Supplementary Information for Transient and Local Expression of Chemokine and Immune Checkpoint Traps to Treat Pancreatic Cancer

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1 Supplementary Information for Transient and Local Expression of Chemokine and Immune Checkpoint Traps to Treat Pancreatic Cancer Lei Miao 1, Jingjing Li 2, Qi Liu 1,4, Richard Feng 2, Manisit Das 1, C. Michael Lin 1, Tyler J. Goodwin 1, Oleksandra Dorosheva 2, Rihe Liu 2,3*, and Leaf Huang 1,4,* 1 Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, 2 Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, 3 Carolina Center for Genome Sciences, 4 UNC & NCSU Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. * Corresponding authors There s no conflict of interest to disclose 1

2 Supplementary Tables Table S1. Serum biochemical value analysis for tumor bearing mice Sample# BUN mg/dl Creatinine mg/dl AST U/L ALT U/L Healthy 22.0± ± ± ±10.6 PBS 24.0± ± ± ±3.6 Protein trap 33.0± ± ± ±8.9 Combo trap NP 26.0± ± ± ±8.6 Ctrl NP 30.0± ± ± ±4.8 Normal Range Numbers in red indicate that the value is over the normal range Table S2. Whole cell counts of tumor bearing mice treated with different groups Sample# WBC LYMF GRAN MONO HCT RBC HGB PLT Healthy 5.8± ± ± ± ± ± ± ±92.7 PBS 5.5± ± ± ± ± ± ± ±34.7 Protein trap 6.3± ± ± ± ± ± ± ±46.7 Combo trap NP 5.8± ± ± ± ± ± ± ±25.8 Ctrl NP 5.8± ± ± ± ± ± ± ±13.1 * Numbers in red indicate that the value is over the normal range Table S3. Serum biochemical value analysis for normal healthy mice treated with different groups Sample# BUN mg/dl Creatinine mg/dl AST U/L ALT U/L PBS 24.0± ± ± ±3.2 Protein trap 22.0± ± ± ±6.8 Combo trap NP 19.3± ± ± ±3.2 Ctrl NP 19.4± ± ± ±6.5 Normal Range All the numbers are in the normal range Table S4. Whole cell counts of normal healthy mice treated with different groups Sample# HCT RBC HGB PBS 39.2± ± ±0.8 Protein trap 41.5± ± ±0.6 Combo trap NP 36.7± ± ±0.4 Ctrl NP 36.4± ± ±0.7 * Numbers in red indicate that the value is over the normal range 2

3 Table S5. Antibodies Used in the Study Antibodies Company Catalog Application Anti-αSMA Abcam Ab5694 IF Anti-CD31 Abcam Ab28364 IF Anti-SDF1 (CXCL12) Abcam Ab9797 IF Anti-PDL1 Abcam Ab80276 IF Ant CD8α (FITC-conjugated) BD Pharmingen TM flow cyt Ant CD4 BD Pharmingen TM flow cyt (FITC-conjugated) Anti-FOXP3 BD Pharmingen TM flow cyt (PE-conjugated) Anti-CD11b BD Pharmingen TM flow cyt (FITC-conjugated) Anti-Gr1 (Ly-6G and Ly-6C) BD Pharmingen TM flow cyt (PE-conjugated) Anti-CD206 BiogLegend flow cyt (PE-conjugated) Anti-CCR7 BD Pharmingen TM flow cyt (Alexa Fluor 647-conjugated) APC Rat IgG2b, BD Pharmingen TM flow cyt ҝ Isotype Control Anti-Rabbit IgG Cell Signaling 4414 IF, flow cyt (Alex Fluor 647 Conjugate) Goat anti-rabbit IgG-HRP Santa Cruz Sc-2030 WB Anti-RFP Invitrogen R10367 WB Table S6. Primers for real-time PCR used in the study Primer Mouse IFN-γ Mouse IL12α Mouse TNF-α Mouse IL4 Mouse IL10 Mouse GAPDH Applied Biosystems/Ref Mm _m1 Mm _m1 Mm _g1 Mm _m1 Mm _m1 Mm _g1 3

4 Supplementary Figures Supplementary Figure 1. Binding affinity of monomeric PD-L1 trap. The binding of monomeric ligand at different concentrations (blue 0.4µM; red 0.2 µm; cyan 1.0 µm) to immobilizd PD-L1 with an estimated Kd at ~0.6 µm. Supplementary Figure 2. The plasmid map that codes for the secreted monomeric PD-L1 trap. Same vector was also used to assemble the pcxcl12 trap gene. The sequence of the PD-L1 trap used in this project is as follows: 4

