Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T- lymphocytes

Size: px

Start display at page:

Download "Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T- lymphocytes"

Ami Knight
5 years ago
Views:

1 Supporting Online Mteril for Heprnse promotes tumor infiltrtion nd ntitumor ctivity of -redirected T- lymphocytes IgnzioCrun, Brr Svoldo, VlentinHoyos, Gerrit Weer, Ho Liu, Eugene S. Kim, Michel M. Ittmnn, Drio Mrchettind GinpietroDotti* *To whom correspondence should e ddressed. E-mil: gdotti@cm.edu 1 Nture Medicine: doi:1.138/nm.3833

2 Supplementl Figures 65 kd 5 kd 65 kd 5 kd 293T + -ctin FI-T 3d LTE-T in IL-2 (5U/mL) 7d 1d LTE-T in IL-2 (2U/mL) 3d 7d 1d LTE-T in IL-7 (1ng/mL) IL-15 (5ng/mL) 3d 7d 1d #1 #2 fold chnge compred to CD14 + cells LTE-t in IL-2 (5U/mL) LTE-T in IL-2 (5U/mL) + AB serum LTE-T in IL-2 (2U/mL) LTE-T in IL-7 (1ng/mL)+IL-15 (5ng/mL) FI-T ctin Time (d) c 65 kd 5 kd 293T + FI-T Medi with FBS Medi with hab serum hab lone Supplementry Fig. 1. down regultion in LTE-T cells is not cused y specific culture conditions. T lymphocytes were ctivted with immoilized OKT3 (1 μgml) nd nti- CD28 (1 μgml) As, nd then expnded in complete medium supplemented with either 1% FBS or AB serum (5%), nd either IL-2 (5 UmL) or IL-2 (2 UmL) or IL-7 (1 ngml) nd IL- 15 (5 ngml) twice week. Expression of ws ssessed in cell lystes y western lot () nd qrt-pcr () t dy 3, 7 nd 1 fter T-cell ctivtion. (,)Illustrte dt from 2 nd 4 donors, respectively. We lso ssessed whether the presence of FBS in the culture medi ffected expression. We removed FBS from the medi nd used humn AB serum (5%). mrna ws down regulted lso in the presence if hab serum s ssessed y qrt-pcr (). Western lot is not informtive in the presence of hab serum since, s illustrted in (c), hab serum contins. 2 Nture Medicine: doi:1.138/nm.3833

3 T= T= 18h T= 72h T= 1d PBMC CD3 + CD45RA + CD45RO + CD62L + CD45RO + CD62L - PBMC CD3 + CD45RA + CD45RO + CD62L + CD45RO + CD62L - CD3 + CD45RA + CD45RO + CD62L + CD45RO + CD62L - CD3 + CD45RA + CD45RO + CD62L + CD45RO + CD62L - 65 kd 5 kd -ctin CTLs stim. OKT3 Donor#1 EBV-CTLs II stim Donor#1 EBV-CTLs IV stim Donor#2 EBV-CTLs III stim Donor#2 EBV-CTLs VI stim Donor#3 EBV-CTLs III stim Donor#3 EBV-CTLs VI stim 293T 6 kd c % of invsion 5% 4% 3% 2% 5 kd 1% GAPDH % FI-T LTE-T EBV- CTLs Supplementry Fig. 2. expression in specific T-cell susets. ()We seprted nive CD45RA +, centrl-memory CD45RO + CD62L + nd effector-memory CD45RO + CD62L - from the peripherl lood nd ctivted these susets with immoilized OKT3 nd nti-cd28 As nd IL- 2. Cells were collected t different time points fter ctivtion to ssess the expression of y western lot. Totl PBMC were used s positive control. Dt illustrte the trnsition from the inctive (65 kd) to the ctive (5 kd) form of in T-cell susets in representtive donor in whom oth forms of the protein were clerly detectle. ws not detectle y dy 1 fter ctivtion in LTE-T cells regrdless of whether these cells were derived from nive, centrl-memory of effector-memory T cells. ()Epstein Brr Virus-specific cytotoxic T cells (EBV-CTLs) were generted y weekly stimultion with lympholstoid cell lines 1. ws undetectle y western lot in 3 different EBV-CTL lines. 293T cells trnsfected with the vector encoding were used s positive control.(c) Invsion of FI-T cells, LTE-T cells nd estlished EBV-CTLs into the BD BioCotMtrigel TM invsion chmer. Five percent FBS medium ws used s chemottctnt. 3 Nture Medicine: doi:1.138/nm.3833

