CB-1158 Inhibits the Immuno-oncology Target Arginase and Causes an Immune Mediated Anti-Tumor Response

Transcription

1 CB-1158 Inhibits the Immuno-oncology Target Arginase and Causes an Immune Mediated Anti-Tumor Response Francesco Parlati, Ph.D. Calithera Biosciences South San Francisco, CA

2 Arginine Depletion by Arginase Blocks T-cell and NK-cell Activation/Proliferation Arginase-expressing MDSCs/neutrophils are found in many tumor types and are associated with poor prognosis Arginase is an immunosuppressive enzyme Depletes arginine, required for the activation and proliferation of T- and NK-cells Inhibition of arginase in the tumor microenvironment should restore arginine levels leading to T- and NK-cell activation and proliferation Arginine Nutrient sensor pathways expression of TCRz production of IFNg proliferation MDSC/ neutrophil = Arginase T-cell NK-cell

3 Immunosuppressive Myeloid Cells Accumulate in the Tumor Microenvironment Immunosuppressive myeloid cells are present in the blood and tumors of many cancer patients and are associated with poor prognosis Myeloid-derived suppressor cells (MDSCs) immature myeloid cells related to monocytes (M-MDSC) or granulocytes (G-MDSC) Neutrophils/polymorphonuclear cells (PMNs) Tumor-associated macrophages Arginase is a key immunosuppressive enzyme in neutrophils and MDSCs

4 MDSCs are a Common Immunosuppressive Cell Type Across Tumor Histologies Tumor Type Elevated Peripheral MDSCs Suppressive Correlated with: Tumor burden/stage Prognosis Melanoma Head and neck cancer Lung cancer Hepatocellular carcinoma Renal cell carcinoma Bladder carcinoma Prostate cancer Gastrointestinal cancer Glioblastoma Ovarian cancer Breast cancer Pancreatic carcinoma Esophageal Tumor MDSCs observed Hematological malignancies Adapted from Solito et al. (214) Ann NY Acad Sci 1319:47

5 Arginase-expressing Granulocytic MDSCs/PMNs are Present in Multiple Tumor Types Tumor Arginase in MDSCs Peripheral Tumor MDSC Sub-type Breast Granulocytic/Monocytic 1 Prostate Granulocytic 2 RCC Granulocytic 3, 4 NSCLC Granulocytic 5, 6 GBM Granulocytic 7 HNSCC Granulocytic/Monocytic 8 Ref. Pancreatic Granulocytic 9, 1 HCC Monocytic 11 CML Granulocytic 12 Granulocytic markers (CD15+/CD14-) Monocytic marker (CD14+) References 1: deboniface et al. (212) OncoImmunology 1:135 5: Rotondo et al. (29) Int J Cancer 125:887 9: Khaled et al. (214) J Immunol Res 214: : Hossain et al. (215) Clin Cancer Res 21:3771 6: Heuvers et al. (213) Lung Cancer 81:468 1: Porembka et al. (212) Can Imunol Immunother 61:1373 3: Zea et al. (25) Cancer Res 65:344 7: Sippel et al. (211) Clin Cancer Res 17: : Hoechst et al. (28) Gastroenterology 135:234 4: Rodriguez et al. (29) Cancer Res 69:1553 8: Vasquez-Dunddel et al. (213) J Clin Invest 123:158 12: Giallongo et al. (214) Plos One 9:311848

6 Arginase 1 Co-localizes with Tumor-Infiltrating Granulocytes Head and Neck tumor labeled with a-arginase 1 and a-cd15 fluorescent antibodies Arginase 1 localizes with granulocyte marker CD15 in 95% of cases Arginase 1 H&E Overlay (95% overlap) CD15 (granulocytes)

7 Arginase 1 is Expressed in Tumor-Associated Myeloid Cells in Cancer Patients Arginase 1 Staining of Tumor Tissue Microarray Renal Pancreas TNBC NSCLC (adeno) NSCLC (squam) Gastric Colorectal Bladder Ovarian Prostate = arginase 1 positive neutrophil/granulocyte Arginase 1 IHC: Arginase 1 positive granulocytic myeloid cells present in most tumors Tumor cells rarely express Arg1 (except HCC) Normal healthy tissues generally do not express Arg1 with exception for liver and neutrophils

8 Arginase 1 Positive Granulocytes are Abundant in Many Human Cancers A r g i n a s e 1 P o s i t i v e c e l l s / m m L u n g ( S q u a m o u s ) L u n g ( A d e n o ) S t o m a c h B l a d d e r C o l o n E s o p h a g u s S k i n H e a d a n d N e c k B r e a s t P r o s t a t e O v a r y B r e a s t ( n o n - T N B C ) P a n c r e a s K i d n e y Arg 1 positive cells in tumor tissue (IHC)

