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Therapeutic efficacy of the F8-IL2 immunocytokine in a metastatic mouse model of lung adenocarcinoma Sébastien Wieckowski a , Teresa Hemmerle b,c , Spasenja Savic Prince d , Béatrice Dolder Schlienger a , Sven Hillinger e , Dario Neri b,c , Alfred Zippelius a,f,∗ a
Cancer Immunology and Biology, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland c Philochem AG, Libernstrasse 3, CH-8093 Otelfingen, Switzerland d Institute of Pathology, University Hospital Basel, Schönebeinstrasse 40, CH-4003 Basel, Switzerland e Department of Thoracic Surgery, University Hospital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland f Medical Oncology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland b
a r t i c l e
i n f o
Article history: Received 13 November 2014 Received in revised form 16 January 2015 Accepted 24 January 2015 Keywords: Mouse Human Lung cancer Immunocytokine CD8 T-cells Immunofluorescence
a b s t r a c t Objectives: Antibody–cytokine fusion proteins (immunocytokines) represent a novel class of armed antibodies in oncology. In particular, IL2- and TNF-based immunocytokines targeting the EDB domain of fibronectin and the A1 domain of tenascin-C have demonstrated promising anti-tumor activity and are currently investigated in Phase I and Phase II clinical trials. To advance the development of immunocytokines for NSCLC, we here report on the therapeutic efficacy of F8-IL2, an immunocytokine directed against the alternatively spliced EDA domain of fibronectin in a fully immunocompetent, orthotopic model of NSCLC, and the characterization of the target antigen expression in human NSCLC specimens. Materials and methods: We evaluated the therapeutic efficacy of the F8-IL2 immunocytokine utilizing a K-ras mutant, p53 deficient metastatic mouse model of NSCLC derived from the latest generation of genetically engineered and conditional tumor models. In parallel, we assessed the presence of the EDA domain of fibronectin by immunofluorescence in lung biopsies obtained from patients with NSCLC. Results: The EDA domain of fibronectin was broadly expressed in lung metastases obtained from our model. Treatment with F8-IL2 induced substantial local changes within immune effector cell populations and demonstrated promising therapeutic efficacy as monotherapy. The target of F8-IL2, the EDA domain of fibronectin, was present in all human lung adenocarcinoma specimens tested. Conclusion: Both the therapeutic efficacy in a metastatic mouse model of NSCLC and the extensive presence of the EDA domain of fibronectin in human NSCLC biopsies support the rational development of therapies based on the F8-IL2 immunocytokine for the treatment of NSCLC. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is one of the most frequently mutated cancer types and mutated peptides could, in principle, be recognized by cytotoxic T-cells [1]. Though NSCLC has traditionally been considered to be “non-immunogenic”, immunotherapy is now attracting increasing attention as promising therapeutic option. However,
Abbreviations: EDA/EDB, extra-domain A/extra-domain B; GEMM, genetically engineered mouse model; KP, k-ras mutant, p53 deficient; KPCL, KP cell-line; NSCLC, non-small-cell lung cancer. ∗ Corresponding author at: Department of Biomedicine, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland. Tel.: +41 61 265 5074. E-mail address: [email protected] (A. Zippelius).
early clinical immunotherapy trials have yielded mixed results with ambiguous clinical benefits [2]. Recent efforts have mainly focused on therapeutic vaccination approaches targeting lung tumor antigens and inhibitory receptors on T-cells, e.g. CTLA-4 and PD-1. In particular, the latter approach harnesses cellular immunity against the tumor by overcoming tumor-induced immune dysfunction and has demonstrated meaningful response rates, sustained clinical benefits with exceptional survival rates, and good tolerability in early clinical trials [3,4]. Cytokine-based therapies aim at activating the anti-tumor properties of host immune system that are virtually diminished by the tumor microenvironment [5]. Interleukin-2 (IL2) has been approved for the systemic treatment of metastatic melanoma and renal cell cancer [6–8]. However, severe side-effects such as vascular leakage syndrome have prevented the systemic application
http://dx.doi.org/10.1016/j.lungcan.2015.01.019 0169-5002/© 2015 Elsevier Ireland Ltd. All rights reserved.
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of high therapeutic doses; thereby limiting its clinical use [8–10]. The rapid clearance of IL2 also limits its effectiveness. Localized application of IL2 and related cytokines is a very effective therapy for cancers such as bladder carcinoma [11,12]. In this regard, antibody-based delivery of cytokine to the tumor site may concentrate IL2 onto neoplastic lesions while sparing healthy tissues. Antibody–cytokine fusion proteins based on IL2 and TNF-␣ are particularly active in immunocompetent mice models of cancer [13]. In particular, splice isoforms of fibronectin that contain the extradomain A (EDA) or the extra-domain B (EDB), and a splice isoform of tenascin-C that contains the domain A1 (recognized by the F8, L19 and F16 antibodies, respectively), have proven to be useful targets for antibody-based delivery applications. These markers are accessible and abundant in the majority of tumors, but virtually undetectable in most normal adult tissues [14]. Two IL2 fusion proteins (L19-IL2 and F16-IL2) and one TNF fusion protein (L19TNF) are currently being investigated in Phase II clinical trials in oncology. The activity of immunocytokines such as L19-IL2 and F16-IL2 has been studied extensively using xenograft and transplantable tumor models of cancer, albeit often in immunocompromised mice [13]. However, cancer models derived from xenografts, transplantable tumor fragments, or early generation genetically engineered mouse tumor models (GEMM) do not fully reflect the situation in patients, Singh and colleagues recently demonstrated that a novel conditional GEMM of NSCLC that contains an active oncogenic mutation in k-ras and loss-of-function in p53 faithfully models human clinical responses [15,16]. Here we evaluated the therapeutic efficacy of F8-IL2 in a mouse model derived from this GEMM of NSCLC and established in our laboratory to assess the potential of F8-IL2 therapy in a metastatic model closely related to the human disease. Importantly, the F8 antibody binds to both murine and human EDA domain with the same affinity [17], supporting correlations between preclinical studies and clinical development. Although the EDB domain of fibronectin and the A1 domain of tenascin-C have been detected in human NSCLC specimens [18], the potential of IL2-based immunocytokines directed against the EDA domain of fibronectin has not yet been assessed in this tumor entity. We also assessed the presence of the target of F8-IL2, the EDA domain of fibronectin, in fresh-frozen biopsies of human NSCLC obtained after thoracic surgery. 2. Material and methods 2.1. Proteins and mouse strains Biotinylated F8 and KSF small immunoproteins and F8-IL2 fusion protein were expressed from stable monoclonal cell lines in CHO cells, purified from cell culture supernatant and analyzed as reported previously [19]. K-rasLSL-G12D/+ ;p53fl/fl (B6) transgenic mice were bred in house and 6-week female C57BL/6 mice were obtained from Janvier (Le-Genest, France). All animal experiments were performed in accordance with institutional guidelines and were approved by the Regional Veterinary Office Kantonales Veterinäramt Basel. 2.2. Generation of the KP cell line and metastatic mouse model of NSCLC Lung tumors were initiated in a Cre recombinase-controlled (Cre/LoxP) genetic model of human NSCLC (KP model) using the activation of oncogenic K-ras (K-rasLSL-G12D ) in combination with the loss of function of p53 (p53fl/fl ). 5 × 107 PFU of Cre recombinase-expressing adenovirus were administered by intratracheal instillation, as described previously [20].
