Pompe disease is a lysosomal storage disorder caused by a deficiency in the enzyme acid α-glucosidase (GAA). The consequence of GAA deficiency is muscle inflammation, disruption of muscle tissue, and impaired function of heart and skeletal muscle. Although the advent of enzyme replacement therapy (ERT) for Pompe disease has had a dramatic impact on the life expectancy of babies who are born with this disorder, treatment advances are still needed. Current therapy for Pompe disease is based on early detection of the genetic defect and infusions of the recombinant human enzyme acid α-glucosidase (rhGAA) to prevent glycogen accumulation. Pompe-affected children who do not express endogenous GAA (cross-reactive immunologic material; CRIM) and undergo myozyme treatment develop high-titer anti-drug-antibodies (ADA) because they are not immunologically tolerant to GAA. ADA decrease GAA enzyme uptake by muscle and/or inhibit its activity. High ADA titers correlate with poor outcomes, and even though ERT has prolonged the life of Pompe disease babies, most CRIM-negative Pompe infants who have complete GAA deficiency will eventually succumb to the disease if they are not treated with tolerance-inducing drugs.
The development of experimental immune tolerance regimens to inhibit ADA against life-saving enzyme replacement therapy is an active area of investigation. Current approaches for mitigating GAA-ADA are based on treatment with methotrexate (MTX) to inhibit the proliferation of lymphocytes and Rituximab (Rituxan) to deplete antibody (Ab)-producing B cells.1-3 These approaches, however, share significant limitations. Namely, these treatments are not effective in eliminating long-lived plasma cells, thus the timing of intervention in patients experiencing an ADA response is critical. Additional pharmacological agents that suppress antibody production by long-lived plasma cells might be of use, such as a drug currently in use for plasma cell leukemia and multiple myeloma, Bortezomib (Velcade).4 However, the long-term effects of Rituximab and Bortezomib, that both suppress the immune system systemically, are as yet unknown. Finally, the association between the use of Rituximab and development of certain infections has been reported.5
In addition to these strategies aimed at inhibiting proliferation or eliminating lymphocyte subsets participating in the ADA response are those aimed at modulating the immune system to become tolerant to the therapeutic protein. IVIG has been shown to be associated with modulation of the regulatory T cell axis, including induction of nTregs;6 reduction of IL-17,7 and by enhancing the suppressive function of Tregs.8 It has thus been applied with much success in a number of autoimmune diseases. A recent report describes the clinical outcomes in two Pompe patients who had received prolonged Rituximab therapy for ADA who were also placed on chronic IVIG in an effort to decrease infectious complications. The addition of IVIG may have provided an additional immunomodulatory benefit in promoting tolerance to the GAA therapy.9
Therapies that safely and permanently harness the immune system to induce long-lasting and specific tolerance in Pompe disease children will address a critical unmet medical with broad-reaching implications for other replacement-protein therapies that are also limited by ADA (the Lysosomal Storage Disorders, Hemophilia A and B, etc.).10-12
Autoreactive T cells with moderate T cell receptor (TCR) affinity are known to escape deletion in the thymus to circulate in the periphery where they function as ‘natural’ regulatory T cells (nTreg) by suppressing immunity against self-antigens.13 Induced Tregs (iTregs), also known as adaptive Tregs, are generated from circulating T effector cells; these cells perform similar functions but have more plasticity. It has become increasingly clear that both nTregs and iTregs contribute to immune regulation in the periphery and that their presence, or absence, contributes to the induction of tolerance and the development of autoimmunity and inflammation, respectively.
One of the most fundamental questions about nTreg cells has been their antigen-specificity. We surmised that autologous proteins, such as immunoglobulin G (lgG), contain nTreg epitopes. The presence of nTreg epitopes in lgG might explain why immunoglobulins, which undergo somatic hypermutation in the periphery, do not elicit the expected immune response against the new ‘foreign’ hypervariable sequences. After discovering highly promiscuous MHC class II epitopes in the constant region of IgG, we proposed that these epitopes were nTreg epitopes (Tregitopes)14 that provide inherent inhibitory signals to counter-balance any stimulatory signals that might result from neo-epitopes expressed in Ab hypervariable region.15 Two independent publications support our hypothesis: Ephrem and colleagues showed that intravenous immunoglobulin G (IVIg) induces nTreg,6 and Anthony and Ravetch demonstrated the a linkage between immunoglobulin binding to surface receptors that are associated with antigen-processing pathways, and Treg induction by IVIg.16,17 We describe here our acquired knowledge on the ability of Tregitope peptides to reduce T cell and T cell-dependent antibody responses, induce regulatory T cells, and lessen disease scores in animal models of inflammatory disease.18
Tregitopes have the following four characteristics: 1) Their sequences are highly conserved in IgG sequences; 2) They bind to MHC class II promiscuously; 3) In response to Tregitopes, T cells that exhibit a T regulatory phenotype (CD4+CD25+ FoxP3+) expand in vitro and in vivo; and 4) Co-incubation of Tregitopes with target autoantigens such as pre-proinsulin inhibits effector T cell (Teff) proliferation in vitro and suppresses the secretion of effector cytokines (De Groot et al., unpublished and 18).
