Resistance to Durvalumab and Durvalumab plus Tremelimumab Is Associated with Functional STK11 Mutations in Patients with Non-Small Cell Lung Cancer and Is Reversed by STAT3 Knockdown.

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.

HER2-targeted antibody-drug conjugate induces host immunity against cancer stem cells.

We previously tested HER2-targeted antibody-drug conjugates (ADCs) in immunocompromised (SCID) mice, precluding evaluation of host immunity, impact on cancer stem cells (CSCs), and potential benefit when combined with PD-L1 blockade. In this study, we tested HER2-targeted ADC in two immunocompetent mouse tumor models. HER2-targeted ADC specifically inhibited the growth of HER2-expressing tumors, prolonged animal survival, and reduced HER2+ and PD-L1+ cells. ADC + anti-PD-L1 antibody augmented therapeutic efficacy, modulated immune gene signatures, increased the number and function of CD3+ and CD19+ tumor-infiltrating lymphocytes (TILs), induced tumor antigen-specific immunological memory, stimulated B cell activation, differentiation, and IgG1 production both systemically and in the tumor microenvironment. In addition, ADC therapy modulated T cell subsets and their activation in TILs. Furthermore, HER2-targeted ADC reduced the number and tumorigenicity of ALDHhi CSCs. This study demonstrates that HER2-targeted ADC effectively targets ALDHhi CSCs and this effect is augmented by co-administration of anti-PD-L1 antibody.

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR.

A Novel Murine GITR Ligand Fusion Protein Induces Antitumor Activity as a Monotherapy That Is Further Enhanced in Combination with an OX40 Agonist.

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy.Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors.Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment.Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR.

CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas.

Individual or combinations of somatic mutations found in genes from colorectal cancers can redirect the effects of chemotherapy and targeted agents on cancer cell survival and, consequently, on clinical outcome. Novel therapeutics with mechanisms of action that are independent of mutational status would therefore fulfill a current unmet clinical need. Here the CEA and CD3 bispecific single-chain antibody MEDI-565 (also known as MT111 and AMG 211) was evaluated for its ability to activate T cells both in vitro and in vivo and to kill human tumor cell lines harboring various somatic mutations commonly found in colorectal cancers. MEDI-565 specifically bound to normal and malignant tissues in a CEA-specific manner, and only killed CEA positive cells. The BiTE® antibody construct mediated T cell-directed killing of CEA positive tumor cells within 6 hours, at low effector-to-target ratios which were independent of high concentrations of soluble CEA. The potency of in vitro lysis was dependent on CEA antigen density but independent of the mutational status in cancer cell lines. Importantly, individual or combinations of mutated KRAS and BRAF oncogenes, activating PI3KCA mutations, loss of PTEN expression, and loss-of-function mutations in TP53 did not reduce the activity in vitro. MEDI-565 also prevented growth of human xenograft tumors which harbored various mutations. These findings suggest that MEDI-565 represents a potential treatment option for patients with CEA positive tumors of diverse origin, including those with individual or combinations of somatic mutations that may be less responsive to chemotherapy and other targeted agents.

The CEA/CD3-bispecific antibody MEDI-565 (MT111) binds a nonlinear epitope in the full-length but not a short splice variant of CEA.

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326-349 and 388-410, with critical residues F(326), T(328), N(333), V(388), G(389), P(390), E(392), I(408), and N(410). Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA.

Potent control of tumor growth by CEA/CD3-bispecific single-chain antibody constructs that are not competitively inhibited by soluble CEA.

Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.

TDAG51 is an ERK signaling target that opposes ERK-mediated HME16C mammary epithelial cell transformation.

INTRODUCTION: Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor (RalGEF). Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ErbB2. Here we investigated which signal transduction pathways downstream of Ras contribute to EGFR-dependent transformation of telomerase-immortalized mammary epithelial cells HME16C. Furthermore, we examined whether a highly transcriptionally regulated ERK pathway target, PHLDA1 (TDAG51), suggested to be a tumor suppressor in breast cancer and melanoma, might modulate the transformation process. METHODS: Cellular transformation of human mammary epithelial cells by downstream Ras signal transduction pathways was examined using anchorage-independent growth assays in the presence and absence of EGFR inhibition. TDAG51 protein expression was down-regulated by interfering small hairpin RNA (shRNA), and the effects on cell proliferation and death were examined in Ras pathway-transformed breast epithelial cells. RESULTS: Activation of both the ERK and PI3K signaling pathways was sufficient to induce cellular transformation, which was accompanied by up-regulation of EGFR ligands, suggesting autocrine EGFR stimulation during the transformation process. Only activation of the ERK pathway was sufficient to transform cells in the presence of EGFR inhibition and was sufficient for tumorigenesis in xenografts. Up-regulation of the PHLDA1 gene product, TDAG51, was found to correlate with persistent ERK activation and anchorage-independent growth in the absence or presence of EGFR inhibition. Knockdown of this putative breast cancer tumor-suppressor gene resulted in increased ERK pathway activation and enhanced matrix-detached cellular proliferation of Ras/Raf transformed cells. CONCLUSION: Our results suggest that multiple Ras signal transduction pathways contribute to mammary epithelial cell transformation, but that the ERK signaling pathway may be a crucial component downstream of EGFR activation during tumorigenesis. Furthermore, persistent activation of ERK signaling up-regulates TDAG51. This event serves as a negative regulator of both Erk activation as well as matrix-detached cellular proliferation and suggests that TDAG51 opposes ERK-mediated transformation in breast epithelial cells.

HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically, HAI-1 is also required for matriptase activation, a process that requires sphingosine 1-phosphate (S1P)-mediated translocation of the protease to cell-cell junctions in human mammary epithelial cells. In the present study, we further explored how HAI-1 regulates this protease. First, we observed that after S1P treatment HAI-1 was cotranslocated with matriptase to cell-cell junctions and that the cellular ratio of HAI-1 to matriptase was maintained during this process. However, when this ratio was changed by cell treatment with HAI-1 small interfering RNA or anti-HAI-1 MAb M19, spontaneous activation of matriptase occurred in the absence of S1P-induced translocation; S1P-induced matriptase activation was also enhanced. These results support a role for HAI-1 in protection of cell from uncontrolled matriptase activation. We next expressed matriptase, either alone or with HAI-1 in breast cancer cells that do not endogenously express either protein. A defect in matriptase trafficking to the cell surface occurred if wild-type matriptase was expressed in the absence of HAI-1; this defect appeared to result from matriptase toxicity to cells. Coexpression with matriptase of wild-type HAI-1, but not HAI-1 mutants altered in its Kunitz domain 1, corrected the trafficking defect. In contrast, catalytically defective matriptase mutants were normal in their trafficking in the absence of HAI-1. These results are also consistent with a role for HAI-1 to prevent inappropriate matriptase proteolytic activity during its protein synthesis and trafficking. Taken together, these results support multiple roles for HAI-1 to regulate matriptase, including its proper expression, intracellular trafficking, activation, and inhibition.

Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts.

Sphingosine 1-phosphate (S1P), a bioactive phospholipid, simultaneously induces actin cytoskeletal rearrangements and activation of matriptase, a membrane-associated serine protease in human mammary epithelial cells. In this study, we used a monoclonal antibody selective for activated, two-chain matriptase to examine the functional relationship between these two S1P-induced events. Ten minutes after exposure of 184 A1N4 mammary epithelial cells to S1P, matriptase was observed to accumulate at cell-cell contacts. Activated matriptase first began to appear as small spots at cell-cell contacts, and then its deposits elongated along cell-cell contacts. Concomitantly, S1P induced assembly of adherens junctions and subcortical actin belts. Matriptase localization was observed to be coincident with markers of adherens junctions at cell-cell contacts but likely not to be incorporated into the tightly bound adhesion plaque. Disruption of subcortical actin belt formation and prevention of adherens junction assembly led to prevention of accumulation and activation of the protease at cell-cell contacts. These data suggest that S1P-induced accumulation and activation of matriptase depend on the S1P-induced adherens junction assembly. Although MAb M32, directed against one of the low-density lipoprotein receptor class A domains of matriptase, blocked S1P-induced activation of the enzyme, the antibody had no effect on S1P-induced actin cytoskeletal rearrangement. Together, these data indicate that actin cytoskeletal rearrangement is necessary but not sufficient for S1P-induced activation of matriptase at cell-cell contacts. The coupling of matriptase activation to adherens junction assembly and actin cytoskeletal rearrangement may serve to ensure tight control of matriptase activity, restricted to cell-cell junctions of mammary epithelial cells.

Characterization of matriptase expression in normal human tissues.

Matriptase is a type II transmembrane serine protease that has been implicated in the progression of epithelium-derived tumors. The role of this protease in the biology of normal epithelial cells remains to be elucidated. Matriptase mRNA has been detected by Northern analysis in tissues rich in epithelial cells, and the protein is expressed in vivo in normal and cancerous breast, ovarian, and colon tissues. However, a systematic analysis of the distribution of matriptase protein and mRNA in normal human tissues rich in epithelium has not been reported. In this study we characterized the expression of the protease in a wide variety of normal human tissues using a tissue microarray and whole tissue specimens. Significant immunoreactivity and mRNA expression were detected in the epithelial components of most epithelium-containing tissues. Matriptase expression was found in all types of epithelium, including columnar, pseudostratified columnar, cuboidal, and squamous. Distinct spatial distributions of reactivity were observed in the microanatomy of certain tissues, however. This suggests that although matriptase is broadly expressed among many types of epithelial cells, its activity within a tissue may be regulated in part at the protein and mRNA levels during the differentiation of selected epithelia.

Possible role of matriptase in the diagnosis of ovarian cancer.

There are increasing data that suggest a role for the serine protease matriptase and its inhibitor, hepatocyte growth factor activator inhibitor-1, in the pathogenesis and progression of ovarian cancer. This review will discuss the matriptase/inhibitor system in the context of ovarian cancer and examine the possibility that this system might be a useful therapeutic and/or diagnostic target in this disease.

The activation of matriptase requires its noncatalytic domains, serine protease domain, and its cognate inhibitor.

The activation of matriptase requires proteolytic cleavage at a canonical activation motif that converts the enzyme from a one-chain zymogen to an active, two-chain protease. In this study, matriptase bearing a mutation in its catalytic triad was unable to undergo this activational cleavage, suggesting that the activating cleavage occurs via a transactivation mechanism where interaction between matriptase zymogen molecules leads to activation of the protease. Using additional point and deletion mutants, we showed that activation of matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the serine protease domain, and intact low density lipoprotein receptor class A domains. Its cognate inhibitor, hepatocyte growth factor activator inhibitor-1, may also participate in the activation of matriptase, based on the observation that matriptase activation did not occur when the protease was co-expressed with hepatocyte growth factor activator inhibitor-1 mutated in its low density lipoprotein receptor class A domain. These results suggest that besides matriptase catalytic activity, matriptase activation requires post-translational modification of the protease, intact noncatalytic domains, and its cognate inhibitor.

Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: correlation with clinical outcome and tumor clinicopathological parameters.

PURPOSE: Matriptase is a type II transmembrane serine protease expressed by cells of surface epithelial origin, including epithelial ovarian tumor cells. Matriptase cleaves and activates proteins implicated in the progression of ovarian cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine the expression of matriptase, and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), in epithelial ovarian cancer and to assign clinicopathological correlations. EXPERIMENTAL DESIGN: We have determined by immunohistochemistry the expression of matriptase and HAI-1 in 54 epithelial ovarian cancers. Statistical analyses of immunohistochemistry expression data with clinical outcome and clinicopathological parameters were then performed. RESULTS: Of 54 tumors tested, 39 (72%) and 11 (20%) were positive for matriptase and for HAI-1, respectively. All HAI-1-positive tumors were also matriptase positive. Analysis of clinicopathological parameters demonstrated a loss of matriptase associated with stage III/IV tumors as compared with stage I/II tumors (P = 0.030). There was also a loss of HAI-1 expression associated with stage III/IV tumors (P = 0.039). Of 34 stage I/II tumors, 28 (82%) stained positive for matriptase, and 10 (29%) stained positive for HAI-1; 10 (29%) tumors showed coexpression. Of 20 stage III/IV tumors, however, 11 stained positive for matriptase (55%), only 1 of which coexpressed HAI-1 (P = 0.039). CONCLUSIONS: Advanced-stage ovarian tumors that express matriptase are more likely to do so in the absence of its inhibitor, HAI-1, indicating that an imbalance in the matriptase:HAI-1 ratio could be important in the development of advanced disease. Such an imbalance could promote the proteolytic activity of matriptase and, consequently, a more invasive phenotype.