Graphical Abstract Highlights d Anti-4-1BB IgG2a depletes intratumoral Treg cells; IgG1 promotes CD8 T cell function d The efficacy of anti-4-1BB mIgG1 and anti-4-1BB mIgG2a depends on different FcgRs d Optimal tumor therapy requires sequential anti-4-1BB IgG2a and IgG1 or PD-1 blockade d Hinge-engineered anti-4-1BB mIgG2a/h2B mAb harnesses both mechanisms of action

Growing evidence points to the potential of agonistic anti-CD40 mAbs as adjuvants for vaccination against cancer. These appear to act by maturing dendritic cells (DCs) and allowing them to prime CD8 cytotoxic T lymphocytes (CTLs). Although it is well established that optimal T-cell priming requires costimulation via B7:CD28, recent studies emphasize the contribution of TNF receptors to this process. To understand how anti-CD40 mAbs trigger effective antitumor immunity, we investigated the role of TNFR superfamily members CD27 and 4-1BB in the generation of this immunity and showed that, although partially dependent on 4-1BB:4-1BBL engagement, it is completely reliant on CD27:CD70 interactions. Importantly, blocking CD70, and to some extent 4-1BBL, during anti-CD40 treatment prevented accumulation of tumor-reactive T cells and subsequent tumor protection. However, it did not influence changes in DC number, phenotype, nor the activity of CTLs once immunity was established. We conclude that CD27: IntroductionInteractions between members of the TNF receptor (TNFR) superfamily and their ligands play an important role in providing costimulation at several stages during the development of an effective antigen-specific CD8 T-cell response. [1][2][3] Early in the response, the ligation of CD40 on dendritic cells (DCs) by its ligand, CD154, induces the maturation of DCs and potentiates their ability to stimulate antigen-specific naive CD8 T cells. [4][5][6] Conversely, the absence of DC maturation, for example, during presentation of self-or tumor-associated antigens, leads to the induction of T-cell tolerance. 7 Thus, antigen presentation by immature DCs maintains peripheral tolerance to self-tissues as well as tumors. Agonistic anti-CD40 mAb, which is a potent mimic of the natural ligand, CD154, has been shown to promote T-cell-mediated immunity in a number of settings, including vaccination, and treatment of tumors. [8][9][10][11] The success achieved with agonistic anti-CD40 mAbs in preclinical models has recently led to clinical evaluation of anti-human CD40 mAbs as a potential treatment for cancer. 12,13 It is assumed that anti-CD40 mAbs trigger the maturation, or licensing of DCs which subsequently leads to the priming of tumor-specific CD8 T cells. Identifying the critical changes in DCs during their CD40-triggered maturation is therefore key to understanding the mechanism of action of anti-CD40 mAbs. CD40-induced maturation of DCs is characterized by an increase in their expression of adhesion and costimulatory molecules, including ICAM-1, B7.1, B7.2, CD70, and 4-1BB ligand (4-1BBL) as well as cytokines. [14][15][16][17][18][19] Although initial antigen-specific cytotoxic T lymphocyte (CTL) activation and proliferation depends on the CD28:B7 engagement, 20,21 subsequent expansion and survival of effector and memory T cells are controlled by additional costimulatory interactions and cytokines. Two receptors that appear central in maintaining CD8 T-cell responses are the TNFR superfamily members 4-1BB (CD137) 3,22...

10013 Background: We tested dose-reduced scIL2 in combination with DB-LTI and oral isotretinoin and evaluated toxicity and efficacy in high-risk neuroblastoma patients (EudraCT:2006-001489-17). Methods: High-risk patients (stage 4 ≥1y; stage 4 < 1y with MYCN amplification (MNA); stage 2, 3, 0-21y with MNA) received high intensity induction (rapid COJEC or N5-MSKC and TVD for insufficient response), surgery, high dose therapy with busulfan/melphalan and local radiotherapy. Patients ≤9 months between diagnosis and HDT/SCT who achieved at least a partial response prior to HDT/SCT and without progression thereafter were randomized to receive up to 5 cycles of 100mg/m2 DB-LTI (d8-17) ± 3x106 IU/m2 scIL2 (d1-5; d8, d10, d12, d14, d16) and 160mg/m2 oral isotretinoin (d19-32). Results: Between 04/2014 and 06/2018, 408 patients from 18 countries were randomized. Median follow-up is 1.8 years. Stage, age, MNA, induction treatments and remission status were well balanced between randomization arms. The 2yrs-EFS and -OS for DB-LTI (205 pts) vs. DB-LTI&scIL2 (203 pts) was 64%±4%vs63%±5% (p = 0.844) and 83%±3%vs74%±4% (p = 0.337). For patients in CR the 2yrs-EFS was 69%±5% for DB and 66%±6% for DB&scIL2. Patients with evaluable disease prior DB or DB&scIL2, the end of treatment response rate was 57% (26% CR, 31% PR) vs 52% (27% CR, 25% PR) with 2yrs-EFS rates of 58%±7% and 64% ±8%, respectively. Grade 3&4 toxicity was lower in the group with DB vs DB&scIL2 for fever (14%vs31%, p < 0.001) and pain (7%vs18%, p = 0.005), and no significant difference was seen for general condition (17%vs22%,ns), allergy (3%vs3%,ns), capillary leak (4%vs8%,ns), liver enzyme elevation (20%vs27%, ns) and neurological toxicities (2%vs2%,ns). Conclusions: We previously reported grade 3&4 toxicity to DB short-term infusion (STI) ± 10x6x106IU/m2 scIL2 for general condition (16%vs41%, p = 0.000), fever (14%vs40%, p = 0.000), allergic reaction (10%vs20%, p = p = 0.006), capillary leak (4%vs15%, p = 0.004), liver enzyme elevation (17%vs23%, ns), central neurotoxicity (3%vs8%, p = 0.034) and pain (16%vs26%, p = 0.048). Our results indicate that DB-LTI and dose-reduced scIL2 clearly reduced the toxicity profile, but showed absence of benefits of scIL2. DB-LTI achieved 2yrs-EFS in line with DB-STI (Ladenstein, Lancet Oncology 2018; Yu, NEJM, 2010) and a response rate > 50% supporting its use as standard of care IT. Clinical trial information: EudraCT:2006-001489-17.