We have more recently demonstrated that APCs present Tregitopes to Treg, engage feedback mechanisms promoting a tolerogenic APC phenotype, induce Treg expansion, and modulate antigen-specific effector T cell responses (De Groot De Groot et al., unpublished). Proportions of APC expressing MHC II, CD80, and CD86 are suppressed, consistent with reported effects of IVIg19 and of the IgG-derived peptide hCDR1.20 Moreover, we have observed significant increases in proportions of IL-10-producing CD4+CD25+ FoxP3-expressing Treg in the presence of Tregitopes. The basic mechanism of Tregitope tolerance induction is currently proposed to be as follows: 1) APC present Tregitopes to nTreg, 2) nTreg are activated to proliferate and produce IL-10, 3) nTreg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and 4) nTreg and tolerogenic APC together suppress antigen-specific T cell responses (De Groot et al., unpublished).
Modulation of T cell responses with Tregitopes may contribute to the design of new approaches for the treatment of autoimmune and inflammatory diseases via expansion of Tregs in vivo or ex vivo. Experience with IVIg gives an example of the therapeutic potential of this approach, and evidence is accumulating that Tregitopes provide beneficial immunomodulatory effects that in many respects parallel IVIg. Within our collaborative Tregitope network, we have performed a number of studies to probe potential therapeutic applications of Tregitopes in mouse models of MS (EAE), cardiac transplant, diabetes (NOD), antigen-induced airway hyper-responsiveness, and modulation of viral vector immunogenicity in adeno-associated virus (AAV)-mediated gene transfer.21 Together, the results obtained in these models show that Tregitopes co-administered with proteins suppress antigen-specific T cell and antibody responses, and induce expansion of functional Tregs. Side-by-side in vivo comparisons of Tregitope with IVIg have been performed in the autoimmune EAE model (Khoury and Elyaman, personal communication) and antigen-induced allergic airway disease (Mazer and Massoud, personal communication), demonstrating that IVIg effects can be replicated by Tregitope administration. Adaptation or incorporation of Tregitopes in drug design may aid in the design of improved tolerance-inducing therapies, and safer, more effective protein therapeutics.
Natural (n)Treg participate in central tolerance by controlling immune responses to autologous proteins, and in peripheral tolerance by stimulating induced (i)Treg cells.22 Induction of iTreg is associated with sustained tolerance to transplants, allergens and autologous proteins.13,23,24 Tolerance can be induced with non-depleting anti-CD4 antibodies25,26 and with AAV vector-mediated expression of antigens in the liver,27 including rhGAA.28-30 IVIg, a source of Tregitopes, induces tolerance in a number of different settings such as solid organ transplant,31 eradication of FVIII and FIX inhibitory antibodies in hemophilia patients,32-34 and autoimmune diseases such as systemic lupus erythematosus (SLE35), idiopathic thrombocytopenic purpura (ITP) and chronic inflammatory demyelinating polyneuropathy (CIDP).36-41 Of relevance to the goal of inducing tolerance to rhGAA, IVIg has been used to reduce ADA in Pompe patients undergoing Myozyme treatment.9 See Table 1 for a list of experiments that have been performed with Tregitope, the human disease parallel, and a comment on current therapy for that condition (highlighting the potential role of Tregitope in the future therapy of the condition).
