“First Results of a Head-to-Head Trial of Pegunigalsidase Alfa vs. Agalsidase Beta in Fabry Disease: 2-year Results of the Phase 3 Randomized, Double-blind, BALANCE Study,” Poster Presentation By Eric L. Wallace, M.D.

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First Results of a Head-to-Head Trial of Pegunigalsidase Alfa vs Agalsidase Beta in Fabry Disease: 2-year Results of the Phase 3 Randomized, Double-blind, BALANCE Study

Eric L. Wallace1, Ozlem Goker-Alpan2, William R. Wilcox3, Myrl Holida4, John Bernat4, Nicola Longo5, Derralynn Hughes6, Pilar Giraldo7, Maria Judit Molnar8, Damara Ortiz9, Robert J. Hopkin10, Camilla Tøndel11, Aleš Linhart12, Patrick Deegan13, Ana Jovanovic14, Michael Muriello15,

Bruce A. Barshop16, Virginia Kimonis17, Bojan Vujkovac18, Albina Nowak19, Tarekegn G. Hiwot20, Antonio Pisani21, Dominique P. Germain22, Ilkka Kantola23, Jasmine Knoll24, Ankit Mehta25, Stephen Waldek26, Einat Almon27, Sari Alon27, Raul Chertkoff27, Rossana Rocco28, David G. Warnock1

1The University of Alabama at Birmingham, Birmingham, AL, USA; 2Lysosomal and Rare Disorders Research and Treatment Center, Inc., Fairfax, VA, USA; 3Department of Human Genetics, Emory University, Atlanta, GA, USA; 4University of Iowa Hospitals and Clinics, Iowa City, IA, USA; 5University of Utah, Center for Clinical Translational Sciences, Salt Lake City, UT, USA; 6Lysosomal Storage Disorders Unit, Royal Free London NHS Foundation Trust and University College London, London, UK; 7Quirónsalud Hospital, Zaragoza, Spain; 8Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, HUN; 9UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA; 10Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; 11University of Bergen, Department of Clinical Science, Bergen, NOR; 12Vseobecna fakultni nemocnice v Praze, Praha, CZE; 13Addenbrooke's Hospital, Cambridge University Hospitals, Cambridge, UK; 14Mark Holland Metabolic Unit, Northern Care Alliance NHS Foundation Trust, Greater Manchester, UK; 15Department of Pediatrics - Genetics Curative Network Building, Milwaukee, WI, USA; 16University of California San Diego, La Jolla, CA, USA; 17University of California, Irvine Institute for Clinical and Translational Science, CA, USA; 18Slovenj Gradec General Hospital, Slovenj Gradec, SI; 19University Hospital of Psychiatry Zürich, Department of Internal Medicine, CHE; 20Queen Elizabeth Hospital Birmingham, Edgbaston, Birmingham West Midlands, UK; 21Azienda Ospedaliera Universitaria "Federico II", Dipartimento di Sanita Pubblica, Napoli, ITA; 22Division of Medical Genetics, University of Versailles, Garches, FRA; 23Department of Medicine, Turku University Hospital, Turku, FIN; 24Phoenix Children's Hospital, Phoenix, AZ, USA; 25Baylor University Medical Center, Dallas, TX, USA; 26University of Sunderland, Sunderland, UK; 27Protalix Biotherapeutics, Carmiel, ISL; 28Chiesi Farmaceutici S.p.A., Parma, ITA.

Introduction

• Chronic kidney disease is a main consequence of Fabry disease (FD) and is one of the leading causes of death in male patients with FD1
• Current therapeutic approaches for FD include reduction of accumulated glycosphingolipids to stabilize renal function using enzyme replacement therapy (ERT) or chaperone therapy (ie, migalastat)2,3

• Unmet needs include complications
Figure 1. PEGylated recombinant
related to progressive clinical decline
α-galactosidase A
with deteriorating renal function and
immunogenicity with ERT2,3	114 kDa molecule
• Pegunigalsidase alfa is a
novel PEGylated recombinant	α-Gal A	α-Gal A
α-galactosidase A ERT in	subunit	subunit
development to treat FD (Figure 1)
• Pegunigalsidase alfa is designed
to offer prolonged half-life, reduced
incidence of ADAs, and potentially
improved tolerability compared with
available ERTs1,4
• The molecule is a covalently linked
homodimer composed of:
- 2 plant cell-derived subunits of	~2 kDa PEG cross-linker

