Non-neutralizing antibodies against factor VIII and risk of inhibitor

1

TITLE: Non-neutralizing antibodies against factor VIII and risk of inhibitor

development in patients with severe hemophilia A.

Short Title: Anti-FVIII non-neutralizing antibodies and inhibitor.

Antonino Cannavò1, Carla Valsecchi1, Isabella Garagiola2, Roberta Palla2, Pier Mannuccio

Mannucci1, Frits R. Rosendaal3, Flora Peyvandi1-2.

1Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca'

Granda Ospedale Maggiore Policlinico and Luigi Villa Foundation, Milan, Italy; 2Department

of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; and

3Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The

Netherlands

Corresponding Author:

Flora Peyvandi, MD PhD. Angelo Bianchi Bonomi Hemophilia and Thrombosis Center,

IRCCS Ca’ Granda Maggiore Policlinico Hospital Foundation and Department of

Pathophysiology and Transplantation, University of Milan, via Pace 9, 20122 Milan, Italy at

Tel: +39 02 55 03 4456, fax +39 02 54 100 125.

e-mail: [email protected]

Text count: 2785 words

Abstract count: 232 words

Figure/table count: 1 figure/3 tables.

Reference count: 51

Blood First Edition Paper, prepublished online December 29, 2016; DOI 10.1182/blood-2016-06-720086

Copyright © 2016 American Society of Hematology

For personal use only.on April 3, 2018. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

2

Key points:

Non-neutralizing antibodies against factor VIII are detected in untreated or minimally

treated patients with hemophilia A.

The presence of non-neutralizing antibodies is associated with a substantially

increased risk of inhibitor development.

BLOOD/2016/720086

Keywords: severe hemophilia A, inhibitor, non-neutralizing antibody, previously

untreated patient.




3

ABSTRACT

The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is the major

complication in hemophilia A. Non-neutralizing antibodies (NNAs) have not only been

detected in hemophilia patients but also in unaffected individuals. The aim of this study was

to assess the prevalence of NNAs in a cohort of previously untreated or minimally treated

patients with hemophilia A, and to evaluate whether their presence is associated with the

development of inhibitors. Plasma samples of 237 patients with severe hemophilia A enrolled

in the SIPPET trial were collected before any exposure to FVIII concentrates and analyzed

for the presence of anti-FVIII NNAs. Patients were followed for the development of

neutralizing antibodies. NNAs were found in 18/237 (7.6%) of patients at screening, with a

clear age gradient. Of those with NNAs, seven patients subsequently developed an inhibitor,

for a cumulative incidence of 45.4% (95% confidence interval (CI95) 19.5-71.3%), whereas

among those without NNAs 64/219 (29%) developed an inhibitor (cumulative incidence

34.0%, CI95 27.1-40.9%). In Cox regression, patients with NNAs at screening had a 83%

higher incidence of inhibitor development than patients without NNAs (hazard ratio (HR)

1.83, CI95 0.84-3.99). For high-titer inhibitors, the rate was almost 3-fold increased (HR

2.74, CI95 1.23-6.12). These associations did not materially change after adjustment. The

presence of anti-FVIII NNAs in patients with severe hemophilia A not previously exposed to

FVIII concentrates is associated with an increased incidence of inhibitors.




4

INTRODUCTION

The development of alloantibodies neutralizing factor VIII (FVIII) coagulant activity

(inhibitors) represents the main complication of treatment of hemophilia A. It occurs in

approximately one third of previously untreated patients (PUPs) and causes substantial

morbidity, mortality and costs for the healthcare system1-3. The causes for inhibitor

development are not fully understood, but some risk factors have been identified4-9 .

FVIII inhibitors consist of a polyclonal population of antibodies that are targeted to multiple

antigenic sites within the A2, A3 and C2 domains of the protein10. In addition to inhibitors,

anti-FVIII antibodies are present in healthy individuals and patients affected with hemophilia

A without exerting coagulant inhibitory activity. Several laboratory platforms for the

detection of total FVIII binding antibodies have been developed, based upon immunoblotting

assay, fluorescence immune assay and enzyme-linked immunosorbent assay (ELISA)11-31.

With these assays non-neutralizing antibodies (NNAs) directed towards non-functional FVIII

epitopes that escape detection by means of functional assays have been identified. Their

prevalence is approximately 2-3% in healthy individuals16,18,21,24, while estimates in

hemophilia patients with different degrees of severity of disease vary widely, from 12% to

54%13-22,24-25,29-31. Although NNAs and inhibitors are not distinguishable based on their

isotypes, clonality and epitopes32, recent data published by Hofbauer33 indicate that anti-

FVIII IgG with inhibitory activity have an up to 100-fold higher affinity for FVIII than those

without inhibitory activity. Based on studies on cross-reactivity it has been also suggested

that FVIII inhibitors in hemophilia A patients originate from the expansion of a natural anti-

FVIII clone of B lymphocytes that exists before any treatment with FVIII and secretes anti-

FVIII antibodies similar to the natural antibodies found in healthy individuals. Furthermore,




5

FVIII inhibitors seem to be produced by a B cell clone that has undergone an antigen-driven

affinity-maturation and hypermutation of the V-region33. With this as background, what is not

known pertaining to NNAs is their presence before any exposure to FVIII concentrates and

their relationship with subsequent inhibitor development, even though Boylan et al.15 already

suggested that NNAs could be an early sign of subsequent inhibitor development. Currently

available data on NNAs stem from large but heterogeneous cohorts of multi-transfused

patients, some with previous but then eradicated inhibitors, and others during immune

tolerance induction for inhibitor eradication. Furthermore, the majority of studies were cross-

sectional in design, and could not assess temporal relations. In particular, none of the

previous studies was performed on plasma samples collected before any FVIII concentrate

exposure11-31.

The aim of this study was to determine the prevalence and significance of anti-FVIII NNAs

in patients enrolled in the prospective randomized SIPPET study who were screened for

neutralizing and non-neutralizing anti-FVIII antibodies before any treatment with FVIII

concentrates.

MATERIAL AND METHODS

Patients:

This was a cohort study performed in the frame of SIPPET randomized trial35. Patients aged

<6 years, affected with severe hemophilia A, never exposed to FVIII concentrates, not or

minimally exposed (less than 5 EDs) to blood components (whole blood, fresh frozen plasma,

packed red cells, platelets or cryoprecipitate) and inhibitor negative by modified Bethesda

assay were included and then randomized to treatment with a single plasma-derived

(pdFVIII) or recombinant (rFVIII) FVIII concentrate. Patients were followed and monitored




6

at scheduled time-points for FVIII inhibitor occurrence35. Data were collected on family

history of hemophilia and inhibitor, age at screening, country site, FVIII gene mutations and

FVIII sources (for more details, see reference35); FVIII antigen levels (measured at screening

by Asserachrom VIII:C Ag, Diagnostica Stago, Asnières sur Seine, France) were categorized

as <1% and ≥ 1%. Minimally treated patients (MTPs) were defined as those exposed to less

than 5 EDs with blood components before screening, whereas previously untreated patients

(PUPs) were those never exposed to blood components. Screening was defined as the time of

inclusion in the SIPPET trial, before any exposure to FVIII concentrate. At this time a blood

sample was collected and patients were screened both for the presence of inhibitory

antibodies using the Bethesda assay with the Nijmegen modification and stored for future

determination of the presence of NNAs using an ELISA assay. Plasma samples collected at

screening were available for 237 patients and were tested for anti-FVIII NNAs.

Approval was obtained from medical ethics committee at each study center. Parents or

guardians of all children provided written informed consent.

Inhibitor testing

Inhibitor testing at screening and follow-up was performed centrally at the Angelo Bianchi

Bonomi Hemophilia and Thrombosis Centre, Milan, Italy, using the Bethesda assay with the

Nijmegen modification35. In case of inhibitor occurrence during follow-up, inhibitor levels

were confirmed on a second sample within 14 days after the first positivity, and patients were

followed for six months to establish whether the inhibitor was transient or persistent.

Non-neutralizing antibody testing

ELISA plates (96-wells NUNC Maxisorp, St. Louis, MO) were coated overnight at 4°C with

1.2 ug/mL of the full length rFVIII product Advate® (Baxter Healthcare Corporation,




7

Westlake Village, CA) previously dialyzed against phosphate buffered saline (PBS). The

plates were then blocked for 2 h at room temperature with PBS containing 5% skim milk

powder (Merck, Kenilworth, NJ). After blocking, the plates were washed 3 times with PBS

0.1% Tween-20 (Sigma, Merck, Kenilworth, NJ). The same washing procedure was

performed between the different incubation steps. Samples were diluted in PBS, 1% skim

milk powder (Merck, Kenilworth, NJ), 0.01% Tween-20 (Sigma, Merck, Kenilworth, NJ),

added to the coated plates and incubated for 2 h at room temperature. After a washing step,

the plates were incubated 1 h at room temperature with horseradish peroxidase (HRP)- anti

human IgG (GE Healthcare, Little Chalfont, UK ) diluted 1/2000 in PBS, 1% skim milk

powder (Merck, Kenilworth, NJ), 0.01% Tween-20. O-phenylenediamine dihydrochloride

(OPD) substrate (Sigma, Merck, Kenilworth, NJ) was added and the reaction stopped by

adding 3 mol/L H₂SO₄. Absorbance was read at 492 and corrected for background at 620 nm.

Each plasma sample was analyzed twice in different assay runs. In case of discrepancy with

values deviating from the range of interassay variability (CV= 25%), a third assay was

performed. The mean of two assay results or of the two closest values in the case of three

assays was used. In the first run all plasma samples were diluted 1/10 and, in the second,

samples with high IgG concentration were further diluted to have an absorbance value that

fell within the linear portion of the standard curve. In each assay, a positive control sample

(plasma with a high-titer inhibitor) diluted 1/40 and a negative control sample (normal pool

plasma) diluted 1/10 were also used.

Standard curve: Total IgG from a plasma of a patient with hemophilia A and a high-titer

inhibitor (600 BU) were purified on a Protein G-Sepharose (GE Healthcare, Little Chalfont,

UK ) column. Specific anti-FVIII IgG were then isolated by using affinity chromatography

on Affi-Gel 10 (Bio-Rad, Hercules, CA) coupled with rFVIII (Advate®), 4000 IU/2mL of

slurry gel. The affinity purified anti-FVIII specific IgGs were checked for purity by means of




8

sodium dodecyl sulphate – poly acrylamide gel electrophoresis (SDS PAGE electrophoresis)

and used in the ELISA assay to construct the standard curve. The cut-off for positive anti-

FVIII NNA was set at 1.64 µg/mL specific anti-FVIII IgG, corresponding to 100% specificity

and 96% of sensitivity in the ROC curve constructed with the results of anti-FVIII IgG

measured in 107 healthy individuals and 101 hemophilia A patients with positive FVIII

inhibitor detected by Bethesda assay (mean 103.9 BU, median 14.5 BU, range: 0.5-1400

BU).

Statistical analysis

Prevalences were estimated as proportions with confidence intervals obtained by the exact

binomial method of Clopper-Pearson. We used odds ratio (OR) and 95% confidence intervals

(CI95) to assess putative determinants of NNA. Analysis by age at screening was done by

stratifying patients in 5 age categories. Independent samples t-test was used to compare age

between the two groups.

Kaplan-Meier survival analyses were performed to assess the cumulative incidence for both

all inhibitors and high-titer inhibitors by NNAs serotype, and the incidence rates were

compared with Cox regression survival analyses taking into account as covariates FVIII gene

mutations (categorized as null- vs non-null mutation)35, FVIII antigen levels, self-reported

family history for inhibitor, trial treatment arm (pdFVIII or rFVIII), age at screening (in

months) and country site. Adjustments in multivariate Cox models were each made

individually, as there were too few events to include all variables in a single multivariate

model. Confidence intervals were derived from this model. Due to the occurrence of two

deaths during the trial35, we performed a sensitivity analysis assuming that both patients had

developed a high-titer inhibitor at the truncated follow-up instead of dying. Statistical

analyses were performed within SPSS, version 23.0 (IBM Corp., Armonk, NY).




9

RESULTS

Characteristics of the study cohort

The mean age at screening was 18.3 months (median 13 months; range: 0-67 months). FVIII

gene causative mutations were detected in 94.1% of the cases (n=223): 185 of them (83%)

were carrying a null-mutation (large deletions, nonsense mutations, inversions and

frameshift). In 107 of 237 patients (45.1%) there was a positive history of hemophilia in

relatives, and 23 (21.5%) reported a positive family history of inhibitor. One hundred and

twenty-one patients (51.1%) were randomized to treatment with pdFVIII and 116 (48.9%) to

rFVIII. Of 237 patients followed for a mean of 27 ED (median 23 days; range: 1-50 days), 71

developed a FVIII inhibitor (30.0%), which in 48 (67.6%) was at high-titer.

Determinants of non-neutralizing antibodies towards FVIII

NNAs were detected at screening in 18/237 patients (7.6%; CI 95 4.9-11.7%). Table 1 shows

the general characteristics of NNA-positive and NNA-negative subjects. Patients with NNAs

were older than those without (mean 27.4 months; median 24 months; range: 2-59 months vs

mean 17.5 months; median 12 months; range 0-67months; p-value: 0.007), and NNA

prevalence clearly increased with age (Table 2). Non-null mutations, measurable FVIII

antigen levels in plasma and a positive family history of inhibitor were also associated with a

higher prevalence of NNAs at screening (Table 2). Previous exposure to blood components

was associated with a reduced prevalence (OR 0.49, CI95 0.17-1.43). For all these

associations, confidence intervals were wide due to the limited sample size, and none

changed after adjustment for age.




10

NNAs and inhibitor development

Among patients positive for NNAs at screening (n = 18), seven subsequently developed an

inhibitor, for a cumulative incidence of 45.4% (CI95 19.5-71.3%), whereas among those

negative (n = 219) 64 developed an inhibitor, for a cumulative incidence of 34.0% (CI95

27.1-40.9%). In the NNA-positive group, all inhibitors were high-titer, whereas in the NNA-

negative group, 41 of 64 inhibitors were high-titer, for a cumulative incidence of 21.9%

(CI95 15.8-28.0%). Importantly, no inhibitor among NNA-positive subjects was transient,

compared with 26.6% (17 out of 64 patients) of inhibitors in the NNA-negative group which

were transient and disappeared within 6 months.

Figure 1 shows the Kaplan-Meier plots for all and high-titer inhibitors by the presence or

absence of NNAs at screening. In univariate Cox regression, presence of NNAs was

associated with an 83% higher incidence of inhibitors than in its absence (hazard ratio (HR)

1.83, CI95 0.84-3.99). For high-titer inhibitors the rate was almost three-fold increased (HR

2.74, CI95 1.23-6.12). In adjusted models, the effect of NNAs on inhibitor development if

anything became stronger (Table 3).

Due to the stringency of our cut-off value for non-neutralizing anti-FVIII antibody positivity

we also analyzed data using a lower cut-off of 1.035 µg/mL, corresponding to the highest

value of the Younden index obtained from the ROC curve (99% of sensitivity, 98% of

specificity) and results did not change: all inhibitors by NNA positivity HR 1.69 (CI95%:

0.97-2.96) and high-titer inhibitors HR 2.33 (CI95% 1.25-4.34) . Two deaths occurred during

the trial, both among NNA-negative patients. We performed a sensitivity analysis assuming

that both patients had developed a high-titer inhibitor at the truncated follow-up instead of

dying. We recalculated the crude hazard ratio by the presence of NNAs and the results did

not change. The hazard ratio by NNAs presence was 1.78 (0.81-3.87) for all inhibitors, and

2.62 (1.18-5.84) for high-titer inhibitors. Severe non-fatal adverse events were nine episodes




11

of intracranial bleeding and two episodes of gastro-intestinal bleeds, all of them occurred in

the NNA-negative group. Per protocol these adverse events were not considered a reason for

withdrawal of the study, and subjects continued to be treated and completed the study.

Further sensitivity analyses were performed for patients who were censored before reaching

the predefined end-point of three years follow-up or 50 EDs, and no major deviations from

the overall findings were found.

DISCUSSION

We studied a cohort of 237 patients with severe hemophilia A, previously untreated and

minimally exposed to blood components and thereby at risk for FVIII inhibitor development

after exposure to FVIII concentrates. A sensitive ELISA assay was used to detect NNAs at

screening before any exposure to FVIII, which were present in 7.6% of patients. Age, non-

null mutation, FVIII antigen levels and family history of inhibitor seemed to influence NNAs

prevalence. Importantly, the presence of NNAs was associated with an increased risk of

inhibitor development, particularly for high-titer inhibitors, with an nearly three-fold

increased incidence rate.

In previously reports, NNA prevalence ranged from 2% to 3% in healthy people16,18,21,24 and

from 12.2% to 53.8% in patients with hemophilia A13-22,24-25,29-30. Several factors, particularly

that all previous studies predominantly included multi-transfused patients, explain this large

variation and the gaps of knowledge on their potential clinical role. For instance, the intron

22 inversion of FVIII mutation was associated with an increased frequency of NNAs in

multi-transfused patients in one study29, which was not confirmed in another20. Among multi-

transfused patients those with NNA were older than those with no NNAs in one study20, but

not in others13,30.




12

Given the increasing prevalence with age projected at 20% at an age of more than 40 months,

NNAs can be considered an age-related phenomenon in patients previously untreated and

minimally exposed to blood components, which may reflect maturation of the immune

system and in particular of the adaptive immune system in growing children37. This finding is

also supported by the increase of natural IgGs during the first years to remain stable with

aging38.

We found a higher prevalence of NNAs at screening in untreated patients with non-null

mutations than those with null mutations. One may hypothesize that patients with minor gene

variations have residual non-functional plasma levels of FVIII antigen, which elicit an

immune response before exposure to concentrates. This view is supported by the association

between detectable FVIII antigen and NNAs. Measurable FVIII antigen in plasma39, potential

contamination with maternal blood during delivery40, and the presence of milk fat globule

epidermal growth factor-FVIII (MFGEGF-F8) (a glycoprotein with a strong homology with

circulating FVIII in breast milk) might all be examples of early exposure41. Possibly, the

presence of NNAs before any specific treatment may be the expression of a natural anti-FVIII

B cell clone, that exists independently to any environmental FVIII exposure42.

Whereas some findings, particular those on determinants of NNAs had considerable

statistical uncertainty and should be confirmed in a larger cohort, the association of NNAs at

screening with subsequent inhibitor development was robust .

In our study, the presence of NNAs at screening in children not previously treated with any

FVIII concentrate was associated with an almost 3-fold increased risk of development of

high-titer inhibitors. In auto-immune diseases, such as systemic lupus erythematosus or

rheumatoid arthritis, the appearance of autoantibodies often precedes the clinical onset of

disease43-45. A hypothetical explanation is that the autoantibody response needs to mature

before acquiring such distinct characteristics as specificity of antigen recognition, or an




13

increase or shift in antigen recognition43-46. Similarly, we found that detectable inhibitors, and

mostly high-titer inhibitors, can be preceded by NNAs.

Furthermore, the effect of NNA on inhibitor development did not change its direction when

adjusted for other variables, but the hazard ratio increased after adjustment for mutation. This

can be explained by the inverse relations of null mutation with NNA presence on the one

hand and inhibitor development on the other.

This study has some limitations. The follow-up of patients started from screening and not

from birth, and therefore patients who died early were missed. Given the low fatality rate of

hemophilia, and the implausibility of a differential death rate by NNAs, this is an unlikely

source of bias.

In conclusion, the development of anti-FVIII inhibitors is a multicausal event and although

several factors have been identified4-9,35,47-51, none of them suffice to reliably predict the risk

for an individual patient. The identification of NNAs as an additional marker for inhibitor

development needs to be validated in further studies and once confirmed it could be useful to

measure NNA immediately at diagnosis or before any exposure to FVIII products. This may

contribute to improve prediction scores, which in turn may lead to individualized

interventions tailored to reduce the risk of inhibitor formation.

Acknowledgements: we thank SIPPET investigators groups for patient recruitment and data

collection. See the appendix for the full list.

Authors’ contributions. A.C., F.R.R. and F.P. designed the study, interpreted data and wrote

the manuscript. C.V. developed and validated ELISA assay, and performed the laboratory

workout. I.G. collected data and did the literature search. R.P. performed data analysis and

contributed in writing the manuscript. P.M.M. critically revised the manuscript. All authors




14

revised critically the work providing substantial input and gave final approval of the version

to be published.

Disclosure of interests. R.P. has received travel support from Pfizer. P.M.M. reports

honoraria for participating at advisory boards or as speaker at satellite symposia and

educational meetings organized by Baxalta, Bayer, Grifols, Kedrion Biopharma, Novo

Nordisk, Biotest and LFB. FP has received honoraria for participating as a speaker at satellite

symposia and educational meetings organized by Bayer, Biotest, CSL Behring, Grifols, Novo

Nordisk, and Sobi and she has received consulting fees from Kedrion Biopharma, LFB and

Octapharma. She is recipient of research grant funding from Alexion, Biotest, Kedrion

Biopharma, and Novo Nordisk received by Fondazione Luigi Villa. She is member of the

Ablynx scientific advisory board.

The other authors have no conflicts to declare.

Funding source. The work was supported by research grants from Fellowship project

Haemophilia Bayer Awards (to Dr Antonino Cannavò, 2014). The funder had no role in study

design, protocol preparation, patient recruitment, data collection, handling, analysis and

interpretation, or writing of the report




15

REFERENCES:

1. Mannucci PM, Tuddenham EG. The hemophilias: from royal genes to gene therapy. N

Engl J Med. 2001;344(23):1773-1779.

2. Peyvandi F, Garagiola I, Young G. The past and future of haemophilia: diagnosis,

treatments, and its complications. Lancet. 2016;388(10040):187-197.

3. Walsh CE, Soucie JM, Miller CH; United States Hemophilia Treatment Center

Network. Impact of inhibitors on hemophilia A mortality in the United States. Am J

Hematol. 2015;90(5):400-405.

4. Gouw SC, van den Berg HM, Oldenburg J, Astermark J, de Groot PG, Margaglione M,

Thompson AR, van Heerde W, Boekhorst J, Miller CH, le Cessie S, van der Bom JG.

F8 gene mutation type and inhibitor development in patients with severe hemophilia A:

systematic review and meta-analysis. Blood. 2012;119(12):2922-2934

5. ter Avest PC, Fischer K, Mancuso ME, Santagostino E, Yuste VJ, van den Berg HM,

van der Bom JG; CANAL Study Group. Risk stratification for inhibitor development at

first treatment for severe hemophilia A: a tool for clinical practice. J Thromb Haemost.

2008;6(12):2048-2054.

6. Oldenburg J, Schroder J, Brackmann HH, Muller-Reible C, Schwaab R, Tuddenham E.

Environmental and genetic factors influencing inhibitor development. Semin Hematol

2004;41(1 Suppl 1):82–88.

7. Astermark J, Oldenburg J, Escobar M, White GC, Berntorp E. The Malmo International

Brother Study (MIBS). Genetic defects and inhibitor development in siblings with

severe hemophilia A. Haematologica 2005;90(7):924–931.




16

8. Gouw SC, van den Berg HM, Fischer K, Auerswald G, Carcao M, Chalmers E,

Chambost H, Kurnik K, Liesner R, Petrini P, Platokouki H, Altisent C, Oldenburg J,

Nolan B, Garrido RP, Mancuso ME, Rafowicz A, Williams M, Clausen N, Middelburg

RA, Ljung R, van der Bom JG; PedNet and Research of determinants of inhibitor

development (RODIN) Study Group. Intensity of factor VIII treatment and inhibitor

development in children with severe hemophilia A: the RODIN study. Blood.

2013;121(20):4046-4055.

9. Iorio A, Halimeh S, Holzhauer S, Goldenberg N, Marchesini E, Marcucci M, Young G,

Bidlingmaier C, Brandao LR, Ettingshausen CE, Gringeri A, Kenet G, Knöfler R, Kreuz

W, Kurnik K, Manner D, Santagostino E, Mannucci PM, Nowak-Göttl U. Rate of

inhibitor development in previously untreated hemophilia A patients treated with

plasma-derived or recombinant factor VIII concentrates: a systematic review. J Thromb

Haemost. 2010;8(6):1256-1265.

10. Lollar, P. Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor

IX. J Thromb Haemost. 2004; 2 (7): 1082–1095.

11. van Helden PM, van den Berg HM, Gouw SC, Kaijen PH, Zuurveld MG, Mauser-

Bunschoten EP, Aalberse RC, Vidarsson G, Voorberg J. IgG subclasses of anti-FVIII

antibodies during immune tolerance induction in patients with hemophilia A. Br J

Haematol. 2008;142(2):644-652.

12. Favaloro EJ, Bonar R, Duncan E, Earl G, Low J, Aboud M, Just S, Sioufi J, Street A,

Marsden K. Misidentification of factor inhibitors by diagnostic haemostasis

laboratories: recognition of pitfalls and elucidation of strategies. A follow up to a large

multicentre evaluation. Pathology. 2007;39(5):504–511.




17

13. Lebreton A, Lapalud P, Chambost H, Biron-Andréani C, Morange PE, Combescure C,

Marquès-Verdier A, Berger C, Schved JF, Granier C, Lavigne-Lissalde G. Prevalence

and epitope specificity of non-neutralising antibodies in a large cohort of hemophilia A

patients without inhibitors. Thromb Haemost. 2011;105(6):954-961

14. Vincent AM, Lillicrap D, Boulanger A, Meilleur C, Amesse C, St-Louis J, Rivard GE.

Non-neutralizing anti-FVIII antibodies: different binding specificity to different

recombinant FVIII concentrates. Haemophilia. 2009;15(1):374-376.

15. Boylan B, Rice AS, Dunn AL, Tarantino MD, Brettler DB, Barrett JC, Miller

CH; Hemophilia Inhibitor Research Study Investigators; Hemophilia Inhibitor Research

Study Investigators. Characterization of the anti-factor VIII immunoglobulin profile in

patients with hemophilia A by use of a fluorescence-based immunoassay. J Thromb

Haemost. 2015;13(1):47-53.

16. Hofbauer CJ, Whelan SF, Hirschler M, Allacher P, Horling FM, Lawo JP, Oldenburg J,

Tiede A, Male C, Windyga J, Greinacher A, Knöbl PN, Schrenk G, Koehn J,

Scheiflinger F, Reipert BM. Affinity of FVIII-specific antibodies reveals major

differences between neutralizing and non-neutralizing antibodies in humans.

Blood. 2015;125(7):1180-1188.

17. Montalvão SA, Tucunduva AC, Siqueira LH, Sambo AL, Medina SS, Ozelo MC A

longitudinal evaluation of anti-FVIII antibodies demonstrated IgG4 subclass is mainly

correlated with high-titre inhibitor in hemophilia A patients. Haemophilia.

2015;21(5):686-692.

18. Whelan SF, Hofbauer CJ, Horling FM, Allacher P, Wolfsegger MJ, Oldenburg J, Male

C, Windyga J, Tiede A, Schwarz HP, Scheiflinger F, Reipert BM.




18

Distinct characteristics of antibody responses against factor VIII in healthy individuals

and in different cohorts of hemophilia A patients. Blood. 2013;121(6):1039-1048.

19. Klintman J, Hillarp A, Berntorp E, Astermark J. Long-term anti-FVIII antibody

response in Bethesda-negative hemophilia A patients receiving continuous replacement

therapy. Br J Haematol. 2013;163(3):385-392.

20. Klintman J, Hillarp A, Donfield S, Berntorp E, Astermark J. Antibody formation and

specificity in Bethesda-negative brother pairs with hemophilia A. Quantitation of anti-

factor VIII antibodies in human plasma. Haemophilia. 2013;19(1):106-112.

21. Krudysz-Amblo J, Parhami-Seren B, Butenas S, Brummel-Ziedins KE, Gomperts

ED, Rivard GE, Mann KG. Quantitation of anti-factor VIII antibodies in human plasma.

Blood. 2009;113(11):2587-2594.

22. Zakarija A, Harris S, Rademaker AW, Brewer J, Krudysz-Amblo J, Butenas S, Mann

KG, Green D. Alloantibodies to factor VIII in hemophilia. Haemophilia. 2011;

17(4):636-640.

23. Gilles JG, Saint-Remy JM. Healthy subjects produce both anti-factor VIII and specific

anti-idiotypic antibodies. J Clin Invest. 1994; 94(4):1496–1505.

24. Batlle J, Gomez E, Rendal E et al. Antibodies to factor VIII in plasma of patients with

hemophilia A and normal subjects. Ann Hematol. 1996;72(5):321–326.

25. Dazzi F, Tison T, Vianello F et al. High incidence of anti-FVIII antibodies against

non-coagulant epitopes in hemophilia A patients: a possible role for the half-life of

transfused FVIII. Br J Haematol. 1996;93(3):688–693.




19

26. Scandella D, Mondorf W, Klinge J. The natural history of the immune response to

exogenous factor VIII in severe hemophilia A. Haemophilia. 1998;4(4):546–551.

27. Kempton CL, Meeks SL, Donald Harvey R, Abshire TC. Evaluation of factor VIII

pharmacokinetics and anti-factor VIII anti-bodies in four boys with hemophilia A and a

poor clinical response to factor VIII. Haemophilia. 2011;17(1):155–156.

28. Martin PG, Sukhu K, Chambers E, Giangrande PL. Evaluation of a novel ELISA

screening test for detection of factor VIII inhibitory antibodies in hemophiliacs. Clin

Lab Haematol. 1999;21(2):125-128.

29. Vianello F, Radossi P, Tison T, Dazzi F, Tagariello G, Davoli PG, Girolami A.

Prevalence of anti-FVIII antibodies in severe haemophilia A patients with inversion of

intron 22. Br J Haematol. 1997;97(4):807-809.

30. Ling M, Duncan EM, Rodgers SE, Street AM, Lloyd JV. Low detection rate of

antibodies to non-functional epitopes on factor VIII in patients with hemophilia A and

negative for inhibitors by Bethesda assay. J Thromb Haemost. 2003;1(12):2548-2553.

31. Hofbauer CJ, Kepa S, Schemper M, Quehenberger P, Reitter-Pfoertner S, Mannhalter C,

Reipert BM, Pabinger I. FVIII-binding IgG modulates FVIII half-life in patients with

severe and moderate hemophilia A without inhibitors. Blood. 2016;128(2):293-296

32. Lacroix-Desmazes S, Misra N, Bayry J, Mohanty D, Kaveri SV, Kazatchkine MD.

Autoantibodies to factor VIII. Autoimmun Rev. 2002;1(1-2):105-110.

33. Hofbauer CJ, Whelan SF, Hirschler M, Allacher P, Horling FM, Lawo JP, Oldenburg J,

Tiede A, Male C, Windyga J, Greinacher A, Knöbl PN, Schrenk G, Koehn J,

Scheiflinger F, Reipert BM. Affinity of FVIII-specific antibodies reveals major




20

differences between neutralizing and nonneutralizing antibodies in humans. Blood.

2015;125(7):1180-1188.

34. Moreau A, Lacroix-Desmazes S, Stieltjes N, Saenko E, Kaveri SV, D'Oiron R, Sultan

Y, Scandella D, Kazatchkine MD. Antibodies to the FVIII light chain that neutralize

FVIII procoagulant activity are present in plasma of nonresponder patients with severe

hemophilia A and in normal polyclonal human IgG. Blood. 2000;95(11):3435-3441.

35. Peyvandi F, Mannucci PM, Garagiola I, El-Beshlawy A, Elalfy M, Ramanan V, Eshghi

P, Hanagavadi S, Varadarajan R, Karimi M, Manglani MV, Ross C, Young G, Seth T,

Apte S, Nayak DM, Santagostino E, Mancuso ME, Sandoval Gonzalez AC, Mahlangu

JN, Bonanad Boix S, Cerqueira M, Ewing NP, Male C, Owaidah T, Soto Arellano V,

Kobrinsky NL, Majumdar S, Perez Garrido R, Sachdeva A, Simpson M, Thomas M,

Zanon E, Antmen B, Kavakli K, Manco-Johnson MJ, Martinez M, Marzouka E,

Mazzucconi MG, Neme D, Palomo Bravo A, Paredes Aguilera R, Prezotti A, Schmitt

K, Wicklund BM, Zulfikar B, Rosendaal FR. A Randomized Trial of Factor VIII and

Neutralizing Antibodies in Hemophilia A. N Engl J Med. 2016;374(21):2054-2064.

36. Verbruggen B, van Heerde W, Novakova I, Lillicrap D, Giles A. A 4% solution of

bovine serum albumin may be used in pace of factor VIII:C deficient plasma in the

control sample in the Nijmegen modification of the Bethesda factor VIII:C inhibitor

assay. Thromb Haemost. 2002;88(2):362-364.

37. Goenka A, Kollmann TR. Development of immunity in early life. J Infect. 2015;7

(Suppl 1): S112-120.




21

38. Lacroix-Desmazes S, Kaveri SV, Mouthon L, Ayouba A, Malanchère E, Coutinho A,

Kazatchkine MD. Self-reactive antibodies (natural autoantibodies) in healthy

individuals. J Immunol Methods. 1998;216(1-2):117-137.

39. Santagostino E, Mancuso ME, Tripodi A, Chantarangkul V, Clerici M, Garagiola I,

Mannucci PM. Severe hemophilia with mild bleeding phenotype: molecular

characterization and global coagulation profile. J Thromb Haemost. 2010;8(4):737-743.

40. Lo YM, Lo ES, Watson N, Noakes L, Sargent IL, Thilaganathan B, Wainscoat JS.

Two-way cell traffic between mother and fetus: biologic and clinical implications.

Blood. 1996;88(11):4390-4395.

41. Larocca D, Peterson JA, Urrea R, Kuniyoshi J, Bistrain AM, Ceriani RL. A Mr 46,000

human milk fat globule protein that is highly expressed in human breast tumours

contains factor VIII-like domains. Cancer Res. 1991;51(18):4994-4998.

42. Gunti S, Notkins AL. Polyreactive Antibodies: Function and Quantification. J Infect

Dis. 2015;212 (Suppl 1):S42-46.

43. Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, Harley

JB. Development of autoantibodies before the clinical onset of systemic lupus

erythematosus. N Engl J Med. 2003;349(16):1526-1533.

44. van der Woude D, Rantapää-Dahlqvist S, Ioan-Facsinay A, Onnekink C, Schwarte CM,

Verpoort KN, Drijfhout JW, Huizinga TW, Toes RE, Pruijn GJ. Epitope spreading of

the anti-citrullinated protein antibody response occurs before disease onset and is

associated with the disease course of early arthritis. Ann Rheum Dis. 2010; 69(8):1554-

1561.




22

45. Recke A, Rose C, Schmidt E, Bröcker EB, Zillikens D, Sitaru C. Transition from

pemphigus foliaceus to bullous pemphigoid: intermolecular B-cell epitope spreading

without IgG subclass shifting. J Am Acad Dermatol. 2009;61(2):333-336.

46. Leslie D, Lipsky P, Notkins AL. Autoantibodies as predictors of disease. J Clin Invest.

2001;108(10):1417-1422.

47. Goudemand J, Rothschild C, Demiguel V, Vinciguerrat C, Lambert T, Chambost H,

Borel-Derlon A, Claeyssens S, Laurian Y, Calvez T. Influence of the type of factor VIII

concentrate on the incidence of factor VIII inhibitors in previously untreated patients

with severe hemophilia A. Blood. 2006;107(1):46–51.

48. Astermark J, Berntorp E, White GC, Kroner BL. The Malmo International Brother

Study (MIBS): further support for genetic predisposition to inhibitor development in

hemophilia patients. Haemophilia. 2001;7(3):267–272.

