3, ?,GG and ?andH).H). 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 RO-1138452 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. INTRODUCTION Immune checkpoint blockade (ICB) using monoclonal antibodies (mAbs) specific to cytotoxic T lymphocyte antigen 4 (CTLA-4) and to programmed cell death 1 (PD-1) or its ligands has emerged as one of the most promising approaches in cancer immunotherapy to invigorate antitumor immunity (1, 2). CTLA-4 is a transmembrane receptor found constitutively on regulatory T cells (Tregs) and is limited in its expression by CD4 and CD8 T cells immediately after engagement of the T cell receptor. CTLA-4 directly competes with CD28 for B7 ligand binding on antigen-presenting cells (APCs), consequently leading to T cell anergy (3). Similarly, surface expression of PD-1 is broadly induced after T cell activation, and PD-1 is thought to RO-1138452 function in peripheral tissues through its binding interactions with PD-1 ligands (PD-L1 and PD-L2) found on many cell subtypes including predominantly, but not limited to, tumor cells and APCs, respectively. After PD-1:ligand engagement, T cell function is dampenedan effect that protects the host during viral infection from immune-mediated tissue destruction leading to T cell exhaustion (3). By blocking these inhibitory pathways using function-blocking mAbs, activation and cytotoxic capabilities of T cells can be restored (1, 3). Although the canonical view on ICB therapy effects is that they are mediated primarily within the tumor microenvironment (TME) by restoring antitumor functions of infiltrating RO-1138452 T cells, evidence of the pleotropic effects of ICB mAbs continues to amass. Specific isotypes of anti-CTLA-4 (aCTLA-4) mAbs [immunoglobulin G2a (IgG2a)] mediate the RO-1138452 depletion of tumor-resident Tregs (trTregs) via antibody-dependent cellular cytotoxicity, although other isotypes (IgG1) do not (4C6). In addition, whereas antiCPD-1 (aPD-1) mAb c-Raf has been shown to restore the effector functions of CD8 and CD4 T cells (7), CD28 stimulation is required for aPD-1 efficacy, suggesting a role of B7-expressing APCs (8). aPD-1 has also been shown to modulate a stem-like CD8 T cell population capable of proliferating and giving rise to T cells of a tumor-killing effector-like phenotype (9C11). Furthermore, PD-L1 expression on tumor cells is not required for disease progression and aPD-1 efficacy in certain cancer types (12C14). Both aCTLA-4 and aPD-1 therapy have also been shown to broaden the repertoire of tumor-specific CD8 T cell clones (15C17), which is associated with improved clinical outcomes (18, 19). Solely blocking checkpoint pathways in the TME may thus not be sufficient to generate high response rates after ICB therapy. To this end, appreciation for lymphoid tissues as critical in the generation of effective immunotherapy responses is increasing.