Figure 3. Study Inclusion and Exclusion Criteria

Main inclusion criteria	Main exclusion criteria
• Symptomatic adults with FD (18-60 years)	• Screening eGFRa 91-120 mL/min/1.73 m2 and
• Linear eGFRa slope more negative than or equal to	historical eGFR >120 mL/min/1.73 m2 during
−2 mL/min/1.73 m2/y	9-18 months before screening
• Treatment with agalsidase beta (1.0 mg/kg E2W) for	• UPCR >0.5 g/g and not treated with an ACEi
≥1 year and ≥80% compliance over the last 6 months	or ARB

aThe chronic kidney disease epidemiology collaboration (CKD-EPI) 2009 equation6 was used to calculate eGFR based on serum creatinine measured at each visit.

ACEi, angiotensin-converting enzyme inhibitors; ARB, angiotensin II receptor block; E2W, every 2 weeks; eGFR, estimated glomerular filtration rate; FD, Fabry disease; UPCR, urinary protein-to-creatinine ratio.

Results

Baseline characteristics

• Mean duration of previous agalsidase beta exposure: ~6 years (range: 1-20 years) (Table 1)
• There were no statistically significant differences in baseline characteristics between the groups, although there was a numerically higher proportion of females and of patients with
  UPCR > 0.5 g/g in the pegunigalsidase alfa arm
  Table 1. Patient Demographics and Baseline Characteristics

Figure 4. Median eGFR Slopes and the 95% CI Limits, Analysis Using Quantile Regressiona

Differenceb (95% CI): -0.359(-2.444c, 1.726)

95%±MedianCI slopeeGFR m(mL/min/1.732/y)					Median: -2.514												Pegunigalsidase alfa
															Agalsidase beta
	-3.788										-1.240
														Median: -2.155
			-3.805													-0.505
-	5	-	4	-	3	-	2	-	1	0	01

aTo determine noninferiority, the annualized median eGFR slopes were analyzed by quantile regression using SAS PROC QUANTREG to obtain the corresponding 95% CI; noninferiority was declared if the lower bound of the CI for the treatment difference (pegunigalsidase alfa - agalsidase beta) was ≥ −3.0 mL/min/1.73 m2/year; b(Pegunigalsidase alfa) − (agalsidase beta); cValue above the predefined noninferiority margin.

CI, confidence interval; eGFR, estimated glomerular filtration rate.

• At baseline, median eGFR values were nearly identical: 73.45 mL/min/1.73 m2 in the

pegunigalsidase alfa arm compared to 74.85 mL/min/1.73 m2 in the agalsidase beta arm

(Figure 5)

• The eGFR change from baseline showed a similar decline between baseline and

24 months in the two arms with a median change of -2.39 (mean [SD]:

-3.60 [10.81]) mL/min/1.73 m2 in the pegunigalsidase alfa arm and −3.20 (mean [SD]:

-1.97 [7.42]) mL/min/1.73 m2 in the agalsidase beta arm

• 1 patient on agalsidase beta discontinued (voluntary withdrawal of consent)
• No deaths were registered during the study

Table 2. Rate of Treatment-emergent Adverse Events

		Pegunigalsidase alfa (n = 52)	Agalsidase beta (n = 25)
	Any TEAE
	Events, n (rate)	561	(572)	406	(817)
	Patients, n (%)	47	(90)	24	(96)
	TEAE related to drug
	Events, n (rate)	42	(43)	76 (153)
	Patients, n (%)	21	(40)	11 (44)
	Serious TEAE related to drug
	Events, n (rate)	1	(1)	0	(0)
	Patients, n (%)	1	(2)	0	(0)
	Related TEAE leading
	to withdrawal
	Events, n (rate)	1	(1)	0	(0)
	Patients, n (%)	1	(2)	0	(0)

The exposure-adjusted rate of TEAEs was calculated as events per 100 exposure-years.

TEAE, treatment-emergent adverse event.

