Imprime PGG triggers a coordinated anti-cancer immune ......TGI
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Imprime+ DC101 DC101 Vehicle 0 2 4 6 8 Relative Fold Change Over Vehicle Ctrls **** TNFα Expression 0.0 0.5 1.0 1.5 Relative Fold Change Over Vehicle Ctrls **** CD206 Expression Imprime+ DC101 DC101 Vehicle 0.0 0.5 1.0 1.5 Relative Fold Change Over Vehicle Ctrls **** ** Bev Vehicle Imprime+ Bev Tumor associated Neutrophils 1000 0 500 ****0.0001 CD86 GMFI Tumor associated Macrophages 200 400 0 600 Bev Vehicle Imprime+ Bev Abstract #601 Imprime PGG triggers a coordinated anti-cancer immune response in concert with anti- angiogenic antibodies, re-polarizing the immune microenvironment to suppress tumor growth Kathryn Fraser, Nadine Ottoson, Xiahong Qiu, Anissa SH Chan, Steven Leonardo, Adria Jonas, Takashi Kangas, Jeremy Graff, Nandita Bose Biothera, Eagan, MN, USA 55121; [email protected] Abstract Background Summary Imprime PGG (Imprime) is a soluble, yeast-derived β-1,3/1,6 glucan in clinical development for the treatment of cancer in combination with other anti-cancer therapies. Imprime acts as a Pathogen Associated Molecular Pattern (PAMP) and can be recognized by cells of the innate immune system. Preclinical data using human whole blood from healthy volunteers show that Imprime binding to innate immune cells triggers a coordinated immune response that includes repolarization of M2 macrophages, activation of neutrophils and maturation of dendritic cells. This response ultimately leads to cross-talk with the adaptive immune system driving T cell expansion and the production of interferon gamma (IFNγ). In a randomized phase 2 clinical study in stage IV non-small cell lung carcinoma (NSCLC) patients, treatment with Imprime plus bevacizumab (bev; anti-VEGF antibody), carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not receiving Imprime. We sought to explore a mechanistic understanding for this promising clinical activity. Angiogenic factors, such as VEGF, not only drive the formation of new leaky vessels but also facilitate the establishment of a suppressive immune microenvironment enabling tumor survival and growth. Recent work has shown that anti-angiogenics not only block neovascularization but may also promote a shift in the immune microenvironment, enabling immune recognition and destruction of the tumor. We therefore sought to evaluate whether Imprime may complement the effect of anti-angiogenics on the immune microenvironment. We tested Imprime in combination with either bev or DC101 (anti-VEGFR2) in distinct NSCLC xenograft models in athymic nude mice. Once tumors reached a mean size of 100mm 3 , mice were treated with Imprime, bev or DC101. H1299 and H441 tumor-bearing mice were used in the bev and DC101 studies, respectively. In the bev study, Imprime plus bev induced >75% tumor growth inhibition in ~50% of mice vs 20% in the bev alone groups. Both macrophages and neutrophils from spleen and tumor tissue of combination- treated mice showed significant upregulation of the activation marker CD86 compared to tissues from bev alone treated mice. Moreover, splenic MDSCs in combination-treated mice showed significantly increased iNOS2 expression with reduced Arg-1 expression compared to bev alone treated mice. Tumors from the Imprime plus bev groups showed significantly reduced expression of the potent immunosuppressor, TGFβ, when compared to tumors from mice treated only with bev-with the greatest reduction evident in the tumors with the greatest growth inhibition. In the H441 tumor-bearing mice treated with Imprime and DC101, a significant suppression of tumor growth compared with DC101 alone was also observed and additional mechanistic studies in this model are ongoing. These data show for the first time that Imprime-based treatment prompts a shift in the immune microenvironment of a tumor in situ, eliciting enhanced tumor growth inhibition in concert with anti-angiogenic therapy. • Imprime is a soluble yeast-derived β-1,3/1,6 glucan immunomodulator (Figure 1) being developed for cancer