9 A rg in a s e 1 (n g /m l) Arginase is Elevated in Cancer Patient Plasma n = N o r m a l H e a d & N e c k B la d d e r M e s o th e lio m a S m a ll C e ll L u n g C R C N S C L C A M L R C C B r e a s t Arg1 protein is elevated in plasma from patients across all histotypes relative to healthy volunteers

10 A rg in in e ( M ) Arginine is Decreased in Cancer Patient Plasma n = N o r m a l H e a d & N e c k M e s o th e lio m a S m a ll C e ll L u n g C R C N S C L C A M L R C C B r e a s t Normal arginine was collected from fasted donors Arginine levels are decreased in plasma from patients across all histotypes relative to healthy volunteers

11 C e l l C o u n t ( % m a x i m a l p r o l i f e r a t i o n ) n o r m a l p l a s m a l e v e l % m a x i m a l p r o l i f e r a t i o n ( C F S E d i l u t i o n ) n o r m a l p l a s m a l e v e l Decreases in Arginine Block Proliferation of Activated T-Cells and NK Cells Normal plasma arginine levels are 5-13 µm in healthy donors Arginine levels <4 µm suppress T-cell proliferation Arginine levels <1 µm suppress NK cell proliferation T-cell proliferation NK cell proliferation C D 4 + CD N K c e l l s 2 2 u n st i mul at e d [ A r g i n i n e ] ( M ) [ A r g i n i n e ] ( M ) CD4+ and CD8+ T-cells isolated from Human PBMCs and stimulated with anti- CD3/CD28 NK cells from healthy donor stimulated with IL-2

12 CB-1158 Inhibits Human Arginase CB-1158 is a potent and orally bioavailable small molecule arginase inhibitor CB-1158 selectively inhibits arginase (minimal off-target activity at 5 M) Arginase Reaction Human Arginase Source IC 5 Arginase 1 (recombinant) Arginase 2 (recombinant) Neutrophil lysate Red blood cell lysate Hepatocyte lysate RCC patient plasma #1 RCC patient plasma #2 98 nm 274 nm 162 nm 116 nm 139 nm 127 nm 174 nm

13 CB-1158 Reverses Human T-cell Immunosuppression by Neutrophils Ex vivo % C e ll D iv is io n (C F S E ) % C e l l D i v i s i o n ( C F S E ) A r g i n i n e ( M ) Neutrophils T-Cells Arginase Arginine Impaired T-Cell Proliferation Neutrophils suppress T-cell proliferation when co-cultured CB-1158 relieves T-cell suppression by neutrophils CB-1158 maintains arginine levels IC 5 = n M F u l l R e c o v e r y 8 7 IC 5 = 2 4 n M T -c e lls T -c e lls + n e u tro p h ils [ C B ] n M [ C B ] n M T-cells activated by anti-cd3/cd28 and proliferation measured after 96 hr

14 CB-1158 Restores Cytokine and Chemokine Receptor Expression in T-cells T r a n s c r i p t F o l d C h a n g e T-Cells Media Only T-Cell Proliferation 2 I F N g C X C R 3 Neutrophils Impaired T-Cell Proliferation 1 CB Neutrophils T-Cell Proliferation. 5 M e d i a O n ly N e u t r o p h i l s C B N e u t r o p h i l s T-cells activated by anti-cd3/cd28 in the presence of conditioned or unconditioned media Genes of interest were quantitated by Q-PCR at 24 h (IFNg) or 48h (CXCR-3)

15 Side Scatter Granulocytic MDSCs from Cancer Patient PBMCs contain Arginase 1 Count Count Count Count Count Count Cancer Patient CD14 + M-MDSC enriched MHCII - Higher arginase levels in G-MDSC PBMCs CD3 - CD14 MHCII CD66b + MHCII - Forward Scatter CD3 CD66 G-MDSC enriched MHCII Arginase 1 Granulocytic MDSCs Monocytic MDSCs

16 Monocytic MDSCs Do Not Suppress T-cell Function A r g i n i n e ( M ) % C e l l D i v i s i o n ( C F S E ) M-MDSCs T-Cells Arginase Arginine T-Cell Proliferation Conditioned media from M-MDSCs does not deplete arginine M-MDSC conditioned media does not suppress T-cell proliferation M e d i a O n ly M - M D S C C o n d i t i o n e d M e d i a T - c e l ls T - c e l ls + M - M D S C s T-cells activated by anti-cd3/cd28 and proliferation measured after 96 hr