A cell line (KPCL) was derived from an adenocarcinoma developed in the KP model 150 days after virus instillation. Cells were maintained in DMEM growth medium supplemented with 10% FCS. Seeding of KPCL cells to the lungs was achieved by tail vein injection of 2.5 × 105 cells in normal C57BL/6 mice (8 weeks). 2.3. Immunofluorescence analysis and H&E staining In the mouse study, lung tissues containing tumors were removed immediately after euthanasia, snap frozen in 2methylbutane and embedded within Tissue-Tek® O.C.T. Compound (Sakura). In the human study, tissue specimens were taken during surgical resection and immediately processed as described previously [18]. In both studies, 10 m-sections were prepared on a Leica CM1950 cryotome. For the detection of Fn EDA, frozen sections were fixed in ice-cold acetone, blocked in 20% FCS (PAA) and stained with biotinylated F8 or KSF specific to hen egg lysozyme (isotype control) and rat anti-CD31 (clone MEC 13.3, BD Pharmingen) in 3% BSA (Sigma-Aldrich). Bound antibodies were detected using streptavidin-Alexa Fluor 488 and goat anti-rat IgG-Alexa Fluor 568 (Life Technologies). Slides were mounted with fluorescent mounting medium (Dako) containing DAPI (0.5 g/mL). In parallel, sections were stained with Harris hematoxylin and eosin (Sigma-Aldrich) followed by an alcohol dehydration series. Images were acquired on a BX63 Apollo microscope (Olympus) and analyzed with ImageJ 1.48 v (NIH). 2.4. Treatment regimen and dosing Starting day 21 after cell transfer, KPCL-injected C57BL/6 mice were randomized and treated with daily intravenous injection of F8-IL2 over 5 days in the lateral tail vein. Animals were examined daily, and body weight was measured twice weekly. Termination criteria, pre-defined specifically for this model, were used for survival analysis. They include lack of mobility, hunching, laborious breathing, ruffled fur, and/or >10% body weight loss from the day of treatment initiation. 2.5. Cell suspension preparation and flow cytometry After treatment, lung tissues were carefully removed, minced and digested. Cell suspensions were obtained by grinding and passing the digested tissues through a 70-m cell strainer. Leucocytes were further enriched by density gradient separation (Histopaque1119; Sigma). Dead cell were identified with the LIVE/DEAD® fixable dead cell stain kit (Invitrogen). Antibody Fc receptor binding was blocked with 10 g/mL anti-CD16/CD32 antibody (Bio X Cell). Cells were subsequently washed in PBS containing 2% FCS, 2 mM EDTA and 0.1% NaN3 , and incubated at 4 ◦ C for 30 min with directly conjugated primary antibodies from BioLegend (CD3-FITC, CD4APC, CD8-PE-Cy7, CD11b-PE, CD25-BV421, CD45-PerCP, F4/80-PE, FoxP3-PE, NK1.1-V421, GranzymeB-PE) and from BD (Ki67-BV421). FoxP3 staining buffer set (eBioscience) was used for staining of intracellular proteins. Counting of absolute cell number was performed on an Accuri C6 cytometer (BD). Data were collected on the CyAn ADP (Coulter) flow cytometer, and analyzed using FlowJo vX software. 2.6. Statistical analysis Tumor infiltration data were compared using two-tailed Student’s t-test (GraphPad Prism 6). Kaplan–Meier curves were tested using the log-rank (Mantel–Cox) test.
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3. Results 3.1. The EDA domain of fibronectin is highly expressed in the KPCL model The KPCL cell-line was derived from an adenocarcinoma generated in the KP GEMM. Lung metastases were analyzed for the presence of the EDA domain of fibronectin (Fn EDA) at different time-points after intravenous transfer of KPCL cells. For immunofluorescence analysis we utilized the F8 small immunoprotein, a homodimeric antibody structure based on the human F8 scFv directed against Fn EDA [19]. Fn EDA, was detected in all mice and at all stages of disease progression (Fig. 1). Small patches of Fn EDA-expressing cells were visible starting at 20 days after tumor cell transfer, when the first atypical lesions appeared (Fig. 1). At 20/30 days after tumor cell transfer, Fn EDA did not colocalize with CD31, a marker for endothelial cells. After 40 days, when the lung tissue was mainly composed of adenocarcinoma lesions, Fn EDA showed a more intense staining as well as vascular expression pattern. KSF specific to hen egg lysozyme (isotype control) did not show any detectable staining (data not shown). These findings show that the EDA domain of fibronectin is a prevalent target for treatment with F8-based immunocytokines in the KPCL model.
3.2. F8-IL2 induces substantial local changes within immune effector cells in the KPCL model Using flow cytometry, we quantified and characterized modifications in the effector cell compartments in the tumor resulting from treatment with F8-IL2 in KPCL mice. The immunocytokine is based on the human IL2 moiety, which is fully active on mouse T-cells [21], indeed murine and human IL2 display a homology of 63% [22]. However, due to the immunogenicity of the human antibody–cytokine construct F8-IL2, an anti-immunocytokine antibody response was detected by surface plasmon resonance in animal sera shortly after treatment initiation (data not shown). To limit the hypersensitivity reaction due to long-term treatment with F8-IL2, the treatment schedule consisted of daily injections for 5 days. The absolute number of total tumor-infiltrating leucocytes increased from 3.6 × 105 ± 1.4 in untreated mice to 9.6 × 105 ± 1.7 5 days after the end of treatment with a dose of 50 g of F8-IL2 (p = 0.0029) (Fig. 2A). Both CD3+ lymphocytes (Fig. 2B) and NK1.1+ natural killer cells (Fig. 2C) accounted for the increase of leucocytes, the proportion of cells increasing respectively from 34.7% ±5.0 and 4.8% ±1.2 in untreated mice to 60.4% ±1.7 (p < 0.001) and 9.6% ±0.3 (p < 0.001) in mice treated with 50 g F8-IL2. In the lymphocyte population, the proportions of CD8+ and CD4+ Tcells increased evenly, the CD8 to CD4 ratio improving slightly but not significantly from 0.9 ± 0.1 to 1.1 ± 0.1 (p = 0.11) upon treatment with F8-IL2 (Fig. 2D). No marked change in the proportion of FoxP3+ CD25high CD4+ regulatory T-cells (2.4% ±0.6 and 2.3% ±0.1 in untreated and treated mice, respectively; p = 0.73) (Fig. 2E) or the number of F4/80+ CD11b+ macrophages (6.6% ±1.7 and 4.0% ±0.3 in untreated and treated mice, respectively; p = 0.10) (Fig. 2F) was seen. Importantly, this shift toward an immunologically permissive microenvironment was accompanied by the acquisition of effector functions. The percentage of intratumoral proliferating CD8+ T-cells, as measured by the nuclear proliferation marker Ki67 (Fig. 2G), and Granzyme B-expressing CD8+ T-cells (Fig. 2H) changed respectively from 18.0% ±2.6 and 2.1% ±0.5 in untreated mice to 90.5% ±1.0 (p < 0.001) and 52.4% ±3.0 (p < 0.001) upon treatment with the immunocytokine. Of note, we observed an increased frequency of total splenocytes, changing from 1.1 × 108 ±0.3 in untreated mice to 4.9 × 108 ±0.6 (p < 0.001), suggesting that F8-IL2 also induced systemic effects (data not shown).