49. Fischer K, Lassila R, Peyvandi F, Calizzani G, Gatt A, Lambert T, Windyga J, Iorio A,

Gilman E, Makris M; EUHASS participants. Inhibitor development in haemophilia

according to concentrate. Four-year results from the European Haemophilia Safety

Surveillance (EUHASS) project. Thromb Haemost. 2015;113(5):968-975.

50. Gouw SC, Van Den Berg HM, Le CS, Van Der Bom JG. Treatment characteristics and

the risk of inhibitor development: a multi-center cohort study among previously

untreated patients with severe hemophilia A. J Thromb Haemost. 2007;5(7):1383–90.

51. Sharathkumar A, Lillicrap D, Blanchette VS, Kern M,Leggo J, Stain AM, Brooker

L, Carcao MD. Intensive exposure to factor VIII is a risk factor for inhibitor

development in mild hemophilia A. J Thromb Haemost 2003;1(6):1228–36.




23

Table 1: General characteristics and prevalence of non-neutralizing antibodies (NNAs) in the study cohort. Crude and age-adjusted odds ratio for the presence of NNAs according to potential determinants.

Characteristic NNA negative

NNA positive

Crude odds ratio (CI 95%) p-value

Age-adjusted odds ratio (CI

95%) p-value

Age at screening (months) Mean 17.5 27.4Median 12 24Max 67 59Min 0 2

FVIII mutation - n (%) null-mutation 173 (93.5) 12 (6.5) 1.0 1.0 non-null mutation 33 (86.8) 5 (13.2) 2.18 (0.72-6.61) 0.167 2.29 (0.74-7.10) 0.153

FVIII antigen levels

< 1% 195 (92.9) 15 (7.1) 1.0 1.0 ≥ 1% 21(87.5) 3 (12.5) 1.86 (0.50-6.94) 0.358 1.71 (0.45-6.50) 0.434

Family history of inhibitors - n (%)

No 69 (95.8) 3 (4.2) 1.0 1.0 Yes 21 (91.3) 2 (8.7) 2.19 (0.34-14.00) 0.407 2.93 (0.41-20.95) 0.284

Previous exposure to blood components (<5) - n (%)

No 123 (90.4) 13 (9.6) 1.0 1.0 Yes 96 (95.0) 5 (5.0) 0.49 (0.17-1.43) 0.193 0.41 (0.14-1.22) 0.110




24

Table 2: Distribution of NNAs over each age category and the corresponding risk estimates for the presence of NNAs.

Age at screening (months)

Total NNA-negative patients n (%)

NNA-positive patients n (%)

Odds ratio (CI95)

p-value*

All 237 219 (92.4) 18 (7.6)

≥ 40 25 20 (80.0) 5 (20.0) 6.50 (1.43-29.52) 0.018

30 - 39 23 20 (87.0) 3 (13.0) 3.90 (0.73-20.80) 0.101

20 - 29 41 38 (92.7) 3 (7.3) 2.05 (0.40-10.65) 0.319

10-19 67 63 (94.0) 4 (6.0) 1.65 (0.36-7.65) 0.331

0 - 9 81 78 (96.3) 3 (3.7) 1.0 ref.

* p-value is the comparison of each age-category to the reference




25

Table 3. Cox regression for inhibitor development by NNA presence. Hazard ratio compares the risk of inhibitor development among those with NNA at screening versus those without. Adjustments in multivariate Cox models were each performed individually.

Hazard ratio (CI95)

Adjustment variable all inhibitor High-titer inhibitorCrude* 1.83 (0.84-3.99) 2.74 (1.23-6.12)Age at screening 1.91 (0.85-4.26) 2.75 (1.20-6.32)Mutation 2.33 (1.05-5.15) 3.53 (1.56-8.00)FVIII antigen level 1.73 (0.79-3.78) 2.95 (1.32-6.60)Countries 2.36 (1.03-5.42) 3.91 (1.62-9.44)Familiar history of inhibitor 1.96 (0.89-4.29) 3.03 (1.35-6.81)Arm of treatment 1.73 (0.79-3.78) 2.61 (1.17-5.84)Exposure to blood components 1.75 (0.80-3.86) 2.75 (1.22-6.22)

Mutation, countries 3.62 (1.49-9.42) 5.76 (2.17-15.26)*un-adjusted model




26

Figures legend

Figure 1: Kaplan-Meier survival curves for inhibitor development by NNA presence. In

figure 1A cumulative incidence for all inhibitor is plotted; in figure 1B cumulative incidence

for high-titer inhibitor is plotted.







doi:10.1182/blood-2016-06-720086Prepublished online December 29, 2016;

Rosendaal and Flora PeyvandiAntonino Cannavò, Carla Valsecchi, Isabella Garagiola, Roberta Palla, Pier Mannuccio Mannucci, Frits R. development in patients with severe hemophilia ANon-neutralizing antibodies against factor VIII and risk of inhibitor

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

digital object identifier (DOIs) and date of initial publication. indexed by PubMed from initial publication. Citations to Advance online articles must include final publication). Advance online articles are citable and establish publication priority; they areappeared in the paper journal (edited, typeset versions may be posted when available prior to Advance online articles have been peer reviewed and accepted for publication but have not yet

Copyright 2011 by The American Society of Hematology; all rights reserved.Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of


http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





Documents

Non-neutralizing antibodies against factor VIII and risk of inhibitor