Pharvaris N : Development of Kallikrein Kinin System Biomarker Assays to Investigate Bradykinin mediated Diseases

Development of Kallikrein-Kinin System Biomarker Assays to Investigate Bradykinin-Mediated Diseases

Evangelia Pardali1, Justyna Nowakowska2, Oliver Domenig3, Grégoire Vuagniaux2, Anne Lesage4

1Pharvaris B.V., Leiden, The Netherlands; 2Former employee of Pharvaris GmbH, Zug, Switzerland; 3Attoquant Diagnostics, Vienna, Austria; 4GrayMatters Consulting, Schilde, Belgium

Introduction

• Activation of the plasma kallikrein-kinin system (KKS) results in cleavage of high-molecular-weight

kininogen (HMWK) and production of vasodilatory kinins, such as bradykinin (BK) and its metabolite

BK1-5.1 Kallidin (KD) is another biologically active kinin produced by tissue kallikrein cleavage of

either HMWK or low-molecular-weight kininogen (LMWK).

• BK is involved in various physiological and pathological processes, including angioedema (AE).2

Differentiating BK-mediated vs histamine-mediated AE and assessing other BK-mediated disorders

Results

• Kinin LC-MS/MS assay in blank matrix met qualification criteria. Within- and between-run accuracy and precision. Coefficient of variation (CV) <15% (Figure 2).

Figure 2. Analysis of quality control (QC) samples for BK1-5,BK1-9 and KD in blank matrix

10000	BK1-5	BK1-9	KD

Results

• Standard curves were prepared by spiking iHMWK or cHMWK in kininogen-deficient plasma
• For both proteins, the back-calculated concentrations of the standards met qualification criteria (Figure 5).

Figure 5. Capillary immunoblotting assay qualification for iHMWK and cHMWK from human plasma

Results

• Acceptable intra-individual variability (CV <15%) for both iHMWK and cHMWK in both liquid PI plasma and control EDTA plasma (Figure 7).

Figure 7. iHMWK and cHMWK intra-individual variability in EDTA plasma and plasma with liquid PI

iHMWK	cHMWK
160	45

by measuring biomarkers produced upon activation of the KKS remains a challenge due to

proteolytic instability of the kinins and limitations of current analytical assays.3

• Establishment of a method to accurately measure BK and related peptides could aid in identifying

BK-mediated AE as well as other BK-mediated diseases.*

Materials and Methods

•	To inhibit ex vivo activation of KKS proteases and proteolytic degradation of BK, a protease
	inhibitor (PI) cocktail was manufactured in a liquid and lyophilized form.
•	Blood samples were collected from healthy volunteers (HV) by Fidelis Research AD in accordance
	with the Declaration of Helsinki and approved by The National Bioethics Committee of Medicines
	and Medical Devices (CNBMDM, protocol no. FRT-19101). All participants gave their written

[pg/mL]	1000	QC high
.conc	100	QC medium
Measured	10	QC low
1

• KD levels were below the limit of detection in all types of human plasma analyzed.
• Liquid PI efficiently inhibited KKS activation and stabilized BK1-9 levels compared with EDTA alone and was superior to lyophilized PI, resulting in low inter- and intra-run variability (CV <15%) for both BK1-5 and BK1-9 (Figure 3).

Figure 3. BK1-5 and BK1-9 inter- and intra-run variability in EDTA plasma and plasma with PI

BK1-5

BK1-9

	1500000		iHMWK
(AUC)			cHMWK
1000000
Signal	500000
			R2 >0.99
	0
	0	1	2	3
	Concentration (μg/mL)

Linearity range of standard curve

iHMWK cHMWK

Concentration range

accessible in human plasma

	EDTA		Liquid PI	g/mL]μ[	EDTA		Liquid PI
g/mL]μ	120				30
[iHMWK	80				KcHMW
40				15
	0					0
	HV1 HV2 HV3 HV1 HV2 HV3		HV1 HV2 HV3 HV1 HV2 HV3
			iHMWK (µg/mL)			cHMWK (µg/mL)
	Plasma	HV 1	HV 2	HV 3	CV %	HV 1	HV 2	HV 3	CV %
	EDTA	113.8	106.8	119.4	<10%	30.1	28.6	29.4	<10%

informed consent before enrollment.

• Plasma was collected using liquid PI or lyophilized PI form, or using ethylenediaminetetraacetic

200	EDTA	Liquid PI	Lyophilized PI

1000	EDTA
800

Liquid PI

Lyophilized PI

i/cHMWK

0.078-2.5 µg/mL

26-140 µg/mL

Liquid PI

109.6

90.3

107.5

<10%

28.5

24.3

26.1

<10%

	acid (EDTA) as a control.
•	An ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS)/MS protocol was
	optimized to measure BK (BK1-9),BK1-5, and KD (Attoquant Diagnostics GmbH).
•	A capillary-based immunoassay was also developed to quantify the cleaved and intact HMWK
	(cHMWK and iHMWK, respectively) (Charnwood Molecular Ltd).

-BK1 [pg/mL]5

100

40

20

0

9-BK1 ][pg/mL

40

20

0

≤ of LLoQ

• Comparable levels of iHMWK and cHMWK were observed in EDTA and liquid PI plasma.
• Plasma with lyophilized PI was not analyzed as it was shown not to stabilize kinins in plasma.
• Acceptable inter-run variability (CV <15%) for both iHMWK and cHMWK in both liquid PI plasma and control EDTA plasma (Figure 6).