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3.3. F8-IL2 improves survival in the KPCL model and toxicity is limited To assess the therapeutic efficacy of F8-IL2 in the KPCL model, we treated tumor-bearing mice starting day 21 after cell transfer with three different doses, 5, 20 and 50 g, selected on the basis of previous studies in mouse cancer models [23,24]. The treatment schedule consisted of daily injections for a total of five doses. We observed a dose-dependent efficacy of F8-IL2 with median survival increasing from 47 days in untreated mice to 48.5 days (p = 0.42), 57 days (p = 0.018) and 59.5 days (p < 0.001) in mice treated with 5, 20 and 50 g of F8-IL2, respectively (Fig. 3A). Animal body weight was monitored during the course of the experiments to estimate the intrinsic toxicity of the treatment. Although the mean body weight dropped by 6.1% shortly after initiation of treatment with 50 g F8IL2 (Fig. 3B), all mice recovered their initial weight within 7 days. Treatment with lower doses of immunocytokine was well tolerated since we did not observe any significant weight loss shortly after end of treatment. The data obtained in the KPCL model are promising as they denote both dose-dependent therapeutic efficacy and high tolerability of single agent F8-IL2. 3.4. The EDA domain of fibronectin is highly expressed in surgical resections from patients with NSCLC The expression and prevalence of Fn EDA in tumor material obtained from patients with NSCLC has not been evaluated so far. One limitation for such analysis is the requirement to use fresh frozen material, as the F8 diabody does not work on paraffin-embedded and formalin-fixed tissues [25]. Eighteen surgically resected adenocarcinoma specimens (11 males, 7 females) obtained from the Division of Thoracic Surgery of the University Hospital of Zürich and from the Institute of Pathology of the Hospital of Basel were collected immediately after surgery and snap-frozen. F8 diabody displayed a strong staining in 18/18 (100%) of the specimens tested by immunofluorescence (Fig. 4). No significant difference in the staining intensity was observed among the histologic grades (data not shown). A particular diffuse fibrillary staining pattern localized exclusively in the stroma was observed in all biopsies. 4. Discussion Although lung cancer is not classically believed to be an immunogenic malignancy, previous analyses of tumor specimens from patients with lung cancer have suggested that immune responses may play an important role in the control of the tumor. For example, increased stromal infiltration of CD4+ and CD8+ T-cells in early-stage NSCLC has been associated with a more favorable prognosis [26,27]. Furthermore, impressive clinical benefits have recently been reported in NSCLC using immunotherapies [3,4]. These findings support the hypothesis that using or manipulating the immune system to generate anti-tumor effects may be a beneficial therapeutic approach in lung cancer. The in vivo targeting performance of immunocytokines mostly correlates to a therapeutic benefit in animal models [13], however testing of this class of agents in reliable animal models remains essential to better predict the complex outcomes of this immunotherapy in a clinical situation. Genetically engineered mouse models may fulfill this need, because (a) they mimic spontaneous disease progression by presenting similar genetic mutations that are found in human cancers, (b) they are fully immunocompetent, which is essential for the in vivo evaluation of agents that aim at modulating the immune system, and (c) they can reliably predict human therapeutic responses to standard-of-care
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Fig. 1. Detection of the EDA domain of fibronectin in developing KPCL tumors. (A) H&E staining displaying the establishment of lung tumors at different time-points after transfer of the KP cell line into C57BL/6 mice (10× magnification). Images at 20× magnification are shown in insets. The first atypical lesions appear between days 20 and 40 (white arrows). Starting day 40, prominent adenocarcinoma nodules are discernible (encircled by dotted white lines). (B) Immunofluorescence analysis of the tumor stroma at different time points after tumor cell transfer (10× magnification). The EDA domain of fibronectin was detected with F8 (green), endothelial cells were stained with anti-CD31 antibody (red) and cell nuclei were marked with DAPI (blue). Images are representative of two individuals. Scale bar = 100 m.
treatment regimens [16]. However, GEMMs also have drawbacks, notably in the NSCLC setting where, in contrast to the human situation, the tumors progress into multifocal disease [15]. Furthermore, in the absence of immunogenic tumor antigens, lymphocyte
infiltration represents only a small fraction of the tumor stroma in the GEMM [28]. Nonetheless, immune tolerance and the development of immune escape reflect critical stages of human tumor development and progression, and underline the physiological
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Fig. 2. Flow cytometry analysis of tumor infiltrating immune cells upon treatment of KP tumor-bearing mice with F8-IL2. (A) The absolute number of total leucocytes was determined on an Accuri cytometer 5 days after end of treatment consisting of daily injection of 50 g F8-IL2 for five total doses, initiated 20 days after tumor cell transfer, and in untreated KPCL mice. The proportion of CD3+ lymphocytes (B) and NK1.1+ natural killer (C) cells amongst live CD45+ cells was determined in single cell suspensions prepared from tumor digests by flow cytometry 5 days after the end of treatment. (D) In parallel, the CD8:CD4 ratio was calculated using the percentages of CD8+ and CD4+ T-cells determined after gating on the CD3+ population. (E) The proportion of FoxP3+ CD25high CD4+ regulatory T-cells was measured within the lymphocyte population. (F) The proportion of F4/80+ CD11b+ macrophages was determined among live CD45+ cells. (G) Gating on CD8+ cells, the proliferation index was determined by measuring the expression of the nuclear proliferation marker Ki67. The right panel shows the flow cytometry data used for analysis. (H) Similarly, the proportion of CD8+ T-cells expressing Granzyme B among total CD8+ cells was determined by flow cytometry. In (A) to (H), the bar represents the mean of n = 6 in untreated mice, and n = 9 in mice treated with F8-IL2.