• The number and rate of IRR events were higher for agalsidase beta than pegunigalsidase
	alfa by ~4-fold and ~8-fold, respectively (p<0.0001; Table 3)
•	Most IRRs were mild or moderate in severity
•	There was a notable drop in the use of premedications at 24 months in both treatment arms

Figure 7. Antidrug Antibodies
A	50%					Pegunigalsidase alfa				IgG positive
positives	30%														Neutralizinga
ADAof	40%
10%
	20%
%	0%	0	2	4	8	12	16	20	26	38	52	64	78	92	104
							Time (weeks)
B	50%						Agalsidase beta					IgG positive
positives	30%														Neutralizinga
ADAof	40%
10%
	20%
%	0%	0	2	4	8	12	16	20	26	38	52	64	78	92	104

Time (weeks)

a% of nAb+ calculated out of total patients in respective treatment arm. ADA, antidrug antibody; nAb+, positive for neutralizing antibodies.

• Treatment-emergent ADA+ rate was lower with pegunigalsidase alfa (6/52, 12%) than

α-galactosidase A linked through	α-Gal A, α-Galactosidase A; PEG, polyethylene glycol.
the ~2 kDa PEG cross-linker,

Age, years

Pegunigalsidase alfa (n = 52)Agalsidase beta (n = 25) p-value

Figure 5. eGFR Values Over Time

- In the pegunigalsidase alfa arm, the mean (min; max) infusion duration was reduced

agalsidase beta (5/25, 20%), despite previous exposure of ~6y to agalsidase beta (Table 4)

resulting in a 114 kDa enzyme

• Pegunigalsidase alfa cleared Gb3 depositions in kidney peritubular capillaries

after 6 months in ERT-naïve adults (dosing groups: 0.2, 1, and 2 mg/kg every

2 weeks [E2W])5

Mean ± SD

Sex, n (%)

Male

Female

UPCR, n (%)

43.9 ± 10.2

1. (56)

1. (44)

45.2 ± 9.6

18 (72)

7 (28)

0.60a

0.19b

	100		Pegunigalsidase alfa
)			Agalsidase beta
2	90
eGFR .73 m
80	74.85	74.48

from 3.08 (0.6; 4.9) h at baseline to 1.56 (1.4; 2.1) h at 24 months; the reduction was

less in the agalsidase beta arm, where the means were 2.96 (2.6; 3.3) h at baseline

and 1.71 (1.4; 3.2) h at 24 months

Table 3. Rate of Infusion-related Reactions

Table 4. Treatment-emergent Antidrug Antibodies

Treatment-emergent	Pegunigalsidase alfa (n = 52)	Agalsidase beta (n = 25)
ADAs, n (%)
Yes	6 (12)	5 (20)

• BRIDGE (NCT03018730; phase 3, open-label, single arm, switch-over from

0.2 mg/kg agalsidase alfa E2W) demonstrated safety and efficacy over 12 months

of pegunigalsidase alfa (1.0 mg/kg E2W) treatment

Objective

• BALANCE (NCT02795676) was a phase 3 noninferiority study that evaluated the efficacy

UPCR ≤ 0.5 g/g

0.5 < UPCR < 1 g/g UPCR ≥ 1 g/g

eGFR, mL/min/1.73 m2 Mean ± SD Median (min, max)

eGFR slope, mL/min/1.73 m2/yd Mean ± SD

Median (min, max)

ADA statuse

Positive

36 (69)

9 (17)

7 (14)

73.5 ± 20.2

73.5 (30.2, 125.9)

−8.0 ± 6.6

−6.7 (−30.5, 6.3)

18 (34.6)

1. (80)

1. (8)
   3 (12)

74.2 ± 21.0

74.9 (34.1, 107.6)

−8.3 ± 4.3

−7.8 (−20.3, −2.8)

8 (32.0)

0.52b

0.82c

0.37c

Median (mL/min/1	70	73.45			69.35
60
	50	0	4	8	12 16 20 24 28 32 36 38 40 44 48 52 56 60 64 68 72 76 78 80 84 88 92 96 100104
					Time (weeks)

eGFR, estimated glomerular filtration rate.