Table 1. Overview of Tregitope in vivo experiments and results
||Human Disease Parallel
||Current Clinical Therapy
||In vivo controls
OVA-specific tolerance in C57BL/6 mice
||Suppression of T cell proliferation; Suppression of OVA-antibody titer
||Enzyme Replacement Therapy
||Immune Tolerance Induction
IVIG CTR similar to Tregitope
|L. Cousens, A. De Groot (EpiVax)
OVA-specific tolerance in DO11.10 TCR Transgenics
||Suppression of T cell proliferation; induction of antigen-specific Tregs
||Enzyme Replacement Therapy
||Immune Tolerance Induction
EAE prevention with Tregitope
|Reduction of EAE symptoms; induction of Tregs
||Copaxone; Interferon β
|OVA peptide (Negative CTR)
IVIG CTR similar to Tregitope
|S.Khoury; W. Elyaman (Brigham)
OVA Allergy Model; Therapy with Tregitope vs. IVIG
||Reduction of allergic response to OVA and airway reactivity, increase in Treg Induction
||Anti-histamines, Immune Tolerance Induction
||Albumin (Negative CTR)
IVIG CTR similar to Tregitope
A. Massoud (McGill)
Tregitope in AAV; TNBS-induced model of inflammatory colitis
||Reduction of clinical and histological severity, increased Treg infiltration in the colon
||Azathiaprine, Anti-TNF Mabs, Steroids
||Saline; No effect
||V. Ferreira, S. van der Marel (UniQure)
Tregitope in AAV for Gene Transfer
||Suppressed CD8+-T cell response to AAV epitope in gene transfer model
||Gene Transfer Vectors currently induce CTL response
||Scrambled Tregitope peptide (Negative CTR)
|F. Mingozzi (CHOP)
Tregitope in T1D (NOD model)
with insulin peptides
|Suppressed development of diabetes; effect enhanced when Tregitope co-delivered with insulin
||Type I Diabetes
||PPI peptides, Tet Tox peptide (Negative CTR) No effect
A. De Groot (EpiVax)
Rohrbach M, Klein A, Köhli-Wiesner A, Veraguth D, Scheer I, Balmer C, et al.
CRIM-negative infantile Pompe disease: 42-month treatment outcome
J Inherit Metab Dis 2010;
33:751-7; PMID: 20882352
; DOI: 10.1007/s10545-010-9209-0
Joseph A, Munroe K, Housman M, Garman R, Richards S.
Immune tolerance induction to enzyme-replacement therapy by co-administration of short-term, low-dose methotrexate in a murine Pompe disease model
Clin Exp Immunol 2008;
152:138-46; PMID: 18307520
; DOI: 10.1111/j.1365-2249.2008.03602.x
Garman RD, Munroe K, Richards SM.
Methotrexate reduces antibody responses to recombinant human alpha-galactosidase A therapy in a mouse model of Fabry disease
Clin Exp Immunol 2004;
137:496-502; PMID: 15320898
; DOI: 10.1111/j.1365-2249.2004.02567.x
Moran E, Carbone F, Augusti V, Patrone F, Ballestrero A, Nencioni A.
Proteasome inhibitors as immunosuppressants: biological rationale and clinical experience
Semin Hematol 2012;
49:270-6; PMID: 22726551
; DOI: 10.1053/j.seminhematol.2012.04.004
Ephrem A, Chamat S, Miquel C, Fisson S, Mouthon L, Caligiuri G, et al.
Expansion of CD4+CD25+ regulatory T cells by intravenous immunoglobulin: a critical factor in controlling experimental autoimmune encephalomyelitis
111:715-22; PMID: 17932250
; DOI: 10.1182/blood-2007-03-079947
Maddur MS, Kaveri SV, Bayry J.
Comparison of different IVIg preparations on IL-17 production by human Th17 cells
Autoimmun Rev 2011;
10:809-10; PMID: 21376142
; DOI: 10.1016/j.autrev.2011.02.007
Kessel A, Ammuri H, Peri R, Pavlotzky ER, Blank M, Shoenfeld Y, et al.
Intravenous immunoglobulin therapy affects T regulatory cells by increasing their suppressive function
J Immunol 2007;
179:5571-5; PMID: 17911644
Messinger YH, Mendelsohn NJ, Rhead W, Dimmock D, Hershkovitz E, Champion M, et al.
Successful immune tolerance induction to enzyme replacement therapy in CRIM-negative infantile Pompe disease
Genet Med 2012;
14:135-42; PMID: 22237443
; DOI: 10.1038/gim.2011.4
Starzyk K, Richards S, Yee J, Smith SE, Kingma W.
The long-term international safety experience of imiglucerase therapy for Gaucher disease
Mol Genet Metab 2007;
90:157-63; PMID: 17079176
; DOI: 10.1016/j.ymgme.2006.09.003
Bluestone JA, Abbas AK.
Natural versus adaptive regulatory T cells
Nat Rev Immunol 2003;
3:253-7; PMID: 12658273
; DOI: 10.1038/nri1032
De Groot AS, Moise L, McMurry JA, Wambre E, Van Overtvelt L, Moingeon P, et al.
Activation of natural regulatory T cells by IgG Fc-derived peptide “Tregitopes”
112:3303-11; PMID: 18660382
; DOI: 10.1182/blood-2008-02-138073
Soukhareva N, Jiang Y, Scott DW.
