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A Novel Immunomodulatory Strategy of Targeting Glyco-Immune Checkpoints with EAGLE TechnologyLizhi Cao1, Adam Petrone1, Wayne Galtlin1, Lihui Xu1, Michele Mayo1, Michal Stanczak2, Jenny Che1, Carolyn Bertozzi3, Karl Normington1, Jeff Brown1, Heinz Läubli2, James Broderick1, Li Peng1

1Palleon Pharmaceuticals, Waltham, MA, USA, 2 Cancer Immunology Laboratory, University Hospital Basel, Switzerland, 3 Department of Chemistry, Stanford University, Stanford, CA, USA

Introduction

Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors to harness the power of the immune system in fighting cancer. However, the majority of patients fail to have durable responses or become resistant to immuno-oncology drugs, highlighting the need to identify new mechanisms of immune evasion in cancer and to develop new therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a novel mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape1-3. Upon ligation of sialylated glycans to ITIM-containing Siglecs on immune cells, this pathway plays a previously unrecognized role in regulating functions of NK cell, macrophages, dendritic cells, monocytes and T-cells in the tumor microenvironment. It suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, priming and activation of anti-cancer T-cell immunity, which may represent a novel mechanism of resistance to current immunotherapy. Here, we described a novel therapeutic modality, a multi-functional antibody-like molecule named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), to inhibit the glyco-immune checkpoint axis in the tumor microenvironment by selectively removing the terminal sialic acids of sialoglycans on tumor cells.

Figure 1. Glyco-immune checkpoints. (Left) Schematic representation of glyco-immune checkpoints axis. At the immunological synapse, Siglecs (sialic acid-binding Ig-like lectins) on immune cells interact with tumor associated sialoglycans and recruit SHP phosphatases, dampening immune responses. Siglecs are expressed on macrophages, monocytes, DC, and NK cells, recognizing a variety of tumor associated glycans which contain a terminal sialic acid. (Right) The role of Siglec/Sialoglycan axis in cancer immunity cycle. The Siglec/Sialoglycan plays roles in cancer cell killing by innate immune cells, cancer antigen presentation, T cell priming and activation, and cancer cell killing by T effector cells. Sialylated glycans, sLex and sTn, were reported to associate with poor outcome in gastric and breast cancer patients.

Glyco-Immune Checkpoints Suppress Multiple Steps in Cancer ImmunityGlyco-Immune Checkpoints in Cancer:

The Siglec/Sialoglycan Axis

Siglec Binding to Hyper-Sialylated Tumor Glycans Blocks Innate & Adaptive Immune Cell Activation

NK CellsMacrophagesEffector T Cells

Dendritic Cells

ImmuneCell

Immuno-SuppressionSiglecs

Hyper-Sialylated Tumor Glycans

Activating Receptor

Immunological Synapse

(-)

(-) ITIM

ITIM

Cancer Cell

Immune Evasion

DAMP/ Tumor Antigen

Adopted from Chen Daniel S. et al. Immunity

Cancer Cell Death and

Antigen Release

Cancer Antigen Presentation

T Cell Priming and Activation

T Cell Infiltration and Killing of Cancer Cells

Evolution Immuno-editing

Innate Immune Cell Killing of Cancer Cells

NK CellsMacrophages

Effector T Cells

Dendritic Cells

Macrophages

Dendritic Cells

Macrophages

Innate Immunity

Adaptive Immunity

Effector T Cells

Tumor

Cancer Immunity Cycle The Roles of Siglec/Sialoglycan Axis

Results

Development of the EAGLE Therapeutic Platform (Enzyme-Antibody Glycan-Ligand Editing)

Bertozzi Lab(Stanford University)

Bacterial Sialidase Chemical ConjugatesIn vitro PoC

^ ^(

Human Sialidase Genetic FusionsIn vivo PoC

Improve CMC Developability

Select Ideal Human Sialidase

Determine EAGLEConfigurations

Cancer cell

Xiao H, Woods EC, Vukojicic P, Bertozzi CR PNAS. 2016

Initial Academic Concept Reduction to Practice

• Developability (yield, purity, stability)

• Intact Fc functions (FcRn and Fc-gamma receptors)

• Dual functions

- Tumor-associated antigen binding arm

- Sialidase arm

- Targeted de-sialylation of tumor cells

Figure 2. Evolution of EAGLE platform. (A) Schematic representation of antibody-sialidase chemical conjugates. Conjugating bacterial sialidase to antibodies which recognize tumor-associated antigens enables targeted removal of sialic acids of sialoglycans on tumor cells4. (B) Development of EAGLE therapeutic platform.

EAGLEs Showed Targeted Cleavage of Terminal Sialic Acids from Her2+ Tumor Cells

Figure 3. FACS staining of desialylation of tumor cells treated with EAGLEs. Compared to the control group of trastuzumab treatment, various EAGLEs (301, 302, and 303) decreased sialic acid levels on cancer cells with high or low levels of Her2. The cleavage of sialic acids on tumor cells are specific, since no sialic acid cleavage was observed for the silent enzyme control (EAGLE-307) and substantially reduced cleavage for the silent Her2-binding (EAGLE-305).

