Volume 24, Issue 12, Pages 2078-2089 (December 2016) Targeted Delivery of an Anti-inflammatory PDE4 Inhibitor to Immune Cells via an Antibody–drug Conjugate  Shan Yu, Aaron D Pearson, Reyna KV Lim, David T Rodgers, Sijia Li, Holly B Parker, Meredith Weglarz, Eric N Hampton, Michael J Bollong, Jiayin Shen, Claudio Zambaldo, Danling Wang, Ashley K Woods, Timothy M Wright, Peter G Schultz, Stephanie A Kazane, Travis S Young, Matthew S Tremblay  Molecular Therapy  Volume 24, Issue 12, Pages 2078-2089 (December 2016) DOI: 10.1038/mt.2016.175 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Generation and characterization of PDE4 inhibitor payload drug. (a) GSK256066 and its derivative linker analogs. (b) PDE4B enzymatic activity inhibition by a range of concentrations of compound 1 to 6 was assessed. (c) THP-1 cells stably transfected with CRE-Luc (THP-1 CRE-Luc cells) were treated with a range of concentrations of compound 1 to 6 for 6 hours, CRE-Luc activity by a range of concentrations of compound 1 to 6 was assessed. (d) THP-1 CRE-Luc cells were treated with a range of concentrations of compound 1 to 6 for 24 hours, and the cell viability was assessed. (e) PDE4B inhibition IC50 of assay (b) and CRE-Luc activity EC50 of assay (c) of compound 1 to 6 was calculated. Results in (b–d) are presented as means ± SD. Molecular Therapy 2016 24, 2078-2089DOI: (10.1038/mt.2016.175) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Characterization of the anti-inflammatory effect of αhuCD11a-PDE4 ADC in vitro. (a) 3-linker was synthesized based on compound 3 for antibody conjugation. This PDE4 inhibitor linker derivative was then conjugated to αhuCD11a-pAcF to generate αhuCD11a-PDE4 ADC. (b) Freshly isolated human PBMCs, monocytes and T cells were treated with various concentrations of αhuCD11a-PDE4, αhuCD11a-pAcF, and GSK256066. The highest concentration on right side for αhuCD11a-PDE4 and αhuCD11a-pAcF was 100 nmol/l, and for GSK256066 was 30 nmol/l. All treatments from right to left are in 1:3 serial dilution. After 7-hour incubation, PBMCs and monocytes were treated with 100 ng/ml LPS and T cells were treated with CD3/CD28 Dynabeads in 1:1 ratio. After 20-hour stimulation, TNFα levels in the cell culture supernatants were assessed by an HTRF assay. TNFα levels were normalized to LPS or CD3/CD28 treatment alone. Results are presented as means ± SD. Molecular Therapy 2016 24, 2078-2089DOI: (10.1038/mt.2016.175) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Characterization of the anti-inflammatory effect of αmuCD11a-PDE4 ADC in vitro. (a) PDE4 inhibitor linker derivative (3-linker-NHS) basing on compound 3 was conjugated to αmuCD11a to generate αmuCD11a-PDE4 ADC. (b) Mouse primary peritoneal cells and mouse embryonic fibroblasts (MEFs) were incubated with αmuCD11a-PDE4, αmuCD11a or Rat IgG control for 7 hours before being fixed and stained with an anti-rat IgG secondary antibody conjugated with Alexa 488 (green). The nuclei was stained with Hoescht (blue). The cells were imaged with confocal microscopy. (c) Mouse primary peritoneal cells were treated with various concentrations of αmuCD11a-PDE4, αmuCD11a, or GSK256066. The highest concentration on the right side for αmuCD11a-PDE4 and αmuCD11a was 900 nmol/l with 1:3 serial dilution from right to left, and for GSK256066 was 1,000 nmol/l with 1:10 serial dilution from right to left. TNFα IC50 values for αmuCD11a-PDE4 and GSK256066 in this assay were calculated and shown in a table. Results are presented as means ± SD. Molecular Therapy 2016 24, 2078-2089DOI: (10.1038/mt.2016.175) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Anti-inflammatory effect of αmuCD11a-PDE4 ADC is mediated through CD11a receptor. (a) A schematic graph showing that αmuCD11a-PDE4 binds CD11a receptor and exerts its anti-inflammatory effect, while occupancy of