Characterization of a new antiasthmatictherapeutic target: synergy between EP2 agonism and anti-IgE through a

biosimilar candidate with omalizumab

Adrián UrbanoDirector: Fernando de Mora Dept. of Pharmacology, Therapeutics

and Toxicology

– Common chronic inflammatory disease in the airways • The most frequent in childhood and adolescence

– Characterized by:• Spasmodic contraction of bronquial muscles

– Triggered by several factors• Shortness of breath (Airflow obstruction)

– Decreased caliber of the bronchi » Airway hyperresponsiveness (AHR)» Mucus hyperproduction» Airway wall remodeling

• Asthma is a global health problem affecting around de 10% of the global population– Allergic asthma (80%):

• Immunologically responses to nonspecific stimuli

INTRODUCTION

Pathology of asthmaAsthma

Normal airway Asthmatic airway

• There is no effective curative or preventive treatment– Inhaled corticosteroids (ICS)– Long acting β2-agonist (LABA)– Glucocorticoides (GC)

• Identification of a new anti-asthmatic target:– EP2 receptor

INTRODUCTION

Need for new anti-asthmatic targets Treatment & Therapeutic target candidate

PGE2Protector effect on asthmatic response

MC EP2 receptor

PGE2-EP2-MC

– Omalizumab (Xolair®)• Humanized monoclonal antibody with anti-IgE function• It binds selectively to free IgE (Fc region)• Prevents mast cell activation (release of inflammatory

mediators)

INTRODUCTION

Need for new anti-asthmatic targets Omalizumab

INTRODUCTION

BiosimilarsWhat is a biosimilar?

INCREASING COMPLEXITY

Biologic Biosimilar

Unlike typical drugs are made from synthetic chemicals, biologics are produced from living organism

Biosimilars are biological products that have been proven to be as safe as

originator biologic drugs

BIOSIMILARS DELIVER COMPARABLE EFFICACY, SAFETY

AND QUALITY RESULTS AS ORIGINATOR BIOLOGICS

Comparability exercise-Quality-Preclinical-Clinical studies

-Are designed from scratch to match areference original biological-Bear in essence the same:

- Active substance-Comes in the samepharmaceutical form-Administered via the same routeand dose-Indications

Thesis hypothesisHYPOTHESIS

“The mast cell EP2 activation in combination with the IgE neutralization by a biosimilar candidate of omalizumab, exerts a synergic potentiating anti-

asthmatic effect”

Omalizumab

IgE

Thesis objectiveOBJECTIVES

Develop an Omalizumab biosimilar candidate and study its effect beside an EP2 agonist in a in vivo and in

vitro model

In order to verify the hypothesis the main objective is:

Sub-objective 1 Sub-objective 2 Sub-objective 3

• Asses in in vivo models • EP2 activity • anti-IgE function

• Design and execute a predictive quality program to assess potential molecules to be biosimilar candidates at early stages.

• Assess the synergic effect of an EP2 agonist and an anti-hIgEbiosimilar candidate.

• In vitro model• In vivo model

Results

A) Validate the protector effect of EP2 in a transgenic model with this receptor overexpressed

B) Asses the anti-IgE activity in a transgenic model expressing hFcεRI

RESULTS

Study 1 (Objective 1)Assess the activity of EP2 and anti-IgE

%[mMCP1]/[Total Prot.] in lung

HDM (n=15)%

[mM

CP1

]/[To

tal P

rot.]

in lu

ng0,00

0,02

0,04

0,06

0,08

HDM(n=16) SF(n=16)SF(n=15)TG (n=30) WT (n=32)

p=0.34

******

CBATEG EP2 L3 2016/2017

Metacholine (mg/mL)

Baseline PBS 1.25 2.5 5 10 20

Rl %

100

120

140

160

180

200

220

240

HDM/TG n=15SF/TG n=14HDM/WT n=16SF/WT n=15

****HDM/WTSF/WT

*

*HDM/TGSF/TG

Fig. 1. A) Airway hyperresponsiveness to methacholine was assessed in BALB/c mice (wildtype and transgenic with EP2 overexpressed) bymeasuring lung resistance (RL) in HDM-sensitized mice. B) Determination of the local lung production of the specific mast cell protein,mMCP-1, in sensitized and non-sensitized, transgenic and wildtype mice by an ELISA assay (*p-value<0.05, **p-value<0.01, ***p-value<0.001and ****p-value<0.0001). HDM (House Dust Mice), SF (Physiological Serum), TG (Transgenic mice), WT (Wildtype mice), mMCP-1(mousemast cell protease 1), PBS (Phosphate Buffered Saline).