Treatment of diabetes in NOD mice by gene transfer of Ig-fusion proteins into B cells: role of T regulatory cells
Cell Immunol 2006;
240:41-6; PMID: 16860296
; DOI: 10.1016/j.cellimm.2006.06.004
Anthony RM, Wermeling F, Karlsson MC, Ravetch JV.
Identification of a receptor required for the anti-inflammatory activity of IVIG
Proc Natl Acad Sci USA 2008;
105:19571-8; PMID: 19036920
; DOI: 10.1073/pnas.0810163105
Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV.
Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc
320:373-6; PMID: 18420934
; DOI: 10.1126/science.1154315
Cousens LP, Najafian N, Mingozzi F, Elyaman W, Mazer B, Moise L, et al.
In vitro and in vivo studies of IgG-derived Treg epitopes (Tregitopes): A promising new tool for tolerance induction and treatment of autoimmunity
J Clin Immunol 2012;
Bayry J, Lacroix-Desmazes S, Carbonneil C, Misra N, Donkova V, Pashov A, et al.
Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin
101:758-65; PMID: 12393386
; DOI: 10.1182/blood-2002-05-1447
Sela U, Sharabi A, Dayan M, Hershkoviz R, Mozes E.
The role of dendritic cells in the mechanism of action of a peptide that ameliorates lupus in murine models
128:e395-405; PMID: 19040426
; DOI: 10.1111/j.1365-2567.2008.02988.x
Hui DJ, Basner-Tschakarjan E, Pien GC, Martin WD, De Groot AS, High KA, et al.
Peptide-Induced Antigen-Specific CD4+CD25+FoxP3+ T Cells Suppress Cytotoxicity T Cell Responses Directed Against the AAV Capsid
Durinovic-Belló I, Rosinger S, Olson JA, Congia M, Ahmad RC, Rickert M, et al.
DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a down-regulatory T helper 2 phenotype
Proc Natl Acad Sci USA 2006;
103:11683-8; PMID: 16868084
; DOI: 10.1073/pnas.0603682103
Sumida T, Kato T, Hasunuma T, Maeda T, Nishioka K, Matsumoto I.
Regulatory T cell epitope recognized by T cells from labial salivary glands of patients with Sjögren’s syndrome
Arthritis Rheum 1997;
40:2271-3; PMID: 9416868
; DOI: 10.1002/art.1780401225
Cobbold SP, Qin SX, Waldmann H.
Reprogramming the immune system for tolerance with monoclonal antibodies
Semin Immunol 1990;
2:377-87; PMID: 2129511
Winsor-Hines D, Merrill C, O’Mahony M, Rao PE, Cobbold SP, Waldmann H, et al.
Induction of immunological tolerance/hyporesponsiveness in baboons with a nondepleting CD4 antibody
J Immunol 2004;
173:4715-23; PMID: 15383608
Mingozzi F, Liu YL, Dobrzynski E, Kaufhold A, Liu JH, Wang Y, et al.
Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer
J Clin Invest 2003;
111:1347-56; PMID: 12727926
Sun B, Bird A, Young SP, Kishnani PS, Chen YT, Koeberl DD.
Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance
Am J Hum Genet 2007;
81:1042-9; PMID: 17924344
; DOI: 10.1086/522236
Sun B, Kulis MD, Young SP, Hobeika AC, Li S, Bird A, et al.
Immunomodulatory gene therapy prevents antibody formation and lethal hypersensitivity reactions in murine pompe disease
Mol Ther 2010;
18:353-60; PMID: 19690517
; DOI: 10.1038/mt.2009.195
Ziegler RJ, Bercury SD, Fidler J, Zhao MA, Foley J, Taksir TV, et al.
Ability of adeno-associated virus serotype 8-mediated hepatic expression of acid alpha-glucosidase to correct the biochemical and motor function deficits of presymptomatic and symptomatic Pompe mice
Hum Gene Ther 2008;
19:609-21; PMID: 18500944
; DOI: 10.1089/hum.2008.010
Jordan SC, Toyoda M, Vo AA.
Regulation of immunity and inflammation by intravenous immunoglobulin: relevance to solid organ transplantation
Expert Rev Clin Immunol 2011;
7:341-8; PMID: 21595600
; DOI: 10.1586/eci.11.10
Muzaffar J, Katragadda L, Haider S, Javed A, Anaissie E, Usmani S.
Rituximab and intravenous immunoglobulin (IVIG) for the management of acquired factor VIII inhibitor in multiple myeloma: case report and review of literature
Int J Hematol 2012;
95:102-6; PMID: 22170228
; DOI: 10.1007/s12185-011-0968-7
Kubisz P, Plamenová I, Hollý P, Stasko J.
