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Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell–mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell–mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy.Significance:DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs.See related commentary by Li et al., p. 1184.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Figure from Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors

Marker genes for the monocyte-macrophage-DC subpopulations identified in Supplementary Figure 5H. Only genes with adjusted p-value < 0.01 and average log fold-change > 0.3 were included. gene, gene name; p_val, p-value calculated based on Wilcoxon test; avg_logFC, log fold-change of the average expression between the given cluster and all other clusters; pct.1, fraction of gene-expressing cells in the given cluster; pct.2, fraction of gene-expressing cells in all other clusters; pct.diff, Difference between the percentage of gene-expressing cells in the given cluster and all other clusters; p_val_adj, Bonferroni adjusted p-value; cluster, cluster annotation based on the gene markers identified; resolution, clustering resolution parameter used in Seurat.

PDF file - 528K, Supplementary Figure 1. BMS908662 enhances human T cell activation in vitro in a concentration-dependent manner. Supplementary Figure 2. BMS908662 inhibits the growth of BRAF mutant tumor cells in vitro. Supplementary Figure 3. BMS908662 enhances murine T cell activation in vitro in a concentration-dependent manner. Supplementary Figure 4. Detection of ERK phosphorylation in activated T cells. Supplementary Figure 5. Gating strategies for OT-1 and ex vivo phosphoflow. Supplementary Figure 6. PLX4720 enhances human T cell activation in vitro in a concentration-dependent manner.

RAF inhibitors selectively block extracellular signal–regulated kinase (ERK) signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T-cell activation, based on the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T-cell activation and signaling in vitro and in vivo. We observe that T-cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade in vivo potentiates T-cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Finally, this combination demonstrates superior antitumor activity, compared with either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T-cell function by potentiating T-cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models. Cancer Immunol Res; 2(1); 70–79. ©2013 AACR.

Migratory CD103+ DCs are the main mediators of cross-priming at the tumor-draining LNs.

Pharmacodynamics of STAT3 ASO in CT26 STK11 KO model. (A) Percentage of CD45+ immune cells among live cells from untreated or drug-treated STK11 KO CT26 tumors as indicated. (B) Percentage of Ly6G+ granulocytic cells among live CD45+ cells. (C) Percentage of intratumoral CD103+ cDC1 dendritic cells among CD45+ immune cells. (D) CD86 MFI of CD103+ cDC1s. (E) Percentage of migratory DC among CD45+ cells in TDLN of tumor engrafted mice. (F) CD86 MFI of these migratory DC in TDLNs. (G) Flow cytometry plots of CD86 levels on TDLN migratory DC. (H) Percentage of CD64+ CD11b+ myeloid cells among live intratumoral CD45+ cells. (I) Flow cytometry plots of CD86 on these CD64+ CD11b+ myeloid cells. (J) Percentages of MHCII+ CD206+ intratumoral myeloid cells. (K) Percentage of MHCII+ CD163+ intratumoral myeloid cells.

Systemic sFlt3L and local intratumoral poly-ICLC expand and mature DCs in B16-OVA bearing mice.

CTLs against the Adpgk neoantigen of MC38 are induced by anti-CD137 and anti-PD-1 mAbs in a fraction of WT mice, but not in Batf3-/- mice.

Supplementary Appendix

Supplementary Figure Legends

Combinations of immunomodulatory anti-CD137 and anti-PD-1 mAbs synergize with sFlt3L and poly-ICLC against grafted B16F10 and B16-OVA melanomas.

Mutations in the STK11 (LKB1) gene regulate resistance to PD-1/PD-L1 blockade. This study evaluated this association in patients with nonsquamous non–small cell lung cancer (NSCLC) enrolled in three phase I/II trials. STK11 mutations were associated with resistance to the anti–PD-L1 antibody durvalumab (alone/with the anti-CTLA4 antibody tremelimumab) independently of KRAS mutational status, highlighting STK11 as a potential driver of resistance to checkpoint blockade. Retrospective assessments of tumor tissue, whole blood, and serum revealed a unique immune phenotype in patients with STK11 mutations, with increased expression of markers associated with neutrophils (i.e., CXCL2, IL6), Th17 contexture (i.e., IL17A), and immune checkpoints. Associated changes were observed in the periphery. Reduction of STAT3 in the tumor microenvironment using an antisense oligonucleotide reversed immunotherapy resistance in preclinical STK11 knockout models. These results suggest that STK11 mutations may hinder response to checkpoint blockade through mechanisms including suppressive myeloid cell biology, which could be reversed by STAT3-targeted therapy.Significance:Patients with nonsquamous STK11-mutant (STK11mut) NSCLC are less likely than STK11 wild-type (STK11wt) patients to respond to anti–PD-L1 ± anti-CTLA4 immunotherapies, and their tumors show increased expression of genes and cytokines that activate STAT3 signaling. Preclinically, STAT3 modulation reverses this resistance, suggesting STAT3-targeted agents as potential combination partners for immunotherapies in STK11mut NSCLC.This article is highlighted in the In This Issue feature, p. 2659

PDF file - 946K, Anti-CTLA-4 mAb treatment fails to extend survival of c-myc OVA tg+

PDF file - 953K, TILs recognize unknown antigens in JMJ cell lines.

