The overall goal of these procedures is to evaluate the specificity and effectiveness of antibody conjugates for accumulating inside target cells and tumors during the initial development stages so that they can ultimately be used in the clinic. This method can help answer key question on the answer the conjugate fields, such as the efficiency of nuclear localization and the overall intercellular accumulation. An advantage of this technique is that via PET imaging researchers can assess the effectiveness and selectivity of candidate antibody conjugates.
The implications of these techniques extend towards therapy and diagnosis of cancer, because ineffective tumor-cell accumulation is a major limitation with these applications for antibody conjugates. After synthesizing a colic-acid nuclear localization sequence antibody conjugate according to the text protocol, treat target TF1A cells with 200 nanomolar 7G3 colic-acid MLS antibody conjugate. Include cells treated with modified control monoclonal antibodies.
Incubate five times 10 to the sixth antigen-positive cells with the conjugates at 37 degrees Celsius for one hour. Then remove the supernatant, and use one milliliter of ice-cold PBS to wash the cells three times. Add fresh medium, and incubate the cells at 37 degrees Celsius for an additional hour.
Next, add 0.5 milliliters of PBS, containing 0.25%trypsin and EDTA, and incubate the cells at 37 degrees Celsius for up to 30 minutes. Then use 1.5 milliliters of RPMI1640 medium with 10%FBS to neutralize the trypsin. Centrifuge the cells at 500 times G for five minutes, remove the supernatant, and use 0.5 milliliters of ice-cold PBS to wash the cells three times again.
Add 0.5 milliliters of 1%PFA and 1%sucrose and PBS to the cells, and place them on ice for 30 minutes to fix them. Then use 0.5 milliliters of ice-cold PBS to wash the cells three times, and centrifuge the samples at 250 times G for five minutes. Next, add 0.15%Triton X-100 and PBS to the cells, and permeablize them on ice for five minutes.
Then, with ice-cold PBS, wash the cells, and repeat the centrifugation. Suspend the cells in 0.1 milliliters of PBS, containing two micrograms per milliliter of an antimirroring FC secondary polyclonal antibody conjugated to Alexa Fluor 647, and incubate the samples in the dark at room temperature for one hour. Centrifuge the cells at 250 times G for five minutes, and use 0.5 milliliters of ice-cold PBS to wash the cells three times.
Then suspend the cells in 0.5 milliliters of PBS. Add 10 micrograms per milliliter of propidium iodide to the cells. Then use mounting medium to mount one times 10 to the fifth cells onto glass slides before covering the sample with a glass cover slip.
Examine the cells with a Plan APO 60X oil-immersion objective, numerical aperture 1.42, on an inverted laser-scanning confocal microscope. Detect PI fluorescence using the 488-nanometer argon laser and the spectral-scanning prism set for 600 to 650 nanometers. For AF 647 fluorescence, use the 633-nanometer helium-neon laser and the spectral-scanning prism set for 650 to 700 nanometers.
Collect images from PI and AF 647 sequentially, 1024 by 1024 pixels, with 2X line averaging, taken at 0.5-micron intervals through the entire cell thickness. Present images as Z projections. To analyze the cells, evaluate and record whether intracellular fluorescence in the cytoplasm is grouped and near the cell surface or diffuse and homogeneous.
Also evaluate the relative fluorescence intensity per cell. After preparing and concentrating radiolabeled colic-acid NLS antibody conjugate according to the text protocol, determine the radiolabeling efficiency by applying 0.5 microliters of 0.1-molar PH5.5 sodium citrate, or ITLC eluent, to an ITLC strip. Form an autoradiograph of the strip, and obtain a digital image.
Carry out densitometry to obtain the proportion of bound and free Copper-64. If free Copper-64 content is greater than 5%concentrate the sample further. For Copper-64 cellular accumulation studies, treat cells with 100 nanomolar of Copper-64-labeled A14 conjugates at 37 degrees Celsius for one, six, and 24 hours, as previously described.
Treat the cells with unmodified A14 to block IL5R alphacytes and at four degrees Celsius to block receptor-mediated internalization. After washing the cells and incubating with fresh medium, as demonstrated earlier in this video, add 0.5 milliliters of PBS containing 0.25%trypsin and EDTA, and incubate at 37 degrees Celsius for 15 minutes. Then, after neutralizing and washing the cells as before, add plasma-membrane lysis buffer to the cells, and incubate them on ice for 10 minutes.
