Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1-GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.
© 2022. The Author(s).

We have recently shown that chemotherapy with immunogenic cell death (ICD)-inducing agents can be advantageously combined with fasting regimens or caloric restriction mimetics (CRMs) to achieve superior tumor growth control via a T cell-dependent mechanism. Here, we show that the blockade of the CD11b-dependent extravasation of myeloid cells blocks such a combination effect as well. Based on the characterization of the myeloid and lymphoid immune infiltrates, including the expression pattern of immune checkpoint proteins (and noting a chemotherapy-induced overexpression of programmed death-ligand 1, PD-L1, on both cancer cells and leukocytes, as well as a reduced frequency of exhausted CD8+ T cells positive for programmed cell death 1 protein, PD-1), we then evaluated the possibility to combine ICD inducers, CRMs and targeting of the PD-1/PD-L1 interaction. While fasting or CRMs failed to improve tumor growth control by PD-1 blockade, ICD inducers alone achieved a partial sensitization to treatment with a PD-1-specific antibody. However, definitive cure of most of the tumor-bearing mice was only achieved by a tritherapy combining (i) ICD inducers exemplified by mitoxantrone and oxaliplatin, (ii) CRMs exemplified by hydroxycitrate and spermidine and substitutable for by fasting, and (iii) immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 interaction. Altogether, these results point to the possibility of synergistic interactions among distinct classes of anticancer agents.
© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.

Agonistic CD40 antibodies activate dendritic cells and can expand and activate tumor-specific T cells. Our purpose was to assess the CD40 agonistic antibody ADC-1013 in the clinical setting including intratumoral administration since preclinical studies have indicated that intratumoral is better than intravenous administration. A Phase I, open label, multicenter study was conducted in patients with advanced solid tumors who had received established treatments. A modified 3 + 3 dose-escalation was applied (every other week dosing). Twenty-three patients were treated with ADC-1013 intratumorally (dosing from 22.5 μg/kg up to 400 μg/kg) or intravenously (dosing at 75 μg/kg). The pharmacodynamic effects observed in the patients were further verified in an hCD40tg mouse model. Adverse events were mostly Common Terminology Criteria for Adverse Events (CTCAE) Grades 1 or 2 and transient. The serum concentration ADC-1013 and cytokine release (MCP-1, TNFα and IL-6) were more pronounced in patients receiving injections in deep metastases compared to patients receiving injections in superficial metastases. Treatment with ADC-1013 resulted in a marked decrease in B cell levels in peripheral blood after 24 h while remaining B cells significantly increased their expression of the cell surface activation marker CD86. Activation of antigen-presenting cells and subsequent activation of T cells were demonstrated in hCD40tg mice. Moreover, ADC-1013 treatment in this mouse model acted synergistically with a PD-1 inhibitor. The results from the first-in-human study of ADC-1013 indicate that intratumoral administration of ADC-1013 into superficial lesions is well tolerated at clinically relevant doses and associated with pharmacodynamic responses.
© 2019 UICC.

Regulatory T cells (Tregs) play a crucial role in controlling autoimmune and inflammatory responses. Recent studies have demonstrated that dendritic cells (DCs) contribute to the homeostasis of peripheral Tregs. Autophagy, a critical pathway for cellular homeostasis, is active in DCs and is upregulated in different inflammatory conditions. We have shown that Tregs are expanded and have phenotypic alterations and impaired suppressive functions in mice with autophagy-deficient DCs. RNA profiling of Tregs revealed that autophagy in DCs is required to stabilize Treg expression signatures. This phenotype is linked to the downregulation of ICOS-Ligand expression in autophagy-deficient DCs, a consequence of the accumulation of ADAM10, the metalloproteinase responsible for its cleavage. Upon inflammation, in antigen-induced arthritis, mice with autophagy-deficient DCs exhibit increased synovial inflammation and cartilage and bone erosion correlating with Treg-to-Th17 conversion. Our data reveal a mechanism that couples autophagy deficiency in DCs to the function, homeostasis, and stability of Tregs.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

Immunogenic cell death (ICD) converts dying cancer cells into a therapeutic vaccine and stimulates antitumor immune responses. Here we unravel the results of an unbiased screen identifying high-dose (10 µM) crizotinib as an ICD-inducing tyrosine kinase inhibitor that has exceptional antineoplastic activity when combined with non-ICD inducing chemotherapeutics like cisplatin. The combination of cisplatin and high-dose crizotinib induces ICD in non-small cell lung carcinoma (NSCLC) cells and effectively controls the growth of distinct (transplantable, carcinogen- or oncogene induced) orthotopic NSCLC models. These anticancer effects are linked to increased T lymphocyte infiltration and are abolished by T cell depletion or interferon-γ neutralization. Crizotinib plus cisplatin leads to an increase in the expression of PD-1 and PD-L1 in tumors, coupled to a strong sensitization of NSCLC to immunotherapy with PD-1 antibodies. Hence, a sequential combination treatment consisting in conventional chemotherapy together with crizotinib, followed by immune checkpoint blockade may be active against NSCLC.