A Technique to Assess the In Vivo Localization of Tumor-Specific Antibodies

Published: November 30, 2023

Abstract

Source: Shivange, G., et al. Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody. J. Vis. Exp. (2020).

This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via imaging.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Expression and purification of antibodies

  1. Maintenance of Chinese hamster ovary (CHO) cells
    1. Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 ˚C shaking at 130 rpm with 5% CO2 using delong Erlenmeyer flasks either glass or disposable.
      NOTE: It is highly recommended to use a baffled flask with a vented cap for increased agitation and to improve gas transfer during shaking conditions. Significantly reduced antibody yield has been obtained with regular flasks (non-baffled) due to limited agitation of the suspension culture.
    2. Maintain the cell number between 1-5 x 106 cells/mL with >95% cell viability. If the cell number increases over 5 x 106 cells/mL, split the cells. Never allow CHO cells to reach below 0.2 x 106 cells/mL.
  2. Transfection of CHO cells
    1. Grow CHO cells in 200 mL of media (2 x 106 cells/mL) in delong Erlenmeyer baffled flasks.
      NOTE: Suspension cultures grown in baffled flasks have always produced higher protein yields vs when grown in non-baffled flasks.
    2. In a 15 mL tube, take 5 mL of CHO FreeStyle media and add 50 μg of VH clone DNA and 75 μg of VL clone DNA. Vortex to mix well.
    3. Incubate the DNA mixture at room temperature for 5 min.
      NOTE: Longer incubation reduces the protein yields.
    4. Add 750 μL of 1 mg/mL polyethyleneimine (PEI) stock to the DNA solution and aggressively vortex the mixture for 30 s. Incubate at room temperature for an additional 5 min.
      NOTE: PEI must be made fresh. Multiple freeze-thaw cycles of PEI reduce overall yield significantly.
    5. Add the entire mixture of DNA and PEI to the cells while manually shaking the flask. Immediately incubate the delong Erlenmeyer baffled flasks with cells at 37 ˚C shaking at 130 rpm.
  3. Expression
    NOTE: The antibody has a secretory signal peptide engineered to its N-terminal end, which helps antibodies to be secreted out into the media.
    1. Grow the transfected cells at 37 ˚C, with shaking at 130 rpm on Day 1.
    2. On Day 2, add 2 mL of 100x anti-clumping agent and 2 mL of 100x anti-bacterial-anti-mycotic solution. Shift the flask to a lower temperature (32-34 ˚C), with shaking at 130 rpm.
    3. On every fifth day, add 10 mL of Tryptone N1 feed, and 2 mL of 100x glutamine supplement.
    4. Keep counting the cells every third day using a hemocytometer after staining an aliquot of cells with a trypan blue stain. Ensure that the cell viability stays above 80%.
    5. On Day 10 or 11, harvest the medium for antibody purification. Spin the culture at 3000 x g, 4 ˚C for 40-60 min and then filter the clear media using 0.22 µM bottle filters.
  4. Purification
    NOTE: Antibody purification is performed using a commercially available Protein-A column (see Table of Materials), using a peristaltic pump.
    1. Equilibrate the column with the two-column volume of binding buffer (20 mM sodium phosphate at pH 7.4).
    2. Pass the filtered media containing antibody (obtained in step 1.3.5) through the column at the flow rate of 1 mL/min.
    3. Wash the column with the two-column volume of binding buffer.
    4. Elute the antibody into 500 μL fractions using 5 mL elution buffer (30 mM sodium acetate at pH 3.4).
    5. Neutralize the pH of the eluted antibody by adding 10 μL of neutralization buffer (3 M sodium acetate at pH 9) per fraction.
      NOTE: Fraction number 3-6 contains most of the antibody. It is advisable to keep all the fractions in case the antibody is eluted in a later fraction than expected.
    6. Measure the concentration of purified antibodies using a spectrophotometer by selecting the default protocol for immunoglobulin G (IgG). The final concentration of the antibody is obtained in mg/mL by considering the molecular weight and absorption coefficient.

2. Fluorescent labeling

NOTE: Antibodies are labeled with the infrared dye that contains an NHS ester reactive group, which couples to proteins and forms a stable conjugate. This reaction is pH-sensitive and works best at pH 8.5. Fluorescent conjugates labeled with the dye display an absorption maximum of 774 nm, and an emission maximum of 789 nm. pH 8.5 is key for effective conjugation.

