Supplementary Methods, Figure Legends 1-4 from GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

Supplementary Table 1 from GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

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 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

Abstract Background: Cachexia is associated with increased inflammatory markers and decreased survival in cancer. Also, elevated GDF-15 has been associated with poor prognosis in several cancer types but its role in cachexia is not well-understood. Methods: We measured body weight change, appetite, plasma GDF-15, and other inflammatory markers in 62 males with cancer-cachexia (CC), 72 non-cachectic cancer subjects (CNC) and 64 non-cancer controls (Co) matched by age, gender and pre-illness body weight. In a subset of patients we also measured grip strength (HGS), appendicular lean body mass (aLBM), ECOG and KPS. Results: GDF-15, IL-6 and IL-8 were increased in CC vs. other groups. Activin and G-CSF were significantly upregulated in CC vs. Co. A subset analyses showed that GDF-15, Activin A and IL-8 were increased in CC vs. CNC in lung cancer patients and that GDF-15, IL-6 and IL-8 were increased in CC patients treated with platinum-based chemotherapy. GDF15, IL-6 and IL-8 levels significantly correlated with 6-month weight loss and with IL-6, IL-1ra, IL-2, IL-4, IL-9, IL-10, IFN, MCP-10, MIP-1a, MIP-1b, TNF-a, VGEF and activin in cancer patients. Analysis in a subset of patients showed that CC had lower grip strength, aLBM, and fat mass; and that ECOG and KPS were lower in CC and CNC compared to controls. GDF-15 and IL-8 correlated negatively with aLBM, HGS and fat mass. Activin correlated negatively with aLBM. Survival analysis showed that GDF-15 and IL-8 predicted survival adjusting for stage and weight change (Cox regression p<0.01, HR 3). Conclusion: GDF-15 and other inflammatory markers are associated with weight loss, decreased muscle mass and strength and poor survival in cancer patients. GDF-15 may serve as a prognostic indicator in cancer patients and be a novel therapeutic target for cancer cachexia. Citation Format: Lerner Lorena, Teresa Hayes, Nianjun Tao, Brian Krieger, Bin Feng, Richard Nicoletti, Ailin Bai, Zhigang Weng, Qing Liu, Maria Isabel Chiu, Jeno Gyuris, Jose M. Garcia. Plasma growth differentiating factor-15 (GDF-15) and other inflammatory markers are associated with weight loss and poor prognosis in cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2013-3504

Abstract Background: We have provided initial evidence1 on the clinical usefulness of the cancer cachexia stages (CCS) proposed by Fearon et al2. However it is still unclear3 if particular molecular phenotypes are also associated with these stages, as well with relevant clinical outcomes. Methods: A candidate list of cytokines (Activin A, Eotaxin, FGF, G-CSF, GDF15, GM-CSF, IFN-g, IL-10, IL-12_p70, IL-13, IL-15, IL-17, IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1_MCAF, MIP-1a, MIP-1b, PDGF-bb, RANTES, TNF-a, VEGF) was measured in 210 blood samples from patients with advanced lung and gastrointestinal cancers, via Luminex and ELISA methods. Non-parametric t-test, Kaplan-Meier and Kruskal-Wallis analyses were used to test the association between cytokines levels with CCS, Patient-Generated Subjective Global Assessment scores (PG-SGA) and survival. Results: Using non-cachectic patients as controls, Activin A and GDF15 were significantly up-regulated in pre-cachectic (p<0.01), cachectic (p<0.05) and refractory cachectic (p<0.001) patients. IL-6, IL-8 and VEGFa were significantly up-regulated only in refractory cachectic (p<0.001) patients. Activin A (p<0.001), GDF15 (p<0.001) and IL-8 (P<0.001) plasma levels correlated with PG-SGA. Quartiles of GDF15 plasma levels identified better survival curves as compared to Activin A, IL-6 and IL-8 quartiles. Conclusions: Activin A and GDF15 appear to be useful aids for the diagnosis of all cachexia stages in advanced cancer. Because of their correlation with nutritional and survival outcomes, these cytokines may also represent useful targets in the development of new compounds for the treatment of cancer cachexia. 1. Vigano A, Del Fabbro E, Bruera E, Borod M. The cachexia clinic: from staging to managing nutritional and functional problems in advancer cancer patients. Critical Reviews in Oncogenesis, 2012 17(3), 293-304 2. Fearon K,Strasser F,Anker SD,Bosaeus I,Bruera E,Fainsinger RL,Jatoi A, Loprinzi C, MacDonald N, Mantovani G, Davis M, Muscaritoli M, Ottery F, Radbruch L, Ravasco P, Walsh D, Wilcock A, Kaasa S, Baracos VE. Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011; 12:489-495. 3. Scheede-Bergdahl, C., Watt, H.L., Trutschnigg, B., Kilgour, R.D., Haggarty, A., Lucar, E., Vigano, A. 2011. Is IL-6 the best pro-inflammatory biomarker of clinical outcomes of cancer cachexia? Clinical Nutrition 31: 85-8. Citation Format: Antonio Vigano, Lorena Lerner, Nianjun Tao, Brian Krieger, Bin Feng, Richard Nicoletti, Qing Liu, Ailin Bai, Zhigang Weng, Thierry Alcindor, Domenico Fuoco, Jeno Gyuris, Maria Isabel Chiu. From bench to bedside: are cytokines still relevant biomarkers for staging cancer cachexia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4650. doi:10.1158/1538-7445.AM2013-4650