relevance of this model in efforts to understand and enhance the engagement of the host anti-tumor immune response, in particular with the help of immunocytokines. We used a metastatic model derived from the KP GEMM, which includes the most important genetic mutations commonly found
in human lung cancer and mimics the metastatic spread of NSCLC. Repeated and continuous administration of a human recombinant construct such as F8-IL2 to mice can induce the production of neutralizing antibodies, which may confound experiments [29]. The rapid disease progression of the KPCL model enables the efficacy
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Fig. 3. Therapeutic activity of F8-IL2 as monotherapy in the KPCL model. (A) Overall survival of C57BL/6 mice treated daily for a total of 5 days with different doses of F8-IL2 starting 20 days after KP tumor cell transfer. Curves were derived from two independent experiments and pooled together (n = 12 in all groups). (B) The animal body weight was monitored for the duration of the experiment. The mean percentage of body weight change from the baseline, fixed at day 15, is plotted for each group (n = 12). In (A) and (B), black curves correspond to untreated mice, and blue, green and red curves correspond to mice treated with 5, 20 and 50 g of F8-IL2, respectively.
of F8-IL2 to be assessed in short duration experiments, thereby reducing the risk of hypersensitivity reactions impacting on results. Although data generated in the KPCL model are very encouraging, to explore the effects of long-term immunocytokine treatment,
the construction of fully murine (and therefore non-immunogenic) immunocytokines is required. Chemotherapy and immunotherapy have been shown to synergize in various models [30–32]. Recently, Gutbrodt and colleagues showed that F8-IL2 had slight therapeutic efficacy in mice bearing C1498 accute myelogenous leukemia cells [33]. Remarkably, the addition of an antibody-drug conjugate led to complete cure which was dependent on CD8+ T-cells and NK cells [33]. Similarly, Moschetta and colleagues have shown that paclitaxel (Taxol© ) enhances the therapeutic efficacy of F8-IL2 by increasing the uptake of F8 diabody in metastatic human melanoma xenografts and the recruitment of NK cells into the tumor [24]. In this article, we report that lymphocytes and NK cells but not macrophages infiltrated the tumor upon treatment of KPCL mice with F8-IL2. In particular, CD8+ T-cells showed locally improved effector functions with higher proliferation index and higher expression of Granzyme B. At the same time, the intratumoral proportion of regulatory T-cells did not significantly increase, yet this phenomenon has been described for recombinant IL2 [34]. These data demonstrate that F8-IL2 is capable to shift the tumor microenvironment toward a favorable immunologic anti-tumor response. In parallel, we observed the systemic expansion of leucocytes, questioning us for the exact mechanisms behind the therapeutic efficacy of F8-IL2. A closer look into the immune cell populations involved in the control of the tumor development, but also into the alterations of the intratumoral blood vessels will be critical to understand the therapeutic response and resistance mediated by F8-IL2 in the KPCL and KP GEM lung tumor models. Splice isoforms of fibronectin and tenascin-C display distinct expression patterns depending on the tumor entity and grade [18,35]. Hypothesizing that the targeting performance of an immunocytokine correlated with the absolute expression of its cognate antigen, the evaluation of the expression of EDA in human NSCLC specimens was essential, although the molecular properties of F8, L19 and F16 antibodies are identical [33,36,37]. The strong expression of EDA in all human specimens tested anticipates the effective targeting of F8-based immunocytokines in NSCLC.
Fig. 4. (A) Detection of the EDA domain of fibronectin in sections derived from 18 surgical resections from patients with NSCLC. Fn EDA was detected with F8 (green) and cell nuclei were marked with DAPI (blue). (B) No signal was detectable using the KSF isotype control. Scale bar = 100 m.
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5. Conclusions F8-IL2 immunocytokine appears to be a promising novel targeted-immunotherapy for the treatment of NSCLC, not only because the targeted antigen is highly expressed in the majority of patient tumor biopsies but also because it induces effector cells within the tumor and significantly extends survival in a mouse model derived from the K-rasLSL-G12D/+ ;p53fl/fl GEMM. Investigations of combination therapies in the KPCL model are required to see if the efficacy demonstrated here with F8-IL2 monotherapy can be further potentiated. Furthermore, gaining mechanistic insights into clinical outcomes and drug resistance mediated by the immunocytokine in the same model will contribute to the rationale development of future clinical trials based on immunocytokines for patients with NSCLC. Author contributions S.W., T.H., D.N. and A.Z. designed research. S.W., T.H. and B.D.S. conducted experiments and analyzed data. S.S.P. analyzed sections from patients with NSCLC. S.W., D.N. and A.Z. wrote the manuscript. Conflict of interest statement Teresa Hemmerle is a consultant for Philochem AG, and Dario Neri is shareholder of Philogen AG, a company that has licensed the F8 antibody from ETH Zürich. No potential conflicts of interest were disclosed by the other authors. Acknowledgments This work was supported by grants from the Swiss National Science Foundation and the Cancer League Basel. References [1] Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al. Signatures of mutational processes in human cancer. Nature 2013;500:415–21. [2] Rangachari D, Brahmer JR. Targeting the immune system in the treatment of non-small-cell lung cancer. Curr Treat Options Oncol 2013;14:580–94. [3] Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 2012;366:2455–65. [4] Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012;366:2443–54. [5] Margolin K. Cytokine therapy in cancer. Expert Opin Biol Ther 2008;8:1495–505. [6] McDermott DF, Regan MM, Clark JI, Flaherty LE, Weiss GR, Logan TF, et al. Randomized phase III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients with metastatic renal cell carcinoma. J Clin Oncol 2005;23:133–41. [7] Negrier S, Escudier B, Lasset C, Douillard JY, Savary J, Chevreau C, et al. Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma. Groupe Francais d’Immunotherapie. N Engl J Med 1998;338:1272–8. [8] Rosenberg SA, Yang JC, White DE, Steinberg SM. Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response. Ann Surg 1998;228:307–19. [9] Rosenberg SA, Lotze MT, Yang JC, Aebersold PM, Linehan WM, Seipp CA, et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 1989;210:474–84. Discussion 484–475. [10] Siegel JP, Puri RK. Interleukin-2 toxicity. J Clin Oncol 1991;9:694–704. [11] Den Otter W, Dobrowolski Z, Bugajski A, Papla B, Van Der Meijden AP, Koten JW, et al. Intravesical interleukin-2 in T1 papillary bladder carcinoma: regression of marker lesion in 8 of 10 patients. J Urol 1998;159:1183–6.