Results

Study 1 (Objective 1)Assessment of MC EP2 receptor role

RL – Lung resistance mMCP1 - Mast cell activityA. B.

Infla

mm

ator

y ce

lls/m

L

0

1e+5

2e+5

3e+5

4e+5

**

*

*

% m

MC

P1 [m

MC

P1]/[

Prot

. Tot

al] i

n lu

ng

0,00

0,02

0,04

0,06

0,08

0,10

*

Fig. 2. A) Differential inflammatory cell count per mL determined from counting at least 300 cells. B) Airway MC activity assessed in mice bymeasuring the local production of mMCP1. (*p-value<0.05, **p-value<0.01, ***p-value<0.001 and ****p-value<0.0001). HDM (House DustMice), SF (Physiological Serum), TG (Transgenic mice), WT (Wildtype mice), mMCP-1(mouse mast cell protease 1), PBS (Phosphate BufferedSaline).

Results

Study 1 (Objective 1)Assessment of MC EP2 receptor role

2,5mg/mL PF Vehicle

Non-Sensitized Sensitized with HDM

2,5mg/mL PF 10mg/mL PF

mMCP1 - Mast cell activity

E M N L E M N L E M N L E M N L E M N L E M N LE M N L E M N L

Transgenic mice Wildtype miceNS NS SensitizedSensitized

B.A. Differential inflammatory cell count

pg m

MC

P-1

in lu

ng /

mg

tota

l pro

tein

0

20

40

60

80

100

hIgE + NP -

OMA -n=6

hIgE +NP +

OMA -n=7

hIgE + NP +

OMA+ 90ugn=4

hIgE + NP +

OMA+ 30ugn=7

***

*

Response Time (min)

base

line

0-2'

2'-4'

4'-6'

6'-8'

8'-10

'10

'-12'

12'-1

4'14

'-16'

16'-1

8'18

'-20'

20'-2

2'22

'-24'

24'-2

6'26

'-28'

28'-3

0'30

'-32'

32'-3

4'34

'-36'

36'-3

8'38

'-40'

40'-4

2'42

'-44'

44'-4

6'46

'-48'

48'-5

0'50

'-52'

52'-5

4'54

'-56'

56'-5

8'58

'-60'

RL (

cm H

2O·s

·mL-1

) [no

rmal

itzad

o]

0,8

1,0

1,2

1,4

1,6

hIgE + / NP - / OMA - n=6hIgE + / NP + / OMA - n=7hIgE + / NP + / OMA + 90ug n=4hIgE + / NP + / OMA + 30ug n=7

Results

Study 1 (Objective 1)Assessment of the anti-IgE activity (PRA protocol)

Grupo tratamiento Grupo tratamiento P valor

IgE+/NP- / OMA - IgE+/NP+/ OMA - ***

IgE+/NP- / OMA - IgE+/NP+/ OMA+ (90µg) ***

IgE+/NP- / OMA - IgE+/NP+/ OMA+ (30µg) 0.1

IgE+/NP+/ OMA - IgE+/NP+/ OMA+ (90µg) ns

IgE+/NP+ / OMA - IgE+/NP+/ OMA+ (30µg) ***

IgE+/NP+/ OMA+ (90µg) IgE+/NP+/ OMA+ (30µg) **

Fig. 3. A) Airway response, bronchospasm, to nebulized NP-BSA through thelung in FCεRI transgenic mice sensitized with NP-BSA and treated withomalizumab following a protocol of passive respiratory anaphylaxis (PRA). B)Airway MC activity assessed by measuring the local production of mMCP1.(*p-value<0.05, **p-value<0.01, ***p-value<0.001 and ****p-value<0.0001).hIgE (human immunoglobulin E), NP (Nitrophenyl), OMA (omalizumab),mMCP-1(mouse mast cell protease 1).

