Global seroprevalence of pre-existing immunity against various AAV serotypes in the hemophilia A population

Gregory Hayes1, Tatiana Andreeva2, Keith Gregg1, Robert Klamroth3, Brandon Hardesty4, Jorja Warren5, Patricia Slev5, Takashi Suzuki6, Natalie Stieltjes7, Flora Peyvandi8, Midori Shima9, Jean-Francois Schved10, Segolene Claeyssens11, Alex Trinh1, Brigitte Pan-Petesch12, Sabine-Marie Castet13, Savita Rangarajan14,

Syed Kazmi14, Doris Quon15, Ismail Haroon Mitha16, Andrew D. Leavitt17, Johannes Oldenburg18,Margareth Castro Ozelo19, Johnny Mahlangu20, Wing Yen Wong1

1BioMarin Pharmaceutical Inc, Novato, USA; 2 City Outpatient Clinic #37, St. Petersburg, Russia;3 Vivantes Hospital in Friedrichshain, Center for Hemophilia and Hemostasis, Berlin, Germany;4Indiana Hemophilia and Thrombosis Center, Indianapolis, USA; 5ARUP Laboratories, Salt Lake City USA; 6Ogikubo Hospital, Tokyo, Japan; 7Cochin Hospital Hemophilia Treatment Center, Paris, France;8Maggiore Polyclinic Hospital General Medicine Hemostasis and Thrombosis, Milan, Italy9Nara Medical University Hospital, Nara, Japan; 10Montpellier University Hospital, Montpellier, France;11Purpan Hospital, Regional Hemophilia Center, Toulouse, France; 12Morvan Hospital, Brest, France;13Pellegrin Hospital, Bordeaux Cedex, France; 14Southampton General Hospital, Southampton, UK;15Los Angeles Orthopedic Hospital, Hemophilia Treatment Center, Los Angeles, USA; 16Worthwhile Clinical Trials, Lakeview Hospital, Benoni, South Africa; 17UCSF Medical Center, San Francisco, USA; 18University Hospital Bonn, Bonn, Germany; 19Campinas University Clinical Hospital, Campina, Brazil20Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg, South Africa

PRESENTING AUTHOR DISCLOSURE

• Gregory Hayes is an employee of BioMarin Pharmaceutical Inc.

VALOCTOCOGENE ROXAPARVOVEC (BMN 270) – CLINICAL PROGRAM

Phase 1/2 Dose Escalation Study Enrollment completed• 15 participants (AAV5-), 4 dose cohorts; UK 201

AAV5+ Phase 1/2 Titer Escalation Study FPI May 2018• 10 participants; 2 titer cohorts (≤500, >500); UK. 203

Global AAV Seroprevalence Study FPI Aug 2017• 820 participants; US, UK, France, Russia, Germany, Italy, Japan, S. Africa, Brazil 901

Non-Interventional Study FPI Feb 2018• N= ~145 AAV5-/HIV- participants, participate for ≥ 6 months, given priority to enroll into GENEr8-1 or GENEr8-2• AAV5+ participants can participate in 902; however, will not be eligible for GENEr8-1 or GENEr8-2• 14 Countries

902

Phase 3 Study with 6×1013 vg/kg dose FPI Dec 2017• N = ~130 participants• 13 Countries

GENEr8-1

Phase 3 Study with 4×1013 vg/kg dose FPI May 2018• N = ~40 participants• 13 Countries

GENEr8-235

110

Advanced Gene Therapy Program for which a Companion Diagnostic (CDx) is being co-developed to select hemophilia A patients without pre-existing antibodies to AAV5 (partnered with ARUP Laboratories)

MULTIPLE METHODS FOR MEASURING PRE-EXISTING AAV IMMUNITYCell-based Transduction Inhibition (TI) Assay

AAV-luciferase

Patient Plasma

HEK293T HEK293T

• Measures ability of plasma to block transduction of a cell line by AAV• Differing amounts of capsid needed for different serotypes can make

TI titer comparisons across capsids more difficult

Anti-AAV Total Antibody (TAb) Assay

AAVx-Ru

AAVx

• Electrochemiluminescent assay (ECLA) on the MSD platform• Ease of qualifying divergent capsid serotype assays in identical format

Passive Immunity Transfer In Vivo

• Immuno-compromised mice are reconstituted with donor plasma or IVIg prior to dosing with AAV

• Ability to detect low neutralizing titers• Often has high variability across animals (low n due

to plasma volumes required)1. Falese L. et al Gene Ther. 2017; 24: 768-7782. Sun L. et al. J Immunol Methods, 2013; 387:114-120

MOST PATIENTS EXHIBIT CONCORDANCE BETWEEN AAV5 TAb & TI

AAV5

TAb

Tite

r

AAV5 Transduction Inhibition (TI) Titer

TAb+ TI+(25%)

TAb- TI+(9%)

TAb+ TI-(4%)

TAb- TI-(62%)

• Majority of hemophilia A patients are negative or positive in both TAb and TI assays (n=253)

• 62% TAb & TI negative • 25% TAb & TI positive

• Patients with detectable titers in only one assay generally exhibit low titer values

• Can efficacy be achieved in TAb+ or TI+ individuals?

AAV5 TOTAL ANTIBODY (TAb) ASSAY PREDICTS BMN 270 EFFICACY

Group (n) Purpose TI Titer TAb Titer

1 (3) Control Negative Negative

2 (4) Non-TAb TI 2 - <5 Negative

3 (3) Non-TAb TI 5 - 10 Negative

4 (5) TAb+ >10 Positive

• Non-human primates exhibit similar levels of pre-existing immunity as humans

• Animals screened with TAb and TI assays, assigned to groups below, and dosed with 6e13 vg/kg valoctocogene roxaparvovec (BMN 270)

TAb- TAb- TAb- TAb+TI- TI+ (2-5) TI+ (5-10) TI>10

72%

Hum

an F

VIII

(ng/

mL)

Group Mean FVIII Protein – Day 29

TI titers in TAb-negative subjects were not associated with reduced transduction or efficacy TAb assay chosen for screening of patients in Ph3 clinical studies and intended as a

companion diagnostic for the program.

