KB174 Reduces Relative Abundance of Multidrug Resistant (MDR) Enterobacteriaceae in Fecal Samples From Patients With Cirrhosis in an Ex Vivo AssayJasmohan S. Bajaj,1 Kelsey Miller,2 Jonathan Lawrence,2 Jie Tan,2 Norma A. Palma,2 Gabrielle LeBlanc,3 Tanya Yatsunenko31Virginia Commonwealth University School of Medicine and Hunter Holmes McGuire VA Medical Center, Richmond, Virginia;2Kaleido Biosciences, Inc, Lexington, Massachusetts; 3Former employees of Kaleido Biosciences, Inc

INTRODUCTION• Patients with liver disease are at risk for spontaneous bacterial peritonitis and other infections1

• Infection occurs in 25% to 35% of patients at admission or during hospitalization (4-5x higher than in the general population), often with multidrug-resistant bacteria1,2

• Bacterial infection increases the probability of death 4-fold in patients with decompensated cirrhosis, with sepsis the number one cause of death in hospitalized patients with cirrhosis3,4

• Many multidrug-resistant infections, including spontaneous bacterial peritonitis, originate as gut-colonizing pathogens, which translocate to the ascitic fluid or other body sites1

• Microbiome Metabolic Therapies (MMTs™) are designed to target the glycan utilization potential of commensal microbiota and not pathogens (e.g. carbapenem-resistant Enterobacteriaceae [CRE]) (Figure 1)5

OBJECTIVE

• KB174 is not directly used by CRE E. coli but causes an enrichment of Parabacteroides, which contributes to the reduction of the relative abundance of E. coli in an ex vivo assay with samples from healthy subjects and patients with cirrhosis

• KB174 led to significant decreases in the relative abundance of multidrug-resistantEnterobacteriaceae as compared with water

PRECISIONscientia provided editorial support

FIGURE 4. RELATIVE ABUNDANCE OF ENTEROBACTERIACEAE IN CIRRHOTIC

SAMPLES AFTER INCUBATION WITH KB174 OR LACTULOSE

RESULTS (CONT)

CONCLUSIONS

ACKNOWLEDGEMENTS

• To determine whether KB174 can reduce the relative abundance of CRE E coli in fecal samples from patients with liver disease in comparison to lactulose

REFERENCES1. Piano S, et al. Infections complicating cirrhosis. Liver Int. 2018;38(suppl 1):126-133.2. Jalan R, et al. Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013. J Hepatol. 2014;60(6):1310-1324.3. Aravanti V, et al. Infections in patients with cirrhosis increase mortality four-fold and should be used in determining prognosis. Gastroenterology. 2010;139(4):

1246-1256.4. Schmidt ML, et al. Decreasing mortality among patients hospitalized with cirrhosis in the United States from 2002 through 2010. Gastroenterology. 2015;148(5):

967-977.e2.5. LeBlanc G, et al. Novel glycans reduce carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococcus (VRE) colonization in an ex vivo

assay by supporting growth of commensal microbiota at the expense of multidrug-resistant (MDR) organisms. Poster presented at: IDWeek 2019; October 2-4, 2019; Washington, DC.

6. Data on file. Kaleido Biosciences Inc.7. Bajaj JS, et al. Microbiome Metabolic Therapies reduce microbiota-associated ammonia in ex vivo fecal samples from healthy subjects and patients with minimal

hepatic encephalopathy and demonstrate improved tolerability over lactulose in a clinical study. Accepted for presentation at: EASL 2020 The International Liver Congress; April 15-19, 2020; London, United Kingdom (postponed to August 2020); and Digestive Disease Week; May 2-5, 2020; Chicago, IL (cancelled). Poster 4178.

8. Bhandari BR, et al. A randomized, double-blind study to evaluate the safety and tolerability of KB174, a novel synthetic glycan, in patients with well-compensated cirrhosis. Accepted for presentation at: EASL 2020 The International Liver Congress; April 15-19, 2020; London, United Kingdom (postponed to August 2020); and Digestive Disease Week; May 2-5, 2020; Chicago, IL (cancelled). Poster 4204.