17 CB-1158 Reverses T-cell Immunosuppression by Cancer Patient-Derived MDSCs % C e l l D i v i s i o n ( C F S E ) % C e l l D i v i s i o n ( C F S E ) A r g i n i n e ( M ) G-MDSCs T-Cells Arginase Arginine Impaired T-Cell Proliferation Conditioned media from G-MDSCs suppress T-cell proliferation CB-1158 relieves T-cell suppression by MDSCs CB-1158 maintains arginine levels IC 5 = n M Fu ll R e c o v e r y 7 6 IC 5 = n M T - c e l ls T - c e l ls G - M D S C s [ C B ] n M [ C B ] n M T-cells activated by anti-cd3/cd28 and proliferation measured after 96 hr G-MDSCs were isolated from a Lung Cancer Patient s PBMCs

18 C B (n m o l/g tis s u e ) A r g in in e (n m o l/g tis s u e ) C B ( M ) A r g in in e ( M ) CB-1158 Elevates Arginine Levels in Mouse Tumors Increasing oral doses of CB-1158 increases drug exposures in plasma and tumor Elevated exposures of CB-1158 increases plasma and tumor arginine levels P la s m a C B P la s m a A rg in in e T u m o r C B T u m o r A rg in in e m g /k g 5 m g /k g 1 m g /k g 2 5 m g /k g V e h ic le 2 5 m g /k g 5 m g /k g 1 m g /k g 2 5 m g /k g C 5 7.B L /6 m ic e b e a rin g L L C tu m o rs. B ID d o s in g, 5 d o s e s (2 h r a fte r la s t d o s e )

19 T u m o r V o l u m e ( m m 3 ) CB-1158 Anti-tumor Efficacy is Immune Based Dose-dependent activity in immuno-competent mice No anti-tumor activity in immuno-compromised mice Syngeneic Lewis Lung Carcinoma (LLC) model C57.BL/6 mice: Intact immune system SCID mice: Immunocompromised D a y s P o s t I m p l a n t D a y s P o s t I m p l a n t V e h i c l e C B m g / k g B I D C B m g / k g B I D C B m g / k g B I D

20 T u m o r V o lu m e (m m 3 ) ) T u m o r V o lu m e (m m 3 ) CB-1158 Anti-tumor Efficacy is Immune Based Single agent activity is observed in the checkpoint refractory LLC model CD8 depletion partially reverses the anti-tumor effects of CB-1158 NK-cell depletion results in near complete reversal of CB-1158 effects Drug is well tolerated with no clinical observations or body weight changes 6 V e h ic le -c o n tr o l C B m g /k g B ID 1 V e h ic le C B m g /k g B ID n s C B C D 8 + c e ll d e p le tio n 8 C B N K c e ll d e p le tio n D a y s P o s t Im p la n t D a y s P o s t Im p la n t

21 CB-1158 Increases T-cell/NK-cell Markers and Interferon-Stimulated Genes T r a n s c r i p t C o u n t T r a n s c r i p t C o u n t T-cell and NK cell markers are elevated Interferon-stimulated genes are induced T-cell and NK-cell markers C D 3 T C R z C D 6 9 I L 2 R B C D 9 4 N C R Interferon response genes I N F A 1 I S G 1 5 U S P 1 8 I R F 5 I R F 7 D d x V e h ic le C B Nanostring analysis of LLC tumors from mice treated with vehicle or CB-1158 (1 mg/kg BID for 14 days)

22 n g / m L CB-1158 Increases Inflammation in the Tumor Microenvironment % o f l i v e c e l l s Cytokine/chemokine changes suggests skewing toward a pro-inflammatory Th1/M1 phenotype Decrease in immuno-suppressive M2 macrophages Increase in CD8 positive T-cells Increase in KI67 + and TCRz positive CD8+ T-cells suggests T-cell proliferation and activation Luminex Flow Cytometry Th1 Cytokines M1 Macrophage Chemokines M2 Macrophage Arg1+ CD68+ CD3+ CD8+ KI67+ CD3+ CD8+ TCRz CD3+ CD8+ I F N - g I L p 7 M I P - 1 M I P V e h i c l e C B % o f C D L L O Q. 1 L L O Q Luminex analysis of LLC tumors from mice treated with vehicle or CB-1158 (2 mg/kg BID for 14 days) Flow cytometry of LLC tumors from mice treated with vehicle or CB-1158 (1 mg/kg BID for 14 days)

23 CB-1158 Enhances Efficacy of Gemcitabine in the LLC Model T u m o r V o l u m e ( m m 3 ) L L C M o d e l 8 V e h i c l e C B , 1 m g / k g B i d G e m c i t a b i n e, 6 m g / k g D a y 6, 1 6 C B G e m c i t a b i n e D a y s P o s t I m p l a n t

