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Pegademase bovine (PeG-ADA) for the treatment

of infants and children with severe combined

immunodeiciency (SCID)

Claire Booth 1,2

H Bobby Gaspar 1,2

1 Centre for Immunodeficiency,

Molecular immunology Unit,

UCL Institute of Child Health,

London, UK; 2 Dept of Clinical

Immunology, Great Ormond Street

Hospital NHS Trust, London, UK

Abstract: Adenosine deaminase deiciency (ADA) is a rare, inherited disorder of purine

metabolism characterized by immunodeiciency, failure to thrive and metabolic abnormalities.

A lack of the enzyme ADA allows accumulation of toxic metabolites causing defects of both

cell mediated and humoral immunity leading to ADA severe combined immune deiciency

(SCID), a condition that can be fatal in early infancy if left untreated. Hematopoietic stem

cell transplant is curative but is dependent on a good donor match. Other therapeutic options

include enzyme replacement therapy (ERT) with pegademase bovine (PEG-ADA) and more

recently gene therapy. PEG-ADA has been used in over 150 patients worldwide and has allowed

stabilization of patients awaiting more deinitive treatment with hematopoietic stem cell trans-

plant. It affords both metabolic detoxiication and protective immune function with patients

remaining clinically well, but immune reconstitution is often suboptimal and may not be long

lived. We discuss the pharmacokinetics, immune reconstitution, effects on systemic disease and

side effects of treatment with PEG-ADA. We also review the long-term outcome of patients

receiving ERT and discuss the role of PEG-ADA in the management of infants and children

with ADA-SCID, alongside other therapeutic options.

Keywords: adenosine deaminase deiciency, PEG-ADA, enzyme replacement therapy, severe

combined immune deiciency (SCID)

Correspondence: HB Gaspar

Molecular Immunology Unit, UCL

Institute of Child Health, 30 Guilford

Street, London WC1N 1EH

Tel + 44 207 905 2319

Fax + 44 207 905 2810

email h.gaspar@ich.ucl.ac.uk

Introduction

Adenosine deaminase (ADA) deficiency is a rare inherited disorder of purine

metabolism characterized by severe and recurrent infection, failure to thrive and meta-

bolic abnormalities. Absence of the enzyme ADA allows accumulation of toxic

metabolites resulting in a complete or partial deiciency of both cell-mediated and

humoral immunity. ADA severe combined immune deiciency (SCID) is fatal within

the irst months of life if left untreated. Hematopoietic stem cell transplant is cura-

tive but dependent on a good donor match. Other therapeutic options include enzyme

replacement therapy (ERT) with pegademase bovine (hereon referred to as PEG-ADA)

and more recently, gene therapy. PEG-ADA was irst used to treat ADA deiciency

in 1986 and to date more than 150 patients worldwide have received this treatment. It

is well tolerated and can restore immune function to protective levels but long-term

follow up suggests that immune recovery is incomplete. The precise role of PEG-

ADA alongside other treatment options is still to be determined but to date it has

allowed stabilization of patients awaiting more deinitive treatment and clinical

well-being in individuals treated for more prolonged periods.

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Biologics: Targets & Therapy 2009:3 349–358

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which permits unrestricted noncommercial use, provided the original work is properly cited.

Booth and Gaspar

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In 1972 Giblett et al 1 described the absence of the enzyme

ADA in erythrocytes in two patients with combined immune

deiciency. Subsequently, with greater awareness, more

patients with similar indings were reported and the clinical

and immunological phenotype of ADA deficiency was

established. Patients can be divided into four phenotypes;

ADA SCID, delayed onset ADA deficiency, late onset

ADA deiciency and partial ADA deiciency. The largest

number of patients (∼85% to 90%) present as ADA SCID

and represent the most severe form of immunodeiciency;

delayed onset presentation accounts for 10% to 15% of cases

and late (or adult) onset deiciency has been identiied in a

handful of individuals. Partial deiciency can be an incidental

inding in a healthy individual where immune function is

normal but ADA expression is abnormal in erythrocytes and

is again extremely rare. The incidence of ADA deiciency

is estimated to be between 1 in 200,000 and 1 in 1,000,000

births 2 and accounts for around 15% to 20% of all SCID cases,

although the true incidence of the disease is undetermined

and may be higher in speciic ethnic populations worldwide.

