Intratumoral Injection of SYNB1891, a Synthetic Biotic Medicine Designed

Intratumoral injection of SYNB1891 A Synthetic Biotic medicine designed to activate the innate immune system. Therapy demonstrates target engagement in humans including intratumoral STING activation. Janku F, MD Anderson Cancer Center; Luke JJ, UPMC Hillman Cancer Center; Brennan AM, Synlogic; Riese RJ, Synlogic; Varterasian M, Pharmaceutical Consultant; Kuhn K, Synlogic; Sokolovska A, Synlogic; Strauss J, Mary Crowley Cancer Research Presented by Filip Janku, MD, PhD Study supported by Synlogic, Inc

American Association for Cancer Research (AACR) April 2021 Introduction and Methods

SYNB1891 Strain Phase 1 First-in-Human Clinical Trial

• Live, modified strain of the probiotic E. coli • Enrolling patients with refractory advanced solid Nissle engineered to produce cyclic tumors or lymphoma dinucleotides (CDN) under hypoxia leading to stimulator of interferon genes (STING)- • Intratumoral (IT) injection of SYNB1891 on Days activation 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. • Preferentially taken up by phagocytic antigen- presenting cells in tumors, activating • Dose escalation planned across 7 cohorts (1x106 complementary innate immune pathways – 1x109 live cells) with Arm 1 consisting of (direct CDN STING activation; cGAS-mediated SYNB1891 as monotherapy, and Arm 2 in STING activation and TLR4/MyD88 activation by combination with atezolizumab the bacterial chassis) SYNB1891 was safe and well-tolerated in heterogenous population

Nov 2020: Interim Analysis IA Updated through 15 Mar 2021 15 Mar 2021: Current Enrollment: Focused on first 11 monotherapy pts No DLTs | No additional CRSs | No 22 patients across 4 sites in the US dosed at 1e6 to 3e7 live cells additional SYNB1891-related SAEs Monotherapy dosed at 1e6 to 1e8 live cells; Mean age 56 yo; 82% female; 82% combination therapy dosed at 1e7 live cells white; all patients progressed on prior oncology therapies Tumor types: melanoma [4], sarcoma [4], esophageal [4], squamous (including 2 head and neck) [4], colon/colorectal [2]; small cell lung, basal cell, bile duct adeno, jejunum adeno 59 IT Doses Administered • Two events of cytokine release syndrome – both resolved within 1 day ✓ No Dose limiting toxicities • 1 injection site reaction/erythema (mod) ✓ No SYNB1891-related infections • No bacteria DNA detected by blood PCR 6 hours after the 1st dose at any ✓ No discontinuations due to adverse events (AEs) dose 2 out of 11 patients with stable disease

Patient Dose Tumor Duration of Response 100-002 1e6 Vulvar melanoma Recist -28% 600-002 1e7 Small Cell Lung Recist -12% Ongoing 100-004 3e6 Basal Cell 100-006 1e7 Squamous Cell 100-005 1e7 Bone chondrosarcoma 200-003 1e6 Liposarcoma 200-004 3e6 Sarcoma - breast 600-003 1e7 Bile duct adeno 200-001 1e6 Synovial Sarcoma

600-001 3e6 Esophageal Clinical progression prior to first restaging 600-004 3e7 Vulvar squamous cell 0 50 100 150 200 250 300 Days

Stable Disease Progressive Disease Serum cytokines suggest systemic inflammatory response with dose escalation

Serum Cytokines: (Baseline, 6 and 24h post-dose): IL-6, TNFa, IFNg IL-1R

antagonist

inflammatory

Pro

1 T T cell 1

- Th

As dose increases cytokine levels also increase Tumor nanostring data suggest target engagement 6 Patients Dosed at 1e6 -1e7 Live Cells

4 Mean± SEM across 6 patients Most upregulated genes across 6 patients in

different categories

n i

l (fold change over baseline) e

s 3

r ISGs – Interferon-Stimulated Genes

v o 2

e CXCL9/10 – Chemokines inducing immune

n a

h cell migration to tumor (Th1, CTLs, NK cells) C

d l

o TNF/R superfamily – induce tumor apoptosis F

0 Granzyme A (GZMA) – induce tumor cytotoxicity

5 1 2 0 9 0 4 2

B A

1 1 1

T T L L

1 D

I I

L F

G C F

F F

I I

S X S

I CD4 – CD4+ T cells

C R

T N

T PD-L2 – Ligand for PD-1 (checkpoint receptor) ISGs Chemokines T cells Cytokines Biopsies pre-treatment – Performed on injected

Patients: 100-002 600-002 lesion in week 4 (7 days after the third weekly dose) Multiplex immunofluorescence staining (IF) for tumor cores

Representative images from the patients: Enhanced T cell signal in “warm” tumors 100-002 600-002 200-003 100-005