4 Fold chnge compred to T= in CD4 + T cells p Fold chnge compred to T= in CD8 + T cells Time (h) Time (d) Time (h) Time (d) p53 Supplementry Fig. 3.p53 is upregulted in LTE-T cells. qrt-pcr of nd p53 in CD4 + nd CD8 + T cells t different time points of culture. Dt summrize mens ± s.d. of 3 donors. Dy 15 shows expression in CD4 + nd CD8 + LTE-T cells cultured for 14 dys, nd re-stimulted with immoilized OKT3 nd nti-cd28 As for 24 hrs. 4 Nture Medicine: doi:1.138/nm.3833

CD3 + LTE-T 1 4 1 3 1 2 1 1 41% 1% 4% 54% 1 1 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 1 4 1 3 1 2 1 1 21% 1% 6% 72% 1 4 1 3 1 2 1 1 97% 1% 2% % 1 1 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 GFP Control (I) 1 4

5 CD3 + LTE-T % 1% 4% 54% % 1% 6% 72% % 1% 2% % GFP Control (I) % 7% 1% 6% % 1% Insert 1% % % 9% ECM 4% 11% % Remining Tumor cell (GFP + ) * Control Insert Chmer (I) ** Supplementry Fig. 4.Epression of in GD2-specific -modified LTE-T cells enhnces ntitumor ctivity in the presence of ECM. (,) Control nd trnsduced LTE-T cells were plted in the upper prt of either ECM ssy or insert ssy, while CHLA-255- GFP + tumorcells were plted in the lower chmer. After dy 3 of culture, cells in the lower chmer were collected to quntify CD3 + T cells nd GFP + tumor cells y flow cytometry. ()Illustrtes representtive dot plots of the ssy, while ()summrizes men ± s.d. of 5 donors, *p=.1, **p= Nture Medicine: doi:1.138/nm.3833

6 (I) Fold expnsion CD8 c % of Control (I) Dy fter trnsduction CD45RA CD45RO CD45RA CCR7 CD62 (I) L CD8 + CD4 + CD45RA + CD45RO + CCR7 + CCR7 + CD45RO d (I) e % + cells 7AAD Resting OKT3 A 1A7 A 1 4 2% 1 4 2% 11% 1 21% 12% 31% % 6% 59% 9% 49% 8% % 1 2% 9% 1 14% 2% 26% % 8% 67% 1% 44% 1% AnnV Resting OKT3 A 1A7 A (I) (I) (I) AnnV + /7AAD - AnnV + /7AAD + AnnV - /7AAD - Supplementry Fig. 5.Expression of in GD2-specific -modified LTE-T cells does not hmper their growth, phenotype or susceptiility to ctivtion induced cell deth. ()Illustrtes the growth kinetics of control, + nd (I) + LTE-T cells. (,c)show the phenotypic nlysis of control nd + nd (I) + LTE-T cells y dy 14 of culture. The gret mjority of LTE-T cells re effector-memory T cells. (d,e)illustrte the susceptiility of control, + nd (I) + LTE-T cells to ctivtion-induced cell deth. By dy 14 of culture, LET-T cells were stimulted with either OKT3Ato ctivte T cells through TCR or the nti-idiotype 1A7 A tht induces cross-linking of the. Apoptosis ws ssessed y Annexin-V nd 7-ADD stining fter 24 hours. Presented dt summrize men ± s.d. of 4 donors. 6 Nture Medicine: doi:1.138/nm.3833

7 Control (I) (I) Time (d) fter LTE-T infusion Time (d) fter LTE-T infusion Supplementry Fig. 6.In vivo imging. () IVIS imging of representtive nimls infused intrperitonelly with LAN-1 tumor cells lelled with fireflyluciferse. Control, + nd (I) + LTE-T cells were infused intrperitonelly y dy 1-12 fter tumor inocultion. ()IVIS imging of representtive nimls surgiclly implnted under the renl cpsulewith CHLA-255 tumor cells lelled with fireflyluciferse. Control, + nd (I) + LTE-T cells were infused intrvenously y dy 7fter tumor inocultion. 7 Nture Medicine: doi:1.138/nm.3833

8 NT (I) Time (d) fter LTE-T infusion 1.x x1 1 5.x1 1 Control (I) 8 2 p/sec/cm 2 /sr 2.5x1 1 6x1 9 4x1 9 2x1 9 p= Time fter LTE-T infusion (d) Supplementry Fig. 7.Expression of in CSPG4-specific -modified LTE-T cells improves the ntitumor effects in n ggressive melnom model. NSG mice were inoculted intrperitonelly with 5x1 5 SENMA cells (CSPG4 + melnom) resuspended in Mtrigel nd lelled with firefly luciferse. By dy 2, mice were infused intrperitonellywith control,.cspg4 (4-1BB costimultion) or.cspg4 (4-1BB costimultion)(i) LTE- T cells (1 7 cells) 2. For these experiments, LTE-T cells were cultured in IL-7 nd IL-15 since these cytokines prolong the persistence of LTE-T cells in NSG mice 3. ()Illustrtes tumor growth in 3 representtive nimls per group, s ssessed y IVIS imging. ()Shows the summry of the tumor ioluminescence of 2 experiments (8 mice per group using 2 donors). 8 Nture Medicine: doi:1.138/nm.3833