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Therapeutic efficacy of the F8-IL2 immunocytokine in a metastatic mouse model of lung adenocarcinoma Sébastien Wieckowski a , Teresa Hemmerle b,c , Spasenja Savic Prince d , Béatrice Dolder Schlienger a , Sven Hillinger e , Dario Neri b,c , Alfred Zippelius a,f,∗ a
Cancer Immunology and Biology, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland c Philochem AG, Libernstrasse 3, CH-8093 Otelfingen, Switzerland d Institute of Pathology, University Hospital Basel, Schönebeinstrasse 40, CH-4003 Basel, Switzerland e Department of Thoracic Surgery, University Hospital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland f Medical Oncology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland b
a r t i c l e
i n f o
Article history: Received 13 November 2014 Received in revised form 16 January 2015 Accepted 24 January 2015 Keywords: Mouse Human Lung cancer Immunocytokine CD8 T-cells Immunofluorescence
a b s t r a c t Objectives: Antibody–cytokine fusion proteins (immunocytokines) represent a novel class of armed antibodies in oncology. In particular, IL2- and TNF-based immunocytokines targeting the EDB domain of fibronectin and the A1 domain of tenascin-C have demonstrated promising anti-tumor activity and are currently investigated in Phase I and Phase II clinical trials. To advance the development of immunocytokines for NSCLC, we here report on the therapeutic efficacy of F8-IL2, an immunocytokine directed against the alternatively spliced EDA domain of fibronectin in a fully immunocompetent, orthotopic model of NSCLC, and the characterization of the target antigen expression in human NSCLC specimens. Materials and methods: We evaluated the therapeutic efficacy of the F8-IL2 immunocytokine utilizing a K-ras mutant, p53 deficient metastatic mouse model of NSCLC derived from the latest generation of genetically engineered and conditional tumor models. In parallel, we assessed the presence of the EDA domain of fibronectin by immunofluorescence in lung biopsies obtained from patients with NSCLC. Results: The EDA domain of fibronectin was broadly expressed in lung metastases obtained from our model. Treatment with F8-IL2 induced substantial local changes within immune effector cell populations and demonstrated promising therapeutic efficacy as monotherapy. The target of F8-IL2, the EDA domain of fibronectin, was present in all human lung adenocarcinoma specimens tested. Conclusion: Both the therapeutic efficacy in a metastatic mouse model of NSCLC and the extensive presence of the EDA domain of fibronectin in human NSCLC biopsies support the rational development of therapies based on the F8-IL2 immunocytokine for the treatment of NSCLC. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is one of the most frequently mutated cancer types and mutated peptides could, in principle, be recognized by cytotoxic T-cells [1]. Though NSCLC has traditionally been considered to be “non-immunogenic”, immunotherapy is now attracting increasing attention as promising therapeutic option. However,
Abbreviations: EDA/EDB, extra-domain A/extra-domain B; GEMM, genetically engineered mouse model; KP, k-ras mutant, p53 deficient; KPCL, KP cell-line; NSCLC, non-small-cell lung cancer. ∗ Corresponding author at: Department of Biomedicine, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland. Tel.: +41 61 265 5074. E-mail address: [email protected] (A. Zippelius).
early clinical immunotherapy trials have yielded mixed results with ambiguous clinical benefits [2]. Recent efforts have mainly focused on therapeutic vaccination approaches targeting lung tumor antigens and inhibitory receptors on T-cells, e.g. CTLA-4 and PD-1. In particular, the latter approach harnesses cellular immunity against the tumor by overcoming tumor-induced immune dysfunction and has demonstrated meaningful response rates, sustained clinical benefits with exceptional survival rates, and good tolerability in early clinical trials [3,4]. Cytokine-based therapies aim at activating the anti-tumor properties of host immune system that are virtually diminished by the tumor microenvironment [5]. Interleukin-2 (IL2) has been approved for the systemic treatment of metastatic melanoma and renal cell cancer [6–8]. However, severe side-effects such as vascular leakage syndrome have prevented the systemic application
http://dx.doi.org/10.1016/j.lungcan.2015.01.019 0169-5002/© 2015 Elsevier Ireland Ltd. All rights reserved.
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of high therapeutic doses; thereby limiting its clinical use [8–10]. The rapid clearance of IL2 also limits its effectiveness. Localized application of IL2 and related cytokines is a very effective therapy for cancers such as bladder carcinoma [11,12]. In this regard, antibody-based delivery of cytokine to the tumor site may concentrate IL2 onto neoplastic lesions while sparing healthy tissues. Antibody–cytokine fusion proteins based on IL2 and TNF-␣ are particularly active in immunocompetent mice models of cancer [13]. In particular, splice isoforms of fibronectin that contain the extradomain A (EDA) or the extra-domain B (EDB), and a splice isoform of tenascin-C that contains the domain A1 (recognized by the F8, L19 and F16 antibodies, respectively), have proven to be useful targets for antibody-based delivery applications. These markers are accessible and abundant in the majority of tumors, but virtually undetectable in most normal adult tissues [14]. Two IL2 fusion proteins (L19-IL2 and F16-IL2) and one TNF fusion protein (L19TNF) are currently being investigated in Phase II clinical trials in oncology. The activity of immunocytokines such as L19-IL2 and F16-IL2 has been studied extensively using xenograft and transplantable tumor models of cancer, albeit often in immunocompromised mice [13]. However, cancer models derived from xenografts, transplantable tumor fragments, or early generation genetically engineered mouse tumor models (GEMM) do not fully reflect the situation in patients, Singh and colleagues recently demonstrated that a novel conditional GEMM of NSCLC that contains an active oncogenic mutation in k-ras and loss-of-function in p53 faithfully models human clinical responses [15,16]. Here we evaluated the therapeutic efficacy of F8-IL2 in a mouse model derived from this GEMM of NSCLC and established in our laboratory to assess the potential of F8-IL2 therapy in a metastatic model closely related to the human disease. Importantly, the F8 antibody binds to both murine and human EDA domain with the same affinity [17], supporting correlations between preclinical studies and clinical development. Although the EDB domain of fibronectin and the A1 domain of tenascin-C have been detected in human NSCLC specimens [18], the potential of IL2-based immunocytokines directed against the EDA domain of fibronectin has not yet been assessed in this tumor entity. We also assessed the presence of the target of F8-IL2, the EDA domain of fibronectin, in fresh-frozen biopsies of human NSCLC obtained after thoracic surgery. 2. Material and methods 2.1. Proteins and mouse strains Biotinylated F8 and KSF small immunoproteins and F8-IL2 fusion protein were expressed from stable monoclonal cell lines in CHO cells, purified from cell culture supernatant and analyzed as reported previously [19]. K-rasLSL-G12D/+ ;p53fl/fl (B6) transgenic mice were bred in house and 6-week female C57BL/6 mice were obtained from Janvier (Le-Genest, France). All animal experiments were performed in accordance with institutional guidelines and were approved by the Regional Veterinary Office Kantonales Veterinäramt Basel. 2.2. Generation of the KP cell line and metastatic mouse model of NSCLC Lung tumors were initiated in a Cre recombinase-controlled (Cre/LoxP) genetic model of human NSCLC (KP model) using the activation of oncogenic K-ras (K-rasLSL-G12D ) in combination with the loss of function of p53 (p53fl/fl ). 5 × 107 PFU of Cre recombinase-expressing adenovirus were administered by intratracheal instillation, as described previously [20].
