effects of anti-dll4 treatment on non-small cell lung...

OncoMed 1

Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors

4652

Alayne Brunner, Fiore Cattaruzza, Wan-Ching Yen, Pete Yeung, Marcus Fischer, Belinda Cancilla, Christopher L. Murriel,

Gilbert O’Young, Raymond Tam, Yu-Wang Liu, Austin Gurney, John Lewicki, Tim Hoey, Min Wang, Ann M. Kapoun

OncoMed Pharmaceuticals, Inc., Redwood City, CA

Background: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers, the

leading cause of cancer-related deaths. Notch signaling has been shown to play an important role in lung

cancer initiation and progression. Delta-like ligand 4 (DLL4) activates the Notch pathway and is

important for cancer stem cell (CSC) survival. Demcizumab (OMP-21M18) is a humanized IgG2 anti-

DLL4 antibody currently being tested in a Phase 2 trial in combination with pemetrexed and carboplatin

for first-line treatment of patients with NSCLC. Previously, OMP-21M18 in combination with its mouse

anti-DLL4 surrogate has been shown to inhibit tumor growth, decrease cancer stem cell frequency, and

cause dysfunctional sprouting of new vessels resulting in an anti-angiogenic effect in patient-derived

tumor xenograft (PDX) models in breast, colon, ovarian, and pancreatic cancers. Here we show results

from NSCLC PDX models.

Methods and Results: Anti-DLL4 treatment was tested in a series of NSCLC PDX models. Because

DLL4 inhibition has been shown to have effects on the tumor as well as the vasculature, the combination

of OMP-21M18 (targeting human DLL4) and 21R30 (antibody targeting mouse DLL4) treatment in the

PDX models was used to model demcizumab treatment in humans. Treatment with anti-DLL4 in

combination with chemotherapy inhibited tumor growth in a series of NSCLC PDX models. Additionally,

a tumorigenicity assay showed a decrease in the frequency of tumor-initiating cells following treatment

with anti-DLL4 and chemotherapy. Gene expression analysis of tumor samples provided insights into the

mechanism of action.

Conclusions: Anti-DLL4 treatment in a panel of NSCLC PDX tumor models in vivo showed inhibition of

tumor growth and a decrease in the frequency of tumor-initiating cells. Mechanism of action and gene

expression analysis of these models treated with anti-DLL4 will be presented. These findings provide

additional evidence supporting demcizumab as an effective treatment for NSCLC patients.

MATERIALS AND METHODS

ABSTRACT IN VIVO TUMOR GROWTH IS INHIBITED WITH ANTI-DLL4

AND COMBINATION TREATMENT IN NSCLC PDX TUMORS

ANTI-DLL4 GENE SIGNATURE IS UP-REGULATED IN

STROMA FROM ANTI-DLL4 TREATED NSCLC PDX TUMORS

ANTI-DLL4 TREATMENT OF NSCLC PDX TUMORS IN

HUMANIZED MICE SHOWS IMMUNE ENGAGEMENT

Stem cell gene sets

are down-regulated

with anti-DLL4

Previously defined

DLL4 gene signature

is up-regulated in

NSCLC stroma

Up-regulated

genes in treated

tumor and stroma

are enriched for

hypoxia gene

signatures

Anti-DLL4 treatment

increases microvessel

density measured by anti-

CD34 immunohistochemistry

NOTCH PATHWAY AND STEM CELL GENES ARE DOWN-

REGULATED BY ANTI-DLL4 IN NSCLC PDX TUMORS

Notch target genes

including HEY1 are

down-regulated with

anti-DLL4

VASCULATURE-RELATED GENES ARE UP-REGULATED IN

STROMA FROM ANTI-DLL4 TREATED NSCLC PDX TUMORS

Vasculature-related genes including previously observed Cldn5 and

Apln are up-regulated in treated stroma

A) Up-regulated angiogenesis genes (Fold change > 1.5 and p-value < 0.05) in at least two comparisons. P-values < 0.05 are marked with *.