treatment in combination with anti-tumor antibodies. • In a randomized phase 2 clinical study, stage IV NSCLC patients treated with Imprime plus the anti-VEGF antibody bevacizumab (bev), carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not receiving Imprime. • Imprime, a pathogen-associated molecular pattern (PAMP), forms an immune complex with endogenous anti-β-glucan antibodies, then binds and primes innate immune cells including macrophages, neutrophils, and DCs (Figure 2). Activation of the above innate cells is central to influencing adaptive immune cell responses. Generating functional and long-lived anti-tumor innate and adaptive immune responses is key to providing durable tumor control. • In addition to blocking neovascularization, anti-angiogenics can promote a shift in the immune microenvironment enabling immune activation. • OBJECTIVE: To evaluate the ability of Imprime to complement the effect of anti- angiogenics on the immune microenvironment in in vivo xenograft models of NSCLC. Results Figure 1: The general structure of yeast-derived Imprime PGG Imprime enhances macrophage, neutrophil and DC activation and function. (A) M1 or M2 macrophages were prepared by culturing Imprime-bound monocytes isolated from whole blood in the presence of M1 or M2-polarizing conditions (XVivo 10 media supplemented with 10% autologous serum and 50 ng/mL recombinant human GM-CSF or M-CSF respectively) for 6 days. Macrophages were subsequently evaluated for phenotype, enhancing CD4 T cell proliferation by CFSE-dilution assay, and IFN-γ production. (B.; first panel) Whole blood was incubated with +/- Imprime for 2hrs and cells were analyzed by flow cytometry. (B.; second panel) Whole blood was treated as in above and neutrophils were isolated by negative selection. Cells were then mixed with Raji cells +/- Rituximab treatment (1μg/ml) at a 25:1 neutrophil:Raji ratio in the presence of luminol (50μM). Cells monitored for production of reactive oxygen species (ROS) measured by relative light units (RLU). (C) MoDC were prepared by culturing Imprime-bound monocytes isolated from whole blood in the presence of XVivo15 media supplemented with 10% autologous serum and 50 ng/mL recombinant human GM-CSF + 50 ng/mL IL-4 conditions conditions for 6 days and then maturing the cells with LPS and TNF-α (50 ng/mL) for 48 hrs. MoDC were subsequently evaluated for phenotype, enhancing CD4/CD8 T cell proliferation by CFSE-dilution assay and IFN-γ production. Figure 4: Splenic macrophages isolated from Imprime +Bev treated mice display a M1-like phenotype. Imprime+ Bev Imprime+ Bev Gated on CD11b+ cells CD68 F4/80 Bev 400 300 350 200 Vehicle iNOS GMFI 250 *0.0108 1000 600 800 200 400 0 CD86 GMFI Bev Vehicle *0.0356 Bev Vehicle Imprime+ Bev Arg1 GMFI 400 300 350 250 **0.0027 Splenic macrophages are M1. At day 20 post tumor injection, spleens were harvested and single cell suspensions were stained with mouse antibodies and analyzed by FACS. GMFI was calculated in Flowjo after gating on CD11b+CD68+F4/80+ cells. Figure 7: Imprime+DC101 treated animals have reduced splenic MDSCs, an increase in activated splenic macrophages, and an increase in Th1-like phenotype on cells within the tumor microenvironment. Imprime+ Bev TGFβ pg/mL 200 400 0 300 100 **0.0048 ***0.0001 Bev Vehicle >50% TGI <50% TGI Immune cells are activated in the tumor microenvironment. (A) At day 37 post tumor injection, spleens were harvested and single cell suspensions were analyzed by FACS. Frequency or GMFI was calculated in Flowjo after gating on indicated cells. (B) Whole tumors were digested and mRNA levels of TNFα, CD206, and TGFβ1 were analyzed by qRT-PCR. Minutes Figure 2: Imprime PGG triggers a coordinated immune response in human ex vivo studies. 