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Purpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen.Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays.Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8+ and CD4+ T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy.Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151–62. ©2013 AACR.

59 probesets from an analysis of blood These are probesets for which the null hypothesis was rejected and the change over time averaged over treatment groups was {greater than or equal to}1.2-fold.

single dose radiotherapy and hypofractionated doses increase the level of CD137 and PD-1 expression on the surface of tumor-infiltrating T cells.

CONSORT diagram.

PDF file - 2976K, HCC cell lines derived from c-myc OVA tg+ mice express OVA and functional antigen presenting molecules.

Radiotherapy reduced the density and absolute numbers of CD4+, CD8+ and CD25+FOXP3+ T cells and Myeloid-derived suppressor cells both in the irradiated and non-irradiated lesions.

Supplementary Information

PDF file - 1620K, Combo3 treatment induces infiltrates of blastic T lymphocytes and increases the CD8/Treg ratio.

Statistical comparisons for figures 1, 2, 4 and supplementary figure 1.

4T1-derived spontaneous lung metastases

The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase casein kinase 2 (CK2) on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages. This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer.Significance: These findings demonstrate the modulatory effects of casein kinase 2 inhibitors on myeloid cell differentiation in the tumor microenvironment, which subsequently synergize with the antitumor effects of checkpoint inhibitor CTLA4. Cancer Res; 78(19); 5644–55. ©2018 AACR.

PDF file - 2166K, Granzyme B, perforin and FasL are upregulated on CD8+ T cells after Combo3 treatment.

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.

Methods Supplement Supplementary Table 1. Patients Included in E-R Analysis Supplementary Table 2. Patients From Studies -006 and -011 Not Included in ER Analysis Supplementary Fig 1. Serum concentration:time profile of urelumab across the dose range evaluated (0.1 to 15 mg/kg) Supplementary Fig 2. Induction of a range of IFN-response genes by urelumab 0.1 mg/kg

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supplementary figure legends and supplementary material and methods section.

Mouse external beam irradiation setting and combinations of radiotherapy and anti-CD137 and/or anti-PD1 immunostimulatory monoclonal antibodies are safe and effective to treat MC38-derived bilateral tumors of which only one of the tumor sites is irradiated.

Ex-vivo irradiation of human carcinoma surgical expecimens gives rise to more intense expression of CD137 and PD-1 on infiltrating lymphocytes.

Although immune checkpoint blockade (ICB) has shown remarkable clinical benefit in a subset of patients with melanoma and lung cancer, most patients experience no durable benefit. The receptor tyrosine kinase AXL is commonly implicated in therapy resistance and may serve as a marker for therapy-refractory tumors, for example in melanoma, as we previously demonstrated. Here, we show that enapotamab vedotin (EnaV), an antibody–drug conjugate targeting AXL, effectively targets tumors that display insensitivity to immunotherapy or tumor-specific T cells in several melanoma and lung cancer models. In addition to its direct tumor cell killing activity, EnaV treatment induced an inflammatory response and immunogenic cell death in tumor cells and promoted the induction of a memory-like phenotype in cytotoxic T cells. Combining EnaV with tumor-specific T cells proved superior to either treatment alone in models of melanoma and lung cancer and induced ICB benefit in models otherwise insensitive to anti–PD-1 treatment. Our findings indicate that targeting AXL-expressing, immunotherapy-resistant tumors with EnaV causes an immune-stimulating tumor microenvironment and enhances sensitivity to ICB, warranting further investigation of this treatment combination.Significance:These findings show that targeting AXL-positive tumor fractions with an antibody–drug conjugate enhances antitumor immunity in several humanized tumor models of melanoma and lung cancer.

A course of radiotherapy plus immunostimulatory monoclonal antibodies anti-CD137 and anti-PD-1 mAb increases CD4 and CD8 T cells in the tumor infiltrate as well as the number of tumor antigen-specific CD8 T cells.

Supplementary Table 1 Differentially expressed genes of PCA3, comparing EnaV-treated tumors to control tumors

PDF file - 1818K, Activated T cells and tumor infiltrating lymphocytes express PD-1, B7-H1 (PD-L1), CD137 and OX40.