- Email: [email protected]
Intratumoural MDSC recruitment by chemotherapeutic agent, 5-FU offsets the anti-tumor activity of immune-checkpoint inhibitor in HCC
abstracts
Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. M. Siurala: Full / Part-time employment: TILT Biotherapeutics Ltd. T. de Gruijl: Advisory / Consultancy: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.
150P
Intratumoural MDSC recruitment by chemotherapeutic agent, 5-FU offsets the anti-tumor activity of immune-checkpoint inhibitor in HCC
T.T. Kwong1, C.H. Wong1, J.Y. Zhou2, A.S-L. Cheng2, A.W.H. Chan3, S.L. Chan1 Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong, China, 2School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China, 3Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China
in order to understand how different lineages of immune cells impacted anti-PD-1 efficacy. Methods: Selected immune cell subpopulations were depleted in four different subcutaneous murine syngeneic models (MC38, CT26.WT, EMT6 and Hepa 1-6) and antiPD-1 (10mg/kg) was administered either following the depletion (only for anti-CD25) or concurrently with the depletion antibodies. The effectiveness of each depletion was assessed by flow cytometry analysis of tumor, blood and spleen at the end of each study. Tumor samples from few sub-groups of MC38 and Hepa1-6 models were processed for proteomics analysis (collaboration with Biognosys). Results: As expected, depletion of CD8þ T cells completely abolished the antitumor effects of anti-PD-1 treatment in MC38, EMT6 and CT26.WT, confirming the crucial roles of CD8þ T cells in tumor killing; Interestingly, anti-PD-1 efficacy in Hepa1-6 was only modestly attenuated by eliminating CD8þ T cells, indicating a vital role of nonCD8þ effector cells in mediating anti-PD-1 efficacy in this particular line. Depletion of NK showed a minor impact on the efficacy of anti-PD-1 treatment, whereas the depletion of macrophages largely promoted the efficacy of anti-PD-1 in MC38 and CT26.WT; however, weakening the effect of anti-PD-1 in the Hepa 1-6 model. Depletion of Treg demonstrated synergistic effects with anti-PD-1 treatment in controlling CT26.WT and EMT6 tumors. Conclusion: Our studies show an unequivocal role of CD8þ T cells in anti-PD-1 induced tumor growth inhibition. However, other immune cell lineages may act differently upon PD-1 blockade release, presumably depending on specific tumor microenvironment. Legal entity responsible for the study: The authors. Funding: CrownBio, Biognosys. Disclosure: All authors have declared no conflicts of interest.
1
Background: Current hepatocellular carcinoma (HCC) immunotherapy trials only yield modest outcome, suggesting that possibly strong intrinsic immunosuppressive network needed to be tackled. Conventional chemotherapies are found to exert immunomodulatory effect on the tumor microenvironment (TME). Our study investigated anti-tumor activity of 5-FU in combination with anti-PD-L1, using pre-clinical HCC mouse model, and evaluated the immunomodulation in TME after drug treatments. Methods: For the orthotopic HCC in vivo experiment, 6-8-week-old male C57BL/6 mice were injected intrahepatically with 5x10ˆ5 RIL-175 lucþ cells. In order to assess its immunomodulatory effect on HCC TME rather than on direct tumor-killing effect, 5FU was administrated (i.p.) 3 times per week at a relative low dose—20mg/kg. Whilst, anti-PD-L1(10mg/kg) was delivered (i.p.) every 5 days. Tumor growth was monitored via in vivo imaging. Tumor, liver, spleen and blood was collected afterwards, followed by immune profiling analysis via flow cytometry. Results: In terms of in vivo imaging results, mice with single anti-PD-L1 treatment showed significant response in tumor growth amongst other groups. Meanwhile 5-FU monotherapy and combined treatment group showed no difference with vehicle group. We found an increased amounts of T lymphocytes and NK cells in the tumor site after anti-PD-L1 single treatment, suggesting that these immune active cells contributed to the anti-tumor effect. Tumor-infiltrating myeloid cells, particularly P-MDSC, elevated upon 5-FU treatment in both single and combined group, suggesting that they may play a role in mediating immunosuppression. Conclusion: This study illustrated that anti-PD-L1 monotherapy enriched tumor-infiltrating T lymphocytes and NK cells, possibly accounting for the deferred tumor growth. However, the accumulation of myeloid cells in 5-FU treated mice hinders the antitumor activity of anti-PD-L1 from this orthotopic HCC mouse model. Therefore, low dose of 5-FU may initiate immunosuppressive mechanism to alter the effectiveness of anti-PD-L1 in HCC. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
