| 1 23 April, 2018

C. Ellen van der Schoot

Department Experimental Immunohematology

Sanquin, Amsterdam, the Netherlands

e.vanderschoot@sanquin.nl

The efficient use of RhIg

| 2 23 April, 2018

Increasing the efficiency of RhIg

1. Increasing the biological activity of RhIg

• Effect of Fc-core glycosylation of anti-D immunoglobulin

2. Decreasing the unnecessary use of RhIg

• Guiding of immunoprophylaxis by fetal RHD typing

Fc-glycans

April 23, 2018 | 3

Asn297

Sialic acid

Galactose

Fucose

Bisected

GlcNac

Glycans affect binding to FcgR

Low Fc-fucosylation

results in 50x

enhanced binding

To FcγRIIIa

Dekkers et al. Front Immun 2017

Fc glycopeptides: Mass spectrometric analysis

| 5 23 April, 2018

Glycan species Glycan structure

IgG1

E293EQYNSTYR301a

P01857b

[M+H]+

No glycan - 1190.5198

G0

2487.9880

G0F

2634.0459

G1

2650.0408

G1F

2796.0987

G0N

2691.0674

G0FN

2837.1253

G2

2812.0936

G2F

2958.1515

G1N

2853.1202

G1FN

2999.1781

G2N

3015.1730

G2FN

3161.2309

Total IgG1

pep pep

pep

pep

pep

pep

pep

G0F G1F

G2F

* *

pep

G1 pep

G2

* *

A

G0

Majority of IgG1 in plasma is fucosylated

Anti-D IgG is low - fucosylated

| 6 23 April, 2018

Total IgG1

Anti-D IgG1

2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 m/z

pep pep

pep

pep

pep

pep

pep

G0F G1F

G2F

pep

pep

pep

G0

G1

G2

pep

G2F pep

G1F

pep

*

*

*

* *

pep

G1 pep

G2

* *

A

B

G0

Fc-fucosylation of anti-D IgG is variable and

related to pathogenicity

| 7 23 April, 2018

F u c o s y la tio n (% )

* * * , p :< 0 .0 0 0 1

0 2 0 4 0 6 0 8 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

T o ta l Ig G

An

ti-D

an

tib

od

y

8 10 12 14 160

20

40

60

80

100

R2=0.467p= 0.010

NK-cell ADCC (% lysis)

An

ti-D

Ig

G1

-fu

co

sy

lati

on

(%

)

0 5 10 15 200

20

40

60

80

100R2=0.289,P: *, 0.037

Haemoglobin (g/dl)

An

ti-D

Ig

G1

-fu

co

sy

lati

on

(%

)

NK-cell ADCC R2=0.467,p=0.010

Neonatal Hb R2=0.289, p=0.038

Fucosylation(%) p<0.0001

Kapur ,Della Valle , Br J Haemat 2016

Fc-fucosylation of anti-D IgG from

hyperimmune donors is variably low

| 8 23 April, 2018

Pre

gnant w

omen

HID

fem

ale

HID

mal

e

0

20

40

60

80

100

An

ti-D

fu

co

syla

tio

n (

%)

NS

**

*

A

Anti-D preparations display variable

decreases in Fc-fucosylation

| 9 23 April, 2018

Rh

oG

am

(a

)

Rh

oG

am

(b

)

Rh

oG

am

(c

)

Pa

rto

bu

lin

SD

F

Rh

op

hy

lac

CG

Rh

op

hy

lac

QC

G

LF

B C

RT

S

D-G

am

Rh

es

on

ati

v

Rh

eD

Qu

in 2

00

7

Rh

eD

Qu

in 2

01

4

0

5

10

15

20

IgG

-bis

ec

tio

n (

%)

50

60

70

80

90

100

Total IgG

Anti-D IgG

IgG

-fu

co

syla

tio

n (

%)

Efficacy of anti-D appears to be related to Fc-

fucosylation

• Human polyclonal anti-D immunoglobulin:

• Low level of fucosylation:

• high ADCC, very rapid RBC clearance, prevented D-immunization.

