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Antibody-Based Inhibition of CSF-1R as a Component of Combination Immunotherapy in Preclinical Models

The colony stimulating factor 1 receptor (CSF-1R) signaling pathway promotes

tumor progression via the recruitment, differentiation, and survival of immuno-

suppressive, M2 polarized, tumor-associated macrophages (TAMs). FivePrime has

developed cabiralizumab (FPA008), an IgG4 antibody against CSF-1R that blocks the

ability of both CSF-1 and IL-34 to bind and activate this receptor, thereby modulating

the immune response to tumorigenesis. In order to investigate the impact of CSF-1R

signaling inhibition in preclinical models, we generated a surrogate antibody,

cmFPA008, that targets mouse CSF-1R and demonstrates equivalent affinity and

ligand-blocking ability as FPA008. Utilizing a combination of flow cytometry and

immunofluorescence analyses, we have identified alterations in the tumor

microenvironment that occur upon CSF-1R inhibition, including significant reduction of

immunosuppressive M2 TAMs and an increase in tumor PD-L1 expression.

Interestingly, we observe an increase in CD8+ T cell number and activation upon TAM

depletion, followed by a subsequent increase in putative monocytic (Cd11b+/Ly6G-

/Ly6C+) and granulocytic (CD11b+/Ly6G+/Ly6c+) MDSC populations. Moreover, we

have used murine syngeneic tumor models to examine the anti-tumor impact of CSF-

1R inhibition in combination with other immuno-oncology agents. Our results show that,

when added to PD-1/PD-L1 blockade, cmFPA008 can significantly enhance anti-tumor

efficacy. We are currently exploring the effects of combining cmFPA008-induced TAM

depletion with additional immuno-oncology agents, including T cell agonists. Our

preclinical results demonstrate that inhibition of the CSF-1R pathway can combine with

various immuno-oncology agents with distinct mechanisms of action. FivePrime has

initiated a clinical trial in collaboration with Bristol-Myers Squibb (BMS) to investigate

the use of cabiralizumab in combination with nivolumab (anti-PD-1, OPDIVO®) in six

different tumor types.

Abstract

Summary and Conclusions

• cmFPA008 significantly reduces MC38 tumor growth as monotherapy and when

administered in combination with anti-PD-1.

• Treatment with cmFPA008 results in depletion of immunosuppressive M2 tumor-

associated macrophages (TAMs) via apoptosis, as well as significant, but transient

changes in CD8+ T cells and putative MDSC populations.

• cmFPA008 monotherapy induces an increase in CD8+ T cells in MC38 primarily at

the tumor periphery where PD-L1 staining is the most intense.

• The anti-tumor activity of cmFPA008 in combination with anti-PD-1 in MC38

correlates with a significant depletion of M2 TAMs, increase in CD8+ T cells, and a

favorable CD8+/CD4+ T cell ratio compared to either therapeutic alone.

#1599

David I. Bellovin, Nebiyu Wondyfraw, Anita Levin, Quinn Walker, Barbara Sennino, Anthony Chao, Amy K.H. Hsu, Emma Masteller, Susan Johnson, Susannah D. Barbee, Robert Sikorski, and Tom Brennan Five Prime Therapeutics, Inc., South San Francisco, CA

Anti-Cancer Mechanism of Action of cabiralizumab

© 2017 Five Prime Therapeutics, Inc. All Rights Reserved.

• Cabiralizumab (FPA008) is a humanized IgG4 anti-CSF-1R that blocks binding of both CSF-1

and IL-34.

• Cabiralizumab inhibits survival of CSF-1R-dependent monocytes and macrophages.

• Tumor-associated macrophages (TAMs) facilitate tumor growth by immuno-suppression,

including direct and indirect inhibition of cytotoxic CD8 T cells.

• Cabiralizumab-mediated inhibition of CSF-1R is anticipated to impact TAM function and/or

viability, thereby reducing immuno-suppression and allowing anti-tumor T cell activation.

• Co-inhibition of CSF-1R and PD-1/PD-L1 signaling is hypothesized to potentiate an anti-tumor

immune response.

Combination of cabiralizumab with anti-PD-1 in cancer immunotherapy

Cabiralizumab (FPA008) blocks the binding of CSF-1 and IL-34 to CSF-1R

cmFPA008 Reduces Growth and M2 TAMs in the MC38 Model The Combination of cmFPA008 and anti-PD-1 Induces Potent Anti-Tumor Activity

• cmFPA008 is a surrogate mouse IgG1 antibody developed at Five Prime that binds to murine

CSF-1R with comparable affinity to cabiralizumab.