  1. Dialyze 0.5 mL of the antibody using a dialysis cassette (0.1-0.5 mL) in 1 L of conjugation buffer (50 mM phosphate buffer at pH 8.5). After 4 h transfer the dialysis cassette to a fresh buffer and dialyze overnight.
  2. Set up a conjugation reaction by adding 0.03 mg of IRDye 800CW per 1 mg of antibody in a reaction volume of 500 μL.
    NOTE: IRDye 800CW is dissolved in DMSO at a concentration of 10 mg/mL.
  3. Carry out labeling reactions for 2 h at 20 °C.
    NOTE: Increasing time beyond 2 hours does not improve the labeling.
  4. Purify labeled conjugates by extensive dialysis against 1x PBS.
  5. Estimate the degree of labeling by measuring the absorbance of the dye at 780 nm and the absorbance of the protein at 280 nm. The dye contribution to the 280 nm signal is 3%.
  6. Calculate the dye/protein ratio using this formula:
    Equation 1
    where 0.03 is a correction factor for the absorbance of the dye used at 280 nm (equal to 3.0 % of its absorbance at 780 nm), εDye and εProtein are molar extinction coefficients for the dye and the protein (antibody) respectively.
    NOTE: εDye is 270,000 M-1 cm-1 and εProtein is 203,000 M-1 cm-1 (for a typical IgG) in a 1:1 mixture of PBS: methanol. Proteins other than IgG may have very different molar extinction coefficients. The use of the correct extinction coefficient for the protein of interest is essential for the accurate determination of the D/P ratio.
  7. Calculate the final protein concentration using this formula:
    Equation 2
    NOTE: Always confirm the antigen-binding efficacy of labeled and unlabeled antibodies using ELISA (Enzyme-Linked Immunosorbent Assay) or flow cytometry before proceeding to in vivo studies.

3. Mouse xenograft studies

  1. Preparation of tumor cells for injection
    1. Grow OVCAR-3 cells in RPMI-1640 Medium, supplemented with 10% of FBS and 1x penicillin-streptomycin.
    2. The day before the injection, subculture cells into new 100 mm culture dishes with 10 mL of complete medium/dish. Use cell number of 0.5-1 x 106 cells/dish.
    3. Incubate cultures at 37 °C temperature, 95% humidity, and 5% CO2 for 20-24 h.
    4. On the day of injection, remove the growth medium from culture dishes. Thoroughly rinse the cell layer with Ca2+/Mg2+ free Dulbecco's phosphate-buffered saline (DPBS) to remove dead cells, cellular debris, and all traces of serum, which may interfere with trypsin action.
    5. Trypsinize the cells by adding 1.0-1.5 mL of Trypsin-EDTA solution to each dish and try to spread the solution by tilting the dish all around followed by incubation at 37 °C for 5-10 min.
      NOTE: Observe cells under an inverted microscope to check the actual trypsinization status. Under trypsinization results in a lower number of cells detaching from the surface of the culture dish, over trypsinization induces cellular stress. So, proper trypsinization is important.
    6. Add 1.0-1.5 mL of complete growth medium to each dish to stop the trypsin action, after that re-suspend cells by pipetting gently.
      NOTE: Gentle pipetting is important to maintain cell health.
    7. Collect the cell suspension into a 15 mL conical tube and spin at 250 x g for 5 min at room temperature.
    8. Collect the cell pellet after removing the supernatant and wash the cells by re-suspending the pellet in 1x DPBS.
    9. Spin the cell suspension at a low speed of 250 x g for 5 min.
    10. Add 500 µL of DPBS and re-suspend cells by gentle pipetting to get single-cell suspension.
    11. Count cells using a hemocytometer or automated cell counter.
      NOTE: For better accuracy, repeat the counting three times and take an average.
    12. Adjust the volume in such a way that the final cell density will be 1 x 108 cells/mL.
  2. Subcutaneous injection of cells to develop mouse xenografts
    NOTE: All procedures should be done in a BSL2 safety cabinet. Athymic Nude Foxn1nu/Foxn1+ mice have been used in the current study.
    1. Take 50 µL of the cell suspension into a 1.5 mL tube and mix with 50 µL of basement membrane matrix medium.
      NOTE: Basement membrane matrix medium tends to form a gel-like state at room temperature, so carefully maintain the cells and the matrix medium mixture on ice. It is advisable to keep tubes, tips, and syringes in the fridge and then transfer them on ice prior to the animal injection.
    2. Agitate the mixture to avoid any cell clumping. Then, take this 100 µL of the cell suspension-matrix medium mixture that contains 5 x 106 cells, into a 1 cc syringe.
    3. Gently lift the skin of the animal to separate the skin from the underlying muscle layer and slowly inject the cell suspension (100 µL) under the skin (5 x 106 cells), with a 26 G needle. Wait for a few seconds before taking the needle out, so that basement matrix medium can form the semi-solid gel-like structure along with cells under the skin, preventing the mixture coming out from the site of injection.
      NOTE: Cells need to be tested prior to injection for any contamination which may harm immunodeficient mice. While injecting do not put the needle too deep into the skin as this may form the tumor deeper than expected.
    4. Keep the animal in a sterile cage and observe for around 20 min.
    5. Observe the mice for 2-3 weeks and allow the tumor to grow up to 500 mm3 in size.

4. Antibody localization using an in vivo imaging system

NOTE: In vivo imaging equipment (see Table of Materials) used in this experiment uses a set of high-efficiency filters and spectral un-mixing algorithms for noninvasive visualization and tracking of cellular and genetic activity within a living organism in real time. The system provides both fluorescence and bioluminescence monitoring capability.

