Taming the TCR: antigen-specific immunotherapeutic agents for autoimmune diseases.

International Reviews of Immunology, Early Online:1–26, 2015 C Informa Healthcare USA, Inc. Copyright ISSN: 0883-0185 print / 1563-5244 online DOI: 10.3109/08830185.2015.1027822

REVIEW

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Taming the TCR: Antigen-Specific Immunotherapeutic Agents for Autoimmune Diseases Evan L. Sauer, Nancy C. Cloake, and Judith M. Greer UQ Centre for Clinical Research, The University of Queensland, Brisbane, Queensland, Australia

Current treatments for autoimmune diseases are typically non-specific anti-inflammatory agents that affect not only the autoreactive cells but also the parts of the immune system that are required to maintain health. There is a need for the development of antigen-specific therapeutic agents that can effectively prevent the autoimmune attack while leaving the rest of the immune system functioning as normal. The simplest way to achieve this is using the autoantigen itself as a tolerizing agent; however, there is some risk involved with administering a potentially pathogenic antigen. In this review, we focus instead on the development and use of modified T cell receptor (TCR) ligands, in which the peptide ligand is modified to change the response by the T cell from a disease inducing to a protective response, and still retain the antigen-specificity necessary to target the autoreactive T cells. We review the use of modified TCR ligands as therapeutic agents in animal models of autoimmunity and in human autoimmune disease, and finally consider how they need to be improved in order to use them effectively in patients with autoimmune disease. Keywords: antigen-specific, autoimmune disease, CD4+ T cell, MHC, T cell receptor

INTRODUCTION Autoimmunity affects up to 10% of the global population, and includes both systemic (e.g. systemic lupus erythematosus (SLE) or Sjögren’s disease) and organ-specific diseases (e.g. Graves’ disease, rheumatoid arthritis (RA), multiple sclerosis (MS)). Current treatments for autoimmune diseases are typically non-specific agents that systemically inhibit inflammatory immune activity. While they show some efficacy, they also alter the ability of the immune system to effectively respond to infectious and neoplastic insults; long-term treatment with these agents can result in severe, sometimes fatal, infections and malignancies. The effectors of autoimmunity include antibodies, B cells, CD8+ T cells and many cells of the innate immune system, but autoreactive CD4+ T cells are thought to lie at the heart of many autoimmune diseases, orchestrating immune attack on the target tissues. A major reason for believing that CD4+ T cells are critical for initiation and/or development of these autoimmune diseases, irrespective of whether the final pathogenic mechanism involves primarily antibodies, T cells, or other cells, is that Accepted 15 February 2015. Address correspondence to Judith M. Greer, UQ Centre for Clinical Research, The University of Queensland, Bldg 71/918, Royal Brisbane & Women’s Hospital, Brisbane, QLD 4029, Australia. E-mail: [email protected]

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FIGURE 1. Associations between class II HLA molecules and selected autoimmune diseases. In each box, the top line shows the disease, and the next line lists putative autoantigens. Information in this figure is compiled from [110, 122, 149, 153–160]. † Several DRB1∗ 04 alleles (04:01, 04:02 and 04:05) confer the highest risk for type 1 diabetes. ‡ Several DRB1∗ 04 alleles (04:01, 04:04 and 04:05) confer the highest risk for rheumatoid arthritis. Abbreviations: AChR: acetylcholine receptor; Arg: arginine; CNS: central nervous system; GAD: Glutamic Acid Decarboxylase; GPI: glucose6-phosphate isomerise; TPO: thyroid peroxidase; TSHR: thyroid stimulating hormone receptor.

many autoimmune diseases occur preferentially in patients carrying specific major histocompatibility complex (MHC) class II molecules (the human leukocyte antigen (HLA) molecules HLA-DR, HLA-DQ or HLA-DP) (Figure 1). Some, but not all, of the targets of those CD4+ T cells are known and, in some autoimmune diseases, the range of antigens targeted by the autoreactive response appears to be focused on a relatively small number of autoantigens, particularly in the early stages of disease. It has therefore been proposed that targeting the interaction International Reviews of Immunology


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between the CD4+ T cells and the specific autoantigen(s) could be a method by which the cells responsible for the development of disease could be removed while leaving the rest of the immune system functioning as normal. The strategy of using unaltered autoantigens to induce tolerance as a consequence of the route of delivery or dosage continues to be explored [1–3], such as in recent phase I clinical trials [4–6]. However, delivery of unaltered autoantigens is intrinsically associated with the risk of exacerbating autoimmune reactivity, and therefore a variety of approaches have been devised to modify autoantigenic peptides so that they still target the interaction between the CD4+ T cells and the specific autoantigen but in such a way that they can manipulate this interaction without activating the T cells to induce a pathogenic response. The review will commence with a brief overview of how CD4+ T cells interact with antigen. Thereafter, the types of modified T cell ligands, the means by which they modify the process of antigen presentation, the evidence for their in vivo utility in animal studies, and the barriers that have so far prevented their effective therapeutic use in humans will be considered.