100 80 60 40 20 0 Reactive Oxygen Species (ROS) in RLU 50 37.5 25 12.5 0 PMN Treatment Tumor Treatment Vehicle Imprime Vehicle Imprime None None Rituximab Rituximab CD11b CD62L CD88 Imprime Vehicle B. A. CD163 CD86 PD-L1 0 10 2 10 3 10 4 10 5 Count Count 0 10 2 10 3 10 4 10 5 Median 58 168 158 Median 86 2434 759 Median 75 2398 2329 Median 88 1102 1253 Median 93 5189 4234 Median 103 439 790 MFI M1 M2 CD4 T cell proliferation Division Index Isotype ctrl staining M1/M2-Imprime M1/M2-Vehicle 1.0 0.8 0.6 0.4 0.2 0.0 NS **** IFNγ (pg/mL) 800 600 400 200 0 NS ** C. 0 5 10 15 20 25 **p = 0.0038 ***p = 0.0004 Count CD80 CD86 CD83 HLA-DR Mean Fluorescence Intensity (MFI) Median 151 4286 7049 Median 223 640 744 Median 162 168 466 Median 151 871 10759 Isotype ctrl staining Imprime Vehicle Allo-CD4 T Cells 0 50 100 150 **p = 0.0026 % Proliferating Cells Allo-CD8 T Cells IFNγ (pg/mL) Figure 3: Anti-tumor efficacy of Imprime PGG with anti- angiogenics in vivo. Xenograft models. (A) Outline of in vivo studies. (B) Graph depicting H1299 tumor volume of individual mice at day 18 post challenge. (C) %Tumor Growth Inhibition (TGI) of H1299 tumors was calculated by %TGI= (1-individual treated mouse/median control mice)*100. (D) Graph depicting H441 tumor volume of individual mice at day 37 post challenge. (E) %TGI of H441 tumors was calculated by %TGI= (1-individual treated mouse/median control mice)*100. A. Figure 6: Imprime+Bev tumors have cells that show a more activated phenotype and a decrease in TGFβ. C. Ex-Vivo human studies: • Imprime enhances human macrophage and MoDC activation and function which leads to crosstalk with the adaptive immune system. • Imprime elicits the generation of Reactive Oxygen Species (ROS) from neutrophils specifically in response to antibody-coated tumor cells. In-Vivo studies: • Imprime PGG treatment in vivo can activate myeloid cells within both the tumor and spleen to orchestrate a profound shift in the immune microenvironment which promotes tumor recognition and suppression. Treatment Groups (2x/week): 1. Vehicle 2. Imprime (1.2mg/ms) 3. Bevacizumab/DC101 (5/10mg/kg IP) 4. Bevacizumab/DC101 (5/10mg/kg IP)+ Imprime (1.2mg/ms IV) Tumors meet mean size of ~100mm 3 start treatment Human NSCLC cells (H1299 or H441) Given s.c. Nude mouse: No T, B cells Control Tumor Endpoint Animals Sacrificed Spleen, Tumors analyzed iNOS GMFI CD86 GMFI Arginase 1 GMFI A. Live/Dead CD11b Bev Vehicle Imprime+ Bev Bev Vehicle Imprime+ Bev PD-L1 GMFI 200 400 0 100 300 iNOS GMFI Arg1 GMFI 1000 1500 500 0 2000 1000 1500 500 0 2000 *0.033 *0.091 *0.029 Bev Vehicle Imprime+ Bev iNOS GMFI Arginase 1 GMFI PD-L1 GMFI Bev Vehicle Imprime+ Bev PD-L1 RFC/ Vehicle Control 10 15 5 0 20 *0.0430 iNOS RFC/ Vehicle Control Bev Vehicle Imprime+ Bev 10 15 5 0 *0.0275 Bev Vehicle Imprime+ Bev Arg1 RFC/ Vehicle Control 1.0 1.5 0.5 0 *0.0320 PD-L1 Expression iNOS Expression Arginase 1 Expression B. CD86 GMFI Myeloid cells from tumors are more activated. Cells were harvested from the tumor using type I collagenase. (A) GMFI was calculated after gating on CD11b+ cells. (B) mRNA levels of PD-L1, iNOS and Arg1 from single cell suspensions were analyzed by qRT-PCR. (C) GMFI was calculated after gating on Ly6g+ CD11b+ (red) or Gr1-CD11b+ (blue) cells. (D) Cells were incubated overnight in XVivo10 media and supernatants were then analyzed for TGFβ concentration by ELISA Ly6g CD11b TGFβ Expression Figure 5: Imprime+Bev splenic MDSC display an increase in iNOS and decrease in Arginase 1. MDSCs show a decrease in Arg1. At day 20 post tumor injection, spleens were harvested and single cell suspensions were stained with mouse antibodies and analyzed by FACS. GMFI was calculated in Flowjo after gating on CD11b+Gr1+ cells. GR1 CD11b Gated on Lymphocytes 600 800 200 400 0 iNOS GMFI Bev Vehicle Imprime+ Bev ***0.0007 Bev Vehicle Imprime+ Bev Arg1 GMFI 600 400 500 300 **0.0022 iNOS GMFI Arginase 1 GMFI Vehicle 0 2500 1500 2000 1000 500 Imprime Bev Day 18 Tumor Volume (mm 3 ) Tumor Volume g ms 0 0 0 0 0 0 Day 18 %TGI 0 80 100 60 40 Bev Imprime+ Bev 20 75% B. C. Imprime+ Bev Tumor Growth Inhibition Imprime+ DC101 0 1500 2000 1000 500 Vehicle Imprime DC101 Day 37 Tumor Volume (mm 3 ) D. E. 0 80 100 60 40 20 Day 37 %TGI *0.0123 *0.0179 75% Imprime+ DC101 DC101 DC101/H441 Xenograft Model Tumor Volume Tumor Growth Inhibition Bevacizumab /H1299 Xenograft Model A. B. CD86 GMFI Macrophages Imprime+ DC101 DC101 Vehicle Imprime+ DC101 DC101 Vehicle l 1 e 0 1 2 3 0.3 0.2 0.1 0 %CD11b+Gr1+ Ly6c+ cells ***0.0003 % Mo-MDSC ol 1 e 0 1 2 3 4 5 %CD11b+Gr1+ Ly6c dim cells 5 4 3 2 1 0 % PMN-MDSC ol 1 e 0 0 0 0 0 80 60 40 20 0 ****0.0001 % Macrophages ****0.0001 Imprime+ DC101 DC101 Vehicle *0.0271 %CD11b+CD68+ F4/80+ cells ol 1 e 0 0 0 0 0 4000 3000 2000 0 1000 Imprime+ DC101 DC101 Vehicle ****0.0001 **0.0051 **0.0028 CD86 GMFI CD11b+ CD68+F4/80+ cells SPLEEN TUMOR TGFβ Expression Imprime+ DC101 DC101 Vehicle
