Supplemental figure legends 1-8, Supplementary tables 1-5, and Supplementary methods

Supplementary Figures S1-S11

AbstractPurpose:While immune checkpoint inhibitors such as anti–PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials.Experimental Design:We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects.Results:A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell–dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti–PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti–PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression.Conclusions:These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

Abstract The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Abstract As a central mediator of TH1 immune responses, interleukin 12 (IL-12) in the tumor microenvironment (TME) plays a key role in driving anti-tumor immunity. Recombinant IL-12 has been reported to promote CD8+ cytotoxic T cell (CTL) dependent anti-tumor immunity in a wide variety of preclinical models. However, systemic recombinant IL-12 was poorly tolerated in early clinical trials. We are therefore investigating whether intra-tumoral (IT) delivery of a messenger RNA encoding IL-12 formulated in lipid nanoparticles can drive local IL-12 expression in solid tumors to induce TME transformation and anti-tumor immunity. Here we report that IT IL-12 mRNA induces dose-dependent local IL-12 protein expression in mice bearing both subcutaneous syngeneic and patient-derived xenograft tumors. In multiple syngeneic tumor models (e.g. MC38, A20), mouse IL-12 mRNA (mIL-12 mRNA) treatment promoted dose-dependent tumor regression and significantly improved overall survival. Flow cytometry and luminex analysis revealed that anti-tumor activity of mIL-12 mRNA was accompanied by transformation of the TME characteristic of a TH1 immune response. IFNγ induction and natural killer cell activation was accompanied by antigen presenting cell maturation, increased CTL numbers and activation, and increased PD-L1 expression. Furthermore, rejection of established MC38 tumors in response to mIL-12 mRNA was CTL-dependent, and animals demonstrated immunity to subsequent re-challenge with MC38 tumor cells. Since mIL-12 mRNA enhanced PD-L1 expression in the TME, we next investigated whether PD-L1 blockade would enhance responses to mIL-12 mRNA. We found that the combination of a single dose of mIL-12 mRNA and PD-L1 blockade led to significantly more complete responses compared to mIL-12 mRNA monotherapy in the PD-L1 resistant MC38 model. Furthermore, A single dose of mIL-12 mRNA induced rejection of un-injected MC38 tumors in a dual flank model, and this was further enhanced by combination with PD-L1 blockade. These preclinical data demonstrate the potential for IT IL-12 mRNA to drive TME transformation into a proinflammatory state consistent with a TH1 immune response and to ignite CTL-dependent anti-tumor immunity, even in a PD-L1 resistant setting. Citation Format: Susannah L. Hewitt, Nadia Luheshi, Dyane Bailey, John Zielinski, Ameya Apte, Faith Musenge, Russell Karp, Grace Adjei, Andrew Leinster, Sushma Gurumurthy, Ankita Mishra, Kristen Arnold, Darren Potz, Robert W. Wilkinson, Ronald Herbst, Joshua P. Frederick. Intratumoral IL-12 mRNA promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1758.

Abstract Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p<0.004). In a separate study, similar tumor regressions were noted in the FGFR-driven GEMM at 1 week of erdafitinib with or without anti-PD-1 treatment, while no such response to these treatments was noted in a KRAS-driven lung cancer GEMM. Immune profiling of tumor specimens revealed high baseline expression of programmed death ligand-1 (PD-L1) expression by IHC and flow cytometry. Following combination treatment, subsequent immunohistochemistry (IHC) analyses showed a significant decrease in Ki67 and PD-L1 positive tumor cells, accompanied by an increase in cluster of differentiation 3 (CD3) positive tumor-infiltrating cells in combination group as compared to control. T cell function is not inhibited by erdafitinib, as measured in vitro by mixed lymphocyte reaction and cytomegalovirus recall assays. Additional changes observed in lung tumors across treatment groups in immune cell infiltration, functionality, and T-cell clonality will be discussed. These data suggest that combination treatment of erdafitinib and PD-1 blockade drives improved survival in FGFR2-driven model of lung cancer by simultaneous inhibition of FGFR pathway in tumor cells and enhancement of anti-tumor immunity. Thus, data here provide rationale for the combined clinical testing of erdafitinib and PD-1 blockade in patients with FGFR-altered lung cancers. Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.