ANTIGEN PROCESSING, PRESENTATION, AND IMMUNOGENICITY CD4+ T cells, after maturation, provide immune surveillance as they circulate between vascular and lymphatic compartments in an inactive state. Immune (and autoimmune) reactions are generated by CD4+ T cells when they encounter their cognate antigen, which must be presented bound to MHC class II molecules on the surface of professional antigen-presenting cells (such as dendritic cells and macrophages) that can provide appropriate co-stimulation. Thus, the range of immune reactions an individual is capable of generating is shaped by both the pool of antigens that are able to be presented and the pool of T cells in circulation. The endocytosis of cellular debris or microbial material, digestion of proteins into peptide fragments, and loading of selected peptides into available MHC class II complexes (Figure 2) shape the antigenic repertoire within the host at any point in time. Here, the interaction between the MHC class II dimer and peptide is clearly critically important: A peptide will only be antigenic in people (or animals) who express MHC class II molecules to which the peptide will bind. The process of antigen binding occurs in late endosomal compartments, and is facilitated by HLA-DM, which encourages firstly the displacement of class II-associated invariant chain peptide (CLIP), which is a placeholder remnant of the invariant chain (Ii) that chaperones newly synthesized MHC class II out of the endoplasmic reticulum, and then of peptides with low binding affinity, thereby ensuring that only peptides with sufficiently high affinity are able to be presented on the surface of antigen-presenting cells (APCs) [7, 8]. Recombination of the T cell receptor (TCR) gene in immature thymocytes and mechanisms of central tolerance, whereby T cells carrying TCRs that have high affinity for autoantigens are deleted, generates a pool of na¨ıve T cells that can have low affinity for autoantigens [9]. There are competing and complementary models describing how ligation of TCR with the peptide presented by the MHC (pMHC) might result in sustained signal transduction and T cell activation; however, the question of which model(s) faithfully describe the reality remains an open area of research. Irrespective of the model, T cell signaling is initiated rapidly, and requires interaction with very few – even perhaps a single – pMHC [9]. Sustained signaling is supported by the formation of an immunological synapse between the T cell and the APC [10, 11]. Evidence suggests that the interaction between an autoreactive T cell and its cognate autoantigen results in aberrant immunological synapses; even so, signal transduction resulting in IL-2 production and T cell activation occurs [12]. C Informa Healthcare USA, Inc. Copyright


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FIGURE 2. Antigen processing and presentation through the MHC class II pathway (see text for details).

T cell activation requires the provision of two signals: the first signal delivered via the TCR, and the second signal provided through costimulatory molecules such as CD28. Signaling through the TCR without co-stimulation induces anergy, in which the T cell becomes refractive to subsequent stimulation; this can act as a tolerogenic mechanism. The first detectable event following signal transduction by the TCR is the phosphorylation of tyrosine-based motifs (ITAMs) within the intracellular regions of the CD3 subunits, which form stable complexes with the TCR. ITAM phosphorylation allows the recruitment of ZAP-70, which is activated by phosphorylation and in turn catalyses the formation of an expanding signaling complex, leading to the recruitment of the MAPK signaling pathways (recruiting JNK, ERK and p38), recruitment of NF-κB, and, through sustained influx of extracellular Ca2+ , recruitment of the NF-AT transcription factors (Figure 3). The coordinated action of these transcription factors drives gene transcription resulting in T cell activation, proliferation and cytokine production [13, 14]. T cell activation is also influenced by the cytokine milieu, and the cytokine environment critically influences whether activated na¨ıve T cells will differentiate into Th1, Th2, Th17 or regulatory T cell (Treg) effector subtypes [15].

APPROACHES TO ANTIGEN-SPECIFIC THERAPIES Altered Peptide Ligands The amino acid sequence of the MHC class II molecule determines the shape of the peptide-binding cleft, and the particular peptides that will bind that MHC class II molecule. The interaction between TCR and peptide–MHC complex is extremely sensitive to even small changes in the MHC molecule, resulting in (with few exceptions) tight restriction of peptide binding to a particular MHC molecule [16]. In contrast, differences in the sequence of the peptide are comparatively well tolerated [17]: Amino acid residues that do not interact directly with either the MHC molecule or the TCR may often be substituted without apparent effect as long as substitutions do not dramatically alter the conformation of the peptide [18, 19]. Amino acid substitutions at positions that interact directly with the TCR are much more likely to affect the trimolecular interaction, either abrogating it, or altering the quality of TCR signal transduction. Peptides that differ at positions that interact with the TCR have been called altered peptide ligands (APLs; Figure 4) [20]. Just as individual T cell clones may respond International Reviews of Immunology



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FIGURE 3. An overview of T cell receptor (TCR) signaling pathways (see text for details).

to a range of TCR ligands, individual antigens and APLs are able to interact with a diverse set of T cell clones bearing unique TCRs and exhibiting different binding affinities. Consequently, T cell responses to antigens and APLs are typically polyclonal, and individual T cell clones selected from a polyclonal antigen-specific T cell population may each respond differently to a particular APL.