B) Anti-DLL4 treated stroma from LU56, LU108, and LU121 is enriched for genes in the angiogenesis gene list using GSEA.

GSEA results for stroma treated with A) Anti-DLL4 compared with control in LU56, LU108, and LU121 and B) Combination treatment compared with

carboplatin/pemetrexed in LU56 and LU108.

References:

TUMORIGENIC CELLS ARE DECREASED WITH ANTI-DLL4

COMBINATION TREATMENT IN NSCLC PDX TUMORS

A) Anti-DLL4 B) Combination

Enrichment analysis was performed for genes down-regulated with a fold change < -1.5 and p-value < 0.05.

B)

Anti-DLL4 inhibits growth of LU121

tumors in huSGM3 mice

CD33+ cells

are down-

regulated

following anti-

DLL4 treatment

1) Ambrogio, C., et al. (2016). "Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma." Nat

Med 22(3): 270-277.

2) Fischer, M., et al. (2011). "Anti-DLL4 inhibits growth and reduces tumor-initiating cell frequency in colorectal tumors with oncogenic KRAS mutations."

Cancer Res 71(5): 1520-1525.

3) Hoey, T., et al. (2009). "DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency." Cell Stem Cell 5(2): 168-177.

4) Kotasek D, et al. (2015). "A Phase 1b Study of the Anti-Cancer Stem Cell Agent Demcizumab, Pemetrexed and Carboplatin in Patients with 1st Line

Non-Squamous Non-Small Cell Lung Cancer (NSCLC). " American Society of Clinical Oncology 2015 Annual Meeting. Chicago, Illinois. Poster.

5) Yen, W. C., et al. (2012). "Anti-DLL4 has broad spectrum activity in pancreatic cancer dependent on targeting DLL4-Notch signaling in both tumor and

vasculature cells." Clin Cancer Res 18(19): 5374-5386.

• The recombinant antibodies demcizumab (OMP-21M18, anti-hDLL4) and OMP-21R30 (anti-mDLL4)

were generated at OncoMed Pharmaceuticals and dosed together (as anti-DLL4) in all experiments.

• The NSCLC tumor biopsies used to establish the tumor xenograft models at OncoMed

Pharmaceuticals were provided from MT Group (OMP-LU56), Molecular Response (OMP-LU108), and

Cooperative Human Tissue Network (OMP-LU121).

• For the efficacy experiments, tumor cells were implanted subcutaneously into NOD-scid mice. Tumors

were allowed to grow to 110-165 mm3 and were randomized into treatment groups. Experimental

groups included treatment with control antibody (1B7.11, 20 mg/kg, q2w), OMP-21M18 and OMP-

21R30 (20 mg/kg, q2w), standard-of-care chemotherapy (carboplatin 25-30 mg/kg, qw and pemetrexed

50-100 mg/kg, 2qw or 3qw), or the combination of antibody and chemotherapy. Average tumor volume

(mm3) +/- SEM is shown.

• In the tumorigenicity experiment, tumors were harvested from experimental efficacy groups, dissociated

into single cell suspensions, counted, and 50 cells were implanted into new recipient mice.

• For gene expression analysis, tumors were snap frozen and RNA was isolated. The quality of RNA and

cDNA was assessed by spectrophotometer and Bioanalyzer, and cDNA was hybridized to Affymetrix

HG-U133 plus 2 and Mouse-430 2.0 microarrays. The raw chip data were quantified and scaled using

the GCOS software package (Affymetrix). Genes differentially expressed between two groups were

identified with the Bayesian t test (Cyber-T) based on the p-value <0.05 and absolute fold change >1.5.

• Gene set enrichment analysis (GSEA; Broad Institute) and hypergeometric enrichment analyses were

performed using the C2 and C6 gene set lists (v4) from the Molecular Signatures Database (MSigDB)

combined with OncoMed-developed gene sets.

• Humanized NOD-scid IL2rgnull (NSG) mice crossed to mice expressing human IL-3, GM-CSF, SCF

(NSGS, huSGM3), and engrafted with human CD34+ hematopoietic stem cells were obtained from The

Jackson Laboratory (Sacramento, CA).