A cell line (KPCL) was derived from an adenocarcinoma developed in the KP model 150 days after virus instillation. Cells were maintained in DMEM growth medium supplemented with 10% FCS. Seeding of KPCL cells to the lungs was achieved by tail vein injection of 2.5 × 105 cells in normal C57BL/6 mice (8 weeks). 2.3. Immunofluorescence analysis and H&E staining In the mouse study, lung tissues containing tumors were removed immediately after euthanasia, snap frozen in 2methylbutane and embedded within Tissue-Tek® O.C.T. Compound (Sakura). In the human study, tissue specimens were taken during surgical resection and immediately processed as described previously [18]. In both studies, 10 m-sections were prepared on a Leica CM1950 cryotome. For the detection of Fn EDA, frozen sections were fixed in ice-cold acetone, blocked in 20% FCS (PAA) and stained with biotinylated F8 or KSF specific to hen egg lysozyme (isotype control) and rat anti-CD31 (clone MEC 13.3, BD Pharmingen) in 3% BSA (Sigma-Aldrich). Bound antibodies were detected using streptavidin-Alexa Fluor 488 and goat anti-rat IgG-Alexa Fluor 568 (Life Technologies). Slides were mounted with fluorescent mounting medium (Dako) containing DAPI (0.5 g/mL). In parallel, sections were stained with Harris hematoxylin and eosin (Sigma-Aldrich) followed by an alcohol dehydration series. Images were acquired on a BX63 Apollo microscope (Olympus) and analyzed with ImageJ 1.48 v (NIH). 2.4. Treatment regimen and dosing Starting day 21 after cell transfer, KPCL-injected C57BL/6 mice were randomized and treated with daily intravenous injection of F8-IL2 over 5 days in the lateral tail vein. Animals were examined daily, and body weight was measured twice weekly. Termination criteria, pre-defined specifically for this model, were used for survival analysis. They include lack of mobility, hunching, laborious breathing, ruffled fur, and/or >10% body weight loss from the day of treatment initiation. 2.5. Cell suspension preparation and flow cytometry After treatment, lung tissues were carefully removed, minced and digested. Cell suspensions were obtained by grinding and passing the digested tissues through a 70-m cell strainer. Leucocytes were further enriched by density gradient separation (Histopaque1119; Sigma). Dead cell were identified with the LIVE/DEAD® fixable dead cell stain kit (Invitrogen). Antibody Fc receptor binding was blocked with 10 g/mL anti-CD16/CD32 antibody (Bio X Cell). Cells were subsequently washed in PBS containing 2% FCS, 2 mM EDTA and 0.1% NaN3 , and incubated at 4 ◦ C for 30 min with directly conjugated primary antibodies from BioLegend (CD3-FITC, CD4APC, CD8-PE-Cy7, CD11b-PE, CD25-BV421, CD45-PerCP, F4/80-PE, FoxP3-PE, NK1.1-V421, GranzymeB-PE) and from BD (Ki67-BV421). FoxP3 staining buffer set (eBioscience) was used for staining of intracellular proteins. Counting of absolute cell number was performed on an Accuri C6 cytometer (BD). Data were collected on the CyAn ADP (Coulter) flow cytometer, and analyzed using FlowJo vX software. 2.6. Statistical analysis Tumor infiltration data were compared using two-tailed Student’s t-test (GraphPad Prism 6). Kaplan–Meier curves were tested using the log-rank (Mantel–Cox) test.
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3. Results 3.1. The EDA domain of fibronectin is highly expressed in the KPCL model The KPCL cell-line was derived from an adenocarcinoma generated in the KP GEMM. Lung metastases were analyzed for the presence of the EDA domain of fibronectin (Fn EDA) at different time-points after intravenous transfer of KPCL cells. For immunofluorescence analysis we utilized the F8 small immunoprotein, a homodimeric antibody structure based on the human F8 scFv directed against Fn EDA [19]. Fn EDA, was detected in all mice and at all stages of disease progression (Fig. 1). Small patches of Fn EDA-expressing cells were visible starting at 20 days after tumor cell transfer, when the first atypical lesions appeared (Fig. 1). At 20/30 days after tumor cell transfer, Fn EDA did not colocalize with CD31, a marker for endothelial cells. After 40 days, when the lung tissue was mainly composed of adenocarcinoma lesions, Fn EDA showed a more intense staining as well as vascular expression pattern. KSF specific to hen egg lysozyme (isotype control) did not show any detectable staining (data not shown). These findings show that the EDA domain of fibronectin is a prevalent target for treatment with F8-based immunocytokines in the KPCL model.
3.2. F8-IL2 induces substantial local changes within immune effector cells in the KPCL model Using flow cytometry, we quantified and characterized modifications in the effector cell compartments in the tumor resulting from treatment with F8-IL2 in KPCL mice. The immunocytokine is based on the human IL2 moiety, which is fully active on mouse T-cells [21], indeed murine and human IL2 display a homology of 63% [22]. However, due to the immunogenicity of the human antibody–cytokine construct F8-IL2, an anti-immunocytokine antibody response was detected by surface plasmon resonance in animal sera shortly after treatment initiation (data not shown). To limit the hypersensitivity reaction due to long-term treatment with F8-IL2, the treatment schedule consisted of daily injections for 5 days. The absolute number of total tumor-infiltrating leucocytes increased from 3.6 × 105 ± 1.4 in untreated mice to 9.6 × 105 ± 1.7 5 days after the end of treatment with a dose of 50 g of F8-IL2 (p = 0.0029) (Fig. 2A). Both CD3+ lymphocytes (Fig. 2B) and NK1.1+ natural killer cells (Fig. 2C) accounted for the increase of leucocytes, the proportion of cells increasing respectively from 34.7% ±5.0 and 4.8% ±1.2 in untreated mice to 60.4% ±1.7 (p < 0.001) and 9.6% ±0.3 (p < 0.001) in mice treated with 50 g F8-IL2. In the lymphocyte population, the proportions of CD8+ and CD4+ Tcells increased evenly, the CD8 to CD4 ratio improving slightly but not significantly from 0.9 ± 0.1 to 1.1 ± 0.1 (p = 0.11) upon treatment with F8-IL2 (Fig. 2D). No marked change in the proportion of FoxP3+ CD25high CD4+ regulatory T-cells (2.4% ±0.6 and 2.3% ±0.1 in untreated and treated mice, respectively; p = 0.73) (Fig. 2E) or the number of F4/80+ CD11b+ macrophages (6.6% ±1.7 and 4.0% ±0.3 in untreated and treated mice, respectively; p = 0.10) (Fig. 2F) was seen. Importantly, this shift toward an immunologically permissive microenvironment was accompanied by the acquisition of effector functions. The percentage of intratumoral proliferating CD8+ T-cells, as measured by the nuclear proliferation marker Ki67 (Fig. 2G), and Granzyme B-expressing CD8+ T-cells (Fig. 2H) changed respectively from 18.0% ±2.6 and 2.1% ±0.5 in untreated mice to 90.5% ±1.0 (p < 0.001) and 52.4% ±3.0 (p < 0.001) upon treatment with the immunocytokine. Of note, we observed an increased frequency of total splenocytes, changing from 1.1 × 108 ±0.3 in untreated mice to 4.9 × 108 ±0.6 (p < 0.001), suggesting that F8-IL2 also induced systemic effects (data not shown).