Figure 6. iHMWK and cHMWK inter-run variability in EDTA plasma and plasma with PI

• Stability of iHMWK and cHMWK was analyzed in EDTA control and liquid PI plasma following 1 or 2 cycles of FT.
• Liquid PI had no apparent effect on levels of iHMWK & cHMWK following FT as compared with EDTA (Figure 8).

Figure 8. Effects of freeze and thaw on iHMWK and cHMWK levels in EDTA plasma and plasma

• Qualification of the UPLC-MS/MS and capillary-based immunoassay was performed using plasma

Run 1Run 2Run 1Run 2Run 1Run 2

Run 1Run 2Run 1Run 2Run 1Run 2

iHMWK

cHMWK

with PI

iHMWK

cHMWK

from HVs collected under different conditions.

Results

• Calibration curves were prepared by spiking different kinin peptides in surrogate blank matrix.
• For all tested kinins, the back-calculated concentrations of the calibrator standards were within ±15% of the nominal value and met qualification criteria (Figure 1).

Figure 1. Calibration curves of BK1-5,BK1-9, and KD

	BK1-5		BK1-9
Plasma	Mean (pg/mL)	CV %	Mean (pg/mL)	CV %
EDTA	172.8	<10%	925.0	<10%
Liquid PI	23.6	<10%	21.7	<10%
Lyophilized PI	5.4	<10%	Not applicable	Not applicable

• Liquid but not lyophilized PI efficiently inhibited ex vivo KKS activation and stabilized BK1-9 following 2 cycles of freeze and thaw (FT) as compared with EDTA (Figure 4).

	200			60
	EDTA	Liquid PI		EDTA	Liquid PI
g/mL]μ[	150		g/mL]μ[	45
100		30
iHMWK		cHMWK
			Run 1
50		15	Run 2
	0			0	Run 3
	HV1 HV2 HV3	HV1 HV2 HV3		HV1 HV2 HV3	HV1 HV2 HV3

	200
	EDTA
g/mL]μ	150
100
[
iHMWK
50
	0
	HV1

		60
Liquid PI	g/mL]μ	EDTA	Liquid PI
	45
	[	30
	cHMWK	No FT
	15	1x FT
		2x FT
		0
HV2 HV3 HV1 HV2 HV3		HV1 HV2 HV3	HV1 HV2 HV3

Measured conc. [pg/mL]

10000 1000 100 10 1

BK1-5

BK1-9

KD

R2 >0.99

1	10	100	1000 10000
	Nominal conc. [pg/mL]

	Linearity range of quantification
	LLoQ	ULoQ
BK1-5	5 pg/mL	10,240 pg/mL
BK1-9	5 pg/mL	10,240 pg/mL
KD	20 pg/mL	10,240 pg/mL

LLoQ: lower limit of quantification; ULoQ: upper limit of quantification

Figure 4. Effects of freeze and thaw on BK peptide stability

	150	BK1-5		8000	BK1-9
	Liquid PI Lyophilized PI		Liquid PI Lyophilized PI
	EDTA		EDTA
				6000
[pg/mL]	100		[pg/mL]	4000
	2000
5-1	50		1-9	40	≤ of LLoQ
BK			BK
		20
	0			0
	No 1x 2x No 1x 2x No 1x 2x

	iHMWK		cHMWK
Plasma	Mean (µg/mL)	CV %	Mean (µg/mL)	CV %
EDTA	113.4	5.5%	29.4	2.6%
Liquid PI	102.5	10.4%	26.3	8.1%

Conclusions

Plasma

EDTA

Liquid PI

	iHMWK (µg/mL)				cHMWK (µg/mL)
No FT		1xFT	2xFT		No FT	1x FT		2xFT
113.9		115.2	107.7		27.5	27.8		26.6
105.6		99.0	97.2		25.2	23.4		22.4

References

1. Kaplan AP, et al. Adv Immunol. 2014;121:41-89.2. Maurer M, et al. Clin Rev Allergy Immunol. 2021;61:40-9.

1. Kaplan AP, et al. Front Med (Lausanne). 2017;4:206.

						No 1x 2x No 1x 2x No 1x 2x
		BK1-5 (pg/mL)				BK1-9 (pg/mL)
Plasma	No FT		1xFT		2xFT		No FT		1x FT		2xFT
EDTA	83.1		82.9		90.8	2417.3		4260.9		5942.8
Liquid PI	27.4		27.7		27.4	34.0		35.3		38.9
Lyophilized PI	5.3		5.8		5.9	<5.0		<5.0		<5.0

• The liquid PI cocktail was shown to be more efficacious in inhibiting non-specific activation of KKS and generation of BK1-9 as compared to EDTA without PI.
• Liquid PI was shown to be superior to lyophilized PI in inhibiting degradation of BK1-9.
• The established qualified KKS biomarker assays can be used to reliably measure KKS biomarkers in human plasma.
• These assays could become key tools for identifying, studying, and managing BK-mediated diseases, including BK-mediated AE.

COI: E.P.: employee of Pharvaris, holds stock in Pharvaris; J.N.: former employee of Pharvaris; O.D.: CEO of Attoquant Diagnostics GmbH; G.V.: former employee of Pharvaris; A.L.: employee of GrayMatters Consulting and consultant to Pharvaris, holds stocks/stock options in Pharvaris; advisor to Kosa Pharma.

*Funding statement: Funding towards this study was provided by Pharvaris.