Plasma lyso-Gb3 was stable and overlapped between arms

• High overlap of plasma lyso-Gb3 levels over time between both arms (Figure 6)

Overall IRR Events, n (rate) Patients, n (%)

Mild or moderate IRR Events, n (rate) Patients, n (%)

Severe IRR Events, n (rate) Patients, n (%)

Pegunigalsidase alfa (n = 52)

13 (0.5)

11 (21.2)

12 (0.5)

11 (21.2)

1 (0)

1 (1.9)

Agalsidase beta (n = 25)

1. (3.9)

1. (24.0)

1. (3.9)

1. (24.0)

1. (0)

1. (0)

Titer boosteda,b	3 (50)	2 (40)
De novoa,c	3 (50)	3 (60)
No	46 (89)	20 (80)

a% calculated out of patients with treatment-emergent ADAs; bTiter at least 4-fold baseline values; cIf the patient was ADA- at baseline and became ADA+ at any subsequent time.

ADA, antidrug antibody.

Conclusions

• Pegunigalsidase alfa showed comparable efficacy to agalsidase beta based

and safety of pegunigalsidase alfa (1.0 mg/kg E2W) compared with agalsidase beta

(1.0 mg/kg E2W) in patients with FD previously treated with agalsidase beta who also

had deteriorating renal function

Methods

Negative

Plasma lyso-Gb3, nM Mean ± SD Median (min, max)

Previous agalsidase beta exposure, mo

Mean ± SD

Min, max

34 (65.4)

1. ± 27.3

15.2 (0.8, 143.9)

1. ± 48.0 12.6, 236.9

17 (68.0)

1. ± 35.4

17.6 (2.1, 142.0)

1. ± 41.3 27.6, 168.3

0.82b

0.58c

0.25a

• Plasma lyso-Gb3 median change from baseline was stable with pegunigalsidase alfa

(1.15 nM) and agalsidase beta (−1.50 nM)

Figure 6. Plasma Lyso-Gb3 Concentration Over Time

(nM)	180	Pegunigalsidase alfa
160	Agalsidase beta
140
Gb3
120

The rate of IRRs was calculated as events per 100 infusions.

IRR, infusion-related reaction.

Prevalence of antidrug antibodies

•	Reduction in ADA+ patients in pegunigalsidase alfa arm from 35% (18/52) to 23% (11/47)
	(Figure 7A) and in agalsidase beta arm from 32% (8/25) to 26% (6/23) (Figure 7B)
•	Reduction in patients positive for neutralizing antibodies (nAb+) in pegunigalsidase alfa
	arm from 33% (17/52) to 15% (7/48) (Figure 7A) and slight decrease in agalsidase beta

on eGFR annualized slope (a key measure of FD progression), eGFR change, and

plasma lyso-Gb3 level

• Overall, patients who switched to pegunigalsidase alfa showed improved

tolerability and immunogenicity profiles

• Most patients who completed the study from both arms (97%: 45/47 on

pegunigalsidase alfa and 24/24 on agalsidase beta) opted to continue or initiate

treatment with 1 mg/kg E2W pegunigalsidase alfa in an open-labelextension

Figure 2. BALANCE Study Design

	2:1 Randomization	Exposed to treatment	Primary efficacy
	N = 78a	n = 77	analysis
	(stratification by		(noninferiority)
	(24 months)
	UPCR < or ≥ 1 g/g)
				Pegunigalsidase alfa 1 mg/kg E2W
	Pegunigalsidase alfa	n = 52a
Screening	n = 53		ITT (n = 77)
n = 127		beta
Agalsidase		PP (n = 72)
	n = 25	Agalsidase beta 1 mg/kg E2W
				n = 25

a1 Patient withdrew consent prior to the first dose.

E2W, every 2 weeks; ITT, intent-to-treat; PP, per protocol; UPCR, urinary protein-to-creatinine ratio.

aT-test; bPearson chi-squared; cWilcoxon; deGFR slope at baseline was based on historical, screening, and baseline serum creatinine and was more positive than -2 mL/min/1.73 m2/y for some patients; eAt screening, each sample was tested for reactivity to both drugs, but due to the high cross-reactivity, only data for the assigned drug are presented.