Transcript of Imprime PGG triggers a coordinated anti-cancer immune ......TGI
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Abstract #601
Imprime PGG triggers a coordinated anti-cancer immune response in concert with anti-angiogenic antibodies, re-polarizing the immune microenvironment to suppress tumor growth Kathryn Fraser, Nadine Ottoson, Xiahong Qiu, Anissa SH Chan, Steven Leonardo, Adria Jonas, Takashi Kangas, Jeremy Graff, Nandita Bose
Biothera, Eagan, MN, USA 55121; [email protected]
Abstract
Background
Summary
Imprime PGG (Imprime) is a soluble, yeast-derived β-1,3/1,6 glucan in clinical development for the treatment of cancer in combination with other anti-cancer therapies. Imprime acts as a Pathogen Associated Molecular Pattern (PAMP) and can be recognized by cells of the innate immune system. Preclinical data using human whole blood from healthy volunteers show that Imprime binding to innate immune cells triggers a coordinated immune response that includes repolarization of M2 macrophages, activation of neutrophils and maturation of dendritic cells. This response ultimately leads to cross-talk with the adaptive immune system driving T cell expansion and the production of interferon gamma (IFNγ). In a randomized phase 2 clinical study in stage IV non-small cell lung carcinoma (NSCLC) patients, treatment with Imprime plus bevacizumab (bev; anti-VEGF antibody), carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not receiving Imprime. We sought to explore a mechanistic understanding for this promising clinical activity. Angiogenic factors, such as VEGF, not only drive the formation of new leaky vessels but also facilitate the establishment of a suppressive immune microenvironment enabling tumor survival and growth. Recent work has shown that anti-angiogenics not only block neovascularization but may also promote a shift in the immune microenvironment, enabling immune recognition and destruction of the tumor. We therefore sought to evaluate whether Imprime may complement the effect of anti-angiogenics on the immune microenvironment. We tested Imprime in combination with either bev or DC101 (anti-VEGFR2) in distinct NSCLC xenograft models in athymic nude mice. Once tumors reached a mean size of 100mm3, mice were treated with Imprime, bev or DC101. H1299 and H441 tumor-bearing mice were used in the bev and DC101 studies, respectively. In the bev study, Imprime plus bev induced >75% tumor growth inhibition in ~50% of mice vs 20% in the bev alone groups. Both macrophages and neutrophils from spleen and tumor tissue of combination-treated mice showed significant upregulation of the activation marker CD86 compared to tissues from bev alone treated mice. Moreover, splenic MDSCs in combination-treated mice showed significantly increased iNOS2 expression with reduced Arg-1 expression compared to bev alone treated mice. Tumors from the Imprime plus bev groups showed significantly reduced expression of the potent immunosuppressor, TGFβ, when compared to tumors from mice treated only with bev-with the greatest reduction evident in the tumors with the greatest growth inhibition. In the H441 tumor-bearing mice treated with Imprime and DC101, a significant suppression of tumor growth compared with DC101 alone was also observed and additional mechanistic studies in this model are ongoing. These data show for the first time that Imprime-based treatment prompts a shift in the immune microenvironment of a tumor in situ, eliciting enhanced tumor growth inhibition in concert with anti-angiogenic therapy.