• Moab anti-D produced in human B cell lines (BRAD3, BRAD5)

• Medium level of fucosylation

• medium ADCC, fast clearance, prevented D-immunization in 93% subjects

•

• Moab anti-D produced in human-mouse hetero hybridoma: (Fog1, AD1, G7,

G12)

• High level of fucosylation

• low ADCC, variable and slow clearance, stimulated D-immunization.

| 10 23 April, 2018 Kumpel et al. submitted

Conclusion (1)

Production of RhIg from hyperimmune donors with low fucosylated anti-D Ig

might result in more potent RhIg

| 11 23 April, 2018

Guiding Rh immunoprophylaxis by fetal RHD

typing

• 40% of D-neg women carry a D-neg child and do not need Rh-Ig

• Fetal DNA is present in low concentration in maternal plasma from week 5

| 12 23 April, 2018 Illustration: Lo, 2007

placenta plasma

of pregnant woman

Mixture of DNA

from mother and

DNA from fetus

Dutch screening program

Pregnancy

12th week

ABO typing

D typing

IEAscreen

27th week

ABO typing

D typing

IEAscreen

Fetal RHD

typing

30th week

Antenatal anti-D Ig

prophylaxis if RHD-

positive fetus

After birth

Postnatal anti-D Ig

prophylaxis if fetal RHD

typing was positive

During first 15 months:

Cord blood analysis

Technical approach

• Centralized at one laboratory (Sanquin, Amsterdam)

• DNA isolation from 1 ml of EDTA plasma in 27th week of

pregnancy

• First 15 months comparison with cord blood

DNA

Purification

DNA

PCR

Setup

Amplification

Detection

Report

Electronic

Result

Plasma

Pipetting

DNA

23 april 2018 | 15

Design fetal RHD typing

RHDexon 1 exon 10 exon 10 exon 1

RHCERHDexon 1 exon 10 exon 10 exon 1

RHCE

RHD-PCR Multiplex

exon 5: not amplified in majority of RHD variants

in Caucasians: RHD*DVI

in Blacks: RHD*Ψ; RHD*01N.06 and RHD*03N.01 (r’s)

exon 7: present in most RHD variants

First 25.500 samples:

Diagnostic accuracy of at least 99.1%

CB-serology

Plasma-PCR

Pos Neg

Pos 15,826 225

(0.87%)

Neg 9

(0.03%) 9,740

| 16 de Haas et al. BMJ 2016

Systemic analysis of false results

• False negative fetal RHD typing:

• No antenatal prophylaxis : 0.61% risk of alloimmunization

• No postnatal prophylaxis: 17% risk of alloimmunization

• False positive fetal RHD typing:

• Unneccessary RhIg is given, no clinical consequences

| 17 Thurik et al. Prenat Diagn 2016

Stegman et al. Brit J Haematol 2016

Potential effect of PCR guided ante- and

postnatal immunoprophylaxis

Unnecessary

antenatal anti-

D

No antenatal

anti-D, while

at risk

No postnatal

anti-D, while

at risk

Old program

(no PCR, only cord

blood)

38,3% 0% 0,09%

New program

(only PCR, no cord

blood)

0,43% 0,03% 0,03%

Accuracy of implemented fetal RHD typing

(Denmark, the Netherlands, Finland)

Samples TRUE Pos

False Pos

TRUE Neg

FALSE Neg

sensitivity %

specificity %

Clausen et al., 2014

12688 7636 41 4706 11 99.86 99.14

de Haas et al., 2016

25789 15816 225 9739 9 99.94 97.74

Haimila et al., 2017

10814 7080 7 3640 1 99.99 99.81

Total 49291 21 99.93

| 19 23 April, 2018

van der Schoot et al. Curr Opin Hem 2017

Conclusion (2)

Implementation of fetal RHD typing has resulted in decreased

unnecessary use of antenatal RhIg, without loss of efficiency of the

program

| 20 23 April, 2018

Acknowledgements

• Fc-glycosylation

• Gestur Vidarsson

• Rick Kapur

• Gillian Dekkers

• Lussy Della Valle

• Myrthe Sonneveld

• Manfred Wuhrer (LUMC, Leiden)

• Belinda Kumpel (Bristol)

• Fetal RHD genotyping

• Masja de Haas

• Barbera Veldhuisen

• Florentine Thurik

• Peter Scheffer

• Aicha Ait Soussan

• Tamara Stegman

• Lieve Christiaens (UMCU, Utrecht)

| 21 23 April, 2018