Preclinical evaluation of the recombinant dendritic cell ... AACR 2019.pdfExicure, Inc, Skokie, IL...
1
0 20 40 60 80 100 0 50 100 Survival Days post tumor injection Percent Survival Saline CDX-301 muAST-008 (1mg/kg) muAST-008 (3mg/kg) CDX-301 + muAST-008 (1mg/kg) CDX-301 + muAST-008 (3mg/kg) p=0.0092 p=0.0336 p=0.0339 Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival. We observed a significant increase in the percentage of splenic CD103 + cDCs from the addition of muAST-008 to CDX-301 treatment. In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combined with an agonist CD40 antibody. These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumor immunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers. BACKGROUND: CDX-301 BACKGROUND: AST-008 Dendritic cells (DCs) are often rare or completely missing from tumors and are necessary for anti-tumor immunity. CDX-301 is the soluble recombinant human protein form of the Fms-related tyrosine kinase 3 ligand (Flt3L), a hematopoietic cytokine. Flt3 receptor (CD135) is expressed on hematopoietic stem cells, early progenitor cells, immature thymocytes, and steady state dendritic cells. CDX-301 has shown an ability to increase the number of DC precursors and DCs in blood and tissue, including the CD141 + (BDCA3 + ) cDC subset in humans and the corresponding CD103 + cDC1 subset in mice. In humans and mice, the intratumoral CD141 + /CD103 + DCs are important for antigen cross-presentation to T cells and correlate with improved outcomes for multiple tumor types. Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA Lawrence J. Thomas 2 , Li-Zhen He 1 , Lauren E. Gergel 2 , Eric M. Forsberg 2 , Elizabeth Q. Do 2 , James M. Boyer 2 , April R. Baronas 2 , Mallary Rocheleau 2 , Kathleen M. Borrelli 2 , Anna Wasiuk 1 , Jeffrey Weidlick, Henry C. Marsh, Jr. 2 , Bart R. Anderson 3 , SubbaRao Nallagatla 3 , Richard Kang 3 , Ekambar R. Kandimalla 3 and Tibor Keler 1 Celldex Therapeutics, Inc., 1 Hampton, NJ 08827 and 2 Needham, MA 02494, and 3 Exicure, Inc, Skokie, IL 60077 #3217 Scan QR code for a copy of this poster. Toll-like receptor 9 (TLR9) belongs to the family of pattern recognition receptors in the innate immune system and is predominately expressed in B cells and plasmacytoid dendritic cells (pDCs). CpG dinucleotides present in specific nucleic acid sequence contexts induce immune responses via stimulation of TLR9. Spherical Nucleic Acids (SNAs) are a novel class of therapeutic agents with oligonucleotides densely packed and radially oriented around a spherical liposomal nanoparticle. As a result of the 3D architecture, SNAs exhibit increased cellular uptake and primarily accumulate in endosomes where TLR9 is expressed. AST-008 and muAST-008 are human- and mouse-selective TLR9- agonist SNAs TLR9 agonist SNAs induce Th1-type cytokine production, plasmacytoid dendritic cell (pDC) maturation, and pDC and B cell activation. Subsequent activation of T cells and NK cells results in tumor killing and release of tumor-associated antigens leading to tumor-specific adaptive immunity. In addition, other therapies can be combined with SNAs to enhance the cancer therapy. EFFICACY IN MC38 MODEL DC EXPANSION cDC SUBTYPES B A DC PROLIFERATION AND ACTIVATION DC ACTIVATION A. Female C57BL/6 mice received 10 6 MC38 cells s.c. in one flank on Day 0. Animal were divided into Group 1, receiving saline on days 9, 16, 23 and 30 p.t.; Group 2, receiving 5μg CDX-301 on days 2-8 i.p.; Group 3, receiving 1mg/kg muAST-008 on days 9, 16, 23 and 30 p.t.; Group 4, receiving 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t.; Group 5, receiving 5μg CDX-301 on days 2-8 i.p. and 1mg/kg muAST-008 on days 9, 16, 23 and 30 p.t.; and Group 6, receiving 5μg CDX-301 on days 2-8 i.p. and 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t. B. Female C57BL/6 mice received 10 6 MC38 cells s.c. in both flanks on Day 0. Animal were divided into Group 1, receiving saline on days 9, 16, 23 and 30 p.t.; Group 2, receiving 5μg CDX-301 on days 2-8 i.p.; Group 3, receiving 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t. in the left flank only; and Group 4, receiving 5μg CDX-301 on days 2-8 i.p. and 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t. in the left flank only. CONCLUSIONS DC proliferation and activation. C57BL/6 mice were treated with CDX-301 (2.5 ug, i.p.) daily for 7 days, and muAST-008 (50 ug, s.c.) or agonist anti-CD40 antibody FGK45 (50 ug, i.p.) on day 7, as indicated. On day 9 spleens were collected and processed into a single cell suspension, and stained for DC subset and activation markers. DC subsets were defined as follows; cDC = CD11c ++ MHC-II ++ , pDC = CD11c + MHC-II + B220 + . 