Time (d) fter LTE-T infusion 7 1 17 24 31 Control 19 19(I) c Percent of survivl 1 Control 8 19 19(I) 6 4 2 2 4 6 8 Time fter LTE-T infusion (d) CD3 d % Control 75% 68% (I) p/sec/cm 2 /sr 5x1 9 5x1 8

(I) LTE-T LTE-T infused in collected from mice infused mice Supplementry Fig. 8.Expression of in CD19-specific + LTE-T cells hs noeffects in lymphoid mlignncies.

cd19 (4-1BB costimultion)(i) LTE-T cells (1 7 cells). For these experiments, LTE-T cells were cultured in IL-7 nd IL-15 since these cytokines prolong the persistence of LTE-T cells in NSG mice 3.

9 Time (d) fter LTE-T infusion Control 19 19(I) c Percent of survivl 1 Control (I) Time fter LTE-T infusion (d) CD3 d % Control 75% 68% (I) p/sec/cm 2 /sr 5x1 9 5x1 8 5x1 7 5x1 6 5x Control 19 19(I) Time fter LTE-T infusion (d) e fold chnge compred to CD14 + cells FI-T Cont. (I) Cont. (I) LTE-T LTE-T infused in collected from mice infused mice Supplementry Fig. 8.Expression of in CD19-specific + LTE-T cells hs noeffects in lymphoid mlignncies. NSG mice were inoculted intrvenously with 2x1 6 Dudi (CD19 + lymphom) lelled with firefly luciferse. By dy 3, mice were infused intrvenously with control,.cd19 (4-1BB costimultion) or.cd19 (4-1BB costimultion)(i) LTE-T cells (1 7 cells). For these experiments, LTE-T cells were cultured in IL-7 nd IL-15 since these cytokines prolong the persistence of LTE-T cells in NSG mice 3. ()Illustrtes the tumor growth in representtive nimls s ssessed y IVIS imging. ()Shows the summry of the tumor ioluminescenceof 2 experiments (1 mice per group using 2 donors). (c)illustrtes survivl of the infused mice. (d)illustrtes representtive phenotype for the expression of collected T cells. (e)mice were inoculted intrvenously with control, + nd (I) + LTE-T cells specific for the CD19 ntigen nd euthnized y dy 3 fter infusion. Humn T cells were collected from the spleen of these mice nd enriched y CD45 mgnetic selection. mrna mesured y qrt-pcr showed tht remins upregulted in (I) + LTE-T cells compred to control or + LTE-T cells, in LTE-T cells collected from the spleen of the infused mice y dy 3. Dt summrizes the nlysis of 3 nimls per group. 9 Nture Medicine: doi:1.138/nm.3833

10 Control (I) Control (I) Lung Liver Brin Supplementry Fig. 9A.Expression of does not ffect LTE-T cell iodistriutionin vivo.control, + nd (I) + LTE-T cells were infused vi til injection in NSGmice. Mice were euthnized 24 hours fter T-cell infusion to mesure erly pssge T-cell iodistriution in lung, liver nd rin. (,)Illustrte hemtoxylin nd eosin stining (2X mgnifiction) nd nti-humn CD3 stining (4X mgnifiction). Representtive imginesof 5 mice per group. The lind fshion nlysis of the pthologist (M.M.I.) descries scttered CD3 + T cells in liver nd lungs with no differences etween the 3 groups. 1 Nture Medicine: doi:1.138/nm.3833

11 Crun et l. 6h 24 h 72 h 12 dys (I) Liver Lung Spleen Kidney 2X (I) c Positive control (I) Supplementry Fig. 9B.Expression of does not ffect LTE-T cell iodistriutionin vivo. ()(I)+ nd + LTE-T cells were lelled with the vector encoding GFP-firefly luciferse nd then infused vi til injection in NSGmice. T-cell iodistriution ws evluted y in vivo imging t indicted time points fter LTE-T cell infusion. Tissues were collected from infused mice y dy 12 or 19 fter LTE-T cell infusion nd stined with hemtoxylin nd eosin () nd nti-cd3 ntiody (c) (2X mgnifiction). Humn tonsil sections were used s positive control for CD3 stining.the lind fshion nlysis of the pthologist (M.M.I.) descries no signs of toxicity or differences in T-cell infiltrtion etween the two groups. 11 Nture Medicine: doi:1.138/nm.3833