• MC38-bearing mice were administered a single dose of cmFPA008 at 20 mg/kg, and tumors were

analyzed via flow cytometry and immunofluorescence to examine changes in TILs.

• cmFPA008 significantly reduces MC38 tumor volume beginning on Study Day 18.

• Immunosuppressive M2 macrophages (TAMs; Cd11b+/Ly6G-/Ly6C-/F4/80+/Cd206+) are depleted

within 48 hours of cmFPA008 dosing, with near complete depletion 4 days after treatment.

• TAMs begin to repopulate the tumor microenvironment after Day 17, corresponding with

reduced cmFPA008 drug exposure.

• Statistical analysis was performed by 2-tailed t-test, (* p < 0.05, ** p < 0.01, *** p < 0.001).

• cmFPA008 treatment induces an increase in tumor PD-L1 expression near the tumor periphery.

• The expression of PD-L1 in the tumor periphery is correlated with an increase in CD8+ T cells at

the tumor margin.

• cmFPA008 and anti-PD-1 together induce a greater reduction in MC38 tumor volume than either

therapeutic alone.

• We have shown previously that cmFPA008 and anti-PD-1 exert superior anti-tumor activity in

combination with gemcitabine in a preclinical model of pancreatic ductal adenocarcinoma

(Bellovin, et. al., SITC Annual Meeting, 2015).

• Statistical analysis by one-way ANOVA (** p < 0.01, *** p < 0.001).

• cmFPA008 significantly reduces the number of Cd11b+ myeloid cells within 2 days of dosing.

• Statistical analysis by 2-tailed t-test, (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

• Putative monocytic (Cd11b+/Ly6G+/Ly6C+) and granulocytic (CD11b+/Ly6G+/Ly6c+) MDSC

populations increase transiently following cmFPA008-induced reduction in M2 TAMs and return to

baseline by end of the study.

• Studies to assess whether a similar increase in other in vivo tumor models are ongoing.

• CD8+ T cells increase significantly but transiently, following cmFPA008 treatment.

• The combination of cmFPA008 and anti-PD-1 synergistically enhances the number of CD8+ T

cells in MC38 tumors as assessed by flow cytometry.

• Combination therapy results in a significant increase in the relative abundance of CD8+ versus

regulatory T cells (CD4+/CD25+) in MC38 tumors.

• Statistical analysis by one-way ANOVA (* p < 0.01, ** p < 0.05, *** p < 0.001, **** p < 0.0001).

• Data is representative of multiple independent experiments.

CD8+ T cells CD11b+ Myeloid Cells Mo-MDSCs G-MDSCs

M2 TAMs CD8+ T cells Teff/Treg Ratio

• cmFPA008 treatment results in depletion of immunosuppressive M2 TAMs from MC38 tumors, as

shown by reduction in F4/80 immunofluorescence, with kinetics similar to those demonstrated via

flow cytometry.

• TAMs undergo apoptosis following cmFPA008 administration, as demonstrated by the presence of

activated caspase-3 staining in F4/80+ cells within tumors.

MC38 Tumor Growth cmFPA008 PK M2 TAMs

cmFPA008 Induces a Transient Increase in MDSCs in MC38

Study Design

Day 7

~100 mm3

Day 0

Implant MC38

tumor cells

Subcutaneous

MC38 Model

C57Bl/6 mice

Day 10 Day 14 Day 18

anti-PD-1

Anti-Tumor

Activity

cmFPA008

anti-PD-1 anti-PD-1

Day 22

TIL Analysis

via Flow

cmFPA008

Day 12

TIL Analysis

Via IF/IHC

Increased Tumor CD8+ T cells

after cmFPA008 treatment (5d)

MC38 Tumor Volume

Study Day 18

IgG

cmFPA008

PD-L1 CD3

Tumor Tumor

Tumor Tumor

Skin Skin

Skin Skin

mIgG1 Control 2d after cmFPA008 5d after cmFPA008 7d after cmFPA008

F4/80 Activated Caspase-3

5d after cmFPA008

DAPI

F4/80

TAMs are Depleted via cmFPA008-Induced Apoptosis