Annexin-A1: A New Immunomodulation / Checkpoint...
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Cell Proliferation: A panel of cancer cell lines were treated with MDX-124, a commercial anti-ANXA1 antibody or IgG isotype control (0-10 μM) for 72 hours. Cell viability was measured via MTT assay (n = 3). Statistical significance was calculated via Mann-Whitney test and indicated by ***p<0.001 and ****p<0.0001 (MDX-124 vs IgG) or oo p<0.01 and ooo p<0.001 (MDX-124 vs commercial antibody). Imaging Flow Cytometry: Samples from each cell line were prepared for ImageStreamX® analysis. Multispectral images of cells in brightfield, green (MDX-124) and red (nuclear staining – DRAQ5) were captured. Cellular localization of MDX-124 was determined using the Ideas™ imaging analysis software. In-vivo Efficacy Study: MDX-124 was evaluated in a syngeneic orthotopic 4T1-luc TNBC mouse model. BALB/c mice (n = 12) were randomized to treatment groups and dosed with vehicle control (PBS) or MDX-124 (1 mg/kg) by IV injection. Statistical significance was calculated via 2-tailed unpaired T-test and indicated by *p<0.05 and **p<0.01. Annexin-A1 (ANXA1) is a member of the annexin protein superfamily that binds acidic phospholipids in a calcium-dependent manner. Increased expression of ANXA1 has been shown in a range of cancers including pancreatic, renal, triple-negative breast (TNBC) and bladder. ANXA1 has been demonstrated to influence cancer cell proliferation, angiogenesis and migration (1) , as well exerting immunomodulatory effects on T-cells, macrophages and dendritic cells (2,3) . We have developed a humanized monoclonal antibody targeting ANXA1 (MDX-124) and here we present data showing its anti-proliferative effect on a panel of cancer cell lines which correlates with ANXA1 expression. Furthermore, we have shown efficacy in a mouse model of triple negative breast cancer. METHODS Presented at Transdisciplinary Cancer Interception: Leveraging Biology to Improve Prevention and Detection Organized by Huntsman Cancer Institute at the University of Utah and Nature Reviews Cancer. March 9-11, 2020. Salt Lake City, Utah, USA Annexin-A1: A New Immunomodulation / Checkpoint Pathway in Cancer Fiona C Dempsey 1 , Hussein Al-Ali 2 , Scott J Crichton 1 , Henry C W Hays 1 , Christopher N Parris 2 , Christopher B Wood 1 . 1 Medannex Ltd, Edinburgh, UK; 2 Anglia Ruskin University, Cambridge, UK. INTRODUCTION Our results demonstrate that MDX-124 can suppress cancer cell growth through targeted inhibition of ANXA1. This indicates that MDX-124 is a promising first-in-class monoclonal antibody therapeutic against cancers overexpressing ANXA1. Further studies are under way to understand the mechanism of action of MDX-124. CONCLUSIONS RESULTS RESULTS References: (1) Boudhraa et al., (2016). Clinical Science, 130 (4): 205–220. (2) Tu et al., (2017). Pharmacological Research, 119: 278–288. (3) Moraes et al., (2018). Cell Adhesion & Migration, 12 (5), 417–423. Incubation of a panel of breast, colorectal, pancreatic and ovarian cancer cell lines with MDX-124 (0-10 µM) for 72 hours resulted in a statistically significant reduction in cell proliferation (Figure 1). MDX-124 was then compared to an IgG isotype control or a commercial anti-ANXA1 polyclonal antibody in a selection of cancer cell lines. MDX-124 demonstrated a statistically significant 3-fold increase in potency versus control or commercial antibody. Representative data obtained are shown in Figure 2. 