12 MMP1- Sup 5 MMP2- Sup 25 MMP3- Sup dys dys dys MMP7- Sup dys MMP9- Sup dys IL-2 (5U/mL) IL-2 (2U/mL) IL-7 (1ng/mL) nd IL-15 (5ng/mL) 25 MMP1- Sup 5 MMP1- MMP12-Sup MMP13- Sup dys dys dys Supplementry Fig. 1A.Anlysis of mtrix metllo-proteses (MMPs) in T lymphocytes. Superntnts were collected from T lymphocytes otined 3, 7 nd 1 dys fter ctivtion with OKT3 nd nti-cd28 mas in the presence of IL-2 (5 UmL), or IL-2 (2 UmL) or IL-7 nd IL- 15 (1 ngml nd 5 ngml, respectively). Detection of MMs ws performed using Milliplex Mp kit (Millipore). Dt summrizes mens ± s.d. of 3 donors. 12 Nture Medicine: doi:1.138/nm.3833

13 5 MMP1- Lyste MMP2- Lyste MMP3- Lyste dys dys dys dys MMP7- Lyste MMP9- Lyste dys Lyste of FI-T IL-2 (5U/mL) IL-2 (2U/mL) IL-7 (1ng/mL) nd IL-15 (5ng/mL) MMP1- Lyste Non detectle MMP12- Lyste 2 5 dys dys dys MMP13- Lyste Supplementry Fig. 1B.Anlysis of mtrix metllo-proteses (MMPs) in T lymphocytes.cell lystes were otined from T lymphocytes t dys 3, 7 nd 1 fter ctivtion with OKT3 nd nti-cd28 mas in the presence of IL-2 (5 UmL), or IL-2 (2 UmL) or IL-7 nd IL-15 (1 ngml nd 5 ngml, respectively). The lck rs represent the nlysis of lystes otined from FI-T. Detection of MMs ws performed using Milliplex Mp kit (Millipore). Dt summrizes mens ± s.d.of 3 donors. 13 Nture Medicine: doi:1.138/nm.3833

14 Loction on promoter Primer p-1 sense p-1 ntisense p-2 sense p-2 ntisense p-3 sense p-3 ntisense p-4 sense p-4 ntisense p-5 sense p-5 ntisense Sequence 5'-GAAGCATAAGTGGGTGGATCTC-3' 5'-GTCACCCAGGTTGGAATACAGT-3' 5'-CATGTAGACCACAAGGATGCAC-3' 5'-GATTTCACCATGTCTGTCAGGA-3' 5'-TTTTTGTAGAGATGGGGCTTCA-3' 5'-TGTACCACCAATAAGGCAACAA-3' 5'-TTCACATCCCGATTCTGACA-3' 5'-TTGCCAAATTTCTCCTCTGC-3' 5'-GAGGAAGGGATGAATACTCCA-3' 5'-CTACTTCCTTGCTCGCTTTCC-3' Supplementry Fig. 11.Set of primers used in ChIP nlysis to evlute p53 inding to promoter. Loction of primers reltive to the origin of the promoter is lso indicted. 14 Nture Medicine: doi:1.138/nm.3833

15 SFG.(I)eGFP LTR h IRES egfp LTR SFG.14G2.CD28.OX4z(I) (GD2 specific) LTR 14G2 CD28 OX4 ζ IRES SFG.14G2.CD28.OX4z (GD2 specific) LTR LTR 14G2 CD28 OX4 ζ LTR Supplementry Fig. 12.Schemtic representtion of the retrovirl vectors used to trnsduce T lymphocytes. 15 Nture Medicine: doi:1.138/nm.3833

16 Reference List 1. Smith,C.A. et l. Production of geneticlly modified Epstein-Brr virus-specific cytotoxic T cells for doptive trnsfer to ptients t high risk of EBV-ssocited lymphoprolifertive disese. J. Hemtother.4, (1995). 2. Geldres,C. et l. T lymphocytes redirected ginst the chondroitin sulfte proteoglycn-4 control the growth of multiple solid tumors oth in vitro nd in vivo. Clin. Cncer Res.2, (214). 3. Xu,Y. et l. Closely relted T-memory stem cells correlte with in vivo expnsion of.cd19-t cells nd re preserved y IL-7 nd IL-15. Blood123, (214). 16 Nture Medicine: doi:1.138/nm.3833

Supplementary figure 1

Supplementary figure 1 Supplementry figure 1 Dy 8 post LCMV infection Vsculr Assoc. Prenchym Dy 3 post LCMV infection 1 5 6.7.29 1 4 1 3 1 2 88.9 4.16 1 2 1 3 1 4 1 5 1 5 1.59 5.97 1 4 1 3 1 2 21.4 71 1 2 1 3 1 4 1 5 1 5.59.22