piccione e, rudnick j, mikesell g, daine-matsuoka b, walter k, liu … · 2018-10-18 · 3mm amp is...

A novel CD73-blocking antibody reduces production of immunosuppressive adenosine and restores T cell functionPiccione E, Rudnick J, Mikesell G, Daine-Matsuoka B, Walter K, Liu L, Hotson A, Miller R, McCaffery ICorvus Pharmaceuticals, Burlingame, CA 94010, USA.

CPX-006 AND CPX-016 BIND TO CD73 AND REDUCE ADENOSINE PRODUCTION

Figure 1 (A) MDA-MB-231 cells were incubated with CPX-006 or CPX-016 over a range of concentrations and antibody binding was detected with a PE-conjugated secondary antibody and flow cytometry analysis. Mean fluorescence intensity (MFI) of PE signal is reported. (B) MDA-MB-231 parental cells or CD73 CRISPR knockout cells were incubated with the indicated antibodies or APCP, a small molecule inhibitor of CD73 enzymatic activity, prior to addition of 250 μM AMP. Phosphate levels were measured in the cell culture supernatant using the Sensolyte Malachite Green phosphate assay kit.

INTRODUCTION

Figure 3 (A) Schematic of experimental design. PBMC were isolated from healthy donors and labeled with Cell Trace Violet prior to culture with 1 μg/mL anti-CD3 and anti-CD28, 200 units/mL IL-2, 3mM AMP, and antibody treatment as indicat-ed. After 4 days in culture, IFN-gamma levels in the supernatant were measured by AlphaLISA. Proliferation of CD3+ T cells was followed by flow cytometry analysis of Cell Trace Violet dilution and was defined by gating relative to unstimulated PBMC. (B) T cell proliferation for a representative donor. (C) T cell proliferation for 13 donors treated with 500nM CPX-006 or CPX-016 or 890nM isotype control. (D) IFN-gamma production (reported as raw AlphaLISA values) for a representative donor. (E) IFN-gamma production for 6 donors treated as described in panel (C).

CPX-006 AND CPX-016 BIND TO DISTINCT EPITOPES ON CD73

MDA-MB-231 cells+

APCP (non-hydrolyzable substrate mimic)

CPX-006or

CPX-016

Secondary antibody staining and analysis of binding by

flow cytometry

CPX-006 CPX-016

CPX-006 CPX-016

Figure 2 MDA-MB-231 cells were pre-incubated with APCP, a non-hydrolyzable substrate mimic for CD73. Cells were subsequently incubated with a titration of CPX-006 or CPX-016 prior to staining with PE-conjugated anti-human secondary antibody. Antibody binding was assessed by flow cytometry and mean fluorescence intensity (MFI) for PE was determined.

Binding to MDA-MB-231 CD73 Catalytic ActivityA B

CPX-006 REVERSES SUPPRESSIVE EFFECTS OF AMP ON T CELL ACTIVITY

A

CB

D E

Measure proliferation of CD3+ T cells or

IFN-gamma secretionHuman PBMClabeled with Cell Trace Violet

4 days

+ anti-CD3/anti-CD28+ 3mM AMP+ Antibody

A B

C D

CPI-444 PHASE 1/1B CLINICAL TRIAL DESIGN

CD73 IS HIGHLY EXPRESSED IN MULTIPLE TUMOR TYPES

CPX-006 REVERSES SUPPRESSIVE EFFECTS OF AMP ACROSS A BROAD RANGE OF CD73 EXPRESSION

Figure 5 CD73 expression was analyzed by immunohistochemical staining of tissues from renal cell cancer (n=62), NSCLC (n=68), melanoma (n=68), and breast cancer (n=94). Six representative tissues are shown from each histology.

Figure 4 (A, B) CD73 expression levels in each donor were determined by flow cytometry and were plotted as a function of the effect of CPX-006 or CPX-016 (500nM) on restoring T cell proliferation in the presence of AMP as reported in Figure 3C. (C) MDA-MB-231 cells were engineered to reduce or eliminate CD73 expression via shRNA or CRISPR, respectively. CD73 protein levels were measured by flow cytometry analysis. (D) CD73 catalytic activity was measured for each cell line in the presence of 1μM CPX-006 or CPX-016 or 1mM APCP as described Figure 1B.

CONCLUSIONS

CONCLUSIONS

Figure 6 Effects of CPX-006 and CPI-444 on T cell proliferation and IFN-gamma secretion were measured as described in Figure 3A. (A) T cell proliferation for 10 donors treated with 500nM CPX-006, 10μM CPI-444, 500nM CPX-006 + 10μM CPI-444, or vehicle (890nM isotype control + 0.1% DMSO). (B) IFN-gamma production (reported as raw AlphaLISA values) for 6 donors treated as described in panel (A).