151P
Investigation of the mechanism of action of anti-PD-1 treatment by systematic depletion of different immune cell populations in syngeneic models
L. Bourre1, Y. Jin2, J. Muntel3, H. Yu4, K. Beeler3, R. Bruderer3, Y. Shan2, A. Xiaoyu An2, D. Xuesong Ouyang2, H. Qixiang Li2 1 Scientific Engagement, Crown Bioscience Inc., San Diego, CA, USA, 2Pharmacology, Crown Bioscience Inc, San Diego, CA, USA, 3Proteomic, Biognosys AG, Schlieren, Switzerland, 4Commercial, Biognosys AG, Schlieren, Switzerland
152P
Replicative potency of oncolytic VSV-GP differentially shapes the immune signature in three distinct syngeneic tumour models
G. Wollmann1, B. Spiesschaert2, K. Das3, L-M. Schreiber3, P. Erlmann2, B. Stierstorfer4, D. von Laer3, P. Mueller5, C. Urbiola3 1 Virology - CD, Medical University Innsbruck, Innsbruck, Austria, 2CIIM, Boehringer Ingelheim, Innsbruck, Austria, 3Virology, Medical University Innsbruck, Innsbruck, Austria, 4DDS, Boehringer Ingelheim, Biberach, Germany, 5CIIM, Boehringer Ingelheim, Biberach, Germany Background: Oncolytic viruses (OV) induce potent antitumor treatment effects not only via direct tumour infection and lysis but also via activation of innate and adaptive immune responses. As such, they show strong synergism in combination with various immunotherapy strategies. Here we address the question to what extend intratumoral viral replication of the vesicular stomatitis virus variant VSV-GP affects the tumour microenvironment of three different syngeneic mouse tumour models B16, TRAMP and LLC. Methods: To model tumours with higher permissivity for OVs we generated interferon receptor deficient cells (TRAMP-IFNAR1-/- and LLC1-IFNAR1-/-). Bio-luminescent imaging was used to assess intratumoral virus propagation. Efficacy of VSV-GP was assessed in vivo in syngeneic C57BL/6 mice. The tumour micro environment was V studied using flow cytometry, histology, multiplex cytokine ELISA and NanoString transcriptome analysis. Results: Intratumoral viral propagation was enhanced in the IFNAR1-/- tumours compared to B16. In LLC1-IFNAR1-/- tumours, VSV-GP treatment resulted in significant upregulation of over 300 immune-related genes, increase in tumour infiltrating lymphocytes and expression of pro-inflammatory cytokines. In contrast, immune activation markers in virus replication-restricted B16 tumours were only slightly increased. Yet, based on transcriptional analysis, we found a common VSV-GP treatment-associated immune gene signature. Furthermore, we have observed that VSV-GP induces an up-regulation of inflammatory cytokines in all tumour models, such as type-I IFN, IFN-c and TNF-a, which correlates with an increase of the T-cell infiltration in the tumour. Other cytokines, such as IL-6 or IL-10, were found to be differently regulated depending on the model. Conclusion: In conclusion, we present a number of immune-activating consequences of virotherapy treatment in syngeneic tumour models with varying degree of virus propagation. Higher virus activity in the tumour qualitatively and quantitatively shapes the tumour microenvironment differently compared to tumours with restricted virus activity. Legal entity responsible for the study: The authors. Funding: Christian Doppler Research Association. Disclosure: G. Wollmann: Advisory / Consultancy, Research grant / Funding (self): Boehringer R
Ingelheim. B. Spiesschaert: Full / Part-time employment: Boehringer Ingelheim. P. Erlmann: Full / Part-time employment: Boehringer Ingelheim. B. Stierstorfer: Full / Part-time employment: Boehringer Ingelheim. D. von Laer: Advisory / Consultancy: Boehringer Ingelheim; Shareholder / Stockholder / Stock options, Licensing / Royalties: ViraTherapeutics. P. Mueller: Full / Part-time employment: Boehringer Ingelheim. All other authors have declared no conflicts of interest.
Background: Human cancers are heterogenic and thus response to immune checkpoint inhibitors (ICIs) such as anti-PD-1 or anti-PD-L1 antibody treatments can be vastly different, with only 20% of treated patients responding and some initial responders developing resistance. However, the mechanism of action (MOA) underlying these differences has yet to be revealed. In this study we treated syngeneic tumor-bearing mice with anti-PD-1 antibody in combination with targeted immune cell depletion
xi54 | Tumour Microenvironment
Volume 30 | Supplement 11 | December 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_11/mdz452.021/5675207 by Murdoch University Library user on 17 February 2020
Cytokine content changes and TIL reactivity was assessed during culture by flow cytometry or interferon (IFN) gamma(g) enzyme-linked immune sorbent assay. Results: Treatment of short-term cultures with oncolytic adenovirus coding for TNFa and IL-2 introduced profound changes within the microenvironment, which were characterized by an increase in proinflammatory cytokines and decrease in suppressive cytokines. Further benefits were seen in T-cell depleted ovarian cancer single-cell suspensions infected with cytokine-coding oncolytic adenovirus, which enabled significant production of IFNg by autologous TILs. Such levels were not seen in co-cultures where no virus or the backbone oncolytic adenovirus (no cytokine transgenes) was added. Conclusion: These data illustrate the potential of oncolytic adenovirus coding for TNFa and IL-2 to rewire the ovarian tumour microenvironment for effective TIL antitumour reactivity. This approach may improve the efficacy of adoptive TIL therapy in ovarian cancer patients, thus warranting further clinical investigation. Legal entity responsible for the study: TILT Biotherapeutics Ltd. Funding: TILT Biotherapeutics Ltd. Disclosure: J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. V. Cervera-
Annals of Oncology
Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. M. Siurala: Full / Part-time employment: TILT Biotherapeutics Ltd. T. de Gruijl: Advisory / Consultancy: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.
150P
Intratumoural MDSC recruitment by chemotherapeutic agent, 5-FU offsets the anti-tumor activity of immune-checkpoint inhibitor in HCC
T.T. Kwong1, C.H. Wong1, J.Y. Zhou2, A.S-L. Cheng2, A.W.H. Chan3, S.L. Chan1 Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong, China, 2School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China, 3Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China
in order to understand how different lineages of immune cells impacted anti-PD-1 efficacy. Methods: Selected immune cell subpopulations were depleted in four different subcutaneous murine syngeneic models (MC38, CT26.WT, EMT6 and Hepa 1-6) and antiPD-1 (10mg/kg) was administered either following the depletion (only for anti-CD25) or concurrently with the depletion antibodies. The effectiveness of each depletion was assessed by flow cytometry analysis of tumor, blood and spleen at the end of each study. Tumor samples from few sub-groups of MC38 and Hepa1-6 models were processed for proteomics analysis (collaboration with Biognosys). Results: As expected, depletion of CD8þ T cells completely abolished the antitumor effects of anti-PD-1 treatment in MC38, EMT6 and CT26.WT, confirming the crucial roles of CD8þ T cells in tumor killing; Interestingly, anti-PD-1 efficacy in Hepa1-6 was only modestly attenuated by eliminating CD8þ T cells, indicating a vital role of nonCD8þ effector cells in mediating anti-PD-1 efficacy in this particular line. Depletion of NK showed a minor impact on the efficacy of anti-PD-1 treatment, whereas the depletion of macrophages largely promoted the efficacy of anti-PD-1 in MC38 and CT26.WT; however, weakening the effect of anti-PD-1 in the Hepa 1-6 model. Depletion of Treg demonstrated synergistic effects with anti-PD-1 treatment in controlling CT26.WT and EMT6 tumors. Conclusion: Our studies show an unequivocal role of CD8þ T cells in anti-PD-1 induced tumor growth inhibition. However, other immune cell lineages may act differently upon PD-1 blockade release, presumably depending on specific tumor microenvironment. Legal entity responsible for the study: The authors. Funding: CrownBio, Biognosys. Disclosure: All authors have declared no conflicts of interest.
1
Background: Current hepatocellular carcinoma (HCC) immunotherapy trials only yield modest outcome, suggesting that possibly strong intrinsic immunosuppressive network needed to be tackled. Conventional chemotherapies are found to exert immunomodulatory effect on the tumor microenvironment (TME). Our study investigated anti-tumor activity of 5-FU in combination with anti-PD-L1, using pre-clinical HCC mouse model, and evaluated the immunomodulation in TME after drug treatments. Methods: For the orthotopic HCC in vivo experiment, 6-8-week-old male C57BL/6 mice were injected intrahepatically with 5x10ˆ5 RIL-175 lucþ cells. In order to assess its immunomodulatory effect on HCC TME rather than on direct tumor-killing effect, 5FU was administrated (i.p.) 3 times per week at a relative low dose—20mg/kg. Whilst, anti-PD-L1(10mg/kg) was delivered (i.p.) every 5 days. Tumor growth was monitored via in vivo imaging. Tumor, liver, spleen and blood was collected afterwards, followed by immune profiling analysis via flow cytometry. Results: In terms of in vivo imaging results, mice with single anti-PD-L1 treatment showed significant response in tumor growth amongst other groups. Meanwhile 5-FU monotherapy and combined treatment group showed no difference with vehicle group. We found an increased amounts of T lymphocytes and NK cells in the tumor site after anti-PD-L1 single treatment, suggesting that these immune active cells contributed to the anti-tumor effect. Tumor-infiltrating myeloid cells, particularly P-MDSC, elevated upon 5-FU treatment in both single and combined group, suggesting that they may play a role in mediating immunosuppression. Conclusion: This study illustrated that anti-PD-L1 monotherapy enriched tumor-infiltrating T lymphocytes and NK cells, possibly accounting for the deferred tumor growth. However, the accumulation of myeloid cells in 5-FU treated mice hinders the antitumor activity of anti-PD-L1 from this orthotopic HCC mouse model. Therefore, low dose of 5-FU may initiate immunosuppressive mechanism to alter the effectiveness of anti-PD-L1 in HCC. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
151P
Investigation of the mechanism of action of anti-PD-1 treatment by systematic depletion of different immune cell populations in syngeneic models
L. Bourre1, Y. Jin2, J. Muntel3, H. Yu4, K. Beeler3, R. Bruderer3, Y. Shan2, A. Xiaoyu An2, D. Xuesong Ouyang2, H. Qixiang Li2 1 Scientific Engagement, Crown Bioscience Inc., San Diego, CA, USA, 2Pharmacology, Crown Bioscience Inc, San Diego, CA, USA, 3Proteomic, Biognosys AG, Schlieren, Switzerland, 4Commercial, Biognosys AG, Schlieren, Switzerland
152P
Replicative potency of oncolytic VSV-GP differentially shapes the immune signature in three distinct syngeneic tumour models
G. Wollmann1, B. Spiesschaert2, K. Das3, L-M. Schreiber3, P. Erlmann2, B. Stierstorfer4, D. von Laer3, P. Mueller5, C. Urbiola3 1 Virology - CD, Medical University Innsbruck, Innsbruck, Austria, 2CIIM, Boehringer Ingelheim, Innsbruck, Austria, 3Virology, Medical University Innsbruck, Innsbruck, Austria, 4DDS, Boehringer Ingelheim, Biberach, Germany, 5CIIM, Boehringer Ingelheim, Biberach, Germany Background: Oncolytic viruses (OV) induce potent antitumor treatment effects not only via direct tumour infection and lysis but also via activation of innate and adaptive immune responses. As such, they show strong synergism in combination with various immunotherapy strategies. Here we address the question to what extend intratumoral viral replication of the vesicular stomatitis virus variant VSV-GP affects the tumour microenvironment of three different syngeneic mouse tumour models B16, TRAMP and LLC. Methods: To model tumours with higher permissivity for OVs we generated interferon receptor deficient cells (TRAMP-IFNAR1-/- and LLC1-IFNAR1-/-). Bio-luminescent imaging was used to assess intratumoral virus propagation. Efficacy of VSV-GP was assessed in vivo in syngeneic C57BL/6 