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3.3. F8-IL2 improves survival in the KPCL model and toxicity is limited To assess the therapeutic efficacy of F8-IL2 in the KPCL model, we treated tumor-bearing mice starting day 21 after cell transfer with three different doses, 5, 20 and 50 g, selected on the basis of previous studies in mouse cancer models [23,24]. The treatment schedule consisted of daily injections for a total of five doses. We observed a dose-dependent efficacy of F8-IL2 with median survival increasing from 47 days in untreated mice to 48.5 days (p = 0.42), 57 days (p = 0.018) and 59.5 days (p < 0.001) in mice treated with 5, 20 and 50 g of F8-IL2, respectively (Fig. 3A). Animal body weight was monitored during the course of the experiments to estimate the intrinsic toxicity of the treatment. Although the mean body weight dropped by 6.1% shortly after initiation of treatment with 50 g F8IL2 (Fig. 3B), all mice recovered their initial weight within 7 days. Treatment with lower doses of immunocytokine was well tolerated since we did not observe any significant weight loss shortly after end of treatment. The data obtained in the KPCL model are promising as they denote both dose-dependent therapeutic efficacy and high tolerability of single agent F8-IL2. 3.4. The EDA domain of fibronectin is highly expressed in surgical resections from patients with NSCLC The expression and prevalence of Fn EDA in tumor material obtained from patients with NSCLC has not been evaluated so far. One limitation for such analysis is the requirement to use fresh frozen material, as the F8 diabody does not work on paraffin-embedded and formalin-fixed tissues [25]. Eighteen surgically resected adenocarcinoma specimens (11 males, 7 females) obtained from the Division of Thoracic Surgery of the University Hospital of Zürich and from the Institute of Pathology of the Hospital of Basel were collected immediately after surgery and snap-frozen. F8 diabody displayed a strong staining in 18/18 (100%) of the specimens tested by immunofluorescence (Fig. 4). No significant difference in the staining intensity was observed among the histologic grades (data not shown). A particular diffuse fibrillary staining pattern localized exclusively in the stroma was observed in all biopsies. 4. Discussion Although lung cancer is not classically believed to be an immunogenic malignancy, previous analyses of tumor specimens from patients with lung cancer have suggested that immune responses may play an important role in the control of the tumor. For example, increased stromal infiltration of CD4+ and CD8+ T-cells in early-stage NSCLC has been associated with a more favorable prognosis [26,27]. Furthermore, impressive clinical benefits have recently been reported in NSCLC using immunotherapies [3,4]. These findings support the hypothesis that using or manipulating the immune system to generate anti-tumor effects may be a beneficial therapeutic approach in lung cancer. The in vivo targeting performance of immunocytokines mostly correlates to a therapeutic benefit in animal models [13], however testing of this class of agents in reliable animal models remains essential to better predict the complex outcomes of this immunotherapy in a clinical situation. Genetically engineered mouse models may fulfill this need, because (a) they mimic spontaneous disease progression by presenting similar genetic mutations that are found in human cancers, (b) they are fully immunocompetent, which is essential for the in vivo evaluation of agents that aim at modulating the immune system, and (c) they can reliably predict human therapeutic responses to standard-of-care
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Fig. 1. Detection of the EDA domain of fibronectin in developing KPCL tumors. (A) H&E staining displaying the establishment of lung tumors at different time-points after transfer of the KP cell line into C57BL/6 mice (10× magnification). Images at 20× magnification are shown in insets. The first atypical lesions appear between days 20 and 40 (white arrows). Starting day 40, prominent adenocarcinoma nodules are discernible (encircled by dotted white lines). (B) Immunofluorescence analysis of the tumor stroma at different time points after tumor cell transfer (10× magnification). The EDA domain of fibronectin was detected with F8 (green), endothelial cells were stained with anti-CD31 antibody (red) and cell nuclei were marked with DAPI (blue). Images are representative of two individuals. Scale bar = 100 m.
treatment regimens [16]. However, GEMMs also have drawbacks, notably in the NSCLC setting where, in contrast to the human situation, the tumors progress into multifocal disease [15]. Furthermore, in the absence of immunogenic tumor antigens, lymphocyte
infiltration represents only a small fraction of the tumor stroma in the GEMM [28]. Nonetheless, immune tolerance and the development of immune escape reflect critical stages of human tumor development and progression, and underline the physiological
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Fig. 2. Flow cytometry analysis of tumor infiltrating immune cells upon treatment of KP tumor-bearing mice with F8-IL2. (A) The absolute number of total leucocytes was determined on an Accuri cytometer 5 days after end of treatment consisting of daily injection of 50 g F8-IL2 for five total doses, initiated 20 days after tumor cell transfer, and in untreated KPCL mice. The proportion of CD3+ lymphocytes (B) and NK1.1+ natural killer (C) cells amongst live CD45+ cells was determined in single cell suspensions prepared from tumor digests by flow cytometry 5 days after the end of treatment. (D) In parallel, the CD8:CD4 ratio was calculated using the percentages of CD8+ and CD4+ T-cells determined after gating on the CD3+ population. (E) The proportion of FoxP3+ CD25high CD4+ regulatory T-cells was measured within the lymphocyte population. (F) The proportion of F4/80+ CD11b+ macrophages was determined among live CD45+ cells. (G) Gating on CD8+ cells, the proliferation index was determined by measuring the expression of the nuclear proliferation marker Ki67. The right panel shows the flow cytometry data used for analysis. (H) Similarly, the proportion of CD8+ T-cells expressing Granzyme B among total CD8+ cells was determined by flow cytometry. In (A) to (H), the bar represents the mean of n = 6 in untreated mice, and n = 9 in mice treated with F8-IL2.