NO TOXICITY OBSERVED WITH CPX-006 IN NON-HUMAN PRIMATES

CPX-006 AUGMENTS A2AR ANTAGONIST, CPI-444

Antibody-mediated Inhibition of CD73 Activity on MDA-MB-231 Cells Engineered with Varying Levels of CD73 Expression

parental scrambledshRNA

shRNA D shRNA C shRNA B CRISPRKO

CD7

3 M

FI

CD73 Expression on Engineered MDA-MB-231 Cell Lines

Renal Cell Cancer Breast Cancer

Melanoma Lung Cancer

T cell proliferation(representative donor)

IFN-gamma secretion (representative donor)

T cell proliferation

IFN-gamma secretion A BT cell proliferation

• We describe a unique Type 1 anti-CD73 antibody that binds to the active site of CD73 and completely inhibits CD73 activity in contrast to all other described antibodies which bind to the N-terminal lobe (Type 2)

• CPX-006 inhibits AMP-mediated suppression of T cell proliferation and IFN-gamma secretion and effects are augmented with CPI-444

• CPX-006 inhibits the immune-suppressive activity of CD73 completely even at high levels of CD73 found in tumors, in contrast to CPX-016

• CPX-006 is well-tolerated with no observed toxicities in multi-dose non-human primate studies where complete CD73 occupancy is achieved

• IND for CPX-006 is planned for Q1 2018

• Cynomolgus monkeys dosed weekly for 5 consecutive weeks at 10, 40, or 120 mg/kg

• No changes observed in clinical chemistry, gross pathology, organ weights, histopathology

• Complete CD73 coverage is achieved with CPX-006 at all doses tested

Figure 7 CD73 occupancy was determined by measuring ex vivo staining of CD8+ T cells with Alexa-Fluor 647 labeled CPX-006. Ratio of AF647 staining on CD73+/CD73- cells is reported.

Figure adapted from Antonioli et al, Nat Rev Cancer. 20131

ATP

ATP

AMP

AMP Adenosine

CD39

CD39CD73

CD73

CD26 Inosine

Cancer cell

ADANT

LysosomesTransport vesiclesNucleotide channelsInjury or lysisExocytosis

MDSC

Population expansionImmunosuppressive effects

Dendritic cell

Tumor antigencross-presentation

NK cell

Inhibition oftumor cell lysis

Macrophage

M2 polarizationVEGF production

A2A ReceptorA2A Receptor A2A Receptor

T cell

TH1 cellsTREG cells

A2A Receptor A2A Receptor

ATP

ATP

ADENOSINE IS GENERATED BY CD73 AND CREATES AN IMMUNOSUPPRESSIVE TUMOR MICROENVIRONMENT

CD73 IS AN ECTOENZYME THAT CONVERTS AMP TO ADENOSINE

Figure adapted from Allard et al, Expert Opin Ther Targets. 20142

AMP

Adenosine

lipid bilayerGPI anchor

Cytosol

COOH

NH2

Open Conformation

Closed Conformation

active sitemetal ion binding sites

• Switching from the open to closed conformation is required for catalytic activity3

• CD73 is overexpressed in multiple tumor types and high expression associates with poor prognosis4,5,6

• CD73 inhibition has demonstrated anti-tumor activity as a single agent and in combination with other IO agents in preclinical models4,5,6

donor 1donor 2donor 3donor 4donor 5donor 6donor 7donor 8donor 9donor 10donor 11donor 12donor 13

donor 1donor 7donor 9donor 10donor 14donor 15

donor 1donor 2donor 4donor 5donor 6donor 8donor 10donor 11donor 12donor 13donor 14

donor 1donor 2donor 3donor 4donor 5donor 6donor 7donor 8donor 9donor 10

donor 8donor 9donor 10donor 11donor 12donor 13

3mM AMP is supra-physiological. Adenosine levels have been reported to be 1-10 µM.7

Type I anti-CD73

Type 2 anti-CD73

Type I anti-CD73

Type 2 anti-CD73

ATP

Pro-inflammatory stimulus

REFERENCES1. Antonioli et al. Nat Rev Cancer. 13, 842-857, 2013.2. Allard et al, Expert Opin Ther Targets. 18, 1-19, 2014.3. Knapp et al, Structure. 20, 2161-73, 2012.4. reviewed by Allard et al, Immunotherapy. 8, 145-163, 2016.5. reviewed by Antonioli et al, Trends Cancer. 2, 95-109, 2016.6. reviewed by Allard et al, Immunological Reviews, 276, 121-144, 2017.7. Blay et al. Cancer Research. 57, 2602-2605, 1997.

no AMP isotype+

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CD73 score(%CD73+ cells x MFI)