M. Angela Aznar

Evolving synergistic combinations of targeted immunotherapies to combat cancer

Immunotherapy has now been clinically validated as an effective treatment for many cancers. There is tremendous potential for synergistic combinations of immunotherapy agents and for combining immunotherapy agents with conventional cancer treatments. Clinical trials combining blockade of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) may serve as a paradigm to guide future approaches to immuno-oncology combination therapy. In this Review, we discuss progress in the synergistic design of immune-targeting combination therapies and highlight the challenges involved in tailoring such strategies to provide maximal benefit to patients.

Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1+Lin− hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.

Abscopal Effects of Radiotherapy Are Enhanced by Combined Immunostimulatory mAbs and Are Dependent on CD8 T Cells and Crosspriming

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.

Deciphering CD137 (4‐1BB) signaling in T‐cell costimulation for translation into successful cancer immunotherapy

CD137 (4‐1BB, TNF‐receptor superfamily 9) is a surface glycoprotein of the TNFR family which can be induced on a variety of leukocyte subsets. On T and NK cells, CD137 is expressed following activation and, if ligated by its natural ligand (CD137L), conveys polyubiquitination‐mediated signals via TNF receptor associated factor 2 that inhibit apoptosis, while enhancing proliferation and effector functions. CD137 thus behaves as a bona fide inducible costimulatory molecule. These functional properties of CD137 can be exploited in cancer immunotherapy by systemic administration of agonist monoclonal antibodies, which increase anticancer CTLs and enhance NK‐cell‐mediated antibody‐dependent cell‐mediated cytotoxicity. Reportedly, anti‐CD137 mAb and adoptive T‐cell therapy strongly synergize, since (i) CD137 expression can be used to select the T cells endowed with the best activities against the tumor, (ii) costimulation of the lymphocyte cultures to be used in adoptive T‐cell therapy can be done with CD137 agonist antibodies or CD137L, and (iii) synergistic effects upon coadministration of T cells and antibodies are readily observed in mouse models. Furthermore, the signaling cytoplasmic tail of CD137 is a key component of anti‐CD19 chimeric antigen receptors that are used to redirect T cells against leukemia and lymphoma in the clinic. Ongoing phase II clinical trials with agonist antibodies and the presence of CD137 sequence in these successful chimeric antigen receptors highlight the importance of CD137 in oncoimmunology.

Virotherapy with a Semliki Forest Virus–Based Vector Encoding IL12 Synergizes with PD-1/PD-L1 Blockade

Quetglas and colleagues report that intratumoral injection of cytolytic nonreplicative Semliki Forest virus vector expressing IL12, along with systemic administration of anti-PD-1/PD-L1 antibodies, induced regression of both virally injected and distal tumors and synergistically prolonged survival in mouse tumor models. Virotherapy and checkpoint inhibitors can be combined for the treatment of cancer with complementarity and potential for synergistic effects. We have developed a cytolytic but nonreplicative viral vector system based on Semliki Forest virus that encodes IL12 (SFV-IL12). Following direct intratumoral injection, infected cells release transgenic IL12, die, and elicit an inflammatory response triggered by both abundantly copied viral RNA and IL12. In difficult-to-treat mouse cancer models, such as those derived from MC38 and bilateral B16-OVA, SFV-IL12 synergized with an anti–PD-1 monoclonal antibody (mAb) to induce tumor regression and prolong survival. Similar synergistic effects were attained upon PD-L1 blockade. Combined SFV-IL12 + anti–PD-1 mAb treatment only marginally increased the elicited cytotoxic T-lymphocyte response over SFV-IL12 as a single agent, at least when measured by in vivo killing assays. In contrast, we observed that SFV-IL12 treatment induced expression of PD-L1 on tumor cells in an IFNγ-dependent fashion. PD-L1–mediated adaptive resistance thereby provides a mechanistic explanation of the observed synergistic effects achieved by the SFV-IL12 + anti–PD-1 mAb combination. Cancer Immunol Res; 3(5); 449–54. ©2015 AACR.