Abstract Immunologic responses to cancer involve complex interactions of cells within the tumor microenvironment (TME). These interactions are mediated in part through co-stimulatory and inhibitory transmembrane proteins, cytokines and chemokines. Direct intratumoral (ITu) administration of messenger RNA results in synthesis of proteins that can prime and enhance an anti-cancer immune response. Priming, expansion and migration of anti-cancer T-cell clones may also result in a systemic or ‘abscopal’ response in distal lesions. There is mounting preclinical evidence that OX40-OX40L signaling boosts anti-cancer immunity via co-stimulatory activity on T-cells. We generated mRNA constructs encoding OX40L with miR-122 binding sites that have been shown to suppress protein translation in hepatocytes yet not within tumor cells. A single ITu injection of OX40L mRNA, formulated in a lipid nanoparticle, resulted in transfection of up to 25% of live tumor cells, predominantly cancer and myeloid cells. OX40L protein persisted for 7 days after mRNA injection, and a resulting increase in tumor T-cells was observed. Repeat dosing of OX40L mRNA induced complete responses in a model that is sensitive to immune-meditated therapies, such as anti-PD1/PD-L1 blockade, yet was ineffective in a related model that might better represent an immunologically barren or immunosuppressive TME. The OX40L-resistant tumor model was also resistant to anti-PD1 treatment and was shown to contain fewer infiltrating immune cells. As this model may represent a subset of human cancers that do not respond well to checkpoint inhibitors, it was used to test various mRNA constructs designed to cooperatively harness components of both the innate and adaptive immune systems in igniting a productive anti-cancer response. Both IL-36γ and IL-23 have established roles in mediating immune responses in humans and have been implicated in driving various inflammatory diseases. IL-36γ activates innate immune cells and promotes Th1 responses, whereas IL-23 has been implicated in Th1/Th17 immunity as well as in the modulation of antigen presenting cells. Repeated weekly intratumoral co-administration of mRNAs encoding IL-23 and IL-36γ resulted in durable complete responses in both anti-PD1-sensitive and -resistant tumors in a synergistic fashion. A further modest improvement in outcomes was observed when the IL-23/36γ mRNA doublet was co-administered with OX40L mRNA. Further pharmacodynamic and mechanism of action studies with this combination are underway. ITu injection of mRNA encoding combinations of co-stimulatory proteins and cytokines represents a novel platform for cancer immunotherapy, and may be able to induce responses in patient tumors that are resistant to checkpoint inhibition. The co-formulated combination of mRNAs encoding human OX40L, IL-23 and IL-36γ is planned to enter clinical trials in 2017. Citation Format: Josh P. Frederick, Ailin Bai, Susannah Hewitt, Russ Karp, John Zielinski, Kana Ichikawa, Ameya Apte, Dyane Bailey, Kristen Arnold, Sam Farlow, Darren Potz, Bo Ying, Graham MacLean, Kerry Benenato, Maja Sedic, Steve M. Kelsey. Durable efficacy and anti-cancer immunity following intratumoral administration of messenger RNAs encoding IL-36γ, IL-23 and OX40L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1607. doi:10.1158/1538-7445.AM2017-1607

This study describes an imaging strategy based on glow stick chemistry to non-invasively image oxidative stress and reactive oxygen species (ROS) production in living animals.

Background Automated scanning devices and image analysis software provide a means to overcome the limitations of manual semiquantitative scoring of immunohistochemistry. Common drawbacks to automated imaging systems include an inability to classify tissue type and an inability to segregate cytoplasmic and nuclear staining. Methods Immunohistochemistry for the membranous marker α-catenin, the cytoplasmic marker stathmin and the nuclear marker Ki-67 was performed on tissue microarrays (TMA) of archival formalin-fixed paraffin-embedded tissue comprising 471 (α-catenin and stathmin) and 511 (Ki-67) cases of prostate adenocarcinoma. These TMA were quantitatively analysed using two commercially available automated image analysers, the Ariol SL-50 system and the Nuance system from CRi. Both systems use brightfield microscopy for automated, unbiased and standardised quantification of immunohistochemistry, while the Nuance system has spectral deconvolution capabilities. Results Overall concordance between scores from both systems was excellent (r=0.90; 0.83–0.95). The software associated with the multispectral imager allowed accurate automated classification of tissue type into epithelial glandular structures and stroma, and a single-step segmentation of staining into cytoplasmic or nuclear compartments allowing independent evaluation of these areas. The Nuance system, however, was not able to distinguish reliably between tumour and non-tumour tissue. In addition, variance in the labour and time required for analysis between the two systems was also noted. Conclusion Despite limitations, this study suggests some beneficial role for the use of a multispectral imaging system in automated analysis of immunohistochemistry.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant β-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of β-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and β-catenin. All LAM cases were positive for β-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results indicate that β-catenin is a useful marker of LAM and may be clinically useful in the diagnostic setting.