Ex vivo assay: fecal samples from 25 patients with cirrhosis (collected in Kaleido study S005) who were not on lactulose or rifaximin and from 19 healthy controls were processed into suspensions. CRE E. coli*was added to each of the fecal slurries. Samples were incubated anaerobically for 45 hours at 37°C in mega medium without the addition of TWEEN 80 (Sigma-Aldrich) or meat extract. KB174 or lactulose was added as a carbon source, and samples incubated with water in the absence of added carbon source served as a control. Genomic DNA was extracted from all samples in the study and used for shallow shotgun sequencing*ID: Antibiotic Resistance Isolate Bank 0001. Resistance mechanisms: aac(6')Ib-cr, aadA5, dfrA17, KPC-3, mph(A), OXA-1, sul1, tet(A). Source: Centers for Disease Control and Prevention Enterobacterales Carbapenem Breakpoint panel

RESULTS• KB174 does not support the growth of CRE E. coli above what is measured in the background media

with no added carbon source, suggesting E. coli cannot use this MMT • Despite significant differences in community composition between healthy and cirrhotic fecal

communities, incubation with KB174 led to significant decreases in the relative abundance of multidrug-resistant Enterobacteriaceae as compared with water (mean percent decrease, 43% [P<.001] and 50% [P<.001], respectively) (Figure 3)

FIGURE 3. RELATIVE ABUNDANCE OF E COLI IN HEALTHY OR CIRRHOTIC SAMPLES AFTER INCUBATION WITH KB174, LACTULOSE, OR WATER

KB174 Lactulose

Structure

Glycan type Oligosaccharide mixture Dimer

Molecular formula H-[C6H10O5]n-OHWhere n=9 on average C12H24O12

Sugar types Glucose, galactose Galactose, fructose

Bond types Multiple alpha and beta 1,1-; 1,2-; 1,3-; 1,4-; and 1,6- Only 1 beta-1,4-

Branching Yes No

Amount of carbohydrate-activated enzymes required for fermentation Large Small

TABLE 1. STRUCTURAL COMPLEXITY OF KB174 AS COMPARED WITH LACTULOSE

FIGURE 5. PERCENT CHANGE IN PARABACTEROIDES RELATIVE ABUNDANCE IN HEALTHY OR CIRRHOTIC SAMPLES AFTER

INCUBATION WITH KB174 OR LACTULOSE

• A greater reduction in relative abundance of Enterobacteriaceae was seen in 68% (17 of 25) of samples tested from patients with cirrhosis when incubated with KB174 compared with lactulose (Figure 4)

• Reduction in abundance of Enterobacteriaceae was compensated by significant increases of Parabacteroides in samples from both healthy controls and patients with cirrhosis for KB174, which shows KB174 and lactulose reduce pathogen levels through different commensal pathways (Figure 5)

Healthy Cirrhosis

KB174 Lactulose KB174 Lactulose

Mean reduction (vs water)

-43% -41% -50% -33%

P value (vs water) <.0001 <.0001 <.0001 <.0001

P value (vs compound) 0.601 0.005

a19 healthy microbiomes and 25 cirrhosis microbiomes were tested (1 sample from each patient) with CRE E coli spiked in at start of experiment. Average reduction is relative to water (with no added carbon source).

FIGURE 1. MMTS ARE DESIGNED TO TARGET THE GLYCAN UTILIZATION POTENTIAL OF COMMENSAL BACTERIA

EASL | The Digital International Liver Congress; August 27-29, 2020. Poster 4178.