Vulvar melanoma SCLC Liposarcoma Chrondrosarcoma

Baseline SYNB1891

DAPI (nucleus), CD4+ cells, CD8+ cells Patient 100-002: Stable disease at 7 months with ensuing progressive disease Metastatic Vulvar Melanoma previously treated with Nivolumab Strong upregulation of INF pathway & T cell Stable disease, prior Nivolumab response 63-yo Female dosed with 1e6 cells Interferon- Cytokines T cell Previous Treatment: Local resection, nivolumab (see note) Stimulated genesChemokines Functions 4 4 3 Adverse Events: Itching, Hypoglycemia, Anxiety, Atrial fibrillation ISG15 IL2 CD3D IFIT1 IL12B CD3E IFI27 IL1B CD3G Patient history: On nivolumab from 30 Jan 2019 to 06 Nov 2019 with PD IFIT2 IL1A CD4

IFITM1 IL18 CD8A n o

3 i

Subject started treatment (C1D1) with SYNB1891 on 10 Jul 2020. IFIH1 IL4 CD8B t 3

s l

IFI35 IL5 FOXP3 u G 2

KIT/PDGFRA/KDR Amplification. ATM Deletion g e S IL6 CD28

ISG20 r I

IFI16 IL12A CD80 p U IFNAR2 IL10 CD86 IFITM2 CXCL11 CTLA4 2 2 IFNAR1 TNF PD-1 IFNL1 TNFRSF18 PD-L1 Tumor reduction (cm/%) IFNA7 TNFRSF9 PD-L2 1

IFNB1 TNFSF12 LAG3 n

o i Sum of Measurements GZMA 1 t

s IFNA17 TNFRSF4 1

l u

N IFNA2 TNFAIP3 GZMB

g F

Percent Change e I

IFNL2 TNFRSF1B GZMH r

IFNA8 TNFSF13B GZMK n w

IFNA1 TNFSF10 GZMM o

1 1

NanoString data analysis (Baseline tumor vs Cycle 2 Day 1 tumor): • Multiple ISGs ↑ - STING pathway engagement • IL-10, IL-12A and CXCL-11 upregulation • Small changes in tumor T cell compartment Patient 600-002: imaging results indicate stable disease at >7 months Small cell lung cancer previously treated with Pembrolizumab Strong upregulation of INF pathway & T cell Stable disease, prior Pembro response 55-yo Female dosed with 1e7 cells Interferon- Antigen T cell Stimulated genes Processing Functions Previous Treatments: Etoposide/carboplatin, Pegzilarginase, 4 4 ISG15 HLA-A CD3D Pembrolizumab IFIT1 HLA-B CD3E 4 IFI27 HLA-C CD3G Adverse Events: Hyponatremia – mild, not related, Bradycardia – mild, CD4 IFIT2 TAP1

CD8A n

related IFITM1 TAP2 o

3 i

IFIH1 CD8B t 3 a

s PSMB10 l

IFI35 FOXP3 u 3 G

PSMB7 g e

S ISG20 CD28 r Patient History: On pembrolizumab for 14 months: 13 Mar 2019 to 27 May I PSMB8 IFI16 CD80 p PSMB9 U 2020 with PD . Subject started treatment (C1D1) with SYNB1891 on 01 Jul IFNAR2 CD86 PSMD7 IFITM2 CTLA4 2 2 2020 HLA-DMA IFNAR1 PD-1 2 HLA-DMB IFNL1 PD-L1 Tumor reduction (cm/%) IFNA7 HLA-DPA1 PD-L2 HLA-DPB1

IFNB1 LAG3 n

o i HLA-DRA 1 t

s IFNA17 GZMA 1

l u

Sum of Measurements N IFNA2 HLA-DRB3 GZMB 1

F e I CD1B

IFNL2 GZMH r

Percent Change IFNA8 CD1C GZMK n w

IFNA1 CD1D GZMM o

6 6 Cytokines: Modest ↑ in serum INFγ an IL-1Rα but not in IL-6 or TNF- α NanoString STING: Multiple ISGs upregulated Chassis: ↑chemokines, cytokines and TLRs genes T cell compartment: ↑↑ antigen processing and T cell function genes SYNB1891 safe and well tolerated, data supports continued study

Dose levels through 1e7 live cells SYNB1891 is safe and well-tolerated Evidence of durable stable disease demonstrate target engagement as as an intratumoral injection in a was seen in 2 patients and was assessed by increases in serum heterogenous population. associated with upregulation genes cytokines, upregulation of ISGs and tied to immune activation and No dose limiting toxicities or presence of tumor infiltrating increased intratumoral lymphocytes infections lymphocytes

These data support continued dose escalation in the monotherapy arm and combination arm with atezolizumab