Intratumoral Delivery of Immunotherapy—Act Locally, Think Globally

Immune mechanisms have evolved to cope with local entry of microbes acting in a confined fashion but eventually inducing systemic immune memory. Indeed, in situ delivery of a number of agents into tumors can mimic in the malignant tissue the phenomena that control intracellular infection leading to the killing of infected cells. Vascular endothelium activation and lymphocyte attraction, together with dendritic cell–mediated cross-priming, are the key elements. Intratumoral therapy with pathogen-associated molecular patterns or recombinant viruses is being tested in the clinic. Cell therapies can be also delivered intratumorally, including infusion of autologous dendritic cells and even tumor-reactive T lymphocytes. Intralesional virotherapy with an HSV vector expressing GM-CSF has been recently approved by the Food and Drug Administration for the treatment of unresectable melanoma. Immunomodulatory monoclonal Abs have also been successfully applied intratumorally in animal models. Local delivery means less systemic toxicity while focusing the immune response on the malignancy and the affected draining lymph nodes.

Successful Immunotherapy against a Transplantable Mouse Squamous Lung Carcinoma with Anti–PD-1 and Anti-CD137 Monoclonal Antibodies

Introduction: Anti–programmed cell death 1 (anti–PD‐1) and anti–programmed cell death ligand 1 (PD‐L1) antagonist monoclonal antibodies (mAbs) against metastatic non–small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. Methods: We generated a transplantable squamous cell carcinoma cell line (UN‐SCC680AJ) from a lung tumor induced by chronic N‐nitroso‐tris‐chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti‐CD137 and/or anti–PD‐1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. Results: In syngeneic mice, large 12‐day–established tumors derived from the transplantable cell line variant UN‐SCC680AJ were amenable to curative treatment with anti–PD‐1, anti–PD‐L1, or anti‐CD137 immunostimulatory mAbs. Single‐agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti–PD‐1 plus anti‐CD137 achieved complete rejections. Tumor cells expressed weak baseline PD‐L1 on the plasma membrane, but this could be readily induced by interferon‐&ggr;. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co‐expressing CD137 and PD‐1 were prominent. Conclusions: These promising results advocate the use of combined anti–PD‐1/PD‐L1 plus anti‐CD137 mAb immunotherapy for the treatment of squamous non–small cell lung cancer in the clinical setting.

Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism.

ABSTRACT Hypoxia is a common feature in solid tumors that has been implicated in immune evasion. Previous studies from our group have shown that hypoxia upregulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O2) promotes CD137 transcription. However, the resulting mRNA is predominantly an alternatively spliced form that encodes for a soluble variant, lacking the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. Indeed, silencing CD137 with shRNA renders more immunogenic tumor-cell variants upon inoculation to immunocompetent mice but which readily grafted on immunodeficient or CD8+ T-cell-depleted mice. These mechanisms are interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L.

CD137 (4-1BB) costimulation modifies DNA methylation in CD8+ T-cell relevant genes.

CD137(4-1BB) ligation with agonist monoclonal antibodies used in immunotherapy induces long-term sequence-specific CpG DNA-methylation patterns in human CD8+ T lymphocytes. These epigenetic changes give rise to mRNA and protein expression changes in key T-cell genes and regulatory factors. CD137 (4-1BB) costimulation imprints long-term changes that instruct the ultimate behavior of T cells that have previously experienced CD137 ligation. Epigenetic changes could provide a suitable mechanism for these long-term consequences. Genome-wide DNA methylation arrays were carried out on human peripheral blood CD8+ T lymphocytes stimulated with agonist monoclonal antibody to CD137, including urelumab, which is in phase I/II clinical trials for cancer immunotherapy. Several genes showed consistent methylation patterns in response to CD137 costimulation, which were confirmed by pyrosequencing in a series of healthy donors. CD96, HHLA2, CCR5, CXCR5, and CCL5 were among the immune-related genes regulated by differential DNA methylation, leading to changes in mRNA and protein expression. These genes are also differentially methylated in naïve versus antigen-experienced CD8+ T cells. The transcription factor TCF1 and the microRNA miR-21 were regulated by DNA methylation upon CD137 costimulation. Such gene-expression regulatory factors can, in turn, broaden the effects of DNA methylation by controlling expression of their target genes. Overall, chromatin remodeling is postulated to leave CD137-costimulated T lymphocytes poised to differentially respond upon subsequent antigen recognition. Accordingly, CD137 connects costimulation during priming to genome-wide DNA methylation and chromatin reprogramming. Cancer Immunol Res; 6(1); 69–78. ©2017 AACR.

For Whom the Cell Tolls? Intratumoral Treatment Links Innate and Adaptive Immunity

Intratumoral immunotherapy can potentially modulate the tumor microenvironment (TME) and potentiate the effects of concomitant or sequential systemic immunotherapies. Intratumoral administration of different Toll-like receptor agonists, including TLR4, can potentiate these effects through innate and adaptive immunity connection. See related article by Bhatia et al., p. 1185