Download - Preclinical evaluation of the recombinant dendritic cell ... AACR 2019.pdfExicure, Inc, Skokie, IL 60077 # 3217. Scan QR code for a copy of this poster. Toll-like receptor 9 (TLR9)

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Page 1: Preclinical evaluation of the recombinant dendritic cell ... AACR 2019.pdfExicure, Inc, Skokie, IL 60077 # 3217. Scan QR code for a copy of this poster. Toll-like receptor 9 (TLR9)

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S u rv iv a l

D a y s p o s t tu m o r in je c t io n

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rviv

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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)

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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+.

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CELLS CDX-301 muAST-008

CD103+ out of cDCsCD8a+ out of cDCsCD11b+ out of cDC

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cDC% pDC%

cDC absolute # in spleen pDC absolute # in spleen

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CDX-301 D1-7

D7 AST008FGK45

AST008+FGK45

D9 CDX301CDX301+ASTCDX301+FGKCDX301+AST+FGKbaseline

CD40 MFI in cCDsCD80 MFI in cCDs

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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

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4545

αCD40αCD40D7

muAST-008FGK45

muAST-008 + FGK45

CDX-301

CDX-301 + muAST-008

CDX-301 + αCD40

CDX-301 + muAST-008 + αCD40

Baseline