.5 mL of immobilized pepsin (3000 U/mg), pre-equilibrated with digestion buffer. The preliminary experiments, which include the optimization conditions for enzymatic digestion, are reported elsewhere . Post preparation, the antibody fragments were purified using Amicon filtration devices (MWCO 30,000 Da) and stored in phosphate buffer saline (PBS, 20 mM, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Toward Understanding the Binding Synergy of Trastuzumab and Pertuzumab to Human Epidermal Growth Factor Receptor 2

2021

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Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast, gastric, esophageal, ovarian, and endometrial cancer. Combination therapy using trastuzumab and pertuzumab antibodies targeting HER2 has shown better survival outcomes in breast cancer patients. In the quest to understand the synergistic effect observed due to combination therapy, trastuzumab, pertuzumab, and their F(ab′)2 fragments were labeled with radioisotope and fluorescent probes. Detailed in vitro studies to understand binding synergism in HER2 overexpressing cell lines were done. Antibodies and their F(ab’)2 fragments prepared by enzyme digestion with pepsin were radiolabeled with iodine-125. In vitro binding studies to evaluate immunoreactivity, specificity, affinity, and binding synergism between radiolabeled trastuzumab, pertuzumab, and their F(ab′)2 fragments were carried out. Synergism was observed by 20–30% enhanced uptake of radiolabeled pertuzumab and its F(ab′)2 fragments in the presence of excess of unlabeled trastuzumab or F(ab′)2-trastuzumab. However, uptake of radiolabeled trastuzumab was not enhanced in the presence of excess pertuzumab or its fragments; rather inhibition or competition in binding to HER2 was observed. Studies using fluorescent antibodies by flow cytometry confirmed enhanced binding of pertuzumab in the presence of trastuzumab. Live cell tracking was done to give insights into the binding synergy and fate of fluorescent antibodies . Colocalization of antibodies on HER2 followed by internalization in the cells was observed. The radiolabeled immunoconjugates served as an important tool for experimental characterization of interaction between pertuzumab and trastuzumab to HER2. Studies with fluorescent antibodies corroborated the binding data and provided evidence of colocalization and internalization of both the antibodies in HER2-positive cells.

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“…111 In, 125 I, 67 Ga) for radiation treatment of tumours (Aghevlian et al 2017 ; Ku et al 2019 ). Trastuzumab labeled with the β-particle emitter, 177 Lu was cytotoxic in vitro to HER2-positive human SK-BR-3 and MDA-MB-453 human BC cells and SK-OV-3 ovarian cancer cells (Sharma et al 2020 ). Treatment of athymic mice with intraperitoneal (i.p.)…”

Section: Introductionmentioning

confidence: 99%

Formulation of a kit under Good Manufacturing Practices (GMP) for preparing [111In]In-BnDTPA-trastuzumab-NLS injection: a theranostic agent for imaging and Meitner-Auger Electron (MAE) radioimmunotherapy of HER2-positive breast cancer

Chan

Prozzo

Aghevlian

et al. 2022

EJNMMI radiopharm. chem.

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Background 111In[In]-BnDTPA-trastuzumab-NLS is a radiopharmaceutical with theranostic applications for imaging and Meitner-Auger electron (MAE) radioimmunotherapy (RIT) of HER2-positive breast cancer (BC). Nuclear localization sequence (NLS) peptides route the radiopharmaceutical to the nucleus of HER2-positive BC cells following receptor-mediated internalization for RIT with subcellular range MAEs. The γ-photons emitted by 111In permit tumour imaging by SPECT. Our aim was to formulate a kit under Good Manufacturing Practices conditions to prepare 111In[In]-BnDTPA-trastuzumab-NLS injection for a first-in-human clinical trial. Results Trastuzumab was derivatized with p-SCN-BnDTPA to introduce Bn-DTPA for complexing 111In, then modified with maleimide groups for conjugation to the thiol on cysteine in NLS peptides [CGYGPKKKRKVGG]. BnDTPA-trastuzumab-NLS (5 mg in 1.0 mL of 0.05 M ammonium acetate buffer, pH 5.5) was dispensed into unit dose sterile glass vials to produce kits for labeling with 100–165 MBq of 111In[In]Cl3. The kits met specifications for protein concentration (4.5–5.5 mg/mL), volume (0.95–1.05 mL), pH (5.5–6.0), appearance (clear, pale-yellow, particulate-free), BnDTPA substitution level (2.0–7.0 BnDTPA/trastuzumab), purity and homogeneity (SDS-PAGE and SE-HPLC), 111In labeling efficiency (> 90%), binding to HER2-positive SK-BR-3 human breast cancer cells (Ka = 1–8 × 108 L/mmol; Bmax = 0.5–2 × 106 sites/cell), NLS peptide conjugation (upward band shift on SDS-PAGE), sterility (USP Sterility Test) and endotoxins (USP Bacterial Endotoxins Test). 111In-BnDTPA-trastuzumab-NLS injection met specifications for pH (5.5–6.5), radiochemical purity (≥ 90%), radionuclide purity (≥ 99%), appearance (clear, colourless, particle-free) and sterility (retrospective USP Sterility Test). Kits were stable stored at 2–8 °C for up to 661 days (d) meeting all key specifications. Protein concentration remained within or just slightly greater than the specification for up to 139 d. 111In[In]-BnDTPA-trastuzumab-NLS injection was stable for up to 24 h. An expiry of 180 d was assigned for the kits and 8 h for the final radiopharmaceutical. Conclusion A kit was formulated under GMP conditions for preparing 111In[In]-BnDTPA-trastuzumab-NLS injection. This radiopharmaceutical was safely administered to 4 patients with HER2-positive BC to trace the uptake of trastuzumab into brain metastases before and after MRI-guided focused ultrasound (MRIg-FUS) by SPECT imaging.

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ComparativeIn VitroCytotoxicity Studies of¹⁷⁷Lu-CHX-A″-DTPA-Trastuzumab and¹⁷⁷Lu-CHX-A″-DTPA-F(ab′)₂-Trastuzumab in HER2-Positive Cancer Cell Lines

Cited by 6 publications

References 57 publications

Comprehensive Analysis of the Expression and Prognosis for ITGBs: Identification of ITGB5 as a Biomarker of Poor Prognosis and Correlated with Immune Infiltrates in Gastric Cancer

Comprehensive Analysis of the Expression and Prognosis for ITGBs: Identification of ITGB5 as a Biomarker of Poor Prognosis and Correlated with Immune Infiltrates in Gastric Cancer

Toward Understanding the Binding Synergy of Trastuzumab and Pertuzumab to Human Epidermal Growth Factor Receptor 2

Formulation of a kit under Good Manufacturing Practices (GMP) for preparing [111In]In-BnDTPA-trastuzumab-NLS injection: a theranostic agent for imaging and Meitner-Auger Electron (MAE) radioimmunotherapy of HER2-positive breast cancer

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