CD11a receptor by αmuCD11a blocks αmuCD11a-PDE4 from accessing to CD11a receptor, thus abrogating αmuCD11a-PDE4's anti-inflammatory effect. (b) Mouse peritoneal cells were treated with different concentrations of αmuCD11a as indicated for 30 minutes before αmuCD11a-PDE4 or GSK256066 was added. After 7-hour incubation, cells were stimulated with 100 ng/ml LPS for 20 hours. The TNFα content in the supernatant was assessed by an HTRF assay. The results are normalized to LPS treatment alone and are presented as means ± SD. (c) Peritoneal macrophages or MEFs were stained with IgG control, CD11a (2D7) or αmuCD11a antibodies conjugated with Alexa 488 and were analyzed by flow cytometry. (d) Mouse primary peritoneal cells (upper panel) or MEFs (lower panel) were treated with various concentrations of αmuCD11a-PDE4, αmuCD11a, rat IgG control or GSK256066. The highest concentration from right side for αmuCD11a-PDE4, αmuCD11a and IgG control was 300 nmol/l with 1:3 serial dilution from right to left, and for GSK256066 was 1,000 nmol/l with 1:10 serial dilution from right to left. The results are normalized to LPS treatment alone, and are presented as means ± SD. Molecular Therapy 2016 24, 2078-2089DOI: (10.1038/mt.2016.175) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Mouse surrogate αmuCD11a-PDE4 inhibitor ADC reduces inflammation in a mouse carrageenan air pouch model. (a) A schematic graph showing timelines for air pouch generation and drug administration. (b) αmuCD11a (M17 clone) levels in the diluted air pouch exudate and serum samples were assessed using an anti-rat IgG ELISA. (c) Exudate cytokines including TNFα, IFNγ, IL-6, IL-12p70, MCP-1, and IL-10 were assessed by a mouse inflammatory CBA assay kit. The results are presented as means ± SEM. n = 6 per group. (d) Exudate cells were stained with appropriate antibodies and subjected to flow cytometry analysis. The live cells that were CD45-positive and live/dead stain negative were designated as live CD45+ cells, within which the percentage of different cell populations were analyzed, including CD3+ T cells, CD11b+ Ly6C+ F4/80- monocytes, CD11b+ Ly6C+ F4/80+ macrophages, CD11b+ Ly6G+ Ly6Cint neutrophils. (e) Binding of αmuCD11a (M17 clone) on different exudate immune cell population was assessed by anti-rat IgG stain. (f) CD11a expression on different immune cell populations in the exudate was assessed by flow cytometry. Molecular Therapy 2016 24, 2078-2089DOI: (10.1038/mt.2016.175) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 αmuCD11a-PDE4 and αmuCD11a modulate the composition and activation of immune cell population in the spleen. (a) Spleens of mice in the experiment as shown in Figure 5 were collected. The single cell suspension was stained with appropriate antibodies and subjected to flow cytometry analysis. The cells that were CD45-positive and live/dead stain negative were designated as live CD45+ cells, within which the percentage of different cell populations were analyzed, including CD3+ T cells, CD11b+ Ly6C+ F4/80- monocytes, CD11b+ Ly6C+ F4/80+ macrophages, CD11b+ Ly6G+ Ly6Cint neutrophils. (b) Actual numbers of different immune cells in the spleen as calculated basing on the total numbers of cells of each spleen sample. (c) Percentage of different subsets of CD3+ T cell population with differential CD25 and CD69 expression. (d) Binding of αmuCD11a (M17 clone) on different exudate immune cell population was assessed by anti-rat IgG stain. (e) CD11a expression on different immune cell populations in the exudate was assessed by flow cytometry. (f) Binding of αmuCD11a (M17 clone) on CD45+ splenic cells that express different levels of CD11a from mice that were treated with αmuCD11a-PDE4 and αmuCD11a. Molecular Therapy 2016 24, 2078-2089DOI: (10.1038/mt.2016.175) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions