Pre-existing antibodies to biologics: Predictive for treatment outcome?

Claus H. NielsenProfessor, Ph.D., M.D., M.Sc.  

Institute for Inflammation ResearchDept. of Infectious Diseases and RheumatologyCopenhagen University Hospital Rigshospitalet

Appearance of serum sickness-like reactions during treatment with rituximab

• In a study on Sjögrens disease HACAs were formed in 4 of 8 patients, 3 of these developed SSLR (Pijpe J, Arthritis Rheum 2005)

• In children with ITP, RTX caused SSLRs in 5 out of 60 (Bennet et al. Blood 2006; Wang et al., J Pediatr 2005).

• In a study on Graves’ disease 3 of 10 patients developed SSLR (el Fassi et al.,J Endocrin Invest, 2011)

Arthralgia, skin eruptions, fever, lymphadenopathy

Serum sickness like reactions (SSLR)

Anti-IgG/IgA/IgM

Demonstration of circulating complement-opsonized immune complexes

IgM

-25

0

25

50

75

100

125

A

P=0.01

P= 0.01

#5

#3 #10M

FI

IgG

0

100

200

300

B

P= 0.008

P= 0.01

#5

#3#10

MF

I

C3

+ RTX

+ RTX

- RTX

0

100

200

C

P= 0.003

P= 0.04

P=0.004

#5

#3 #10

M

FI

An anti-C3-capture ELISA developed with anti-IgG/IgA/IgM confirmed

El Fassi et al. J Endocrinol Invest 2011; 34: e163-e167

Is there a pre-existing immune response to rituximab?

How can you develop antibody responseswhen no B cells are present?

Ongoing production of pre-existing antibodies by plasma cells?

Basic conundrum

Study on endometrial protein PP14: 81% of male sera were false-positive

Analyte

Coating Ab

Detecting Ab

False-positive reactions in two-site immunoassays

Coating Ab

Detecting Ab

False-positive reactions in two-site immunoassays

Cross-binding anti-mouse Ig Ab

Prevalence of antibodies causing false-positive reactions

Kricka et al. Clin Chem 1999; 45: 942-56

HAAAs: Human anti-animal antibodies

HAMAs: Human anti-mouse antibodies

HACAs: Human anti-chimeric antibodies

HAHAs: Human anti-human antibodies

Heterophilic antibodies

What are they?

HAAAs versus Heterophilic Antibodies

Heterophilic antibodiesImmunoglobulin Group I

Anti-Fab(85% of positive

samples)

Group IIAnti-Fc

(15% of positive samples)

Negative samples

Mouse +++ +++ -

Rat +++ - -

Goat +++ - -

Horse +++ +++ -

Rabbit - +++ -

Cow +++ +++ -

BSA (neg. Control) - - -

Hennig et al.

Anaphylactic reactions to cetuximabcaused by pre-existing IgE antibodies to the α-gal epitope

All humans have IgG antibodies specific for the oligosaccharide galactose-α-1,3-galactose, which is closely related to substances in the ABO blood group

Produced in mouse hybridoma cells

Chung et al., N Engl J Med 2008; 358;1109-17

Our assay

Coating: F(Ab’)2 of IFX, RTX or murine IgG

Proband serum/plasma

Anti-human γ-chainbiotin

HRP-straptavidin

IgG antibodies against mouse Fab, anti-IFX Fab and anti–RTX Fab are common in human plasma

T-helpercells

Anti-mouse Fab, anti-IFX Fab and anti–RTX Fab correlate

r2=0.90, p < 0.0001r2=0.90, p < 0.0001 r2=0.90, p < 0.0001

Mouse IgGcolumn

Anti-IF

X F(a

b')20

200

400

600

800

1000

mU

/L

IFX F(ab')2

Infliximab column

Anti-IF

X F(a

b')20

200

400

600

800

1000

mU

/L

IFX F(ab')2

Cross-reactivity between anti-IFX and anti-mIgG

Anti-m

ouse Ig

G

0

200

400

600

800

1000

mU

/L

Mouse IgG F(ab')2

Anti-m

ouse Ig

G

0

200

400

600

800

1000

mU

/L

Mouse IgG F(ab')2

29 patients with Crohn’s disease treated with infliximab

21 patients with ulcerative colitis treated with infliximab

Measurement of pre-existing anti-infliximab-Fab Absand clinical responses

Clinical study

Observational, retrospective, single center study

Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83

Pre-existing anti-IFX Fab IgG: Prevalence in IBD patients naïve to ‘biologics’

Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83

8

Pre-existing anti-IFX Fab IgG: Association with 1 year remission on IFX

Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83

9

Pre-existing anti-IFX Fab IgG:Levels associated with 1 year remission in CD

Anti-IFX Fab Ab cut-off

mU/l

Sensitivity

% [95%CI]

Specificity

% [95%CI]

Identification of patients in remission <439 100 [63–100] 67 [43–85]

Identification of patients not in remission ≥61 100 [84–100] 25% [3–65]

Optimal differentiation between patients in

remission or not (sensitivity=specificity)

233 75 [35–97] 76 [53–92]

Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83

Primary vs. secondary non-responders

Other modalitiesOther TNF-inhibitor

Primary non-responder

TNF-α inhibitor

Primary responder

Secondary non-responder

Continue TNF-α inhibitor

1/3

1/3

1/3

Probably patients without ”TNF-driven disease”

Ususally loss of response due to ADAsPre-existing antibodies indicate priming

10

Efficacy by anti-IFX Fab Abs

Crohn's disease

Primary non-response Loss of response Remisson

10

100

1000

10000p=0.397

p=0.05

p<0.01

n=8 n=7 n=8

A

308308

692

91

Pre

-exi

stin

g an

ti-IF

X F

ab A

b (m

U/l)

Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83

Antigen receptor (BCR)

B cell

Heterophilic BCR

Mouse IgG

Pre-existing T-cell responses may support development of drug-neutralizing antibodies

Th cell

T-helper cell with specificity for xenogenic peptide

TCRMHC II

Pre-existing T-cell responses may support development of drug-neutralizing antibodies

BCR recognizingidiotype of drug

Drug

B cell Th cell

T-helper cell with specificity for xenogenic peptide

TCRMHC II

10

All patients

Safety by anti-IFX Fab Abs

Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83

”Positive samples”

Meta-analysis by Xu & Rup

31 studies: 1331 patients & 499 controls

What are the antigenic determinants?

Glycosylation

Xenogenic part

F(ab’)2

Origin of pre-existing antibodies?

Heterophilic antibodiesImmunoglobulin Group I

Anti-Fab(85% of positive

samples)

Group IIAnti-Fc

(15% of positive samples)

Negative samples

Mouse +++ +++ -

Rat +++ - -

Goat +++ - -

Horse +++ +++ -

Rabbit - +++ -

Cow +++ +++ -

BSA (neg. Control) - - -

Hennig et al.

Bovine IgGcolumn

1 10 1000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

OD

490

nm

Dilution 1:x

Eluate

Cross-reactivity between cow IgG, mouse Fab and infliximab

1 10 1000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

OD

490

nm

Dilution 1:x

Eluate

Anti-IFX Fab

Anti-mouse IgG Fab

Origin: Possibly vaccines

Diphte

ria to

xoid

I (u

sed u

ntil 1

990)

Tetan

us to

xoid

I (u

sed u

ntil 1

990)

Ovalb

umin

)(neg

ativ

e co

ntrol)

Bovine

IgG (p

ositiv

e co

ntrol)

Havrix

(Hap

atiti

s A)

Diphte

ria to

xoid

II (u

sed a

fter1

990)

Pneum

ococc

al p

olysa

cchar

ide

vacc

ine

Haem

ophilus

influ

enza

e b

Human

pap

illom

a vi

rus

Tetan

us to

xoid

II (u

sed a

fter1

990)

Conclusions regarding pre-existing antibodies

• exist against chimeric antibodies (IFX, RTX, CTX)

• are presumably ”heterophilic antibodies”

• apparently predict outcome of infliximab therapy in Crohn’s disease

• may cause IgG- or IgE-mediated infusion reactions

• may be induced by vaccination

Everybody at Institute for Inflammation Research

Thanks to:

Yaseelan Palarasah, Christina Aniol-Nielsen, Casper Steenholdt, Børge Teisner