EAGLE Reduced Tumor Cell Surface Sialic Acids Levels in vivo

Figure 4. Systematic delivery of EAGLE cleaved sialic acids from tumors in vivo. (A) Experiment design of EAGLE treatment. (B) FACS analysis of surface sialic acid levels of tumors.

A

B

Multiple EAGLE Formats Achieved Single Agent Complete Regressions in Preclinical Tumor Models

Figure 5. Efficacy studies of EAGLEs in syngeneic breast cancer EMT6-Her2 tumor model. Wild type BALB/c mice (n = 8 per group) were injected s.c. EMT6-Her2 cells. When tumor sizes reached about ~120 mm3, mice were treated with EAGLE-301, 302, 303, Trastuzumab, or vehicle control twice a week for 3 weeks at 10mg/kg. The tumor growth was measured twice a week.(A) Tumor growth curves of mean tumor sizes of each group. (B) Tumor growth curves of individual mice.

A

B

EAGLE Showed Sialidase-Dependent Efficacy, Induced Anti-Tumor Immunological Memory, and Increased Immune Cell Infiltration

A

B

C

Figure 7. Efficacy studies of EAGLE in orthotopic syngeneic breast cancer tumor model. (A) EAGLE demonstrated sialidase-dependent anti-tumor activity. Wild type BALB/c mice (n = 6 per group) were inoculated with EMT6-Her2 cells into mammary fat and treated with EAGLE-302 and its loss-of function control when tumor sizes reached about ~250 mm3. EAGLEs and trastuzumab were dosed twice a week for 2 weeks at 10mg/kg. (B) Cured mice from EAGLE treatment in experiment (A) rejected re-challenge of EMT6-Her2 and EMT6 cells. (C) FACS analysis of tumor infiltrating lymphocytes after EAGLE treatment. EAGLE increased T cell infiltration and activation.

The Mechanism of Action of EAGLE Involves Innate and Adaptive Immunity

EAGLE has Monotherapy Efficacy Comparable to the Combination of α-PD1 and α-CTLA4 in the “Cold” Tumor B16 Model

Figure 6. EAGLE demonstrated single agent anti-tumor activity in B16D5-Her2 syngeneic tumor model. Wild type C57BL/6 mice (n = 6 per group) were inoculated with B16D5-Her2 cells subcutaneously and treated with EAGLE-302, the combination of anti-CTLA4 and anti-PD-1, or Trastuzumab when tumor sizes reached about ~100 mm3. All antibodies were dosed twice a week for 2 weeks at 10mg/kg. Each line represented a tumor growth curve of an individual mouse.

Cold Tumor B16 Model (B16D5-Her2)

EAGLE-302: No weight lossα-PD1 + α-CTLA4: Weight loss

α-CTLA4 + α-PD1

EAGLE-302

Trastuzumab

Tumor ~350 mm3

Single dose (10mg/kg)

24h (p.i.) 72h (p.i.)

Sacrifice & FACS analysis

FACS Analysis: Measure of Tumor Cells’ De-Sialylation

↑PNA = ↑Galactose Residues =

De-sialylation↓MAL II = ↓sialic acids =

De-sialylation

Breast Cancer EMT6-Her2 Syngeneic Tumor Model

Intra mammary implantation of EMT6-Her2 Balb/c

A

B

Figure 8. The MoA of EAGLE involves NK cells, macrophages, and CD8+ T-cells. Wild type BALB/c mice (n = 8 per group) were injected s.c. EMT6-Her2 cells. When tumor sizes reached about ~120 mm3, mice were treated on the same days as EAGLE-302 with either anti-mouse NK1.1 (10 mg/kg) to deplete natural killer cells, liposomal clodronate (0.5 mg/mouse, three times a week for two weeks) to deplete macrophages, or anti-mouse CD8α (10 mg/kg) to deplete CD8+ T cells. (A) Tumor growth curves of mean tumor sizes of each group. (B) Tumor growth curves of individual mice.

EAGLE Has Significant Combination Efficacy with T-Cell Checkpoint Inhibition

Figure 9. EAGLE achieved 100% cures in breast cancer EMT6-Her2 orthotopic model in combination with PD-1/PD-L1 inhibitions. Wild type BALB/c mice (n = 6 per group) were inoculated with EMT6-Her2 cells into mammary fat. When tumor sizes reached about ~250 mm3, mice were treated with EAGLE-302, anti- PD1 mAb, the combination of EAGLE-302 and anti-PD1 mAb, or controls of EAGLE-loss-of function and Trastuzumab twice a week for 2 weeks at 10mg/kg.

No Change in Blood Counts No Impact to Platelets Modest Changes in Liver EnzymesNo Weight Loss

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EAGLE-Her2 Pilot Rat Toxicity Study Showed No Major Events

Figure 10. Rat 14-day toxicity study for EAGLE-Her2. EAGLE-302 was given by intravenous administration on days 1, 4, 8 and 12 to Sprague-Dawley rats. There were no significant changes in body weights, white or red blood cell counts, or histopathological findings.