Pathogens have smaller number andencode distinct repertoire of

glycosidases

Potential stereochemical and regiochemical possibilities of

carbohydrates make them the most structurally diverse biopolymer known

MMTs are structurally diverse ensembles of complex carbohydrates

(glycans), enabled by Kaleido’ssynthetic chemistry platform

• KB174, an MMT, has previously been shown to:• Reduce the relative abundance of CRE E coli ex vivo in fecal samples from healthy subjects6

• Reduce the amount of ammonia produced ex vivo in fecal samples from patients with hepatic encephalopathy7

• Be safe and generally well-tolerated in patients with well-compensated cirrhosis, and reduce the urinary excretion of a biomarker of ammonia produced by the gut microbiome8

• Lactulose, a nonabsorbed disaccharide available to the gut microbiota, is the current first-line treatment for hepatic encephalopathy

• Certain prebiotic fibers (e.g. fructooligosaccharides) promote pathogen growth in ex vivo culture (internal data) so evaluation of lactulose and comparison to KB174 is a relevant study

• We hypothesize that commensal bacteria will outcompete pathogens in the gut when fed an MMT, leading to a reduced risk of infection for the host. Moreover, we hypothesize that KB174 will show increased performance over lactulose in part due to the larger structural complexity of KB174 vs. lactulose (Table 1)

Number of Glycosidases0

Bacteroides ovatusBacteroides thetaiotaomicron

Bacteroides xylanisolvensBacteroides dorei

Bacteroides fragilisClostridium cellulovorans

Parabacteroides distasonisKlebsiella oxytoca

Enterobacter aerogenes

Klebsiella pneumoniaeBifidobacterium infantis

Enterococcus faecium

Akkermansia muciniphilaEnterobacter cloacae

Salmonella entericaCitrobacter freundii

Escherichia coliRuminococcus sp

Bifidobacterium breveClostridioides difficile

Bifidobacterium bifidumEnterococcus faecium

Citrobacter koseriEnterococcus faecalisRuminococcus obeum

Pseudomonas aeruginosaRuminococcus bromii

Staphylococcus aureusProteus mirabilis

PC2 (12% VariationExplained)

PC1

(42%

Var

iatio

n E

xpla

ined

)

CommensalsPathogens

100 200 300

METHODSSingle strain assay: to confirm that KB174 is not used by CRE E. coli,* the CRE strain was incubated anaerobically with KB174 in monoculture, and OD600 was measured over time to generate growth curves

RORORO

RORO

RORO

RO

RORO

RO RO

RORORO

RORO

RO

RO

RO

RO

RO

RO

OR

OR

OR

OR

OR

OR

OR

OROROR

OROROROR

+

or

;

;

;

;

;

;OR

O

OO

O O

O

O

O

O

O

OROR OR

RO

O

OHO

HOHO

HOHO

OH

OH

OHO

a-1,2; a-1,3; a-1,4; a-1,6b-1,2; b-1,3; b-1,4; b-1,6

HOHO

Glycosylacceptors

8 Possible head-tail dimers:

Glycosyldonor

HO OHHO

O

METHODS (CONT)

Each R group is as defined above

• A reduction in the abundance of pathogenic bacteria in the gut (including decolonization) has the potential to reduce the risk of infection in susceptible populations. This ex vivo data supports, in conjunction with ammonia reduction7, a multi-pronged mechanism of action to treat HE

• The totality of data on KB174 activity supports its inclusion in a clinical study in people with cirrhosis (NCT03855956)

SUMMARY

Compound

Rel

ativ

e Ab

unda

nce

0.4

0.6

0.2

Healthy Cirrhosis

KB174Lactulose KB174LactuloseWater Water

Escherichia

Bond distributions

CatalystMono-/Oligo-/Poly-/

saccharide

25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 110

5

0

-5

-10

-15

-20

Patient

Cha

nge

in R

elat

ive

Abun

danc

e of

Ent

erob

acte

riace

ae, %

-25

-30

-35

-40

-45

-50

-55

-60

-65

-70

-80

-75 KB174Lactulose

-200

0

200

400

600

800

1000

1200

Healthy Cirrhosis

Perc

ent C

hang

ea

KB174

Lactulose