FIGURE 4. Particular amino acids within an antigen (grey circles) contribute to interactions with MHC class II and the TCR (arrows). Substitutions at positions that interact with the TCR create typical APLs, while substitutions at positions that interact with MHC class II create MVPs. C Informa Healthcare USA, Inc. Copyright


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Altering the quality of TCR ligation can affect T cell activation. Because of this, APLs are often described according to the functional outcomes that they induce in T cell clones. APLs may function as strong or weak agonists, or as partial agonists if they induce some, but not all, of the T cell effector functions that are generated by the cognate ligand. Partial agonists can induce cytokine production in the absence of proliferation [21], altered patterns of cytokine production, or induction of anergy [22]. APLs can also antagonize T cell activation [23, 24] via the generation of a dominant-negative signal which overrides T cell activation, rather than by competition for TCR binding, as is seen in “classical” antagonism [25, 26]. Anergy and antagonism are sensitive to the cytokine environment; both can be overcome by exogenous IL-2 [22, 27, 28]. The antagonistic properties of certain APLs, in particular, have been seen as a means to therapeutically suppress pathogenic T cell-mediated autoimmune responses [29]. Differential signaling though the TCR has been observed at the earliest detectable stages of signal transduction: stimulation with APLs induces a qualitatively different pattern of CD3 ITAM phosphorylation than stimulation with agonists, characterized by upregulation of partially phosphorylated TCR ζ , little induction of fully phosphorylated TCR ζ or CD3ε, and absence of ZAP-70 phosphorylation [30–34]. Expression of TCR ζ proteins containing mutations at individual tyrosine residues demonstrated that antagonistic APLs induce phosphorylation of only one of the two tyrosine residues within each of the three TCR ζ ITAMs [33], and that over-expression of this partially phosphorylated form of TCR ζ is sufficient to provide an inhibitory signal [35]. How such a signal is generated is incompletely understood, but may involve a change in the balance of the activities of competing protein kinases and phosphatases, either through failure to recruit protein kinases to the early TCR signalosome, by enhanced recruitment of phosphatases, or both. APL antagonism can occur in Th cells lacking CD4 [36, 37], and the pattern of CD3 phosphorylation induced by antagonistic APLs can be reproduced by mutating CD4 so that it cannot bind to MHC class II [31], suggesting that antagonism may involve failure to recruit CD4-associated Lck. Others have observed that blocking antibodies against CD4 interfere with both T cell agonism and antagonism [38]. Antagonistic APLs (unlike agonists) induce association of the phosphatase SHP-1 with Lck-CD4, co-localizing SHP-1 with the TCR at the interface between T cells and APCs [39]; abolition of SHP-1 activity abrogates APL antagonism [40]. The roles of other kinases and phosphatases in APL antagonism have not been investigated. A smaller proportion of total cellular CD4, CD28 and PKC θ is reported to occur in the immunological synapses formed by antagonized versus agonized T cells [41]. APL antagonism permits the formation of stable conjugates of T cells and APCs [41–43], although synapse formation is compromised (evident in low MHC density and abnormal patterns of ICAM-1 clustering), leading to the induction of intermittent Ca2+ flux [43], very limited Ca2+ signaling in T cells, and interference with the strong, stable Ca2+ signals induced by agonist ligands [44, 45]. The irregular Ca2+ signal observed during APL antagonism is relatively insensitive to the inhibition of PLC-γ 1 activation [45], and antagonistic APLs inhibit inositol phosphate hydrolysis [46], suggesting that alternative signaling pathways may be involved in triggering the release of Ca2+ from internal stores. Consistent with aberrant Ca2+ signaling is reduced, but observable, nuclear translocation of Nuclear Factor of Activated T-cells (NFAT) transcription factors [47]. The activity of other signaling molecules, such as SLP-76 and Vav, and activation of JNK-1 via Rac do not appear to be affected by APL antagonism [41]. MHC Variant Peptides (MVP) Altering an antigen by introducing amino acid substitutions at positions responsible for binding of the peptide to the MHC class II (Figure 4) can be used to manipulate International Reviews of Immunology



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the stability of the antigen:MHC class II complex and antigenicity (functional avidity). As this strategy makes no changes to the residues of the antigen that contact the TCR, these MVPs should theoretically act upon the same population of T cells as the unaltered antigen, decreasing the likelihood that an MVP might inadvertently cross react with and agonize autoreactive T cells. Even so, some consideration must be taken in their design to ensure that alterations do not force a change in the binding register of the antigen [48]. The affinity of MVP for MHC class II generally (although imperfectly) correlates with antigenicity in vitro: those with high affinity act as superagonists for antigen-specific T cells [49], while those with low affinity act as partial agonists, or have low or no stimulatory capacity [48, 50, 51]. Partial agonist MVPs can also induce T cell lytic activity against target cells in the absence of T cell proliferation [48]. It has been reported that the immunogenicity of an antigen correlates with the rate of dissociation of the antigen:MHC class II complex: amino acid substitutions that increase the dissociation rate decrease immunogenicity [52]. However, others have reported that antigens and MVPs which form unstable complexes are just as immunogenic as those that form stable complexes [53, 54] but expand a population of T cells utilizing a limited range of high-affinity TCRs [53]. There are instances in which MVPs appear to act upon different T cell populations than the unaltered antigen [52, 55], or upon different T cell populations than other MVPs based on the same antigen [51]. These results suggest that certain substitutions at MHC-binding residues may alter the interaction between the antigen:MHC class II complex and the TCR, which could occur through the repositioning of the antigen in the binding groove of the MHC molecule. For example, substitution of asparagine for glutamine at residue 73 of Hb64–76 (which occupies the P6 pocket of I-Ek ) displaced ˚ relative to the position of the adjacent TCR contact residues of the peptide by up to 2 A, native antigen in the MHC class II molecule [55]. In this case, the native antigen and the MVP shared similar affinities and dissociation rates for I-Ek , but the association rate for binding a cognate TCR was three-fold higher for the MVP: the MVP is therefore effectively acting as a classical APL, not as an MVP. Soluble MHC–Peptide Complexes Another antigen-specific strategy that has been explored in animal models of autoimmune disease is the use of soluble MHC–antigen complexes (sMAgs). These complexes, which interact with the T cell without requiring APCs, are designed to activate the TCR in the absence of co-stimulation. Early generations of sMAgs used purified MHC class II loaded non-covalently with antigen, which typically resulted in less than 50% occupancy of the MHC molecule [56, 57]. Subsequently, recombinant expression has been used to create a diverse range of sMAgs with improved stability and a range of valencies. Monovalent sMAgs have been stabilized by linking the extracellular domains of MHC class II to the leucine zipper domains of Fos and Jun [58–61] or by covalently coupling the MHC class II α 1 and β 1 domains together to create a set of sMAgs dubbed “recombinant T cell ligands” (RTLs), [62, 63]. Others have generated bivalent sMAgs by coupling the extracellular domains of MHC class II to the IgG Fc domain [59, 60, 64–66]. The valence of an sMAg appears to be a major factor in determining its effect. Occupancy has been improved by covalently linking antigen to the MHC class II β1 domain [58–60, 62, 63, 65]. The interaction between sMAgs and T cells is both antigen-specific [67, 68] and MHC-restricted [58, 69]. In vitro studies suggest that the valence of an sMAg directly affects the avidity of this interaction, and thereby its potency in generating a T cell response. Bivalent sMAgs on an Ig scaffold have off-rates that are 20–25 times lower than monovalent sMAgs that contain leucine zippers [59]. Likewise, sMAgs generated as dimers, trimers and tetramers elicit proportionally greater degrees of T cell activation, C Informa Healthcare USA, Inc. Copyright


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as measured by the downregulation of CD3 and the upregulation of activation markers CD69 and CD25 [67]. Increasing the valence and dose of sMAgs progressively shifts the functional effects of T cell activation across the spectrum from a Th2 response to a Th1 response to apoptosis (activation-induced cell death) [70]. On the other hand, monovalent sMAgs bind cognate TCR with low avidity [71, 72] and are only able to weakly induce activation processes (if at all) in vitro unless immobilized [67, 73, 74]. When immobilized, the monovalent sMAgs can induce phosphorylation of TCRζ and ZAP70 and trigger the release Ca2+ from intracellular stores, although they cannot induce influx of extracellular Ca2+ [73]. Bivalent sMAgs that have been constructed by attaching extracellular MHC class II domains to the Fc region of IgG can bind to Fc receptors on APCs, which could result in their presentation to T cells with concomitant co-stimulation [75]. However, this is not necessarily their standard mode of action, since competition for Fc receptor binding does not diminish the T cell response to sMAgs [64, 76], bivalent sMAgs lacking the Cterminus of the Fc region retain efficacy [65], and T cell proliferation can be induced by sMAgs in the absence of APCs [67, 77]. Monovalent RTLs form aggregates in solution unless modified [78], potentially inducing T cell signaling through cross-linking TCRs. However, as site-directed mutation of the external face of the RTL to prevent aggregation does not diminish their biological effects, it is not clear whether they affect T cell activation through this mechanism alone [78]. RTLs were found to bind in an antigen-independent manner to a ligand present on CD19+ B cells, CD11b+ macrophages/dendritic cells, and CD11c+ dendritic cells [79]; this ligand has been identified as the invariant chain (CD74) [80]. T cell proliferation induced by incubation with free antigen and CD11b+ macrophages was suppressed by the presence of RTL; in contrast, RTLs could not suppress T cell proliferation in response to CD19+ APCs [79]. At least in this scenario, T cell unresponsiveness is not being induced by free RTL but by the specific interaction between cognate T cells, RTL and a macrophage/dendritic cell population, and this may be a more accurate representation of the mechanism by which RTL (but not necessarily dimeric sMAgs) exert their effects in vivo. As noted above, stimulation of T cells with sMAgs can induce processes associated with T cell activation. Stimulation of cognate T cells with low doses (5 μg/mL) of the bivalent sMAg “DEF” induced the production of IL-2, and subsequent stimulation with antigen-pulsed APCs enhanced the proliferation and the production of IL-4 by DEFtreated cells [64]. Higher doses of DEF (50 μg/mL) rendered cognate T cells anergic, without evidence of apoptosis [81]. Here CD4 and Lck were displaced from lipid rafts, TCRζ was incompletely phosphorylated, and ZAP-70 was poorly recruited to the TCR in both raft and non-raft domains. Further downstream, anergic T cells showed reduced DNA binding of transcription factors, including c-Maf, NFAT and AP-1 (but not GATA3), and reduced expression of c-MAF, NFAT2, STAT4, STAT6 and T-bet (but not GATA3, nor NFAT1, which was upregulated) [81]. Others report that T cells anergized by sMAgs go on to become apoptotic within five days [77]. Peptide-Coupled Cells or Nanoparticles One long-recognized strategy to induce autoantigen-specific unresponsiveness is to couple autoantigen(s) to apoptotic cells (reviewed in [82]). For this, carrier cells are pulsed with peptide in the presence of a chemical cross-linker, such as 1-ethyl-3(3dimethylaminopropyl)-carbodiimide (ECDI), and then delivered intravenously. The uptake of antigen-coupled apoptotic cells by macrophages in the marginal zone of the spleen upregulates IL-10 production, which in turn upregulates the immunomodulatory co-stimulatory molecule, PD-L1, which is essential for the induction of tolerance to the coupled antigens [83]. By contrast, the maintenance of tolerance is dependent International Reviews of Immunology


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on the induction of Tregs [83]. Peptide-coupled synthetic biodegradable nanoparticles have been found to induce tolerance by similar mechanisms [82]. Studies of Modified Peptides in Animal Models of Autoimmune Disease Several well-established animal models have been used to test the effects of modified peptides on the induction and treatment of autoimmune disease. The most frequently used model in these studies has been experimental autoimmune encephalomyelitis (EAE), which reproduces many of the neuropathological and immunological characteristics of MS [84, 85]. Both EAE and MS are characterized by inflammation and demyelination of the central nervous system (CNS) white matter. EAE is typically induced by the injection of antigens derived from myelin proteins, such as myelin proteolipid protein (PLP), myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG), or by the adoptive transfer of myelin antigen-specific CD4+ T cells [86, 87]. Similarly, to study the immunological features of RA, a range of collageninduced arthritis (CIA) animal models have been used [88], in which mice or rats are injected with collagen peptides in adjuvant, or T cells specific for such peptides are transferred into recipients to induce disease. One of the few spontaneously occurring autoimmune diseases in non-transgenic animals occurs in female non-obese diabetic (NOD) mice, which spontaneously develop insulitis at the age of 12- to 14 week [89], and which are routinely used as an animal model of type 1 diabetes (T1D). In comparison with other autoimmune diseases noted above, myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), are considered to be primarily B cell-mediated autoimmune diseases, caused by antibodies specific for the acetylcholine receptor (AChR) that induce degradation of the receptor, activation of complement cascade, and destruction of the post-synaptic membrane at the neuromuscular junction, resulting in a functional reduction of AChR availability [90]. Antibody production does, however, appear to be dependent on T cell help [90]; thus, targeting autoreactive T cells by using antigen-specific therapeutic agents has been suggested as a viable strategy for ameliorating MG. APLs in Animal Models of Autoimmune Disease Various APLs have been used to treat all of the animal models noted above; these studies are summarized in Table 1. While most of the APLs were able to antagonize antigenspecific T cell clones in vitro, this was not the main mechanism by which they acted in vivo. Instead, most of the APL expanded independent populations of T cells that had immunoregulatory functions in vivo or produced regulatory cytokines that acted on the disease-inducing cells via bystander suppression. Importantly, a consistent observation was that the effects of APLs on monoclonal T cell populations in vitro often poorly corresponded to their effects on polyclonal T cell populations in vivo. MVPs for Treatment of EAE and CIA Peripheral tolerance can be induced by treatment with MVPs that have reduced affinity for MHC class II. This approach has been adopted in two EAE models: PLP139–151 induced EAE in SJL mice and MOG35–55 -induced EAE in C57Bl/6 mice. In the SJL model, pre-immunization with the MVP reduced the severity of PLP139–151 -induced EAE, while multiple treatments with MVP after the onset of clinical EAE reduced the severity of the chronic disease course without inducing hypersensitivity [91]. In the C57Bl/6 model, treatment of MOG35–55 -specific T cells with the MVP “45D” prior to adoptive transfer reduced their encephalitogenicity [50]. In this case, the MVP induced hyporesponsiveness in the polyclonal T cell population, characterized by a reduced capacity to produce IL-2 upon subsequent stimulation (which could not be restored by exogenous IL-2) but continued capacity to produce IFN-γ and IL-4 [50, 92]. In fact, the C Informa Healthcare USA, Inc. Copyright

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Effects of APL in animal models of autoimmune disease.

