In recent years, antibodies have shown significant potential in new** research across a variety of diseases. In the field of cancer**, research into antibody-drug conjugates (ADCs) is booming. ADCs use the specificity of monoclonal antibodies (mAbs) to deliver cytotoxic drugs to tumor cells expressing specific markers or antigens in a targeted manner, thereby achieving the death of targeted cells. The breast cancer drug Kadcyla is an ADC drug consisting of trastuzumab (Herceptin), which targets HER2, combined with the cytotoxic drug imetansine (DM1).
Its mechanism of action (MOA) is as follows: DM1 directed transport to HER2-positive cells Trastuzumab-mediated inhibition of the HER2 signaling pathway Inhibition of HER2 extracellular domain shedding from the cell membrane Antibody-mediated ADCC.
In the main mechanism of action of the breast cancer drug KADCYLA, the antibody efficiently binds to HER2 on the cell surface and directs the internalization of the drug, resulting in the intracellular release of the DM1 molecule. Therefore, experimental studies on the binding, internalization, and toxicity of ADC drugs are indispensable. The dynamics of these processes can be captured and analyzed by the Incucyte Real-Time Live-Cell Analysis System.
Incucyte Real-Time Live-Cell Analysis System.
Schematic diagram of the Kadcyla structure.
1. Antibody internalization.
Methods: Incucyte Human FabFluor-pH Antibody Labeling DYE specifically labeled the Fc segment of human IgG in a one-step process, which is pH sensitive, and can achieve the following effect: Once the labeled antibody complexes bind to a specific site, they are internalized by the cell and enter the low pH lysosomal environment. As the pH changes, the dye produces a fluorescent signal, which allows visualization of the internalization of the antibody when using the Incucyte Live-Cell Analysis System.
Kadcyla and trastuzumab showed higher levels of internalization in HER2-positive breast cancer cells AU565 12 hours after the addition of the labeled antibody drug (compared with the IgG-negative control group and the MDA-MB-231 cell group with low HER2 expression). The 6 g ml Kadcyla group had reached a plateau at < 6 h. The 24-hour EC50 values of Kadcyla and trastuzumab were 0., respectively38 μg/ml，0.22 g ml, which exhibited similar binding specificity and internalizing effects. These observations were made by using a live-cell analysis system and a live-cell imager.
2. Cell proliferation.
Methods: Incucyte Nuclight Green Lentivirus reagent stably expressed the green nuclear fluorescence signal in AU565 cells. Sartorius' Incucyte live-cell analysis system captures images in real-time and analyzes the number of nuclei to characterize the state of cell proliferation.
DM1 in Kadcyla is a tubulin inhibitor that inhibits the assembly of microtubules, leading to disruption of the cell cycle, which leads to the cessation of the mito** and then death. When Kadcyla is internalized into lysosomes, DM1 is cleaved and released into the cell, where it functions.
After 72 hours of drug action, kadcyla (15 g mL) showed unhealthy cell morphology and proliferation was significantly inhibited (compared to the IgG-negative control and trastuzumab groups). The IC50 of the inhibitory effect of Kadcyla on cell proliferation is 024 g ml, which is similar to internalized titer and shows maximal cell killing comparable to camptothecin. Use the cell health indicator incucyte annexin v dye (030 g ml, data not shown) assay kadcyla cell apoptosis titer is consistent.
3. Cell cycle.
Methods: Incucyte Cell Cycle Lentivirus reagent stably expressed G1 and S G2 M phase fluorescent indicators in Au565 cells, and the reagent did not affect the normal function of cells. The nuclei fluoresce green at the S g2 m phase; Nuclei are colorless in M to G1 phases; Nuclei fluoresce red at the G1 phase; In the G1 to S phases, the nuclei fluoresce yellow (green with red superimposed). The Incucyte Live Cell Analysis System captures images in real time, and the Incucyte Cell-by-Cell Module analyzes the number of nuclei of different colors to characterize cell cycle status.
Under normal circumstances, the percentage of cells in different phases of the AU565 cell cycle is green (S g2 m, 40 3%), red (G1, 24 2%), yellow (G1 S, 15 2%), or colorless (M g1, 20 4%). In the first 24 hours of Kadcyla (3 g ML)-treated AU565 cells, there was a decrease in red blood cells (down to 13%) and an increase in colorless cells (up to 27%), indicating cessation of the M G1 phase cycle. At 36 h, the images mainly show green cells (24%) and non-fluorescent cells (52%), which, together with their increasingly rounded morphology, suggests that cycle arrest occurs near the mitomatous stage. After 48 hours, the cells appear to be morphologically unhealthy and are dying. Isotype comparators IgG or trastuzumab (data not shown) had little effect.
4、adcc
Methods: AU565 target cells stably expressing green nuclear fluorescence signal were cultured with NK cells at a 5:1 ratio of efficacy to target, and different concentrations of Kadcyla were added to different sample groups. Incucyte annexin v nir dye is added to the medium to characterize apoptosis signals. The Incucyte Live-Cell Analysis System captures images in real time to analyze the number of target cells and apoptosis signals to characterize ADCC effects. To demonstrate the combined efficacy of KADCYLA, its ability to kill target cells by conventional ADCCs (in combination with direct killing) was compared to its direct cytotoxic effects (monoculture) in a single culture.
In both monoculture and co-culture wells, there was a concentration-related decrease in green fluorescence, indicating Kadcyla-treated target cell death. It is evident from the images that the additional ADCC effect is stronger than the direct cytotoxic effect alone in the presence of NK. ADCC kills target cells faster and is about 25 times stronger (EC50: direct cytotoxicity 0.).27 g ml, 0 for ADCC011 μg/ml）。The Annexin V signal exhibits similar data. The data at 96 h showed that the ADCC co-culture model had improved target cell clearance, which was consistent with the high clinical efficacy of Kadcyla on tumor cell clearance.
In summary, the Incucyte Live-Cell Analysis System is an example of the use of KADCyla as an ADC drug, which enables simple and clear visualization and quantification of KADCYLA's antibody internalization, cell proliferation, cell cycle, and ADCC effects.
Kadcyla binds specifically to HER2-expressing cells and is internalized with similar efficiency and potency to trastuzumab.
Unlike trastuzumab, Kadcyla, once internalized, leads to cell-directed death by releasing cytotoxic load DM1, a potent tubulin inhibitor.
The combined effect of Kadcyla-mediated ADCC activity and direct killing improves the efficiency of tumor cell clearance.
Why Choose the Incucyte Real-Time Live-Cell Analysis System?
The reason for choosing the Incucyte Real-Time Live-Cell Analysis System comes down to its multiple benefits:
The system allows for continuous observation within the incubator for weeks with shooting intervals as short as a few minutes, greatly reducing the need for manual handling and preventing possible harm to cells from over-intervention. This guarantees the capture of critical information, such as the monitoring of cell cycle dynamics mentioned in the article, which allows for visual observation of cell arrest.
The Incucyte is equipped with 6 positions, each of which can be independently programmed, and is compatible with a wide range of plates and dishes for high-throughput experiments. Whether it is a variety of experimental combinations such as immunosuppressants and efficacy-target ratios, Incucyte can complete the detection of all experimental groups at one time.