supplementary data mpd-1.fchis control dna mp5 mp7 mp23 figure s1. enriched aptamers pull down...
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Supplementary Data
mPD
-1.F
cHIS
Con
trol
DN
AM
P5
MP7
MP2
3
Figure S1. Enriched aptamers pull down mPD-1.FcHIS. Biotinylated versions of MP5, MP7, and MP23 when immobilized onto streptavidin magnetic beads immunoprecipitated mPD-1.FcHIS. Bound mPD-1.FcHIS was identified by western blotting using anti-HIS HRP antibody. An irrelevant DNA sequence used as a negative control did not pull down mPD-1.FcHIS.
Anti-PD-1 mAb 29F.1A12
Anti-PD-1 mAb RMPI-14
MP5 MP7
PD-1
Isotype Control mAb
cSeq
Figure S2. Aptamers MP5 and MP7 bind PD-1 expressing cells. Pre-blocked P815 mouse mastocytoma cells were stained with FAM labelled aptamers or FITC labelled antibodies as detailed (see Methods). Histograms shown are of the reagent used (blue) relative to unstained P815 cells (red). Both MP5, MP7, and two known anti-PD-1 antibodies bound P815 cells, while an irrelevant aptamer and isotype matched antibody showed negligible staining.
CF
SE
-lo
w%
anti-
CD3
anti-
CD3 +
mPD-L
1.Fc
MP5
MP7
30
35
40
45
50
55
a b
c d
Figure S4. MP7 restores anti-CD3 induced PD-L1 suppressed lymphocyte proliferation. CFSE labelled splenocytes were stimulated by plate bound anti-CD3 antibody in the presence or absence of PD-L1 and CFSE dilution profiles analysed by flow cytometry. (a) Comparison of CFSE dilution profiles from splenocytes cultured in anti-CD3 coated wells (2g/ml), or wells with anti-CD3 and PD-L1.Fc (15g/mL). Effect of anti-PD-1 aptamer MP7 (b) or MP5 (c) on PD-L1 suppressed proliferation. Note increased amount of CFSE-low% in MP7 treated lymphocytes. (d) Summary of experiment. Each histogram represents the % of CFSE-low cells in each group of duplicate wells ± SD.
IL2
SF
U/2
x105
cell
sUnst
imula
ted
PBS M
P5M
P70
100
200
300
1g/mL anti-CD3
Figure S3. Anti-PD-1 DNA Aptamers do not stimulate IL-2 secretion in the absence of PD-L1 signalling. Aptamers were added to primary splenocytes which were undergoing polyclonal stimulation from plate-bound anti-CD3 antibody, and the changes in IL-2 secretion monitored by ELISPOT. Histograms represent mean SFU ± SD (n=2).
% P
D-1
Blo
cke
d
No Apta
mer
PEG-MP7
MP7
mAb
0
25
50
75
100
Figure S6. PEGylation does not disrupt the ability of MP7 to block the PD-1:PD-L1 interaction. PEGylated and non-PEGylated MP7 similarly inhibit the binding of soluble mPD-1.FcHIS from binding to plate-bound mPD-L1.Fc in a competitive ELISA experiment. Each histogram represents the % PD-1 blocked from binding PD-L1 ± SD (n=3).
Figure S5. Purification of PEGylated aptamers. The PEGylation reaction products were prepared in 0.1M TEAA and purified by reverse phase HPLC using a C18 OST semi-preparative column. The bound PEGylated aptamers were eluted using a linear gradient going from 5-90% CH3CN over a 60-minute period. Absorbance (UV260nm) was used to track elution of free nucleic acid and nucleic acid conjugates. The second peak at 22 minutes corresponds to unreacted aptamer and the last peak (38 minutes) corresponds to PEGylated aptamer.
Column: xBridge C18 OST (10x50mm)Mobile Phase A: 0.1M TEAA + 5% CH3CNMobile Phase B: 0.1M TEAA + 90% CH3CNGradient: 0-100%B over 60minFlow Rate: 2.2 mL/min
MP7
PEG MP7NHS
0 1 4 7
20µg Tx (i.p.)
Day
Experimental Groups
1. PBS 2. PEG-N54
3. PEG-MP7 4. RMPI-14 mAb
MC38.CEA (i.p.)
14
a
PBSN54
MP7
mAb
0
20
40
60
Nu
mb
er o
f P
erit
on
eal
Tu
mo
r N
od
ule
s
NS
******
PBSN54
MP7
mAb
0
500
1000
1500
Tu
mo
r V
olu
me
(m
m3)
*
******
b
c d
Figure S7. PEGylated MP7 suppresses growth of aggressive disseminated PD-L1+ colon carcinoma MC38.CEA cells in-vivo. (a) Experimental outline of in-vivo tumor model. MC38.CEA cells were injected i.p. into groups of C57Bl/6 mice (n=5; day 0) and groups of animals were subsequently treated with either RMPI-14 mAb, PEG-MP7 or a CEA-specific aptamer PEG-N54 on days 1, 4, and 7. (b) Photographs highlighting tumor burden in the intraperitoneal cavity of each group after 14 days. The total number of tumor nodules (c) and cumulative volume of each tumor (d) in the intraperitoneal cavity of each animal was quantified upon necropsy. Treatment with the anti-PD-1 aptamer MP7 or monoclonal antibody resulted in significant reductions in tumor burden. *P<0.05, ***P<0.001, NS=not significant.
PBS PEG-N54
PEG-MP7 RMPI-14
IFNα
IL12
β-actin
CpG -
PEGMP7
Figure S7. PEGylated MP7 does not induce transcription of genes associated with TLR-9 triggering. Agarose gel electrophoresis analysis of IFNα, IL12, and β-actin RT-PCR reactions. RNA was isolated from RAW264.7 mouse macrophages treated with CpG ODN 1585, PEG-MP7, or left untreated, with only CpG treatment showing upregulated transcription levels of theseTLR9 associated cytokines.