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial–stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.

Background> The Met (Hepatocyte Growth Factor Receptor, HGFR) tyrosine kinase can stimulate cancer cell migration and invasion as well as proliferation and survival: these biological functions are consistent with a role for Met in the metastatic process. Methods> We investigated the role of Met in a lymph node-derived lung cancer cell line (H1993) and a line derived from the primary lung tumor of the same patient (H2073), as well as in tissue samples from 18 primary NSCLCs and their paired brain metastases and from primary 22 NSCLCs without metastases. The two groups of patients had matched clinical-pathological features, were never treated with epidermal growth factor receptor (EGFR) inhibitors and were followed-up for an average of 35 months. Met was investigated by immunohistochemistry (IHC) and FISH. IHC for EGFR and Erbb2 and wide screen mutation analysis by Oncomap were also performed in all the cases. Results> While Met was found to be active and amplified in the metastatic line H1993, the H2073 line did not harbor Met activation or amplification. The inhibition of Met by shRNA as well as by specific small molecule inhibitors resulted in the arrest of both migration and growth of H1993 but had not effect on H2073. In contrast, EGFR was activated in primary tumor-derived H2073 cells and not in H1993 cells, and EGFR inhibition severely limited the proliferation only of H2073 cells. The analysis of the metastatic NSCLCs and their paired brain metastases showed that both phosphorylation and expression of Met were heterogeneous and focal in primary tumors but were markedly enriched in the brain metastases (p=0.031, p=0.002 respectively). In contrast, neither EGFR not Erbb2 expression were up-regulated in the metastases. Met activation and expression were significantly associated with shorter metastasis-free survival (p=0.041 and p=0.011 respectively). High Met copy number gain was detected in a subset of brain metastases (27%) and paired primary lung tumors (13%), but bona fide Met amplification as well as Met mutations were never observed. Finally we observed that 70% of the samples with activated Met also carried KRAS mutations. Conclusions>(1) Met activation in metastatic NSCLC never treated with EGFR inhibitors is not driven by genetic mutations and/or amplification of Met; (2) treatment with Met inhibitors of NSCLC patients harboring activated Met may prevent metastatic spread; (3) the presence of RAS mutations in a significant number of Met-activated cases may affect the strategy of pathway inhibition. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4808.

Breast carcinoma cells with the CD44+/CD24low phenotype have been reported to exhibit 'cancer stem cell' (CSC) characteristics on the basis of their enhanced tumorigenicity and self-renewal potential in immunodeficient mice. We used immunohistochemistry to study the expression of these proteins in whole tissue sections of human breast carcinoma. We found that the fraction of CD44v6+ cells is higher in estrogen receptor-positive carcinomas after neoadjuvant chemotherapy. We also performed double immunohistochemistry for CD44v6 and for the proliferation marker Ki67. We found that the relative number of quiescent carcinoma cells is higher in the CD44v6+ population than in the CD44v6− population in specific carcinoma subtypes. We then used quantum dots and spectral imaging to increase the number of antigens that could be visualized in a single tissue section. We found that anti-CD44v6 and CD24 antibodies that were directly conjugated to quantum dots retained their ability to recognize antigen in formalin-fixed, paraffin-embedded tissue sections. We then performed triple staining for CD44v6, CD24 and Ki67 to assess the proliferation of each sub-population of breast carcinoma cells. Our results identify differences between CD44v6-positive and CD44v6-negative breast carcinoma cells in vivo and provide a proof of principle that quantum dot-conjugated antibodies can be used to study specific sub-populations of cancer cells defined by multiple markers in a single tissue section.

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that 14C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic β-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of β-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed β-catenin. A strong significant association between FASN and cytoplasmic (stabilized) β-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous β-catenin signal (P<0.001, Spearman's ρ=0.33). We propose that cytoplasmic stabilization of β-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.