0 5 10 0 20 40 60 80 100 120 Antibody Concentration (µM) Cell Viability (% signal rela1ve to untreated) Colorectal (Caco-2) Colorectal (SW480) Colorectal (HCT 116) Pancreatic (PANC-1) Pancreatic (MIA PaCa-2) Pancreatic (BxPC-3) Ovarian (A2780) Breast (HCC1806) Ovarian (A2780adr) Ovarian (A2780cis) Breast (MCF-7 TMR) Breast (MCF-7) Figure 1. Anti-proliferative effect of MDX-124 in a panel of cancer cell lines. 0.0 2.5 5.0 7.5 10.0 0 20 40 60 80 100 120 Breast (HCC1806) 113% 100% 37% IgG Control Commercial Antibody MDX-124 Figure 2. Anti-proliferative effect of MDX-124 in HCC1806 triple negative breast cancer cells. 0 2 4 6 8 10 12 14 16 0 50 100 150 200 250 Dosing day Tumour volume (mm 3 ) Vehicle (PBS) MDX-124 (1 mg/kg) * ** ** ** Figure 4. MDX-124 reduces 4T1-luc breast tumour growth. Arrows indicate day of dosing. MDX-124 has been assessed for binding affinity to ANXA1 by Biacore and found to have a KD of 3.98 nM. The antibody has demonstrated strong binding to ANXA1, but poor binding to the most structurally similar annexin protein (ANXA2) by ELISA, indicating specificity to the target molecule. Imaging flow cytometry was used to quantitate the level of ANXA1 protein expression in different cellular compartments of cancer cells treated with MDX-124 (5 μM) for 72 hours. An MDX-124 sensitive cell line (Figure 3A) showed significant ANXA1 protein expression, particularly at the plasma membrane (Figure 3C). This was not observed in an MDX-124 refractory cell line (Figure 3B), which showed little to no observable ANXA1 protein expression (Figure 3D). These data indicate the anti-proliferative effect of MDX-124 correlated with levels of ANXA1 protein expression, particularly at the plasma membrane. Further in-vivo studies evaluated MDX-124 (1 mg/kg) in the syngeneic, orthotopic, 4T1-luc triple- negative breast cancer model. Following dosing once per week we identified a statistically significant reduction in tumor volume compared to the vehicle control (*p<0.05 and **p<0.01), without any associated significant change in body weight (Figure 4). 0.0 2.5 5.0 7.5 10.0 0 20 40 60 80 100 120 Antibody Concentration ( µM) Lung (COR-L23) 100% 87% IgG Control MDX-124 0.0 2.5 5.0 7.5 10.0 0 20 40 60 80 100 120 Antibody Concentration ( µM) Pancreatic (BxPC-3) 91% 50% *** **** **** **** IgG Control MDX-124 A B D C Figure 3. Anti-proliferative effect of MDX-124 correlates with annexin-A1 expression.
Transcript of Annexin-A1: A New Immunomodulation / Checkpoint...
Cell Proliferation: A panel of cancer cell lines were treated with MDX-124, a commercialanti-ANXA1 antibody or IgG isotype control (0-10 μM) for 72 hours. Cell viability wasmeasured via MTT assay (n = 3). Statistical significance was calculated via Mann-Whitneytest and indicated by ***p<0.001 and ****p<0.0001 (MDX-124 vs IgG) or oop<0.01 andooop<0.001 (MDX-124 vs commercial antibody).
Imaging Flow Cytometry: Samples from each cell line were prepared for ImageStreamX®analysis. Multispectral images of cells in brightfield, green (MDX-124) and red (nuclearstaining – DRAQ5) were captured. Cellular localization of MDX-124 was determined usingthe Ideas™ imaging analysis software.
In-vivo Efficacy Study: MDX-124 was evaluated in a syngeneic orthotopic 4T1-luc TNBCmouse model. BALB/c mice (n = 12) were randomized to treatment groups and dosed withvehicle control (PBS) or MDX-124 (1 mg/kg) by IV injection. Statistical significance wascalculated via 2-tailed unpaired T-test and indicated by *p<0.05 and **p<0.01.