B.

A. RL – Lung resistance

mMCP1 - Mast cell activity

DISCUSSION

Study 1 (Objective 1)Discussion

• The protective effect of EP2 has been validated– Having a high number of EP2 allows a lower airway

response in HDM-sensitized mice due to a decrease in MC activity.

– Blockade the activity of EP2 receptor worsens asthmatic parameter

• PRA model is useful to study the anti-IgE function in transgenic hFcεRI mice.– This protocol can be use to compare the omalizumab

in vivo activity versus a biosimilar candidate.

RESULTS

Study 2 (Objective 2)Predictive Quality Study for a BC with omalizumab

– Composition and physical properties– Primary structure assess– PTMs

• Glycosylation

Predictive Quality Study

Biological Characterization

Physicochemical Characterization

– Biologic activity– Immunochemical properties

RESULTS

Study 2 (Objective 2)Physicochemical characterization

XolairBiosimilar Candidate

pI7.8 7.6

7.71 7.51

7.6 7.41Species more predominant

• IDENTITY (Composition and physical properties)– Primary structure assessment (Aminoacid composition)– Molecular size & Purity

• CE-SDS (Capillar Electrophoresis)

– Isoform pattern (Charge heterogeneity) • IEF (Isoelectric Focusing)

– Glycosylation

Minutes14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

PDA - 220nmXolair NR

PDA - 220nmBC NR

PDA - 220nm PDA - 220nmBC NR

PDA - 220nm20180517A 001

Minutes12 13 14 15 16 17 18 19 20 21 22 23 24

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

PDA - 220nm20180517A 002

PDA - 220nm20180517A 003

PDA - 220nm20180517A 004

PDA - 220nm20180517A 005

Fig. 4. CE-SDS electropherograms of (A) Non-reduced and (B) Reduced antibodies.

Fig. 5. IEF gel image ofXolair and the biosimilarcandidate.

RESULTS

Study 2 (Objective 2)Biological characterization

• Biological activity– In vitro– In vivo

• Immunological activity– Target affinity

» SPR (Surface Plasmon Resonance)

Omalizumab

IgE

RESULTS

Study 2 (Objective 2)Biological activity

Group A Group B Group C

In vitro β-hexosaminidase release assay

In vivoPCA (Passive Cutaneous Anaphylaxis)

Xolair®

Bios. Candidate

Fig. 6. Comparison between Xolair® and biosimilar candidate activity. (A) β-hexosaminidase release assay. (B) Passive Cutaneous Anaphylaxis dosing 2mg of anti-IgEantibody for 300ng of chimeric hIgE. (*p-value<0.05, **p-value<0.01, ***p-value<0.001 and ****p-value<0.0001).

A.B.

RESULTS

Study 2 (Objective 2)Immunological activity

Ligand Ka (1/Ms) Kd (1/s)

Xolair® 2.12·106 0.0024

Biosimilar Candidate 1.28·106 0.0044

-20

30

80

130

180

-100 0 100 200 300 400Tim e s

RU

Res

pons

e

-20

30

80

130

180

-100 0 100 200 300 400Tim e s

RU

Res

pons

e

Fig. 7. Kinetic Analysis of (A) Xolair® and (B) biosimilar candidateusing BiaEval 1.1. Coloured lines correspond to observed results atdifferent analyte concentration and black lines correspond totheoretical adjustment.

A.

B.

• Target affinity– SPR (Surface Plasmon

Resonance)

DISCUSSION

Study 2 (Objective 2)Discussion

• The Biosimilar Candidate:– Have physicochemical differences compared with

the reference product• Due to the post-traductional modifications (PTMs)

– Glycosylation

– Recognize the desired antigen (hIgE )• With a lower affinity than the reference product• Has biological activity in in vitro and in vivo models

– More studies are needed to be done.

RESULTS

Study 3 (Objective 3)Evaluation of the synergic activity in vivo and in vitro

– In vivo• PRA model

– In vitro• β-hexosaminidasa release test

hFcεRI

Thanks for your attention!