ADA, antidrug antibody; eGFR, estimated glomerular filtration rate; lyso-Gb3, globotriaosylsphingosine; max, maximum; min, minimum; mo, months; nM, nanomolar (nmol/L); SD, standard deviation; UPCR, urine protein creatinine ratio.

Noninferiority met: eGFR slope analysis

• The median eGFR slopes during treatment and their 95% CI limits were −2.514 (95% CI: −3.788, −1.240) mL/min/1.73 m2/year with pegunigalsidase alfa and −2.155 (95% CI: −3.805, −0.505) mL/min/1.73 m2/year with agalsidase beta (Figure 4)
• The difference in median eGFR slope for the ITT population between treatment arms, the primary endpoint, was −0.36 mL/min/1.73 m2/year (95% CI: −2.444, 1.726)
• • Noninferiority was achieved: the lower bound of the difference in median eGFR slopes of the treatment arms was larger than the prespecified noninferiority margin of
    −3.0 mL/min/1.73 m2/year
  • The 95% CI included 0, and a high overlap between individual CIs, indicating no significant difference between the treatment arms
  • The analysis was repeated, adjusting the model for gender, and the conclusions remained unchanged

-	100
lyso
80
Plasma	60
40
20
0
	0	6	12	26	38	52	78	104

Time (weeks)

Box and whiskers represent the median and quartiles, with outliers as diamonds and squares for pegunigalsidase alfa and agalsidase beta, respectively; 'X' represents the mean.

Lyso-Gb3, globotriaosylsphingosine; nM, nanomolar (nmol/L).

Safety

• The exposure-adjusted rate of related TEAEs was ~4-fold higher for agalsidase beta than pegunigalsidase alfa (p<0.0001; Table 2)
• 1 patient receiving pegunigalsidase alfa withdrew due to a serious, related TEAE of hypersensitivity (this patient was IgE positive at baseline)
• 5 patients on pegunigalsidase alfa discontinued: 1 withdrew before first infusion, 4 withdrew

throughout the study (n = 2 due to AE, n = 3 voluntary withdrawal of consent)

arm from 28% (7/25) to 25% (6/24) (Figure 7B)

References

1. Schiffmann R et al. Nephrol Dial Transplant. 2009;24: 2102-2111; 2. Muntean C et al. Front Pediatr. 2022; 10: 908657; 3. Lenders M and Brand E. J Am Soc Nephrol. 2018;29:2265-2278; 4. Ruderfer I et al. Bioconjug Chem. 2018;29(5):1630-1639; 5. Schiffmann R et al. J Inherit Metab Dis. 2019;42(3):534- 544; 6. Levey AS et al. Ann Intern Med. 2009;150:604-612.

Acknowledgments

We thank all the patients, their families, and the investigators involved in this study. The authors would like to acknowledge Dr. Anat Sakov for her assistance in the statistical analysis, the Bioanalytical Laboratory at Protalix for assistance with the ADA analysis, and the CHUS laboratory for assistance with the Lyso-Gb3 analysis. The trial was sponsored by Protalix Biotherapeutics. Medical writing support was provided by Melissa Victoria Fernandez, PhD and Marisa DeGuzman, PhD, of Oxford PharmaGenesis, Inc., Newtown, PA, USA, and was funded by Chiesi USA, Inc.