24 CB-1158 Single Agent Efficacy in the Madison 19 Lung Carcinoma Model T u m o r V o l u m e ( m m 3 ) M a d i s o n 1 9 M o d e l 1 V e h i c l e C B m g / k g D a y s P o s t - I m p l a n t

25 T u m o r V o l u m e ( m m 3 ) # L u n g M e t s CB-1158 Enhances Efficacy of apd-1 + actla-4 Efficacy seen with triple combination in 4T1 model 4 T 1 M a m m a r y C a r c i n o m a 1 5 V e h i c l e C B m g / k g 1 5 L u n g M e t a s t a s i s a P D a C T L A C B a P D a C T L A D a y P o s t I m p l a n t CB-1158 dosed orally BID starting Day 2 until study end actla-4 (clone 9H1) dosed IP at 5 mg/kg on Days 2, 5, 8 apd-1 (clone RMPI-14) dosed IP at 5 mg/kg on Days 3, 6, 9 V e h i c l e C B , 1 m g / k g a C T L A a P D 1 C B a C T L A 4 + a P D 1

26 T u m o r V o l u m e ( m m 3 ) CB-1158 Single Agent Efficacy in B16.F1 Model B 1 6. F 1 S y n g e n e i c M o d e l 1 5 V e h i c l e 1 C B m g / k g B I D P = D a y s P o s t I m p l a n t Oral dosing of CB-1158 or Vehicle started on Day 2

27 T u m o r V o l u m e ( m m 3 ) T u m o r V o l u m e ( m m 3 ) NK and CD8 + T-cells are Required for CB mediated Efficacy in the B16 Model Depletion of either CD8 + T-cells or NK cells both fully reverse the efficacy of CB-1158 in the B16 model 1 5 B 1 6. F 1 M o d e l V e h i c l e C B m g / k g B I D 1 5 Day 13 Tumor Volume C B a C D C B a N K D a y s P o s t I m p l a n t V e h i c l e C B m g / k g B I D C B a C D - 8 C B a N K 1. 1

28 T u m o r V o l u m e ( m m 3 ) T u m o r V o l u m e ( m m 3 ) CB-1158 Enhances the Efficacy of IDO Inhibitor B 1 6. F 1 M o d e l V e h i c l e, c o n t r o l 8 C B , 1 m g / k g B I D 1 2 E p a c a d o s t a t, 1 m g / k g B I D 6 C B E p a c a d o s t a t D a y s P o s t I m p l a n t CB-1158 and Epacadostat dosed orally starting on Day 2 V e h i c l e, c o n t r o l C B , 1 m g / k g B i d E p a c a d o s t a t, 1 m g / k g B i d C B E p a c a d o s t a t

29 T u m o r V o l u m e ( m m 3 ) T u m o r V o l u m e ( m m 3 ) T u m o r V o l u m e ( m m 3 ) CB-1158 Enhances the Efficacy of apd-l1 a P D - L B 1 6. F 1 M o d e l V e h i c l e C B , 1 m g / k g B I D a P D - L 1, 5 m g / k g C B a P D - L D a y s P o s t I m p l a n t 2 a P D - L 1 + C B D a y s P o s t I m p l a n t CB-1158: 1 mg/kg PO BID start on Day 1 apd-l1 (1F.9G2): 5 mg/kg IP Days 3, 5, 7, 9, 11, D a y s P o s t I m p l a n t

30 Summary Arginase-expressing MDSCs/neutrophils are present in several tumor types MDSC/neutrophil-derived arginase depletes arginine and causes suppression of T- cells and NK-cells CB-1158 potently inhibits arginase and reverses MDSC/neutrophil induced suppression of T-cell proliferation CB-1158 is orally bioavailable, increases tumor and plasma arginine levels, and has immune-mediated single agent efficacy in syngeneic mouse tumor models CB-1158 increases the anti-tumor efficacy of gemcitabine, checkpoint and IDO inhibitors CB-1158 has potential for activity across a wide range of tumors A Phase I clinical trial in cancer patients has been initiated testing the clinical activity of CB-1158

31 Acknowledgements Biology Francesco Parlati Ethan Emberley Andy MacKinnon Gisele Marguier Silinda Neou Alison Pan Susanne Steggerda Melissa Works Pharmacology Matthew Gross Jason Chen Tony Huang Julie Janes Amani Makkouk Chemistry Jim Li Roland Billedeau Lijing Chen Guy Laidig Tim Stanton DMPK Weiqun Li Tracy Wang Jing Zhang Winter Zhang Pharm. Dev. Peter Shwonek Natalija Sotirovska Scientific Management Team Mark Bennett Eric Sjogren