Centrifuge the plasma-membrane-lysed cells at 90 times G for five minutes. Remove the supernatant, and transfer it to a fresh tube. This represents the cytoplasmic fraction.
Then use ice-cold PBS to wash the nuclei three times, and add the washes to the cytoplasmic fraction. Ensure vials containing the nuclear and cytoplasmic fractions are sealed, then place them in a gamma counter calibrated for Copper-64 in order to convert raw counts to megabecaril. Determine the quality of nuclei isolation by performing Western-Blot analysis for lamin AC, a restricted nuclear protein;and Rab-7, an abundant cytoplasmic protein, on wholesale lysate, nuclear, and cytoplasmic fractions.
Treat five times 10 to the six cells with 500 microliters of radio-amino-precipitation assay, or RIPA buffer, and plasma-membrane lysis buffer containing 1%2%and 4%NP-40. Furthermore, treat the isolated nuclei with 500 microliters of RIPA buffer to obtain isolated nuclear proteins. Add four times the sample volume of cold acetone to the isolated nuclear, cytoplasmic, and wholesale proteins, and incubate for 60 minutes at minus 20 degrees Celsius.
Centrifuge the samples at 13, 000 times G for 10 minutes. Decant the supernatant, being careful not to dislodge the protein pellet. Then add 100 microliters of PBS to dissolve the proteins.
After performing Western Blotting as previously described, cut the membrane in half at the 40-kilodalton molecular-weight marker. Use lamin-AC-specific antibodies to probe the blot containing the higher-molecular-weight proteins, and use Rab-7-specific antibodies to probe the lower half of the membrane. In groups containing four nodskit mice, inject into the tail vein 20 to 30 micrograms of radiolabeled A14 colic-acid NLS antibody conjugate, radiolabeled A14 NLS, and radiolabeled A14.
On the same day, place a cylindrical phantom containing five megabecaril of Copper-64 into the PET scanner to convert the radioactive counts per second into injected dose per gram of tissue. 48 hours later, after anesthetizing the mice according to the text protocol, rapidly transfer a mouse to a PET scanner table in the headfirst prone position with a nosecone for isoflurane. Start the PET data acquisition using a regular sampling-mode setting and an energy window of 250 to 650 kiloelectron volts.
After the scan, remove the mouse from the scanner, and place it back in the cage. As shown in this figure, the arrows show the difference when evaluating antibody conjugate accumulation with and without trypsinization. In cells that are not trypsinized, intracellular accumulation and distribution is difficult to evaluate due to the over expression of the target cell's surface receptors.
The arrows show the difference in intracellular accumulation and distribution of two antibody conjugate candidates when cells are properly trypsinized. Radiolabeled A14 colic-acid NLS antibody conjugate shows nanomolar affinity for IL5R alpha. With increasing concentrations of radiolabeled A14 colic-acid NLS antibody conjugate, specific binding of A14 colic-acid NLS antibody conjugate approached saturation at concentrations of 3.5 nanomolar in both HT-1376 and HT-B9 cells.
Gamma counting reveals that the increase of radioactivity in the nucleus and in the cytoplasm from radiolabeled A14 colic-acid NLS antibody conjugate is specific and increases over time. HT-1376 and HT-B9 cells were incubated with plasma-membrane lysis buffer containing 1%2%or 4%NP-40. For HT-1376 and HT-B9 cells, the lysis buffer containing one or 2%NP-40 Rap-7 was not detected in the nuclear fraction, and lamin AC was not detected in the cytoplasmic fraction.
PET imaging allows for the evaluation of candidate antibody conjugates for their ability to target antigen-positive tumors, red and blue arrows, and their associated biodistribution properties in comparison to the parental non-peptide-modified antibody. One can see the signal accumulation in the tumor to help select on the potential for a developed antibody conjugate. PET imaging also allows for the evaluation of candidate antibody conjugate uptake in healthy tissues such as the liver.
After mastering these techniques, researchers in the field of targeted tumor delivery of molecular payloads will be able to explore additional agents where tumor-cell accumulation is hindered due to endosome entrapment.