Disclosures

EW has consulting agreements and/or grants with Sanofi, Protalix, Chiesi, Idorsia, 4DMT, Amicus, and Natera. OG-Ahas conducted contracted research, received consulting fees, and/or served on advisory boards with Amicus, Freeline, Genentech, Protalix, Sangamo, Sanofi, Takeda, Sangamo, 4DMT, and Avrobio. WRW has been or is currently involved in clinical trials and/or registries with Alexion, Amicus, BioMarin, Chiesi, Freeline, Idorsia, Orphazyme, Pfizer, Protalix, Sanofi, Sangamo, Takeda, and 4DMT. He has received honoraria from Alexion, Amicus, Sanofi, Spark, and Takeda; and research funding from Amicus and Takeda. MH received speaker-relatedfees from Protalix and has been, or is currently, involved in clinical trials with Sanofi, Sangamo, Avrobio, Protalix, and Idorsia (no direct funding is received for these trials as they are institution directed). JB receives research support from Avrobio, BioMarin Pharmaceutical, Chiesi Farmaceutici, Idorsia Pharmaceuticals, Pfizer, Protalix Biotherapeutics, Sangamo Therapeutics, Sanofi, Takeda, Travere Therapeutics; and has received a speaker honorarium from the Fabry Support and Information Group; and has participated in advisory boards for Chiesi USA, Sanofi, and Takeda. NL receives research support from and has participated in advisory boards for Amicus, Astellas, Avrobio, BioMarin Pharmaceutical, Homology, Horizon, Moderna, Pfizer, Protalix Biotherapeutics, PTC Biotherapeutics, Reneo, Sanofi, Takeda, and Ultragenyx (no direct funding is received as they are institution directed). DH has received honoraria for speaking and consulting fees for advisory boards from Protalix, Takeda, Sanofi, Freeline, and Sangamo, administered through University College London consultants, and used in part to support research in lysosomal

study for 60 months (NCT03566017)

storage diseases. PG has been involved in premarketing studies with Genzyme, Protalix, and Idorsia, and has received grants from Sanofi- Genzyme and Takeda; monies received for these activities have been deposited into the Spanish Foundation for the Study and Treatment of Gaucher Disease (FEETEG) to contribute to the development of research in lysosomal storage disorders. MJM, DO, MM, BAB, VK, JK, and TG have no disclosures. RJH has received consulting fees from Alexion, Amicus Therapeutics, Inc., Avrobio, Chiesi, Sangamo, Sanofi/ Genzyme and Takeda; advisory fees from Alexion, Amicus Therapeutics, Inc. and Sanofi/Genzyme; speakers' bureau fees from Alexion and Sanofi/Genzyme and grants/research funding from Alexion, Amicus Therapeutics, Inc., Idorsia, Protalix, Sangamo, Sanofi/Genzyme and Takeda. CT has received honoraria, travel support, and/or participated as an investigator in clinical studies supported by Protalix, Sanofi, Idorsia, Takeda, Amicus, Freeline and Acelink. AL has received consultancy and speaker´s honoraria from Amicus Therapeutics, Sanofi, and Takeda. PD has been a paid consultant with Sanofi; received speaker honoraria from Sanofi and Takeda; and participated in an advisory board with Protalix. AJ has received a grant from Amicus and consultancy and speaker's honoraria from Takeda, Sanofi, Amicus. BJ has received honoraria, travel and accommodation funding from Greenovation Biotech GmbH, Sanofi, Takeda; member of the EU Advisory Board of Fabry Registry, sponsored by Sanofi. AN received lecturing honoraria and research support from Takeda, Amicus, Sanofi Genzyme. AP received travel expenses and grants from Takeda, Sanofi, Amicus and Chiesi. DPG has received consulting honoraria from Idorsia Pharmaceuticals, Sanofi, and Takeda, and speaker honoraria and travel support from Amicus Therapeutics, Sanofi, and Takeda.

IK has received lecture, travel, and consulting fees from Amicus, Chiesi, Bayer, Boehringer-Ingelheim, Sanofi-Genzymeand Takeda-Shire. AM has received non-financialsupport from Chiesi Pharmaceuticals, during the conduct of the study; personal fees from AstraZeneca, personal fees from Bayer Pharmaceuticals, and personal fees from Janssen Pharmaceuticals, outside the submitted work. SW has been a paid consultant to Protalix. EBA, SA, and RC are full-timeemployees of Protalix Biotherapeutics. RR is a full-timeemployee of Chiesi Farmaceutici S.p.A., Parma, Italy. DGW has been or is currently involved in clinical trials and/or registries with Alexion, Amicus, BioMarin, Chiesi, Freeline, Idorsia, Orphazyme, Pfizer, Protalix, Sanofi, Sangamo, Takeda, and 4DMT. He has received honoraria from Alexion,

Amicus, Sanofi, Spark, and Takeda; and research funding from Amicus and Takeda.

WORLDSymposium: 19th Annual Research Meeting | Orlando, FL, US | February 22-26, 2023