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A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cellsurface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4+ T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patientderived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations. [ABSTRACT FROM AUTHOR]

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research. [ABSTRACT FROM AUTHOR]

Twenty-five years ago, we reported that agonist anti-CD137 monoclonal antibodies eradicated transplanted mouse tumors because of enhanced CD8+ T-cell antitumor immunity. Mouse models indicated that anti-CD137 agonist antibodies synergized with various other therapies. In the clinic, the agonist antibody urelumab showed evidence for single-agent activity against melanoma and non-Hodgkin lymphoma but caused severe liver inflammation in a fraction of the patients. CD137's signaling domain is included in approved chimeric antigen receptors conferring persistence and efficacy. A new wave of CD137 agonists targeting tumors, mainly based on bispecific constructs, are in early-phase trials and are showing promising safety and clinical activity., Significance: CD137 (4-1BB) is a costimulatory receptor of T and natural killer lymphocytes whose activity can be exploited in cancer immunotherapy strategies as discovered 25 years ago. Following initial attempts that met unacceptable toxicity, new waves of constructs acting agonistically on CD137 are being developed in patients, offering signs of clinical and pharmacodynamic activity with tolerable safety profiles., (©2022 American Association for Cancer Research.)

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy., (©2022 American Association for Cancer Research.)

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated., Significance: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007., (©2022 American Association for Cancer Research.)

Specific mechanisms by which tumor-infiltrating lymphocytes (TIL) become dysfunctional remain poorly understood. Here, we employed a two-pronged approach using single-cell mass cytometry and tissue imaging technologies to dissect TILs from 25 patients with resectable and 35 patients with advanced non-small cell lung cancer (NSCLC). We identified a burned-out CD8 + TIL subset (Ebo) that specifically accumulated within the tumor microenvironment (TME) but not in adjacent nontumoral tissues. Ebo showed the highest expression of proliferation and activation markers but produced the lowest amount of IFNγ and were the most apoptotic CD8 + TIL subset. Using a humanized patient-derived tumor xenograft model, we demonstrated that Ebo expansion occurred within the TME in a PD-1/B7-H1 pathway-dependent manner. Ebo abundance in baseline tumor tissues was associated with resistance to anti-PD therapy in patients with NSCLC. Our study identifies a dysfunctional TIL subset, with distinct features from previously described exhausted T cells, and implies strategies to overcome immunotherapy resistance. SIGNIFICANCE: We identified a highly proliferative, overactivated, and apoptotic dysfunctional CD8 + tumor-infiltrating subpopulation that is functionally distinct from previously described exhausted T cells. This population is expanded in lung cancer tissues in a PD-1/B7-H1-dependent manner, and its abundance is associated with resistance to cancer immunotherapy, thus becoming a potential tissue biomarker. This article is highlighted in the In This Issue feature, p. 1601 ., (©2021 American Association for Cancer Research.)

Checkpoint inhibitors are being added to standard-of-care chemotherapy in multiple clinical trials. Success has been reported in non-small and small cell lung carcinomas and urothelial, head and neck, gastric, and esophageal cancers, and promising results are already available in triple-negative breast and pancreatic malignancies. The potential mechanisms of synergy include immunogenic tumor cell death, antiangiogenesis, selective depletion of myeloid immunosuppressive cells, and lymphopenia, which reduces regulatory T cells and makes room for proliferation of effector T cells. However, chemotherapy regimens have not been optimized for such combinations, perhaps explaining some recent clinical trial disappointments. Approaches to make the most of chemoimmunotherapy include neoadjuvant and adjuvant schemes. Significance: Immunotherapy of cancer based on PD-1/PD-L1 blockade has prompted a revolution in cancer clinical management. Evidence in phase III clinical trials already supports combinations of immunotherapy with standard-of-care chemotherapy for a number of malignant diseases. This review focuses on such evidence and provides an overview of the potential synergistic mechanisms of action and the opportunities to optimize chemoimmunotherapy regimens., (©2021 American Association for Cancer Research.)

Mechanisms of acquired resistance to immune checkpoint inhibitors (ICI) are poorly understood. We leveraged a collection of 14 ICI-resistant lung cancer samples to investigate whether alterations in genes encoding HLA Class I antigen processing and presentation machinery (APM) components or interferon signaling play a role in acquired resistance to PD-1 or PD-L1 antagonistic antibodies. Recurrent mutations or copy-number changes were not detected in our cohort. In one case, we found acquired homozygous loss of B2M that caused lack of cell-surface HLA Class I expression in the tumor and a matched patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. CRISPR-mediated knockout of B2m in an immunocompetent lung cancer mouse model conferred resistance to PD-1 blockade in vivo , proving its role in resistance to ICIs. These results indicate that HLA Class I APM disruption can mediate escape from ICIs in lung cancer. Significance: As programmed death 1 axis inhibitors are becoming more established in standard treatment algorithms for diverse malignancies, acquired resistance to these therapies is increasingly being encountered. Here, we found that defective antigen processing and presentation can serve as a mechanism of such resistance in lung cancer. Cancer Discov; 7(12); 1420-35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1355 ., (©2017 American Association for Cancer Research.)

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3-dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8 + T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFNγ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8 + TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation. Cancer Res; 76(20); 5994-6005. ©2016 AACR., (©2016 American Association for Cancer Research.)

A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations., (©2015 American Association for Cancer Research.)

Unlabelled: The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by positron emission tomographic (PET) imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) of transplanted colon carcinomas, melanomas, and spontaneous breast adenocarcinomas are CD137 (4-1BB)-positive, as opposed to their counterparts in tumor-draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Importantly, hypoxia does not upregulate CD137 in hypoxia-inducible factor (HIF)-1α-knockout T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating and co-transferred HIF-1α-sufficient T lymphocytes. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies to the tumor tissue. As a result, low-dose intratumoral injections avoid liver inflammation, achieve antitumor systemic effects, and permit synergistic therapeutic effects with PD-L1/B7-H1 blockade., Significance: CD137 (4-1BB) is an important molecular target to augment antitumor immunity. Hypoxia in the tumor microenvironment as sensed by the HIF-1α system increases expression of CD137 on tumor-infiltrating lymphocytes that thereby become selectively responsive to the immunotherapeutic effects of anti-CD137 agonist monoclonal antibodies as those used in ongoing clinical trials.