Experimental model


PLP139–151 -induced EAE in SJL/J mice (peptide presented by I-As ) [161–163]

PLP139–151 -induced EAE in SJL/J mice (peptide presented by I-As ) [164, 165] PLP178–191 -induced EAE in SJL/J mice (peptide presented by I-As ) [166]

TCR contacts W144, H147

APL 144W>L/ 147H>R

144W>Q

F188

188F>A

MBPAc1–9 -induced EAE in PL/J mice (peptide presented by I-Au ) [167]

Q3

3Q>K [168]

MBP87–99 -induced EAE in Lewis rats (peptide presented by RT.D1 ) [169]

K91

91K>A

CII263–272 -induced CIA in Q267, Lewis rats (peptide K270 presented by RT.B1 ) [170, 171]

267Q>A/270 K>A/271 G>A

Insulin B9–23 -induced T1D in NOD mice (peptide presented by I-Ag7 ) [130]

NBI-6024 (16Y>A, 19C>A)

E13, L15, Y16

Effects of APL • Antagonised PLP139–151 -specific T cell clones in vitro. • Reduced incidence of EAE in SJL mice when co-immunized with PLP139–151 (6:1) but not by antagonism; instead, the APL expanded a population of Th2/Th0 T cell clones that could be activated by the native antigen and could be found in the CNS. • Expanded Th2/Th0 clones and IL-10 producing T cells that cross react with the native antigen and recognize L141 as the primary TCR contact residue. • PLP178–191 -specific T cells show minimal proliferation to A188 but A188-specific T cells cross react with PLP178–191 . • A188-specific T cells utilize different TCR Vβ elements than T cells specific for PLP178–191 [E. Sauer, unpublished data]. • Activated A188-specific T cells upregulate expression of IL-4, IL-5, IL-10 and IFN-γ , and transfer of these cells alongside activated PLP178–191 -specific T cells prevented passive transfer of disease. • Protected against EAE in PL/J mice. • Protection in mice correlated with ability of APL to antagonize polyclonal T cell populations, rather than individual T cell clones, in vitro. • Weak cross-reactivity with MBP87–99 -specific T cells; weak antagonist in vitro. • Protected against EAE in Lewis rats/mice when co-immunized with MBP87–99 (1:1) via reduction in production of TNFα and IFN-γ . • Mucosal administration of triple-substituted APL (1:3 compared with CII263–272 ) suppressed progression of CIA in Lewis rats. • Protective effects associated with the induction of CD4+ CD25+ FOXP3+ Tregs, reduced levels of IFN-γ and IL-17, and reduced titres of Th1-associated IgG2a . • Generates polyclonal Th2 T cells response with poor, but measurable, cross-reactivity to unaltered antigen. • 20 mg/kg of APL reduced incidence of clinical signs of disease in NOD mice, even when treatment was started after the development of insulitis. (Continued on next page)

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Antigen-Specific Therapeutics for Autoimmunity TABLE 1.

Effects of APL in animal models of autoimmune disease. (Continued)

Experimental model


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AChRα 195–212 -induced EAMG in SJL/J mice (peptide presented by I-As ) [172] AChRα 259–271 -induced EAMG in BALB/c mice (peptide presented by I-Ad ) [172] AChRα 195–212 -induced EAMG in SJL/J, BALB/c and C57BL/6 mice [173–180]

TCR contacts

APL

M207

207M>A

E262

262E>K

M207, E262

Tandem peptide 207M>A, 262E>K

Effects of APL • Inhibited the in vitro and in vivo (at 10:1 APL:AChRα 195–212 ) proliferative responses of AChRα 195–212 -specific T cells. • Inhibited the in vitro and in vivo (at 10:1 APL:AChRα 259–271 ) proliferative responses of AChRα 259–271 -specific T cells. • Inhibited proliferation of T cell lines specific for AChRα 195–212 or AChRα 259–271 [178]. • When given orally or subcutaneously (at 20:1 APL:AChR), could reverse EAMG manifestations induced in BALB/c mice by AChRα 259–271 -specific T cells [178] or by the immunization of C57BL/6 mice with Torpedo AChR [179]. • Acted as partial agonist [177] to decrease the production of IL2 and IFN-γ and increase the production of TGF-β [180]. • Induced CD4+ CD25+ FOXP3+ [174] and CD8+ CD28− [175] regulatory T cell subsets. • Increases phosphorylation of c-Jun and p38 and upregulates expression of Fas and FasL on CD4+ T cells [173, 176].

AChRα: α chain of the acetylcholine receptor; CIA: collagen-induced arthritis; CII: type 2 collagen; EAE: experimental autoimmune encephalomyelitis; EAMG: experimental autoimmune myasthenia gravis; MBP: myelin basic protein; PLP: myelin proteolipid protein; T1D: type 1 diabetes.

production of IFN-γ was essential for the therapeutic action of MVP, as hyporesponsiveness to subsequent stimulation and protection against EAE could not be reproduced in IFN-γ −/− or IFN-γ R−/− mice [93]. The means by which IFN-γ mediates the MVP-induced protection in this scenario is not completely understood, but a regulatory mechanism that may be involved is negative feedback to TCR signaling by SHP-1. In mice expressing low levels of SHP-1, the MVP failed to induce hyporesponsiveness in SHP-1low , MOG35–55 -specific T cells, and became encephalitogenic [94]. In CIA, an MVP peptide known as A12 has been described, which is derived from the CII259–273 epitope. Although A12 is described in the literature as an APL, the peptide is modified at the major MHC-binding residues for HLA-DR1/DR4 at positions 263 (F→N) and 266 (E→D) [95, 96], thus it is more accurately described as an MVP. A12 inhibited CII-induced disease in HLA-DR1 and HLA-DR4 transgenic mice, largely via induction of A12-specific T cells that produce elevated amounts of IL-4 [95, 97]. This MVP has recently progressed to human trials, as discussed below. sMAgs in T1D and Other Animal Models of Autoimmune Disease Monovalent and bivalent sMAgs have been tested as therapeutic agents in a number of different animal models, including T1D (spontaneous and transgenic models), EAE, C Informa Healthcare USA, Inc. Copyright

