Blockade of immune checkpoints in lymph nodes through locoregional delivery augments cancer immunotherapy

Systemic administration of immune checkpoint blockade (ICB) monoclonal antibodies (mAbs) can unleash antitumor functions of T cells but is associated with variable response rates and off-target toxicities. We hypothesized that antitumor efficacy of ICB is limited by the minimal accumulation of mAb within tissues where antitumor immunity is elicited and regulated, which include the tumor microenvironment (TME) and secondary lymphoid tissues. In contrast to systemic administration, intratumoral and intradermal routes of administration resulted in higher mAb accumulation within both the TME and its draining lymph nodes (LNs) or LNs alone, respectively. The use of either locoregional administration route resulted in pronounced T cell responses from the ICB therapy, which developed in the secondary lymphoid tissues and TME of treated mice. Targeted delivery of mAb to tumor-draining lymph nodes (TdLNs) alone was associated with enhanced antitumor immunity and improved therapeutic effects compared to conventional systemic ICB therapy, and these effects were sustained at reduced mAb doses and comparable to those achieved by intratumoral administration. These data suggest that locoregional routes of administration of ICB mAb can augment ICB therapy by improving immunomodulation within TdLNs.


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Materials and Methods Fig. S1. Changes in CD4 T cell compartment resulting from ICB. Fig. S2. Therapeutic and staining aPD-1 mAbs simultaneously stain PD-1-expressing T cells. Fig. S3. mAb fluorescent labeling, accumulation within dLNs, and binding to LN-resident T cells after administration. Fig. S4. ICB therapy is less effective in larger tumors. Fig. S5. T regs in TME express CTLA-4 at higher frequencies than helper CD4 and CD8 T cells. Fig. S6. ICB therapy modulates CD8 T cells in various tissues leading to effector cell phenotypes. Fig. S7. ICB with vaccination promotes CD4h activation in TdLNs. Fig. S8. ICB directed to TdLNs alone or in combination with the TME enables dose sparing. Fig. S9. Effective ICB therapy in breast cancers requires aCTLA-4. Fig. S10. mAb accumulation in kidneys and lungs proportional to administered dose.

Other Supplementary Material for this manuscript includes the following:
(available at stm.sciencemag.org/cgi/content/full/12/563/eaay3575/DC1) Data file S1 (Microsoft Excel format). Individual data points for merged or averaged data plots.

Analysis of non-fluorescent aCTLA-4 accumulation in dLNs
Twenty four hours after i.p. or i.d. administration of 150 g of aCTLA-4, mice were euthanized and dLNs (axial and brachial) harvested. LNs were placed in optimal cutting temperature (FisherScientific) compound and frozen in 2-methylbutane (SigmaAldrich) solutions using liquid nitrogen. Frozen LNs were sliced using a CryoStar NX70 instrument to 8-10 micrometers.
LN sections were serum blocked followed by staining using an AlexaFluor546 conjugated antihamster secondary antibody (Abcam). LNs sections were then imaged on a Laser Scanning Confocal microscope (Zeiss 700) and processed using Zeiss ZEN Black 2.3 SP1 software.

Tissue and single cell preparations
After collection, tissues were processed by cutting up to circumvent the possibility of cell enzymatic surface receptor degradation. Single cell suspensions were generated by disrupting tissues through a 70-m cell strainer (FisherScientific) using a 1 mL syringe plunger followed by two washes in 20 mL of 1X PBS prior to centrifugation and decanting. Red blood cells were lysed with lysing buffer hybrid-max (Sigma) for 7 min at room temperature followed by quenching in PBS. For ex vivo T cell restimulation in Iscove's modified Dulbecco's medium (ThermoFisher) supplemented with 10% FBS (VWR) and 1% penicillin/streptomycin (VWR) (complete medium) or surface/intracellular staining in PBS, either 30% or 70% of total LN cells, respectively, 2 x 10 6 splenocytes, or 5 x 10 6 tumor cells were plated in 96-well U bottom plates (FisherScientific).

Ex vivo T cell stimulation
Complete medium was spiked with either ovalbumin (10 g/mL, SigmaAldrich) or SIINFEKL (1 g/mL, SigmaAldrich) and lymphocytes or splenocytes were cultured for 3 hours at 37C followed by the addition of brefeldin A (1X: BioLegend) for 3 hours. Cells were harvested, stained, and analyzed by flow cytometry as described above.