In vivo mAb biodistribution studies

mAb fluorescent labeling

Fluorescent labeling of aCTLA-4 (9H10), aPD-1 (RMP1-14), or aCD3 (KT3) mAb was performed using AlexaFluor647-NHS-Ester (ThermoFisher) for 1 hour, and labeled mAb was purified using a Sepharose CL-6B (Cytiva) column. Labeled mAb fractions were pooled and concentrated using a 10 kDa (Millipore) spin filter. 

• AlexaFluor647-NHS-Ester was typically added at 6-molar excess to mAb in buffer (pH ~7.4). Other NHS ester dyes may be used as well.
• After adding dye-labeled mAb to a CL-6B column, add several mL of PBS as ‘dead volume’ and allow to run down the column. Stop the column, add PBS to the top of the column (~20-30mL so that the column does not run dry), start the fraction collector, and re-start the column to begin collecting fractions. Collect 10-15 drops per tube so that dye-labeled mAb may be fully separated from unconjugated dye. 
  • (We use 4mL dead volume, 10-15 drops/tube, and collect ~45-60 tubes, but this will vary based on the column height and volume. Labeled mAb and free dye may be visualized as two bands that separate down the length of the column, make sure to begin collecting fractions before the first band reaches the bottom of the column). 
• In a 96-well plate, add a small sample from each fraction and measure fluorescence intensity using a plate reader (BioTek Synergy H4) and plot as in Fig. S3a to determine mAb-containing fractions.

mAb concentrations was determined using a BCA assay (ThermoFisher).

• Use manufacturer’s instructions for the BCA assay kit.

In vivo mAb biodistribution studies 

On day 5 after tumor implantation, mice were administered aCTLA-4 or aPD-1 mAb.

• Make sure to reserve excess injected mAb for making standard curves to determine mAb tissue concentration.

Fluorescent imaging was performed with an IVIS Spectrum instrument (PerkinElmer) at the injection site over 24 hours. Twenty-four hours after mAb injection, mice were euthanized and tissues were collected for imaging and homogenization. 

• Image live mice or dissected tissues with IVIS Spectrum at the appropriate excitation/emission and camera settings.
• For homogenization: First, add 300uL of PBS to homogenization tubes (“1.4 mm Acid Washed Zirconium Beads, Pre-Filled Tubes”; OPS diagnostics) and weigh each tube. This can be done in advance of animal dissection. After adding each dissected tissue to a homogenization tube, re-weigh to obtain organ weight. 
• Homogenize tissues (“FastPrep-24™ Classic bead beating grinder and lysis system”; MP Biomedicals). Pipet tissue homogenate samples into wells of a 96-well plate and read fluorescence signal on a plate reader (BioTek Synergy H4). 

Concentrations of mAb in homogenized tissues were determined using a standard curve of injected mAb solution in naïve tissue homogenates. Tissue background was subtracted from all measurements.

• Add the reserved fluorescent-labeled mAb injectate to naïve tissue homogenate in the desired concentration range. Separate standard curves were made for each tissue type (i.e., a separate standard curve was used for each of the LN, spleen, tumor, etc.) and applied to the relevant sample.

For biodistribution experiments using aCD3 mAb, naïve mice were injected with 6.25 mg of mAb and euthanized after 1, 5, or 24 hours. LNs were either collagenase-treated for 30 min followed by tissue disruption to form single-cell suspensions (a process described in Supplementary Materials and Methods) or immediately cut up and dispersed into a single-cell suspension to prevent ex vivo T cell labeling. 

• Samples were run on a flow cytometer to assess mAb labeling of dLN cells.