 E. L. Sauer et al.


CIA and EAMG (Table 2). In most of these studies, sMAgs have been created by linking the normally pathogenic peptides to the MHC molecules, but in spontaneous T1D in NOD mice, the greatest clinical efficacy was found using sMAgs created by linking epitopes of GAD-65 that induce protective Th2-focussed responses, particularly responses involving production of IL-10 and TGF-β [66, 75, 98]. Overall, as also observed for the majority of APLs, effective sMAgs appear to rely on induction of IL-10 and/or Tregs to exert their protective effects. Peptide-Coupled Cells in EAE and Diabetes Models Numerous studies, dating back to the early 1990s, demonstrated that intravenous administration of autoantigen-coupled spleen cells could prevent the induction of active EAE [99–101], of adoptive EAE [100, 102], and, when administered after the acute phase of disease, the incidence of subsequent relapses [103–106]. T cells recovered from the peripheral blood of tolerized animals displayed loss of reactivity to the autoantigen, which could be restored by IL-2 [101]. Protection against relapses conferred by the administration of autoantigen-coupled splenocytes at the peak of the active phase of EAE was associated with increased production of TGF-β and IL-10 by cells recovered from the CNS [107]. Protection was dependent on the ECDI-coupled cells being administered intravenously, and on their ability to induce IL-10 production and induce Treg s [83]. In the NOD mouse diabetes model, intravenous administration of insulin-coupled splenocytes shortly after the spontaneous appearance of insulitis led to the remission of disease in half of the cohort, and similar treatment following the adoptive transfer of disease to pre-diabetic mice prevented clinical signs [108]. Even though peptidecoupled splenocytes can induce Tregs, they did not do so in the latter model, anergic mechanisms, dependent on the interaction of programmed death 1 (PD-1) with its ligand PD-L1 [108, 109]. HUMAN STUDIES AND CLINICAL TRIALS APLs and Other Modified Peptides in Multiple Sclerosis In humans, putative autoantigenic epitopes for MS exist within the 83-to-99 region of MBP. This peptide binds strongly to HLA-DRB1∗ 15:01, the HLA molecule carried by approximately 60% of MS patients, but it also binds to several other MHC class II molecules, with the specific epitopes differing slightly in register [110, 111], thereby changing the amino acids that are available to interact with TCRs with obvious implications for the design of APLs and MVPs [112]. In vitro analyses of APLs derived from this epitope showed that some could act as antagonists towards some T cell clones and as agonists or superagonists towards others, while other APLs were capable of antagonizing a broader range of T cell clones [113, 114]. Person-to-person differences in APL responsiveness were also observed: T cell clones derived from different HLA-DRB1∗ 15:01+ MS patients displayed bias towards particular TCR variable gene segments and different patterns of cross-reactivity to MBP83–97 and the APL A91 (numbered according to the human sequence) [115]. In one patient, MBP83–97 and A91 selected cross-reactive T cell clones, demonstrating that both peptides agonized the same pool of T cells, while in another patient MBP83–97 and A91 selected independent, non-cross-reactive T cell clones. The results from these studies demonstrate two critical points which must be considered when using APLs to modulate autoimmune responses in people: firstly, a person’s HLA haplotype will determine how an epitope will be presented and consequently which positions are suitable for alteration when designing an APL, and secondly, a patient’s personal T cell repertoire will shape their response to a particular APL. International Reviews of Immunology



Effects of sMAgs in animal models of autoimmune disease.

Bivalent (I-Ag7 -GAD217–236 or I-Ag7 -GAD290–309 ) constructs on Ig scaffolds (both of the GAD peptides induce a Th2-focussed response) RTL551 (I-Ab -MOG35–55 ) construct

Spontaneous T1D in NOD mice [75, 98, 170]

RTL VG312 (HLA-DR2-MOG35–55 construct)

Bivalent I-Ab - MOG35–55 construct

MOG35–55 -induced EAE in HLA-DR2 transgenic mice (peptide presented by HLA-DR2) [186]

MOG35–55 -induced EAE in C57Bl/6 mice (peptide presented by I-Ab ) [187]

MOG35–55 -induced EAE in C57Bl/6 mice (peptide presented by I-Ab ) [185]

Bivalent (I-Ag7 -peptide 1040–31 [184] construct on Ig scaffold

T1D in TCRα-deficient NOD mice passively transferred with islet-reactive T cells from NOD.BDC2.5 TCR transgenic mice [183]

(Continued on next page)