5 GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCC CATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA CGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAG TGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGA CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAA AATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTG CTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCCACC ATGAAATGGGTCACCTTTATCAGCCTGCTGTTCCTGTTCAGCAGCGCCTACAGCGGATCCG GTCCGCCTACCTTTAGTCCGGCACTGCTGGTTGTTACCGAAGGTGATAATGCAACCTTTAC ATGCAGCTTTAGCAATACCAGCGAAAGCTTTGTTCTGAATTGGTATCGTATGAGCCCGAGC AATCAGACCGATAAACTGGCAGCATTTCCGGAAGATCGTAGCCAGCCTGGTCAGGATAGC CGTTTTCGTGTTACCCAGCTGCCGAATGGTCGTGATTTTCATATGAGCGTTGTTCGTGCAC GTCGTAATGATAGCGGCACCTATCTGTGTGGTGCAATTAGCCTGGCACCGAAAGCACAGA TTAAAGAAAGCCTGCGTGCAGAACTGCGTGTGACCGAACGTCGTGCAGAAGGCCCGCAAC CGCAACCGAAACCGCAGCCGAAACCGGAACCGGAACCGCAACCGCAAGGCGGTTCTGAG GAAGACCCCTGTGCCTGTGAGTCCATACTGAAATTTGAGGCCAAGGTGGAGGGTCTGCTG CAGGCCCTGACCAGGAAGCTGGAAGCTGTGAGCGGGCGGCTGGCTGTCCTGGAGAACAG AATCATCGCGGCCGCTGGCGCCCCTGTGCCTTATCCTGATCCCCTGGAACCTAGAGGCGG CAGCCACCACCACCATCACCACTGATGA Supplementary Figure 3. The competition assay between PD-1 and trimeric PD-L1 trap. PD-L1 immobilized on the biosensor was first saturated with 200 nm of PD-1. The complex was incubated with a mixture of 200 nm PD-1 in the presence (blue) or absence (red) of the trimeric PD-L1 trap. The trimeric ligand still bound well to PD-L1 that was saturated by PD-1, indicating its efficient disruption of the preformed interaction between PD-1 and PD-L1. 5

6 Supplementary Figure 4. Histology (H&E and Masson s trichrome) comparison of PDAC tumors from KPC primary genetic engineered mouse model (KPC), GEM Derived Allograft model (GDA) or allograft model with stable RFP/Luc transfection. All the tumor models resemble patient PDAC with dense stroma structure. Tissue staining data for KPC GEM primary tumor and KPC GDA allograft 1 are provided by Dr. Serguei Kozlov. They have been generated at the NCI-CAR. The corresponding model is to be published elsewhere. 6

7 pgfp LPD /αsma fibroblast Sample 1 Sample 2 Sample 3 pgfp LPD /RFP tumor pgfp LPD /CD31 vessel pgfp LPD /CD45 leukocytes % GFP positive cells in the specific cell type fibroblasts tumor cells blood vessels leukocytes Supplementary Figure 5. Representative images of the expression of GFP LPD in different cell types within the KPC model (n=3 from 3 different mice). The quantification is shown on right. 7

8 Inoculation KPC (RFP/ Luc) 1X Image Injection PBS pctrl LPD pcxcl12 trap LPD / pctrl NP LPD ppdl trap LPD / pctrl LPD pcombo trap LPD Combo trap protein D13 D18 D23 D28 Supplementary Figure 6. IVIS kinetic images of KPC tumor treated with different traps (n=5~7). 8

9 Supplementary Figure 7. IFN-γ ELISPOT assay of splenocytes from mice bearing orthotopic KPC pancreatic cancer with different treatments. A. Spleens were harvested from tumor bearing animals. Splenocytes were re-stimulated with extracts from normal splenocytes (control), KPC cells or KPC cells transduced with RFP and Luciferase markers. Cells secreted IFN-γ were stained with anti-ifn-γ antibody. Results are quantified and shown in the right panel. ns: not significant, *** P < n = 4. B. Spleens were harvested from tumor bearing animals with different treatments: PBS, pcxcl12 trap NPs, ppd-l1 trap NPs and Combo trap NPs. Splenocytes were re-stimulated with extracts from KPC RFP/Luc. Cells secreted IFN-γ were stained with anti-ifn-γ antibody. No significant differences were found among different treatments (n = 4). 9

10 PBS pcombo LPD /Isotype IgG pcombo LPD /an CD4 D15 D18 D21 T u m o r V o lu m e In c re m e n t (V t/v o ) PBS P B S Isotype m o / CIgG/ o mpcombo b o tra p trap N P LPD anti-cd4/ m C D 4 / CpCombo o m b o tra trap p LPD N P * * 3 0 D a y s p o s t in o c u la tio n D26 Supplementary Figure 8. CD4 T cell depletion assay. Mice bearing KPC tumors were pretreated with 3 injection of CD4 mab (200 µg/mice) to deplete CD4 in the mice. Isotype mab were used as control. The efficacy of combo trap NP in mice with or without CD4 depletion were compared and quantified. Plasmid NPs were injected on Day 15, Day 18 and Day 21 respectively. 10

11 Supplementary Figure 9. Accumulation (A) and distribution (B) of DiI labeled LPD NPs in different organs and tumors respectively, from mice bearing KPC98027 allografts treated with either PBS or combo trap NPs. 11

12 Supplementary Figure 10. Collagen coverage within tumors from mice treated with different groups 12