• Imprime is a soluble yeast-derived β-1,3/1,6 glucan immunomodulator (Figure 1)
being developed for cancer treatment in combination with anti-tumor antibodies. • In a randomized phase 2 clinical study, stage IV NSCLC patients treated with
Imprime plus the anti-VEGF antibody bevacizumab (bev), carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not receiving Imprime.
• Imprime, a pathogen-associated molecular pattern (PAMP), forms an immune
complex with endogenous anti-β-glucan antibodies, then binds and primes innate immune cells including macrophages, neutrophils, and DCs (Figure 2). Activation of the above innate cells is central to influencing adaptive immune cell responses. Generating functional and long-lived anti-tumor innate and adaptive immune responses is key to providing durable tumor control.
• In addition to blocking neovascularization, anti-angiogenics can promote a shift in the immune microenvironment enabling immune activation.
• OBJECTIVE: To evaluate the ability of Imprime to complement the effect of anti-
angiogenics on the immune microenvironment in in vivo xenograft models of NSCLC.
Results Figure 1: The general structure of yeast-derived Imprime PGG
Imprime enhances macrophage, neutrophil and DC activation and function. (A) M1 or M2 macrophages were prepared by culturing Imprime-bound monocytes isolated from whole blood in the presence of M1 or M2-polarizing conditions (XVivo 10 media supplemented with 10% autologous serum and 50 ng/mL recombinant human GM-CSF or M-CSF respectively) for 6 days. Macrophages were subsequently evaluated for phenotype, enhancing CD4 T cell proliferation by CFSE-dilution assay, and IFN-γ production. (B.; first panel) Whole blood was incubated with +/-Imprime for 2hrs and cells were analyzed by flow cytometry. (B.; second panel) Whole blood was treated as in above and neutrophils were isolated by negative selection. Cells were then mixed with Raji cells +/- Rituximab treatment (1µg/ml) at a 25:1 neutrophil:Raji ratio in the presence of luminol (50µM). Cells monitored for production of reactive oxygen species (ROS) measured by relative light units (RLU). (C) MoDC were prepared by culturing Imprime-bound monocytes isolated from whole blood in the presence of XVivo15 media supplemented with 10% autologous serum and 50 ng/mL recombinant human GM-CSF + 50 ng/mL IL-4 conditions conditions for 6 days and then maturing the cells with LPS and TNF-α (50 ng/mL) for 48 hrs. MoDC were subsequently evaluated for phenotype, enhancing CD4/CD8 T cell proliferation by CFSE-dilution assay and IFN-γ production.
Figure 4: Splenic macrophages isolated from Imprime+Bev treated mice display a M1-like phenotype.
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Figure 7: Imprime+DC101 treated animals have reduced splenic MDSCs, an increase in activated splenic macrophages, and an increase in Th1-like phenotype on cells within the tumor microenvironment.
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Minutes
Figure 2: Imprime PGG triggers a coordinated immune response in human ex vivo studies.
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Figure 3: Anti-tumor efficacy of Imprime PGG with anti-angiogenics in vivo.
Xenograft models. (A) Outline of in vivo studies. (B) Graph depicting H1299 tumor volume of individual mice at day 18 post challenge. (C) %Tumor Growth Inhibition (TGI) of H1299 tumors was calculated by %TGI= (1-individual treated mouse/median control mice)*100. (D) Graph depicting H441 tumor volume of individual mice at day 37 post challenge. (E) %TGI of H441 tumors was calculated by %TGI= (1-individual treated mouse/median control mice)*100.
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Figure 6: Imprime+Bev tumors have cells that show a more activated phenotype and a decrease in TGFβ.
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Ex-Vivo human studies: • Imprime enhances human macrophage and MoDC activation and function which
leads to crosstalk with the adaptive immune system.
• Imprime elicits the generation of Reactive Oxygen Species (ROS) from neutrophils specifically in response to antibody-coated tumor cells.
In-Vivo studies:
• Imprime PGG treatment in vivo can activate myeloid cells within both the tumor and spleen to orchestrate a profound shift in the immune microenvironment which promotes tumor recognition and suppression.
Treatment Groups (2x/week): 1. Vehicle 2. Imprime (1.2mg/ms) 3. Bevacizumab/DC101 (5/10mg/kg IP) 4. Bevacizumab/DC101 (5/10mg/kg IP)+ Imprime (1.2mg/ms IV)
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Figure 5: Imprime+Bev splenic MDSC display an increase in iNOS and decrease in Arginase 1.
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