Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Summary

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Abstract

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Introduction

FDA approved the first monoclonal antibody targeting CD3 (OKT3, Muromonab) in 1986^1,2. Since then for the next twenty years, there has been a rapid explosion in the field of antibody engineering due to the overwhelming success of antibodies against immune checkpoint inhibitors³. Beside indirect activation of immune system, antibodies are being aimed to directly flag cancer cells to precisely engage immune effector cells, trigger cytotoxicity via death receptor agonist, block tumor cell survival signaling, obstruct angiogenesis (growth of blood vessels), constrain immune checkpoint regulators, deliver radioisotopes, chemotherapy drugs and siRNA as a conjugated agents². In addition, studying the single chain variable fragments (scFv) of various antibodies on the surface of patient derived T-cells and NK cells (CAR-T and CAR-NK) is a fast growing area of clinical investigations for cell-based therapies⁴.

The ultra-high affinity of antibody-based drugs that provides selectivity to antigen expressing tumor cells makes it an attractive agent. Likewise, the targeted delivery and tumor retention of a therapeutic antibody (or a chemical drug) is the key to balance efficacy over toxicity. Therefore, a large number of protein engineering based strategies that include but are not limited to bispecific⁵ and tri-specific antibodies⁶ are being exploited to significantly enhance avidity optimized tumor retention of intravenously (IV) injected therapeutics^5,7. Here, we describe a simple fluorescence-based method to address the tumor and tissue distribution of potentially effective anti-cancer antibodies.

Because animal tissues possess auto-fluorescence when excited in visible spectrum, the antibodies were initially labeled with near Infrared dye (e.g., IRDye 800CW). For proofs of concept investigations, we have made use of folate receptor alpha-1 (FOLR1) targeting antibody called farletuzumab and its derivative called Bispecific anchor Cytotoxicity activator (BaCa)⁷ antibody that co-targets FOLR1 and death receptor-5 (DR5)⁸ in one recombinant antibody. FOLR1 is a well-defined overexpressed target receptor in ovarian and TNBC cancer cells, tumor xenografts and patient tumors⁹. Notably, there are multiple efforts to clinically exploit FOLR1 using antibody-based approaches to engage immune effector cells and antibody drug conjugates (ADC) for ovarian and breast cancers^10,11.

In this methods paper, we cloned, expressed and purified clinical anti-FOLR1 (farletuzumab) along with other control antibodies using CHO expression system. IgG1 isotype and a clinical anti-idiotype mucin-16 antibody called abagovomab¹² were used as negative controls. Following protein-A purification, indicated antibodies were labeled with IRDye 800CW and were administered into the tail vein of nude mice either bearing ovarian tumor xenografts or stably transfected human FOLR1 expressing murine colon cancer xenografts. The antibody localization was tracked by live imaging using in vivo imaging spectrum at multiple different time points⁷. This method does not require any genetic modification or injection of the substrate to enable light emission and is significantly quicker, cost effective and efficient. The general cloning, expression, purification and labeling protocol described below can be applied to any clinical and nonclinical antibody if heavy and light chain sequences are available.

Protocol

All the procedures involving animals handling and tumor xenografts studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) here at the University of Virginia and conform to the relevant regulatory standards

1. Expression and purification of antibodies

1. Maintenance of 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 vented cap for increased agitation and to improve gas transfer during shaking condition. 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 allows 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 additional 5 min.
      NOTE: PEI must be made fresh. Multiple freeze thaw cycle of PEI reduces overall yield significantly.
   5. Add the entire mixture of DNA and PEI on 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 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 the Day 1.
   2. On the Day 2, add 2 mL of 100x anti-clumping agent and 2 mL of 100x anti-bacterial-anti-mycotic solution. Shift the flask to 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 hemocytometer after staining an aliquot of cells with trypan blue stain. Ensure that the cell viability stay above 80%.
   5. On the 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 commercially available Protein-A column (see Table of Materials), using a peristaltic pump.
   1. Equilibrate the column with 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 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 antibody using a spectrophotometer by selecting the default protocol for 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 form 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 fresh buffer and dialyze overnight.
2. Setup 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 h 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 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:
   
   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), ε_Dye and ε_Protein are molar extinction coefficients for the dye and the protein (antibody) respectively.
   NOTE: ε_Dye is 270,000 M^-1 cm^-1 and ε_Protein is 203,000 M^-1 cm^-1 (for a typical IgG) in 1:1 mixture of PBS: methanol. Proteins other than IgG may have very different molar extinction coefficients. Use of correct extinction coefficient for the protein of interest is essential for accurate determination of D/P ratio.
7. Calculate the final protein concentration using this formula:
   
   NOTE: Always confirm the antigen-binding efficacy of labeled and unlabeled antibody 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 in 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 lower number of cell detachment from the surface of 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 low speed 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 hemocytometer or automated cell counter.
       NOTE: For better accuracy, repeat the counting for three times and take an average.
   12. Adjust the volume in such a way so 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 Foxn1^nu/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 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 needs to be tested prior to injection for any contamination which may harm to 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 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. System provides both fluorescence and bioluminescence monitoring capability.

1. Inject 25 μg of dye labeled antibody via 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 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, isoflurane gas supply to anesthetic chamber and set the isoflurane vaporizer valve to 2%.
   3. Transfer the mice to anesthetic chamber and wait till 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 it on the imaging field using 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 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 photographic image with optical fluorescence intensity displayed in units of counts or photons, or in terms of efficiency.
   8. After acquiring the image, transfer the mice from the imaging chamber back to their cage and observe for 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 a 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 antibody.
      NOTE: Data can be further supported with tissue specific ELISA by making use of antigen (FOLR1 in this case) in 96 well assays.

Results

In the described methodology, first we cloned antibodies targeting folate receptor alpha-1 (FOLR1) named farletuzumab, and a bispecific antibody called BaCa consisting of farletuzumab and lexatumumab along with control antibodies such as abagovomab (sequences provided in Supplementary File 1). Details of representative variable heavy (VH) and variable light (VL) domains in DNA clones (pVH, pVL) are shown in Figure 1A. To confirm the positive clones, we carried out colony PCR...

Discussion

Selective and tumor tissue specific delivery of anti-cancer therapeutic agent is the key to measure efficacy and safety of a given targeted therapy¹³. Here we have described a quick and efficient approach to investigate the detailed tissue and tumor distribution of clinical, farletuzumab and a nonclinical BaCa antibody. The described approach is applicable to any newly generated antibody and can be used alongside of a clinically effective antibody (with desired qualities) for its tumor/organ distr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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