mice. The tumour micro environment was V studied using flow cytometry, histology, multiplex cytokine ELISA and NanoString transcriptome analysis. Results: Intratumoral viral propagation was enhanced in the IFNAR1-/- tumours compared to B16. In LLC1-IFNAR1-/- tumours, VSV-GP treatment resulted in significant upregulation of over 300 immune-related genes, increase in tumour infiltrating lymphocytes and expression of pro-inflammatory cytokines. In contrast, immune activation markers in virus replication-restricted B16 tumours were only slightly increased. Yet, based on transcriptional analysis, we found a common VSV-GP treatment-associated immune gene signature. Furthermore, we have observed that VSV-GP induces an up-regulation of inflammatory cytokines in all tumour models, such as type-I IFN, IFN-c and TNF-a, which correlates with an increase of the T-cell infiltration in the tumour. Other cytokines, such as IL-6 or IL-10, were found to be differently regulated depending on the model. Conclusion: In conclusion, we present a number of immune-activating consequences of virotherapy treatment in syngeneic tumour models with varying degree of virus propagation. Higher virus activity in the tumour qualitatively and quantitatively shapes the tumour microenvironment differently compared to tumours with restricted virus activity. Legal entity responsible for the study: The authors. Funding: Christian Doppler Research Association. Disclosure: G. Wollmann: Advisory / Consultancy, Research grant / Funding (self): Boehringer R
Ingelheim. B. Spiesschaert: Full / Part-time employment: Boehringer Ingelheim. P. Erlmann: Full / Part-time employment: Boehringer Ingelheim. B. Stierstorfer: Full / Part-time employment: Boehringer Ingelheim. D. von Laer: Advisory / Consultancy: Boehringer Ingelheim; Shareholder / Stockholder / Stock options, Licensing / Royalties: ViraTherapeutics. P. Mueller: Full / Part-time employment: Boehringer Ingelheim. All other authors have declared no conflicts of interest.
Background: Human cancers are heterogenic and thus response to immune checkpoint inhibitors (ICIs) such as anti-PD-1 or anti-PD-L1 antibody treatments can be vastly different, with only 20% of treated patients responding and some initial responders developing resistance. However, the mechanism of action (MOA) underlying these differences has yet to be revealed. In this study we treated syngeneic tumor-bearing mice with anti-PD-1 antibody in combination with targeted immune cell depletion
xi54 | Tumour Microenvironment
Volume 30 | Supplement 11 | December 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_11/mdz452.021/5675207 by Murdoch University Library user on 17 February 2020
Cytokine content changes and TIL reactivity was assessed during culture by flow cytometry or interferon (IFN) gamma(g) enzyme-linked immune sorbent assay. Results: Treatment of short-term cultures with oncolytic adenovirus coding for TNFa and IL-2 introduced profound changes within the microenvironment, which were characterized by an increase in proinflammatory cytokines and decrease in suppressive cytokines. Further benefits were seen in T-cell depleted ovarian cancer single-cell suspensions infected with cytokine-coding oncolytic adenovirus, which enabled significant production of IFNg by autologous TILs. Such levels were not seen in co-cultures where no virus or the backbone oncolytic adenovirus (no cytokine transgenes) was added. Conclusion: These data illustrate the potential of oncolytic adenovirus coding for TNFa and IL-2 to rewire the ovarian tumour microenvironment for effective TIL antitumour reactivity. This approach may improve the efficacy of adoptive TIL therapy in ovarian cancer patients, thus warranting further clinical investigation. Legal entity responsible for the study: TILT Biotherapeutics Ltd. Funding: TILT Biotherapeutics Ltd. Disclosure: J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. V. Cervera-
Annals of Oncology