Annexin-A1 (ANXA1) is a member of the annexin protein superfamily that binds acidicphospholipids in a calcium-dependent manner. Increased expression of ANXA1 has beenshown in a range of cancers including pancreatic, renal, triple-negative breast (TNBC) andbladder. ANXA1 has been demonstrated to influence cancer cell proliferation, angiogenesisand migration(1), as well exerting immunomodulatory effects on T-cells, macrophages anddendritic cells(2,3). We have developed a humanized monoclonal antibody targeting ANXA1(MDX-124) and here we present data showing its anti-proliferative effect on a panel ofcancer cell lines which correlates with ANXA1 expression. Furthermore, we have shownefficacy in a mouse model of triple negative breast cancer.
METHODS
Presented at Transdisciplinary Cancer Interception: Leveraging Biology to Improve Prevention and Detection Organized by Huntsman Cancer Institute at the University of Utah and Nature Reviews Cancer. March 9-11, 2020. Salt Lake City, Utah, USA
Annexin-A1: A New Immunomodulation / Checkpoint Pathway in Cancer
Fiona C Dempsey1, Hussein Al-Ali2, Scott J Crichton1, Henry C W Hays1, Christopher N Parris2, Christopher B Wood1.1Medannex Ltd, Edinburgh, UK; 2Anglia Ruskin University, Cambridge, UK.
INTRODUCTION
Our results demonstrate that MDX-124 can suppress cancer cell growth through targetedinhibition of ANXA1. This indicates that MDX-124 is a promising first-in-class monoclonalantibody therapeutic against cancers overexpressing ANXA1. Further studies are under wayto understand the mechanism of action of MDX-124.
CONCLUSIONS
RESULTS
RESULTS
References: (1) Boudhraa et al., (2016). Clinical Science, 130 (4): 205–220. (2) Tu et al., (2017). Pharmacological Research, 119: 278–288. (3) Moraes et al., (2018). Cell Adhesion & Migration, 12 (5), 417–423.
Incubation of a panel of breast, colorectal, pancreatic and ovarian cancer cell lines withMDX-124 (0-10 µM) for 72 hours resulted in a statistically significant reduction in cellproliferation (Figure 1). MDX-124 was then compared to an IgG isotype control or acommercial anti-ANXA1 polyclonal antibody in a selection of cancer cell lines. MDX-124demonstrated a statistically significant 3-fold increase in potency versus control orcommercial antibody. Representative data obtained are shown in Figure 2.
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Figure 1. Anti-proliferative effect of MDX-124 in a panel of cancer cell lines.
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Figure 2. Anti-proliferative effect of MDX-124 in HCC1806 triple negative breast cancer cells.
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Figure 4. MDX-124 reduces 4T1-luc breast tumour growth. Arrows indicate day of dosing.
MDX-124 has beenassessed for bindingaffinity to ANXA1 byBiacore and found tohave a KD of 3.98 nM.The antibody hasdemonstrated strongbinding to ANXA1, butpoor binding to themost structurallysimilar annexin protein(ANXA2) by ELISA,indicating specificity tothe target molecule.
Imaging flow cytometrywas used to quantitatethe level of ANXA1protein expression in
different cellular compartments of cancer cells treated with MDX-124 (5 μM) for 72 hours.An MDX-124 sensitive cell line (Figure 3A) showed significant ANXA1 protein expression,particularly at the plasma membrane (Figure 3C). This was not observed in an MDX-124refractory cell line (Figure 3B), which showed little to no observable ANXA1 proteinexpression (Figure 3D). These data indicate the anti-proliferative effect of MDX-124correlated with levels of ANXA1 protein expression, particularly at the plasma membrane.
Further in-vivo studiesevaluated MDX-124 (1mg/kg) in the syngeneic,orthotopic, 4T1-luc triple-negative breast cancermodel. Following dosingonce per week weidentified a statisticallysignificant reduction intumor volume comparedto the vehicle control(*p<0.05 and **p<0.01),without any associatedsignificant change in bodyweight (Figure 4).
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Figure 3. Anti-proliferative effect of MDX-124 correlates with annexin-A1 expression.