  1. Inject 25 μg of dye-labeled antibody via the tail vein.
    1. Anesthetize tumor-bearing mice using 2% isoflurane. Check for the lack of response to pedal reflexes.
    2. Once mice stop moving, dilate the lateral tail vein by applying worm water.
    3. Inject 25 μg (in 100 μL) of labeled antibody using a 1 cc insulin syringe with a 26 G needle.
    4. Similarly, as a negative control, label and inject the non-specific IgG1 Isotype antibody which does not target cancer cells.
  2. Perform in vivo live imaging after 8, 24, 48 h, etc. of antibody injections.
    1. In the associated software, click Initialize located in the control panel, and confirm that the stage temperature is 37 °C.
    2. Turn on the oxygen supply, all the pumps on the anesthesia system, and the isoflurane gas supply to the anesthetic chamber, and set the isoflurane vaporizer valve to 2%.
    3. Transfer the mice to the anesthetic chamber and wait till the mice are completely anesthetized. Apply the eye lubricating ointment to avoid drying of their eyes.
    4. Go to the Control panel, set up the fluorescence imaging through the Imaging Wizard option, and select the excitation at 773 nm and emission at 792 nm.
      NOTE: The default auto exposure settings provide a good fluorescent image. However, the auto exposure preferences can be modified as per the needs.
    5. Transfer the anesthetized mice into the imaging chamber and assemble them on the imaging field using a nose cone. The imaging stage provides the option to accommodate 5 mice at a time.
      NOTE: Have control mice imaged along with the test mice to have a similar amount of exposure and other settings while analysis.
    6. Once everything is ready, select the Acquire option on the control panel for the image acquisition.
    7. With Autoexposure settings the system generates the image within a minute. The generated image is the overlay of fluorescence on the photographic image with optical fluorescence intensity displayed in units of counts or photons or terms of efficiency.
    8. After acquiring the image, transfer the mice from the imaging chamber back to their cage and observe their recovery for 1 to 2 min.
  3. Fine-tune the image further with the tools and functions provided within the software for image analysis. Image analysis tools are in the menu bar and tool palette.
    1. Under Image Adjust, adjust the brightness, contrast, or opacity, and select the color scale.
    2. Use the ROI Tools to specify a region of interest (ROI) in an optical image and measure the signal intensity within the ROI. If needed, export the quantified signal data to spreadsheet software and plot the data.
    3. For follow-up studies, analyze mice necropsies of various tissues (e.g., liver, lung, heart, kidney, spleen, brain, etc.) side-by-side for a detailed non-specific distribution of fluorescently labeled antibodies.
      NOTE: Data can be further supported with tissue-specific ELISA by making use of antigen (FOLR1 in this case) in 96 well assays.

Divulgations

The authors have nothing to disclose.

Materials

FreeStyle CHO media Gibco Life Technologies Cat # 12651-014
Anti-Anti (100X) Gibco Life Technologies Cat # 15240-062
Anti-Clumping Agent Gibco Life Technologies Cat # 01-0057DG
BD Insulin Syringe BD BioSciences Cat #329420
Caliper IVIS Spectrum PerkinElmer Cat #124262
CHO CD EfficientFeed B Gibco Life Technologies Cat #A10240-01
Corning 500 mL DMEM (Dulbecco's Modified Eagle's Medium) Corning Cat # 10-13-CV
Corning 500 mL RPMI 1640 Corning Cat # 10-040-CV
Cy5 conjugated Anti-Human IgG (H+L) Jackson ImmunoResearch Cat # 709-175-149
GlutaMax-I (100X) Gibco Life Technologies Cat # 35050-061
HiPure Plasmid Maxiprep kit Invitrogen Cat # K21007
HiTrap MabSelect SuRe Column GE Healthcare Cat # 11-0034-93
Infusion Takara BioScience STO344
IRDye 800CW NHS Ester LI-COR Cat # 929-70020
Isoflurane, USP Covetrus Cat # 11695-6777-2
Lubricant Eye Ointment Refresh Lacri-Lube Cat #4089
Matrigel Corning Cat # 354234
PEI transfection reagent Thermo Fisher Cat # BMS1003A
Slide-A-Lyzer Dialysis Cassettes Thermo Scientific Cat # 66333
Steritop Vacuum Filters Millipore Express Cat #S2GPT02RE
Trypsin-EDTA Gibco Life Technologies Cat # 15400-054
Experimental Models: Cell lines
Human: OVCAR-3 American Type Culture Collection ATCC HTB-161
Human: CHO-K cells Stable transformed in our lab ATCC CCL-61
Mouse: 4T1 Kind gift from Dr. Chip Landen, UVA
Mouse: MC38 Kind gift from Dr. Suzanne Ostrand-Rosenberg, UMBC Authenticated by STR profiling
Mouse: MC38 hFOLR1 Generated in our laboratory (This paper)
Experimental Models: Animal
Mice: athymic Nude Foxn1nu/Foxn1+ Envigo Multiple Orders
Mice: NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ Jackson Laboratory Multiple Orders

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Citer Cet Article
A Technique to Assess the In Vivo Localization of Tumor-Specific Antibodies. J. Vis. Exp. (Pending Publication), e21800, doi: (2023).

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