Proprietary Hydra Biomarker Assays to Detect Tumor Glyco-Codes in Patients Breast CancerColon CancerNSCLC

Conclusions

References1. Crocker PR, Varki A. Siglecs in the immune system. Immunology. 2001;103:137-45.2. Adams O.J., et al, Targeting sialic acid-Siglec interactions to reverse immune suppression in cancer, Glycobiology, 2018, 28:640-73. Stanczak M., et al., Self-associated molecular patterns mediated cancer immune evasion by engaging Siglecs on T cells, J Clin Invest, 2018, 128: 4912–49234. Xiao H., et al, Glycocalyx editing for cancer immunotherapy, PNAS 2016, 113:10304-10309

• Glyco-immune checkpoints play critical roles in cancer immune evasion • Innate immune response• Adaptive immune response

• EAGLE showed compelling monotherapy efficacy in syngeneic tumor models

• Single agent complete regressions with immunological memory

• Efficacious in cold tumor model • Striking activity in combination with PD-1/PD-L1

• EAGLE offers new opportunities to treat cancer targeting glyco-immune checkpoints

• Overcomes the heterogeneity challenges of tumor sialoglycans

• Disables immunosuppressive glycan functions within tumor microenvironment

• Scalable, potential to transform existing tumor targeting mAbs into immune-modulating agents

Figure 11. Representative Hydra staining of tumor sialoglycan levels in NSCLC, colon cancer, and breast cancer for patient selection.

EAGLE

EAGLE Loss-of-Function

EAGLE

6/6 CR3/6 CR 4/6 CR

Dosing

Anti-PD1 EAGLE + Anti-PD1

Control

Anti-PD1

Control

Anti-PD1 + EAGLE

Dosing Dosing

Single Agent Activity Combination

Breast Cancer EMT6-Her2 Syngeneic Orthotopic Model

3/6 CR

Dosing

Breast Cancer EMT6-Her2 Syngeneic Orthotopic Model

Rechallenge

Cured Mice from EAGLE (n=3)

Complete Rejection of Rechallenge of EMT6-Her2 and EMT6 Cells

EAGLE-302 treated

Naïve mice

vs

(Sialidase Silent)

Vehicle

Trastuzumab

EAGLE-302

EAGLE-302 LoF

0 1 0 2 0 3 0 4 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

D a y s ( p o s t - r a n d o m i z a t i o n )

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mo

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lu

me

(m

m3

,m

ea

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SE

M)

V e h i c l e C o n t r o l

E 3 0 1 ( R a p t o r )

E 3 0 2 ( J a n u s )

E 3 0 3 ( L o b s t e r )

T r a s t u z u m a b

Dosing

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0/8 CR 2/8 CR 3/8 CR 2/8 CR

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0 2 0 4 0 6 0

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D a y s ( p o s t - r a n d o m i z a t i o n )

Tu

mo

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olu

me

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mm

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Breast Cancer EMT6-Her2 Syngeneic Subcutaneous Tumor Model

n=8 mice per groupAntibodies dosed at 10mg/kg

Breast Cancer EMT6-Her2 Syngeneic Subcutaneous Tumor Model

0 2 0 4 0 6 0

0

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3/8 CR0/8 CR 1/8 CR 0/8 CR1/8 CR0 2 0 4 0 6 0

0

5 0 0

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1 5 0 0

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D a y s ( p o s t - r a n d o m i z a t i o n )

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NK Depletion Macrophage Depletion CD8+ T Cell DepletionEAGLETrastuzumab

0 1 0 2 0 3 0 4 0 5 0

0

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D a y s ( p o s t - r a n d o m i z a t i o n )

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mo

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m3

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M)

V e h i c l e C o n t r o l

E 3 0 2

E 3 0 2 + a n t i - m o u s e N K

E 3 0 2 + a n t i - m o u s e C D 8

E 3 0 2 + M O d e p l e t i o n

T r a s t u z u m a b

n=8 mice per groupAntibodies dosed at 10mg/kg

Dosing

MAL II PNA

↑PNA binding signals = ↑Galactose Residues = De-sialylation

↓MAL II binding signals = ↓Sialic acids = De-sialylation

Unstained Cells

Secondary aloneTrastuzumab

EAGLE-301EAGLE-302EAGLE-303

EAGLE-305 (No Her2-binding)EAGLE-307 (Silent Enzyme)

SKBR-3 Cells (Her2 +++)

MAL II PNA

BT-20 Cells (Her2 +)

EAGLE-301(Raptor Format)

EAGLE-302(Janus Format)

FACS Staining of De-sialylation of Tumor Cells by EAGLEs

EAGLE-303(Lobster Format)

EAGLE-307(Silent Sialidase)

EAGLE-305(No-Her2 Binding)

Cartoon Representations of EAGLE Molecules