Abstract Aberrant activation of fibroblast growth factor receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification have been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We identified a broad panel of human cancer cell lines with dysregulated FGFR2 signaling and examined the sensitivity of these human cell lines to monoclonal antibodies specifically targeting FGFR2. These FGFR2 antibodies potently suppressed ligand-induced phosphorylation of FGFR2 and downstream signaling, as well as cell proliferation in vitro. The administration of FGFR2 monoclonal antibodies in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4586. doi:10.1158/1538-7445.AM2011-4586

Abstract Dysregulated fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of human cancers. Aberrant activation of FGF receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification, has been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand-binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We surveyed a broad panel of human cancer cell lines for the dysregulation of FGFR2 signaling and discovered that breast and gastric cancer cell lines harboring FGFR2 amplification predominantly express the IIIb isoform of the receptor. Therefore, we used an FGFR2-IIIb–specific antibody, GP369, to investigate the importance of FGFR2 signaling in vitro and in vivo. GP369 specifically and potently suppressed ligand-induced phosphorylation of FGFR2-IIIb and downstream signaling, as well as FGFR2-driven proliferation in vitro. The administration of GP369 in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support the hypothesis that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies. Cancer Res; 70(19); 7630–9. ©2010 AACR.

Breast cancer is characterised by genomic alterations. We did a multicentre molecular screening study to identify abnormalities in individual patients with the aim of providing targeted therapy matched to individuals' genomic alterations.From June 16, 2011, to July 30, 2012, we recruited patients who had breast cancer with a metastasis accessible for biopsy in 18 centres in France. Comparative genomic hybridisation (CGH) array and Sanger sequencing on PIK3CA (exon 10 and 21) and AKT1 (exon 4) were used to assess metastatic biopsy samples in five centres. Therapeutic targets were decided on the basis of identified genomic alterations. The primary objective was to include 30% of patients in clinical trials testing a targeted therapy and, therefore, the primary outcome was the proportion of patients to whom a targeted therapy could be offered. For the primary endpoint, the analyses were done on the overall population registered for the trial. This trial is registered with ClinicalTrials.gov, number NCT01414933.423 patients were included, and biopsy samples were obtained from 407 (metastatic breast cancer was not found in four). CGH array and Sanger sequencing were feasible in 283 (67%) and 297 (70%) patients, respectively. A targetable genomic alteration was identified in 195 (46%) patients, most frequently in PIK3CA (74 [25%] of 297 identified genomic alterations), CCND1 (53 [19%]), and FGFR1 (36 [13%]). 117 (39%) of 297 patients with genomic tests available presented with rare genomic alterations (defined as occurring in less than 5% of the general population), including AKT1 mutations, and EGFR, MDM2, FGFR2, AKT2, IGF1R, and MET high-level amplifications. Therapy could be personalised in 55 (13%) of 423 patients. Of the 43 patients who were assessable and received targeted therapy, four (9%) had an objective response, and nine others (21%) had stable disease for more than 16 weeks. Serious (grade 3 or higher) adverse events related to biopsy were reported in four (1%) of enrolled patients, including pneumothorax (grade 3, one patient), pain (grade 3, one patient), haematoma (grade 3, one patient), and haemorrhagic shock (grade 3, one patient).Personalisation of medicine for metastatic breast cancer is feasible, including for rare genomic alterations.French National Cancer Institute, Breast Cancer Research Foundation, Odyssea, Operation Parrains Chercheurs.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The t(4;14)(p16.3;q32) translocation found in approximately 15% of patients with multiple myeloma leads to aberrant expression of 2 putative oncogenes, MMSET (a histone methyltransferase) and FGFR3 (a receptor tyrosine kinase). Patients carrying the t(4:14) translocation have a poor prognosis. Previous studies have demonstrated the oncogenic potential of FGFR3 in t(4:14) multiple myeloma, therefore targeting this receptor tyrosine kinase represents an attractive therapeutic opportunity in this patient population. Monoclonal antibodies have been developed to specifically block FGFR3 signaling in response to ligand stimulation. However, some of the tumors that harbor the t(4;14) translocation also carry mutations in FGFR3 that render the receptor constitutively active or hypersensitive to ligand stimulation. Whether antibody treatment is effective on tumors expressing mutant FGFR3 needs to be evaluated in preclinical models. The antagonistic anti-FGFR3 antibody, AV370 potently inhibits ligand-induced proliferation of FDCP cells expressing wild type FGFR3. AV370 also binds to mutant FGFR3 receptor with high affinity and inhibits the proliferation of FDCP cells driven by some mutant variants of the receptor. When tested in xenograft models using multiple myeloma cell lines, AV370 also significantly inhibited the growth of the OPM-2 xenograft that carries the K650E mutation in FGFR3. Although K650E mutation is thought to constitutively activate the receptor, we demonstrated that the tyrosine phosphorylation of this mutant receptor can be further stimulated by its ligands and AV370 could inhibit its tyrosine phosphorylation both in vitro and in vivo. Both cell surface and total FGFR3 level in OPM-2 cells were markedly reduced upon incubation with AV370. A profound receptor dowregulation in OPM-2 xenografts was also observed within 8 hours and the receptor downregulation sustained for at least 72 hours following a single dose of AV370 treatment. Similar receptor downregulation was also observed in another multiple myeloma cell line that carries a different activating mutation. Inhibition of receptor phosphorylation and induction of receptor downregulation by AV370 could bring therapeutic benefits to multiple meyloma patients with t(4:14) translocation. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3775.