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 CELLS CDX-301 muAST-008 CD103 + out of cDCs CD8a + out of cDCs CD11b + out of cDC 0 20 40 60 80 **** **** **** **** **** ** **** * 0 10 20 30 40 **** * *** * **** **** 0 10 20 30 40 50 **** ** **** ** *** cDC% pDC% cDC absolute # in spleen pDC absolute # in spleen 0 5 10 15 **** **** **** * **** **** * *** **** 0 1 2 3 4 **** **** *** *** * * 0 5.0 × 10 6 1.0 × 10 7 1.5 × 10 7 2.0 × 10 7 2.5 × 10 7 **** **** * **** *** **** *** 0 1 × 10 6 2 × 10 6 3 × 10 6 4 × 10 6 5 × 10 6 **** **** ** ** *** **** C57BL/6 Harvest spleen for FACS analysis CDX-301 D1-7 D7 D9 CD40 MFI in cCDs CD80 MFI in cCDs *** **** **** **** **** *** **** 0 500 1000 1500 2000 **** CD86 MFI in cCDs 0 500 1000 1500 2000 2500 * *** *** 0 20 40 60 80 100 **** **** **** **** *** **** CD95 MFI in cCDs 0 500 1000 1500 2000 2500 **** **** *** **** PD-L1 MFI in cCDs *** **** **** 0 500 1000 1500 2000 **** * ** *** CDX-301 Differentiation and expansion of DC precursors Immature DC migration into tumor Immature DC migration into lymph nodes Mature/activated DCs capture and present tumor antigens & secrete IL-12 Mature/activated DCs Activation of tumor- specific T cells TLR agonist CD40 agonist CTL kill tumor cells & secrete IFN-γ Lymph node DCs traffic tumor antigens to lymph nodes Tumor Relative to Control % of Live Splenocytes % of Live Splenocytes (%) (%) (%) D7 muAST-008 FGK45 muAST-008 + FGK45 CDX-301 CDX-301 + muAST-008 CDX-301 + αCD40 CDX-301 + muAST-008 + αCD40 Baseline
Transcript of Preclinical evaluation of the recombinant dendritic cell ... AACR 2019.pdfExicure, Inc, Skokie, IL...
0 2 0 4 0 6 0 8 0 1 0 0
0
5 0
1 0 0
S u rv iv a l
D a y s p o s t tu m o r in je c t io n
Pe
rce
nt
Su
rviv
al
Saline
C D X-301
m uAS T-008 (1m g/kg)
m uAS T-008 (3m g/kg)
C D X -301 + m uA S T-008 (1m g/kg)
C D X -301 + m uA S T-008 (3m g/kg)
p = 0 .0 0 9 2
p = 0 .0 3 3 6
p = 0 .0 3 3 9
Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival.
We observed a significant increase in the percentage of splenic CD103+ cDCs from the addition of muAST-008 to CDX-301 treatment.
In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combinedwith an agonist CD40 antibody.
These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumorimmunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers.
BACKGROUND: CDX-301
BACKGROUND: AST-008
Dendritic cells (DCs) are often rare or completely missing from tumors and arenecessary for anti-tumor immunity.
CDX-301 is the soluble recombinant human protein form of the Fms-relatedtyrosine kinase 3 ligand (Flt3L), a hematopoietic cytokine.
Flt3 receptor (CD135) is expressed on hematopoietic stem cells, earlyprogenitor cells, immature thymocytes, and steady state dendritic cells.
CDX-301 has shown an ability to increase the number of DC precursors andDCs in blood and tissue, including the CD141+(BDCA3+) cDC subset inhumans and the corresponding CD103+ cDC1 subset in mice.
In humans and mice, the intratumoral CD141+/CD103+ DCs are important forantigen cross-presentation to T cells and correlate with improved outcomes formultiple tumor types.
Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNALawrence J. Thomas2, Li-Zhen He1, Lauren E. Gergel2, Eric M. Forsberg2, Elizabeth Q. Do2, James M. Boyer2, April R. Baronas2, Mallary Rocheleau2, Kathleen M. Borrelli2,
Anna Wasiuk1, Jeffrey Weidlick, Henry C. Marsh, Jr. 2, Bart R. Anderson3, SubbaRao Nallagatla3, Richard Kang3, Ekambar R. Kandimalla3 and Tibor Keler1
Celldex Therapeutics, Inc., 1Hampton, NJ 08827 and 2Needham, MA 02494, and 3Exicure, Inc, Skokie, IL 60077#3217 Scan QR code for a
copy of this poster.
Toll-like receptor 9 (TLR9) belongs to thefamily of pattern recognition receptors in theinnate immune system and ispredominately expressed in B cells andplasmacytoid dendritic cells (pDCs).
CpG dinucleotides present in specificnucleic acid sequence contexts induceimmune responses via stimulation of TLR9.
Spherical Nucleic Acids (SNAs) are a novel class of therapeuticagents with oligonucleotides densely packed and radially orientedaround a spherical liposomal nanoparticle.