(dCyd kinase). Increased dAdo levels inactivate the enzyme

S-adenosyl homocysteine hydrolase (SAHH) leading to the

accumulation of S-adenosyl homocysteine (AdoHcy) which

is a potent inhibitor of transmethylation reactions. SAHH

activity in patients with ADA deiciency is usually 5% of

normal levels. 2

Elevated dATP levels have several toxic effects which

may all play a role in lymphotoxicity and dATP levels have

been shown to correlate with clinical severity. It has been

shown in both ADA-deicient patients and murine models that

dATP pool expansion leads to inhibition of ribonucelotide

reductase required for DNA replication and repair 5 and in

induction of apoptosis in immature thymocytes. 2 Because

cells with a high turnover produce more dATP, apoptosis,

which is the fate of most lymphocytes entering the thymus,

also increases the dATP pool within the thymus. Limited

antigen receptor diversity has been demonstrated through

interference of dATP with terminal deoxynycleotidyl trans-

ferase (TdT) 6 and defective signaling pathways may also

impair lymphocyte function. 2 Apoptosis in thymocytes is

also induced by accumulated extracellular adenosine acting

through A(2A) adenosine receptors.

Metabolic abnormalities

ADA is a predominantly cytoplasmic enzyme found in all

tissues of the body where it plays an important role in the

recycling of adenosine after DNA breakdown and speciically

catalyzes the deamination of deoxyadenosine (dAdo) and

adenosine (Ado) to deoxyinosine and inosine respectively. 3

An extracellular form of the enzyme (ecto-ADA) is found

on the surface of many cell types where it binds the cell

surface protein CD26 and speciic adenosine receptors.

Although ubiquitously expressed in keeping with its role as

a ‘housekeeping’ enzyme, ADA expression levels are highest

in the thymus and in lymphocytes where levels are 800- to

1000-fold higher than in erythrocytes. 4 High levels are also

found in the foregut and brain. The lack of ADA results in a

number of metabolic derangements which ultimately lead to

the accumulation of toxic substrates in both intra- and extra-

cellular compartments. The high levels of ADA activity in the

thymus may be a direct consequence of the high level of cell

turnover during thymic lymphocyte selection and the require-

ment for rapid and extensive DNA synthesis. Despite its

importance for many organ systems, it is clear that the lack

of ADA has its most profound manifestations in the immune

system and in lymphocyte development and function.

Lack of ADA activity leads to the accumulation of Ado

and dAdo. Cells compensate for rising levels of dAdo by

converting intracellular dAdo to deoxyadenosine triphos-

phate (dATP) through the action of deoxycytidine kinase

Presentation

Children with ADA SCID present within the irst months

of life with failure to thrive, life threatening infections and

profound lymphopenia. Interstitial pneumonitis is a common

feature and may be caused by a viral pathogen or commonly

Pneumocystis jiroveci but may also be an intrinsic manifes-

tation of ADA deiciency. Pulmonary alveolar proteinosis

(PAP) has been identiied radiologically and histologically

in ADA-deicient patients and is reversible upon treatment

(Grunebaum et al, Abstract at ESID 2008). Results from

broncheoalveolar lavage (BAL) undertaken in ADA SCID

patients with clinical and radiological manifestations of

pneumonitis revealed a high percentage of children in whom

no pathogen was identiied (Gaspar, unpublished data) again

suggesting that pulmonary damage in ADA-deicient patients

is an intrinsic consequence of the disease. Infants may also

suffer from opportunistic infections in other organ systems,

diarrhoea and candidiasis. Clinical examination reveals physi-

cal signs usually related to infection and poor growth and the

absence of lymph nodes and tonsillar tissue due to the lack of

secondary lymphoid organ development. The thymic shadow

may be absent or reduced on chest radiography and blood tests

reveal a profound lymphopenia with greatly reduced numbers

of T, B and NK cells leading to T-B-NK- SCID phenotype.

Functional lymphocyte studies unsurprisingly show absent

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Biologics: Targets & Therapy 2009:3

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PEG-ADA for infants with SCID

proliferation in response to mitogen stimulation and lack of

immunoglobulin production.

Immunodeficiency is the most profound, and in the

majority of cases irst manifestation of ADA deiciency but the

ubiquitous expression of ADA results in abnormalities in other

organ systems. Since untreated children invariably have a short

life expectancy, non-immunological deicits have usually been

recognized once the immunodeiciency has been corrected by

bone marrow transplantation. Abnormal neurological ind-

ings, without evidence of central nervous system infection,

such as developmental delay, abnormal muscle tone and

sensorineural deafness are well described. High levels of ADA

expression have been reported in brain tissue 7 and it is possible

that high levels of dATP are neurotoxic. Cognitive function

is adversely affected by the severity of metabolic derange-

ment at diagnosis and a signiicant inverse correlation has

been shown between dATP levels at diagnosis and IQ score. 8

Signiicant behavioral abnormalities are also seen in these

patients which is not related to the process of transplantation

as demonstrated by a case-matched study. 8 Post mortem

indings in eight children with ADA deiciency revealed

non-lymphoid multisystem pathologic changes, mainly renal

and skeletal. 9 Autoimmune phenomena such as idiopathic

thrombocytopenia and hemolytic anemia are well described. 10

Other rare presenting features include insulin dependent

diabetes mellitus with atopic dermatitis 11 and hepatitis. 12

Delayed onset ADA deiciency is the term applied to

patients who present after the irst year of life and is most

likely related to the speciic ADA mutation which in such

cases results in some residual ADA activity and less severe

dATP expansion and consequently less profound lympho-

penia and immune function. Such patients present with

recurrent infection and lymphopenia but rarely infection with

opportunistic pathogens. Immune dysregulation, autoimmune

and allergic phenomena and cytopenias may also be a feature

of this phenotype. A history of recurrent otitis media, upper

respiratory tract infection or pneumonia is more commonly

seen and often these children do not require hospitalization

initially. Misdiagnosis with other conditions such as allergy

(due to raised eosinophil counts) or antibody deiciency is

not uncommon.

Early diagnosis is essential to prevent the complications

of chronic infection and allow normal growth. Lymphocyte

subsets and functional studies, proliferation studies and

immunoglobulin levels are important but diagnosis rests on

the demonstration of absent or very low plasma or erythro-

cyte ADA activity levels. Raised dATP levels and reduced

SAHH activity in erythrocytes also conirm a diagnosis.

Genetic testing using mutation analysis is available and

over 70 different mutations (mostly missense mutations)

have been identiied 13 involving the ADA gene on chromo-

some 20q13.11. 14 Prenatal diagnosis may be performed by

either mutation analysis or by measurement of enzymatic

activity in trophoblasts from chorionic villus sampling or

amniocentesis. 15

Management options

Currently there are three management options to treat ADA

deiciency: hematopoietic stem cell transplant (HSCT),

ERT and gene therapy. HSCT is a highly successful and

curative procedure if a matched related donor is available

with survival rates of ∼90% but this rate falls to nearer 50%

survival if a matched unrelated donor or haploidentical

transplant is undertaken. 16 Matched family donor transplants

can be successfully undertaken without the need for prior

cytoreductive chemotherapy but transplants from other

donor sources require the use of a chemotherapy regimen.

Immune reconstitution post transplant confers adequate

immunity in terms of lymphocyte numbers and function and

antibody production. Metabolic correction is also seen and

although dATP levels are reduced they remain higher than

in a normal individual (and indeed higher than in patients

on ERT). HSCT is the treatment option of choice if a well

matched family donor can be found. However, HSCT does

not correct the non-immunological manifestations of ADA

deiciency which again suggests that ADA deiciency is a

systemic disease.

More recently somatic gene therapy for the correction

of ADA deiciency has evolved, offering patients another

treatment option. The procedure involves gammaretroviral

mediated introduction of the human ADA gene into autolo-

gous bone marrow progenitors and subsequent infusion of

cells back to the patient following mild nonmyeloablative

chemotherapy. Preliminary results from clinical trials are

encouraging with the majority of patients experiencing

immune reconstitution and metabolic detoxiication with

stable engraftment of transduced hematopoietic cells. 17,18

Importantly there has been little toxicity from the procedure

and to date all patients have survived. Immune reconstitu-

tion appears to be similar to that seen post transplant. Over

25 patients have now been treated at four centers worldwide,

with the most extensive experience in Milan and London.

The protocols differ between the different sites with respect

to the speciic retroviral constructs used for gene transfer

and the conditioning protocols used. Although the majority

of patients have shown improved immune recovery after

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351

Booth and Gaspar

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gene therapy, the level of reconstitution appears variable and

seems to be determined by a number of factors including,

the number of gene corrected progenitor cells re-infused

into the patients, the ability to harvest suficient stem cells

and the degree of thymic reserve at the time of gene therapy.

The kinetics of immune recovery are also delayed when com-

pared to the T cell reconstitution following gene therapy for

the X-linked form of SCID. However a number of patients

show long-term (5 years) complete T cell and humoral

recovery and have been able to discontinue all prophylactic

medication and immunoglobulin replacement therapy. These

pioneering studies suggest that gene therapy can at least in

principle provide effective immune and metabolic recovery.

The success of gene therapy is tempered by the potential for

insertional mutagenesis and leukemic transformation as a

result of using gammaretroviral vectors. 19,20 These compli-

cations have been seen in trials of gene therapy for other

immunodeiciencies but not so far in ADA gene therapy

studies. Gene therapy remains an option if well matched

HSCT donor is not available and is only possible through

recruitment at specialized academic centers.

HSCT but this treatment proved metabolically and clinically

effective in this case.

Later studies did not show such a positive outcome with

regular red blood cell transfusions and in fact, demonstrated

insufficient metabolic correction to allow for immune

recovery, although clinical status did often improve. 25,27 After

the introduction of PEG-ADA a comparative study proved

PEG-ADA to be a superior form of enzyme replacement and

red cell transfusion was rendered obsolete. 27

Enzyme replacement therapy

ERT with PEG-ADA (Adagen ® ; Enzon Inc; obtained outside

USA through Orphan Europe) for the treatment of ADA

deiciency has been available for almost 20 years and has been

designated an orphan drug. The use of PEG-ADA provides

another treatment modality for ADA SCID, however unlike

HSCT or gene therapy it is not a curative therapy but requires

regular intramuscular administration. Nevertheless, the

effectiveness of PEG-ADA in correcting metabolic and

immunological parameters and more importantly in promoting

clinical well-being in patients, makes it an important option

in the care of patients. The question remains as to the timing

and speciic role of the three different treatment modalities

in the treatment of ADA SCID patients and is the subject of

more detailed discussion below.

Red cell transfusions

Originally enzyme replacement was delivered through eryth-

rocyte transfusions after work by Polmar et al demonstrated

addition, in vitro , of calf-intestinal ADA or human erythro-

cyte ADA to ADA deicient patients lymphocytes restored

proliferation after mitogen stimulation. 21 Several patients

were treated with regular transfusions of frozen, irradiated red

blood cells every 2 to 4 weeks. Initial reports were promis-

ing indicating an improvement not only in clinical status but

also in immunological function. Children remained free of

infection for up to 2 years and showed an improvement in

lymphocyte numbers and function. 22,23 Metabolic derange-

ments were also seen to normalize with an increase in ADA

activity, reduction in adenosine and deoxyadenosine levels

and a decrease in erythrocyte dATP. 23–25 However, these

improvements proved to be transient and dependent on

regular transfusions. 25 Long-term transfusion therapy car-

ries with it certain risks including iron overload, exposure

to potential blood borne viral pathogens and the trauma of

repeated cannulation and time in hospital. These are signii-

cant risks in immunodeicient children who already spend

much time in a hospital environment. Bax et al 26 reported a

patient diagnosed in her mid-thirties with ADA deiciency

who was treated for 9 years with autologous erythrocyte

encapsulated ADA infusions (after one year of combined

PEG-ADA injections). The patient was not a candidate for

PEGylation

PEGylation, a process pioneered in the 1970s, involves the

covalent attachment of numerous strands of monomethoxy-

polyethylene glycol (PEG) to a molecule, for example an

enzyme or protein. PEG itself is neither toxic nor immunogenic.

The attachment of PEG to ADA through lysine residues confers

several therapeutically beneicial properties to ADA through

alteration of its physical and chemical properties, mainly due

to an increase in molecular weight. 28,29 The circulating life of

the compound is prolonged from minutes to days as clearance

from the circulation is inhibited. Molecules are protected some-

what from cellular uptake, proteolytic attack, renal clearance,

antibody binding and antigen presentation. PEGylation also

reduces the immunogenicity of a protein which again helps

to extend its circulating life. 28,29 In theory, if a drug or protein

remains in the circulation for a longer period then the frequency

of administration could be reduced thus reducing cost and the

number of injections a patient requires. In 1986, Hershield

et al irst used ADA derived from bovine intestine conjugated

to PEG to treat patients with ADA deiciency. 30

Other PEGylated therapies currently used in clinical

practice include PEGASYS ® (Hoffman-La Roche)

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Biologics: Targets & Therapy 2009:3

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PEG-ADA for infants with SCID

and PEG-Intron ® (Schering-Plough/Enzon) which are

PEGylated alpha-interferons used to treat chronic hepatitis

B and C and PEGylated L-asparaginase (Oncaspar ® ; Enzon)

used for the treatment of acute lymphoblastic leukemia.

The emergence of new variant Creutzfeldt–Jakob disease

(CJD) has led to concerns over the use of bovine ADA as

the source of enzyme for PEG-ADA. Bovine ADA is now

extracted from bovine herds in countries where nvCJD has

not been identiied. There has also been much speculation

over whether a human recombinant form of ADA may be

developed.

It is recommended that trough plasma ADA activity levels

are monitored every 1 to 2 weeks during the irst 2 to 3 months

of treatment, twice a month until 9 months of treatment and

then monthly until 18 to 24 months on PEG-ADA. Once

patients are established on an effective maintenance dose then

plasma ADA levels can be measured every 2 to 4 months

unless there is a change in clinical status.

Immune reconstitution

Immune reconstitution following treatment with PEG-ADA

has to date not been followed in a prospective manner and

therefore formal data are not available. The reasons for this

are varied but most probably due to the rarity of the condi-

tion and the fact that any one center treats only a handful of

patients. The data cited below are therefore taken from ret-

rospective and single-center studies which stretch back over

the last 20 years. Overall, it is clear that immune recovery

is very variable, the reasons for which may be associated

with the underlying clinical condition of the child, the age

at which treatment is started and also the level of residual

thymic activity at the time of PEG-ADA initiation. Up to

20% of patients receiving therapy show no response. 2,33 In

the majority of cases, full immune recovery is seen in the

short term but is followed by waning T cell numbers. 10 In

terms of humoral immunity, continued immunoglobulin

replacement is required in up to 50% of those treated with

long-term PEG-ADA. 16 It is also important to document

that, from the retrospective reports available and from com-

munication with physicians treating patients, despite low

T cell reconstitution and lack of humoral recovery, clinical

well being is maintained and children who have been treated

for many years remain clinically well, free of infection, with

normal growth parameters.

Immune recovery is evidenced initially by a rise in B cell

numbers within a few weeks of starting treatment and is then

followed by a rise in T cell count which may take a number

of months to occur. Symptoms of immune dysregulation such

as hemolytic anemia and immune thrombocytopenia can be

seen during this period and may be related to dysregulated

cellular and humoral recovery. Immature T lymphocytes

appear irst and account for the rise in T cell numbers. Prolif-

erative responses can be detected once mature T lymphocytes

develop demonstrating that ADA-deicient thymic progeni-

tors are able to mature into functional T lymphocytes in the

presence of metabolic correction. 31 Initially these responses

are IL-2 dependent and later become IL-2 independent. 31

Long-term outcome data suggest that mitogen responses

often luctuate within the same patient over time 32 with

Kinetics and dosage

PEG-ADA is administered by intramuscular injection once

or twice weekly. Plasma levels of ADA activity peak 24 to

48 hours after injection and the half life varies from 3 to

more than 6 days. The elimination and pharmacokinetics of

PEG-ADA are variable within the same patient and monitoring

of plasma ADA activity helps to guide dosage and frequency of

administration. Reports also suggest that clearance is enhanced

in younger infants and more severely ill children. 2,31 Cellular

uptake of PEG-ADA is not signiicant but maintaining plasma

ADA levels 100-fold normal levels leads to a reduction in

extracellular Ado and dAdo levels and subsequent normaliza-

tion of intracellular levels 16 through maintenance of equilib-

rium between intra- and extracellular compartments.

Since the irst patients were treated, the dosing regime has

evolved and it is now advised that children should start at a

dose of 60 U/kg/week with biweekly injections until meta-

bolic correction is established (between 1 and 3 months). Once

patients show clinical improvement and biochemical stabi-

lization they can be maintained on a dose of 30 U/kg/week

in a single weekly injection, 32,33 unpub. It is important when

monitoring patients to assess their immune function as well

as metabolic parameters and this should be taken into account

when considering altering the dose of PEG-ADA.

Dosage should be adjusted according to trough levels

of plasma ADA activity. Erythrocyte dATP levels can also

be measured and used to guide treatment. Initial trough

plasma ADA levels (prior to injection) should be maintained

at 50–150 µmol/h/mL (normal range 0.4 µmol/h/mL)

which equates to approximately 4 to 10 times the normal

erythrocyte ADA activity 2 and is required for intial rapid

detoxiication. Once a maintenance dose of 30 U/kg/week

is established, trough plasma ADA levels can be maintained

at 25 to 60 µmol/h/mL. Erythrocyte dATP levels decrease

signiicantly and are maintained at levels below that seen after

HSCT. In addition, SAHH activity is seen to normalize.

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