TAb+ was associated with 72% decrease in hFVIII

1. Long B. et al Mol Therapy Methods & Clin Dev. 2019; 13: 440-452

BIOMARIN GLOBAL AAV SEROPREVALENCE STUDY DESIGN Objective: to provide epidemiological data characterizing pre-existing AAV immunity

among hemophilia A patients • Determine the prevalence of AAV5 capsid antibodies & regional differences that may impact efficacy• Compare seroprevalence and antibody titers against multiple AAV serotypes

• Identical AAV TAb formats specific for AAV2, 5, 6, 8, rh10 with comparable limits of detection (<20ng/mL) were validated at central laboratory

• Determination of titer stability (longitudinal sample analysis)

Study design:• Up to 100 patients enrolled per country in (≤75 adults, ≤25 12-17yo)• Countries: UK, USA, Russia, France, Germany, Japan, Italy, South Africa, Brazil

• Currently enrolled n=453 adults across 17 sites• Approximately 20% of patients returned at months 3 and 6 for follow-up testing

RATES OF AAV5 SEROPREVALENCE VARIES ACROSS COUNTRIES

% A

AV5

TAb

Posi

tive

France Germany Italy Japan Russia

Variability observed in the percentage of hemophilia A patients with pre-existing antibodies to AAV5

Differences represent valid geographic variance of this endemic virus based upon comparisons to healthy controls• e.g. healthy Russia controls = 43% AAV5+

Global weighted average = 31.0% AAV5+(hemophilia A global population-weighted mean)

USAUK

20

40

60

80

40.0 24.7 21.329.9 46.740.030.6

(Pairwise comparison * p-value <0.05)

**

*

*

AAV5 HAS THE LOWEST PREVALENCE ACROSS SEROTYPES TESTED

AAV

Antib

ody

Tite

r

AAV5 AAV2 AAV6 AAV8 AAVrh10

% Positive: 33 54 46 44 44 AAV5 exhibits lowest rate of seroprevalence compared to other serotypes tested

AAV5 exhibits lowest range of antibody titers compared to other serotypes tested

***

(Pairwise comparison to AAV5*** p-value <0.0001, ** p-value =0.0002)

*** *** **

AAV5 TAb RESULT & TITERS REMAIN CONSISTENT OVER 6 MONTHS TIME

Patient samples were collected at Day 0 (enrollment) and at 3 and/or 6 months

94% of patients did not seroconvert over 6 months of follow up time (n=47)

Only 3 patients converted from negative to positive (or vice versa) and all had titers near or below the MRD

Confidence in the MSD-platform based TAb approach to determine pre-existing immunity

• Minimum required dilution = 20 (MRD)

AAV5

Ant

ibod

y Ti

ter

Day 0 Month 3 Month 6

MRD

AAV-(n=23)

AAV+(n=21)

RATES OF AAV+ SEROPREVALENCE INCREASE WITH AGE % of TAb+ hemophilia A patients increases with age for all serotypes tested Probability of becoming AAV5+ is lowest among the serotypes examined

% A

AV T

Ab P

ositi

ve

Age Range

Prob

abili

ty o

f AAV

TAb

+Age

Logistic Regression of Seroprevalence with Age

AAV5

AAV2AAVrh10AAV8AAV6

SUMMARY There are multiple methods of measuring pre-existing AAV immunity which may impact

efficacy Total antibody assay appears to be more discriminating than TI in pre-clinical studies A central lab was used to measure antibodies against 5 different AAV serotypes using an identical

assay format with comparable sensitivities

AAV5 exhibits the lowest seroprevalence among serotypes tested across countries Regional variability in AAV5 seroprevalence across countries % TAb+ of hemophilia A patients and titer range is lowest for AAV5 compared to other serotypes AAV5 TAb results (+/-) and titers remain consistent over 6 months of time Rates of seroprevalence for each capsid increase with age but remain lowest for AAV5

A companion diagnostic is being developed to enable standardized assessment and controlled investigation of the clinical relevance or pre-existing AAV5 antibodies for BMN 270

THANK YOUTo all study participants, advisors, teams and principal investigators

Nara Medical University HospitalNara, Japan

City Outpatient Clinic #37St. Petersburg, Russia

Vivantes Hospital, Center for Hemophilia and Hemostasis Berlin, Germany

Montpellier University HospitalMontpellier, France

Indiana Hemophilia and Thrombosis Treatment CenterIndianapolis, United States

Purpan Hospital, Regional Hemophilia CenterToulouse, FranceMorvan Hospital

Brest, FranceOgikubo Hospital

Tokyo, JapanCochin Hospital, Hemophilia Treatment Center

Paris, FrancePellegrin Hospital

Bordeaux Cedex, FranceMaggiore Polyclinic Hospital

Milan, Italy

Southampton General HospitalSouthampton, UK

Thank you to the BioMarin team

Los Angeles Orthopedic HospitalLos Angeles, USA

Worthwhile Clinical Trials, Lakeview HospitalBenoni, South Africa

University Hospital Bonn, Bonn, Germany

UCSF Medical CenterSan Francisco, United States

Charlotte Maxeke Johannesburg Academic HospitaJohannesburg, South Africa

Campinas University Clinical HospitalCampinas, Brazil

ARUP LaboratoriesSalt Lake City, USA