As a result of the 3D architecture, SNAs exhibit increased cellularuptake and primarily accumulate in endosomes where TLR9 isexpressed.
AST-008 and muAST-008 are human- and mouse-selective TLR9-agonist SNAs
TLR9 agonist SNAs induce Th1-type cytokine production,plasmacytoid dendritic cell (pDC) maturation, and pDC and B cellactivation. Subsequent activation of T cells and NK cells results intumor killing and release of tumor-associated antigens leading totumor-specific adaptive immunity. In addition, other therapies can becombined with SNAs to enhance the cancer therapy.
EFFICACY IN MC38 MODEL
DC EXPANSION cDC SUBTYPES
BA
DC PROLIFERATION AND ACTIVATIONDC ACTIVATION
A. Female C57BL/6 mice received 106 MC38 cells s.c. in one flank on Day 0. Animal were divided into Group 1, receiving saline ondays 9, 16, 23 and 30 p.t.; Group 2, receiving 5µg CDX-301 on days 2-8 i.p.; Group 3, receiving 1mg/kg muAST-008 on days 9, 16,23 and 30 p.t.; Group 4, receiving 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t.; Group 5, receiving 5µg CDX-301 on days 2-8i.p. and 1mg/kg muAST-008 on days 9, 16, 23 and 30 p.t.; and Group 6, receiving 5µg CDX-301 on days 2-8 i.p. and 3mg/kgmuAST-008 on days 9, 16, 23 and 30 p.t.B. Female C57BL/6 mice received 106 MC38 cells s.c. in both flanks on Day 0. Animal were divided into Group 1, receiving saline on days 9, 16, 23 and 30 p.t.; Group 2, receiving 5µg CDX-301 on days 2-8 i.p.; Group 3, receiving 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t. in the left flank only; and Group 4, receiving 5µg CDX-301 on days 2-8 i.p. and 3mg/kg muAST-008 on days 9, 16, 23 and 30 p.t. in the left flank only.
CONCLUSIONS
DC proliferation and activation. C57BL/6 mice were treated with CDX-301 (2.5 ug, i.p.) daily for 7 days, and muAST-008 (50 ug, s.c.) or agonist anti-CD40 antibody FGK45 (50 ug, i.p.) on day 7, as indicated. On day 9 spleens were collected and processed into a single cell suspension, and stained for DC subset and activation markers. DC subsets were defined as follows; cDC = CD11c++MHC-II++, pDC = CD11c+MHC-II+B220+.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
CELLS CDX-301 muAST-008
CD103+ out of cDCsCD8a+ out of cDCsCD11b+ out of cDC
0
20
40
60
80
****
********
**** ****
******
*
0
10
20
30
40 ****
****
* ********
0
10
20
30
40
50 ******
****
*****
cDC% pDC%
cDC absolute # in spleen pDC absolute # in spleen
0
5
10
15
****
****
****
*
****
*****
*******
0
1
2
3
4
********
*** ***
**
0
5.0×106
1.0×107
1.5×107
2.0×107
2.5×107
****
****
*
*******
****
***
0
1×106
2×106
3×106
4×106
5×106****
****
** **
*******
C57BL/6
Harvest spleen for FACS analysis
CDX-301 D1-7
D7 AST008FGK45
AST008+FGK45
D9 CDX301CDX301+ASTCDX301+FGKCDX301+AST+FGKbaseline
CD40 MFI in cCDsCD80 MFI in cCDs
*******
****
********
*******
0
500
1000
1500
2000****
CD86 MFI in cCDs
0
500
1000
1500
2000
2500
*
*** ***
0
20
40
60
80
100
**** **** ***********
****
CD95 MFI in cCDs
0
500
1000
1500
2000
2500****
*******
****PD-L1 MFI in cCDs
*******
****
0
500
1000
1500
2000
****
***
***
CDX-301Differentiation and
expansion of DC precursors
Immature DC migration into
tumor
Immature DC migration into lymph nodes
Mature/activated DCs capture and present tumor antigens & secrete
IL-12
Mature/activated DCs
Activation of tumor-specific T cells
TLR agonist CD40 agonist
CTL kill tumor cells & secrete
IFN-γ
Lymph node
DCs traffic tumor antigens to lymph nodes
TumorRelative to Control
% o
f Liv
e Sp
leno
cyte
s
% o
f Liv
e Sp
leno
cyte
s
(%) (%)(%)
4545
αCD40αCD40D7
muAST-008FGK45
muAST-008 + FGK45
CDX-301
CDX-301 + muAST-008
CDX-301 + αCD40
CDX-301 + muAST-008 + αCD40
Baseline
![[TOC 1]Cover Analytical ReportAnalytical Report June 23, 2017 RE: Drinking Water Jeff Hacker Skokie Park District 9300 Weber Park Place Skokie, IL 60077-4200 Work Order: 17F0612 Dear](https://static.fdocuments.net/doc/165x107/5ff61494fd1dd6153a087cd2/toc-1cover-analytical-analytical-report-june-23-2017-re-drinking-water-jeff.jpg)