relevance of this model in efforts to understand and enhance the engagement of the host anti-tumor immune response, in particular with the help of immunocytokines. We used a metastatic model derived from the KP GEMM, which includes the most important genetic mutations commonly found
in human lung cancer and mimics the metastatic spread of NSCLC. Repeated and continuous administration of a human recombinant construct such as F8-IL2 to mice can induce the production of neutralizing antibodies, which may confound experiments [29]. The rapid disease progression of the KPCL model enables the efficacy
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Fig. 3. Therapeutic activity of F8-IL2 as monotherapy in the KPCL model. (A) Overall survival of C57BL/6 mice treated daily for a total of 5 days with different doses of F8-IL2 starting 20 days after KP tumor cell transfer. Curves were derived from two independent experiments and pooled together (n = 12 in all groups). (B) The animal body weight was monitored for the duration of the experiment. The mean percentage of body weight change from the baseline, fixed at day 15, is plotted for each group (n = 12). In (A) and (B), black curves correspond to untreated mice, and blue, green and red curves correspond to mice treated with 5, 20 and 50 g of F8-IL2, respectively.
of F8-IL2 to be assessed in short duration experiments, thereby reducing the risk of hypersensitivity reactions impacting on results. Although data generated in the KPCL model are very encouraging, to explore the effects of long-term immunocytokine treatment,
the construction of fully murine (and therefore non-immunogenic) immunocytokines is required. Chemotherapy and immunotherapy have been shown to synergize in various models [30–32]. Recently, Gutbrodt and colleagues showed that F8-IL2 had slight therapeutic efficacy in mice bearing C1498 accute myelogenous leukemia cells [33]. Remarkably, the addition of an antibody-drug conjugate led to complete cure which was dependent on CD8+ T-cells and NK cells [33]. Similarly, Moschetta and colleagues have shown that paclitaxel (Taxol© ) enhances the therapeutic efficacy of F8-IL2 by increasing the uptake of F8 diabody in metastatic human melanoma xenografts and the recruitment of NK cells into the tumor [24]. In this article, we report that lymphocytes and NK cells but not macrophages infiltrated the tumor upon treatment of KPCL mice with F8-IL2. In particular, CD8+ T-cells showed locally improved effector functions with higher proliferation index and higher expression of Granzyme B. At the same time, the intratumoral proportion of regulatory T-cells did not significantly increase, yet this phenomenon has been described for recombinant IL2 [34]. These data demonstrate that F8-IL2 is capable to shift the tumor microenvironment toward a favorable immunologic anti-tumor response. In parallel, we observed the systemic expansion of leucocytes, questioning us for the exact mechanisms behind the therapeutic efficacy of F8-IL2. A closer look into the immune cell populations involved in the control of the tumor development, but also into the alterations of the intratumoral blood vessels will be critical to understand the therapeutic response and resistance mediated by F8-IL2 in the KPCL and KP GEM lung tumor models. Splice isoforms of fibronectin and tenascin-C display distinct expression patterns depending on the tumor entity and grade [18,35]. Hypothesizing that the targeting performance of an immunocytokine correlated with the absolute expression of its cognate antigen, the evaluation of the expression of EDA in human NSCLC specimens was essential, although the molecular properties of F8, L19 and F16 antibodies are identical [33,36,37]. The strong expression of EDA in all human specimens tested anticipates the effective targeting of F8-based immunocytokines in NSCLC.
Fig. 4. (A) Detection of the EDA domain of fibronectin in sections derived from 18 surgical resections from patients with NSCLC. Fn EDA was detected with F8 (green) and cell nuclei were marked with DAPI (blue). (B) No signal was detectable using the KSF isotype control. Scale bar = 100 m.
Please cite this article in press as: Wieckowski S, et al. Therapeutic efficacy of the F8-IL2 immunocytokine in a metastatic mouse model of lung adenocarcinoma. Lung Cancer (2015), http://dx.doi.org/10.1016/j.lungcan.2015.01.019
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5. Conclusions F8-IL2 immunocytokine appears to be a promising novel targeted-immunotherapy for the treatment of NSCLC, not only because the targeted antigen is highly expressed in the majority of patient tumor biopsies but also because it induces effector cells within the tumor and significantly extends survival in a mouse model derived from the K-rasLSL-G12D/+ ;p53fl/fl GEMM. Investigations of combination therapies in the KPCL model are required to see if the efficacy demonstrated here with F8-IL2 monotherapy can be further potentiated. Furthermore, gaining mechanistic insights into clinical outcomes and drug resistance mediated by the immunocytokine in the same model will contribute to the rationale development of future clinical trials based on immunocytokines for patients with NSCLC. Author contributions S.W., T.H., D.N. and A.Z. designed research. S.W., T.H. and B.D.S. conducted experiments and analyzed data. S.S.P. analyzed sections from patients with NSCLC. S.W., D.N. and A.Z. wrote the manuscript. Conflict of interest statement Teresa Hemmerle is a consultant for Philochem AG, and Dario Neri is shareholder of Philogen AG, a company that has licensed the F8 antibody from ETH Zürich. No potential conflicts of interest were disclosed by the other authors. Acknowledgments This work was supported by grants from the Swiss National Science Foundation and the Cancer League Basel. References [1] Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al. Signatures of mutational processes in human cancer. Nature 2013;500:415–21. [2] Rangachari D, Brahmer JR. Targeting the immune system in the treatment of non-small-cell lung cancer. Curr Treat Options Oncol 2013;14:580–94. [3] Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 2012;366:2455–65. [4] Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012;366:2443–54. [5] Margolin K. Cytokine therapy in cancer. Expert Opin Biol Ther 2008;8:1495–505. [6] McDermott DF, Regan MM, Clark JI, Flaherty LE, Weiss GR, Logan TF, et al. Randomized phase III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients with metastatic renal cell carcinoma. J Clin Oncol 2005;23:133–41. [7] Negrier S, Escudier B, Lasset C, Douillard JY, Savary J, Chevreau C, et al. Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma. Groupe Francais d’Immunotherapie. N Engl J Med 1998;338:1272–8. [8] Rosenberg SA, Yang JC, White DE, Steinberg SM. Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response. Ann Surg 1998;228:307–19. [9] Rosenberg SA, Lotze MT, Yang JC, Aebersold PM, Linehan WM, Seipp CA, et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 1989;210:474–84. Discussion 484–475. [10] Siegel JP, Puri RK. Interleukin-2 toxicity. J Clin Oncol 1991;9:694–704. [11] Den Otter W, Dobrowolski Z, Bugajski A, Papla B, Van Der Meijden AP, Koten JW, et al. Intravesical interleukin-2 in T1 papillary bladder carcinoma: regression of marker lesion in 8 of 10 patients. J Urol 1998;159:1183–6.
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Please cite this article in press as: Wieckowski S, et al. Therapeutic efficacy of the F8-IL2 immunocytokine in a metastatic mouse model of lung adenocarcinoma. Lung Cancer (2015), http://dx.doi.org/10.1016/j.lungcan.2015.01.019
