Journal: Cancer & Metabolism

Article Title: Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

doi: 10.1186/s40170-022-00279-8

Figure Lengend Snippet: The analysis of humanized antibody variant CA9hu-1(HC4LC4) for its capacity to internalize into cancer cells. Visualization by confocal microscopy revealed punctuated intracellular staining signal after acid strip indicating internalization. Cells were incubated with MAb at 4 °C for 30 min and then in parallel at 4 °C (to prevent internalization) and 37 °C (to trigger internalization) for 3 h. After fixation with methanol, the cells were stained with anti-human Alexa Fluor® 488-conjugated antibody in order to visualize the MAbs. Acid strip before fixation was used to remove the membrane-bound antibody. Scale bar = 20 μm

Article Snippet: C Fit curves determine EC 50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software.

Techniques: Variant Assay, Confocal Microscopy, Staining, Stripping Membranes, Incubation

Journal: Cancer & Metabolism

Article Title: Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

doi: 10.1186/s40170-022-00279-8

Figure Lengend Snippet: A , B Graphical representation of ADCC data. CA9hu-1 and CA9hu-2 antibody variant-dependent cell-mediated cytotoxicity. Comparison of CA IX-negative versus CA IX-positive target cell line samples, n = 2. C Fit curves determine EC 50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software. D Humanized antibody variants showed high ADCC activity in Reporter ADCC assay (Promega) tested on cells BT-20, JIMT-1, and 42MGBA naturally expressing CA IX in hypoxia. The signal of the control was set as 100%. E Humanized antibody variants showed high ADCC activity in comparison with irrelevant therapeutic antibody bevacizumab. This figure clearly shows that humanized antibody variants CA9hu-1 and CA9hu-2 retain the ability to activate the ADCC pathway and to mediate the cytotoxic effect on target cells expressing CA IX. Data in the graph represent the mean ± standard deviation values. Statistical significance of differences was assessed using Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 3

Article Snippet: C Fit curves determine EC 50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software.

Techniques: Variant Assay, Software, Activity Assay, ADCC Assay, Expressing, Standard Deviation

Journal: Cancer & Metabolism

Article Title: Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

doi: 10.1186/s40170-022-00279-8

Figure Lengend Snippet: A The effect of selected humanized antibodies (CA9hu-1(HC4LC4) and CA9hu-2(HC4LC5)) on the viability of analyzed cells ± expressing CA IX (C33-a CA IX versus C33-a neo) in the presence of complement determined via Cell Titer Blue Viability Assay. Cancer cells incubated in the absence of humanized antibodies are marked as “no Ab.” Data in the graph represent mean ± standard deviation values. Statistical significance was assessed using Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 2. B Graphical representation of ADCP data. CA9hu-1 and CA9hu-2 antibody variant-dependent cell-mediated phagocytosis. Comparison of CA IX-negative versus CA IX-positive target cell line samples. Statistical significance was assessed using Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 3

Article Snippet: C Fit curves determine EC 50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software.

Techniques: Expressing, Viability Assay, Incubation, Standard Deviation, Variant Assay

Journal: Cancer & Metabolism

Article Title: Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

doi: 10.1186/s40170-022-00279-8

Figure Lengend Snippet: A Analysis of multicellular aggregation of C33-a CA IX with humanized antibody CA9hu-1 (HC4LC4) and CA9hu-2 (HC4LC5) after 24 h and 72 h on poly-HEMA-coated dishes. C33-a CA IX cells incubated in the absence of humanized antibody are marked as “negative control.” B Analysis of C33-a CA IX cells by propidium iodide staining and flow cytometry after 72 h of treatment with humanized CA9hu-1 (HC4LC4) and CA9hu-2 (HC4LC5) antibody. C33-a CA IX cells incubated in the absence of humanized antibody are marked as “ctrl”

Article Snippet: C Fit curves determine EC 50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software.

Techniques: Incubation, Negative Control, Staining, Flow Cytometry

Journal: Cancer & Metabolism

Article Title: Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

doi: 10.1186/s40170-022-00279-8

Figure Lengend Snippet: Graphical representation of extracellular pH analyzed in C33-a CA IX ( A ) cells after treatment with CA9hu-1 (HC4LC4) or CA9hu-2 (HC4LC5) for 48 h in hypoxia, n = 6, pH values were normalized to cell numbers. B , C The ability of humanized antibodies to inhibit invasion of cancer cells in comparison with no Ab treatment. The invasion ability of C33-a cells expressing CA IX was significantly reduced after the treatment with both humanized antibody variants. Data in the graph represent the mean ± standard deviation values. Statistical significance of differences was assessed using Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 6

Article Snippet: C Fit curves determine EC 50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software.

Techniques: Expressing, Standard Deviation