Successful immune tolerance induction with high-dose coagulation factor VIII and intravenous immunoglobulins in a patient with congenital hemophilia and high-titer inhibitor of coagulation factor VIII despite unfavorable prognosis for the therapy
Med Sci Monit 2009;
15:CS105-11; PMID: 19488019
Klarmann D, Martinez Saguer I, Funk MB, Knoefler R, von Hentig N, Heller C, et al.
Immune tolerance induction with mycophenolate-mofetil in two children with haemophilia B and inhibitor
14:44-9; PMID: 18081836
; DOI: 10.1111/j.1365-2516.2007.01584.x
Zandman-Goddard G, Blank M, Shoenfeld Y.
Intravenous immunoglobulins in systemic lupus erythematosus: from the bench to the bedside
18:884-8; PMID: 19671787
; DOI: 10.1177/0961203309106921
Zandman-Goddard G, Krauthammer A, Levy Y, Langevitz P, Shoenfeld Y. Long-Term Therapy with Intravenous Immunoglobulin is Beneficial in Patients with Autoimmune Diseases. Clin Rev Allergy Immunol 2011; PMID: 21732045 http://www.springerlink.com/content/j614131732668223/
Dykes AC, Walker ID, Lowe GD, Tait RC.
Combined prednisolone and intravenous immunoglobulin treatment for acquired factor VIII inhibitors: a 2-year review
7:160-3; PMID: 11260275
; DOI: 10.1046/j.1365-2516.2001.00489.x
Lozeron P, Adams D.
Advances in the treatment of chronic inflammatory demyelinating neuropathies in 2010
J Neurol 2011;
258:1737-41; PMID: 21713585
; DOI: 10.1007/s00415-011-6143-5
Rajabally YA, Mahdi-Rogers M.
Overview of the pathogenesis and treatment of chronic inflammatory demyelinating polyneuropathy with intravenous immunoglobulins
Elyaman W, Khoury SJ, Scott DW, De Groot AS.
Potential application of tregitopes as immunomodulating agents in multiple sclerosis
Neurol Res Int 2011;
2011:256460; PMID: 21941651
; DOI: 10.1155/2011/256460
Raben N, Jatkar T, Lee A, Lu N, Dwivedi S, Nagaraju K, et al.
Glycogen stored in skeletal but not in cardiac muscle in acid alpha-glucosidase mutant (Pompe) mice is highly resistant to transgene-encoded human enzyme
Mol Ther 2002;
6:601-8; PMID: 12409258
; DOI: 10.1016/S1525-0016(02)90716-1
Cresawn KO, Fraites TJ, Wasserfall C, Atkinson M, Lewis M, Porvasnik S, et al.
Impact of humoral immune response on distribution and efficacy of recombinant adeno-associated virus-derived acid alpha-glucosidase in a model of glycogen storage disease type II
Hum Gene Ther 2005;
16:68-80; PMID: 15703490
; DOI: 10.1089/hum.2005.16.68
Franco LM, Sun B, Yang X, Bird A, Zhang H, Schneider A, et al.
Evasion of immune responses to introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II
Mol Ther 2005;
12:876-84; PMID: 16005263
; DOI: 10.1016/j.ymthe.2005.04.024
Sabatino DE, Mingozzi F, Hui DJ, Chen H, Colosi P, Ertl HC, et al.
Identification of mouse AAV capsid-specific CD8+ T cell epitopes
Mol Ther 2005;
12:1023-33; PMID: 16263332
; DOI: 10.1016/j.ymthe.2005.09.009
Fontenot JD, Gavin MA, Rudensky AY.
Foxp3 programs the development and function of CD4+CD25+ regulatory T cells
Nat Immunol 2003;
4:330-6; PMID: 12612578
; DOI: 10.1038/ni904
Wang R, Wan Q, Kozhaya L, Fujii H, Unutmaz D.
Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression
PLoS ONE 2008;
3:e2705; PMID: 18628982
; DOI: 10.1371/journal.pone.0002705
Wang R, Kozhaya L, Mercer F, Khaitan A, Fujii H, Unutmaz D.
Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells
Proc Natl Acad Sci USA 2009;
106:13439-44; PMID: 19666573
Hori S, Nomura T, Sakaguchi S.
Control of regulatory T cell development by the transcription factor Foxp3
299:1057-61; PMID: 12522256
; DOI: 10.1126/science.1079490
Khattri R, Cox T, Yasayko SA, Ramsdell F.
An essential role for Scurfin in CD4+CD25+ T regulatory cells
Nat Immunol 2003;
4:337-42; PMID: 12612581
; DOI: 10.1038/ni909