• OMP-LU121 cells implanted subcutaneously into HLA-A donor-matched and unmatched huSGM3 mice

were treated with either control IgG2 (10 mg/kg, qw) or OMP-21M18 and OMP-21R30 (10 mg/kg, qw)

antibodies. Post tumor growth analysis of intra-splenic human CD45+ and CD45+CD33+ immune cell

populations was conducted by flow cytometry. One-tailed t test p-values are shown.

• FFPE sections of PDX tumors were stained for murine-specific CD34 or human-specific CD45 by

immunohistochemistry. Digital images were obtained using an Aperio scanner, and image analysis was

performed using Definiens Tissue Studio software. CD34 analysis measured microvessel density.

CD45 analysis measured density of human tumor infiltrating immune cells within each tumor. Data is

expressed as percentage of immune cells compared to all cells within the tumor.

Copies of this poster obtained through Quick Response (QR) code are

for personal use only and many not be reproduced without written

permission from AACR and the authors of this poster.

Control Anti-DLL4 Chemo Combo

Control Anti-DLL4

LU56 anti-DLL4 vs control

LU56 Combo vs chemo


LU108 Combo vs chemo


Down-regulated Notch and stem

cell genes (Fold change < -1.5

and p-value < 0.05) in at least two

comparisons. P-values < 0.05 are

marked with *.

Color Key


LU56 Combo vs chemo


LU108 Combo vs chemo


Color Key A)

Control

SUMMARY

• NSCLC PDX tumors treated with anti-DLL4 show tumor growth inhibition.

• Anti-DLL4 treatment combined with standard-of-care carboplatin/pemetrexed in NSCLC

PDX tumors shows improved tumor growth inhibition.

• Tumor-initiating cells are decreased in NSCLC tumors treated with anti-DLL4 combined

with standard-of-care.

• Notch pathway and stem cell-related genes are down-regulated in NSCLC tumors with

anti-DLL4 and combination treatment.

• Previously-observed stromal genes including many vasculature-related genes are up-

regulated in anti-DLL4 treated NSCLC stroma, consistent with the observed increase in

blood vessel density and modification of hypoxia-related gene expression.

• A proof-of-concept efficacy experiment in humanized mice shows NSCLC PDX tumors

can be grown in huSGM3 mice and treated with anti-DLL4.

• Preliminary results from humanized huSGM3 mice with anti-DLL4 treatment of NSCLC

PDX tumors show up-regulation of splenic and tumor human CD45+ cells as well as

down-regulation of splenic human CD33+ cells, suggesting an increased anti-tumor

immune response.

Control

HYPOXIA-RELATED GENES ARE MODIFIED WITH ANTI-DLL4

TREATMENT IN NSCLC PDX TUMOR AND STROMA

FDR = 0.0487

FDR < 0.0001 FDR < 0.0001

Select hypoxia and blood vessel-related

GSEA gene sets up-regulated in tumor

or stroma treated with anti-DLL4

compared with control in LU56, LU108,

and LU121, and stroma treated with

combination treatment compared with

carboplatin/pemetrexed in LU56 and

LU108.

Harvest and dissociate tumors

Transplant 50 cells into mice

Record tumor volumes at day 54

CD45+ cells are up-regulated

following anti-DLL4 treatment

Anti-DLL4

TUMOR VASCULATURE IS INCREASED FOLLOWING

ANTI-DLL4 TREATMENT IN NSCLC PDX TUMORS

Acknowledgments: Special thanks to James Keck and The Jackson Laboratory, Sacramento, CA for their collaboration with the hu-SGM3 humanized mice, and Akbar

Currimbhoy for help with IHC.

Anti-DLL4

OMP-LU121 Tumor aCD45 IHC

OMP-LU121 Tumor aCD34 IHC

* p < 0.01; ** p < 0.001; *** p < 0.0001. Anti-DLL4 vs control comparisons marked in red. Combination vs chemotherapy comparisons marked in green.

IHC anti-CD34 quantification is expressed

as the number of vessels/mm2.

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