• Treatment of pre-diabetic animals with DEF reduced degree and severity of insulitis. • There was induction of anergy in autoreactive T cells and induction of IL-10 secreting regulatory cells in the pancreas. • Remission of disease was dependent on IL-10. • Single treatment delayed disease onset. • Weekly treatment completely prevented clinical disease, although mice did develop pancreatic immune infiltration. • Majority of transferred T cells underwent apoptosis; remainder produced elevated levels of IL-10, and blocking IL-10 receptor reversed protective effects of treatment. • Mice treated in late pre-clinical stage protected against clinical signs of disease and showed reduced degree of pancreatic inflammation. • Mediated by antigen-specific IL-10-secreting CD4+ Tregs. • Arrested clinical progression and reduced CNS inflammation when administered daily for 5 days from the onset of clinical disease. • Reduced percentage of CD44, CD226 and T-bet expressing CD4+ T cells. • Reduced production of IL-17 and IFN-γ by CNS-infiltrating cells. • Induced long-term tolerance to MOG35–55. • Arrested clinical progression and reduced CNS inflammation when administered daily from the onset of clinical disease. • Reduced production of TNF-α and IFN-γ by T cell cultures in response to MOG35–55 . • Mice treated for 4 days commencing in late pre-clinical stage (day 9) were protected against clinical signs of disease. • Treatment led to the appearance of a population of CD3low TCRlow CD4+ T cells that produced high levels of TGF-β and IL-10. d

Bivalent “DEF” (I-E -HA110–120 construct on Ig scaffold [182])

Effects of sMAg

Type of sMAg

T1D in transgenic mice expressing influenza HA under the control of insulin promoter and a TCR-specific for the immunodominant influenza HLA epitope (spontaneously develop diabetes before 8 weeks of age) [181]

Experimental model

TABLE 2.


 Effects of sMAg

AChRα: α chain of the acetylcholine receptor; CIA: collagen-induced arthritis; CII: type 2 collagen; EAE: experimental autoimmune encephalomyelitis; EAMG: experimental autoimmune myasthenia gravis; GAD: glutamic acid decarboxylase; HA: haemagglutinin; MOG: myelin oligodendrocyte glycoprotein; PLP: myelin proteolipid protein; T1D: type 1 diabetes.

• In vitro, the sMAg reduced IFN-γ and IL-17 production by 2D2 transgenic T cells specific for MOG35–55. PLP139–151 -induced EAE in SJL/J mice (peptide presented by Monomeric RTL401 (I-As -PLP139–151 • Reduced relapse rate and severity of EAE when administered I-As ) [188] construct) s.c. for 8 consecutive days, commencing at onset of clinical signs of EAE. • Induced a cytokine switch from IFN-γ to IL-10 in mononuclear cells infiltrating the CNS (treatment curbed the encephalitogenic potential of PLP139–151 -specific T cells without fully preventing their entry into the CNS). Rat CII-induced CIA in B10.Q mice or in B10.Q × (BALB/c × Bivalent (I-Aq – CII259–273 peptide • I.v. treatment of B10.Q mice just prior to time of onset of B10.Q)F2 mice (chronic relapsing model) (peptide galactosylated at lysine 263) construct arthritis and again 2 weeks later inhibited the development of arthritis. presented by I-Aq ) [61] • 3 × i.v. treatments of B10.Q × (BALB/c × B10.Q)F2 mice in chronic relapsing phase of disease reduced arthritis progression. • sMAg induces population of antigen-specifi regulatory T cells that can exert their effects in a bystander fashion. Bovine CII-induced CIA in DBA/1LacJ mice (peptide Monomeric RTL2001-MII (I-Aq – bovine • Reduced the incidence of the disease, suppressed the clinical presented by I-Aq ) [189] CII257–270 with modification at E257A, and histological signs of CIA and induced long-term G265A, G268A and P269A) construct modulation of T cells specific for the arthritogenic peptide. • Systemically reduced proinflammatory IL-17 and IFN-γ production and significantly increased anti-inflammatory IL-10, IL-13 and FoxP3 gene expression in splenocytes. • Selectively inhibited IL-1β, IL-6 and IL-23 expression in local joint tissue. Bovine CII-induced CIA in DBA/1 mice (peptide presented Bivalent (I-Aq – chick CII257–269 ) • Reduced the severity of CIA in mice by induction of by I-Aq ) [65] construct on IgG3 scaffold Ag-specific hyporesponsiveness. • Treatment of epitope-primed animals reduced ex vivo T cell Rat MHC class II – AChRα 100–116 AChR-induced EAMG in Lewis rats (peptide presented by proliferative response to antigen. RT.B1 ) [190] • I/v administration following onset of clinical signs of EAMG improved survival rate compared with control animals.

Type of sMAg

Effects of sMAgs in animal models of autoimmune disease. (Continued)

Experimental model

TABLE 2.






The design of two phase-II clinical trials of APLs in MS patients did not adequately consider these points [116, 117]. Both trials used identical APLs, based on the sequence of MBP83–99 and modified by amino acid substitution of L in place of F at positions 89, and of A in place of K at position 91, which were assumed to interact with TCRs, and by substitutions at positions 83 and 84 that were designed to improve the peptide’s half-life. Critically, neither trial selected participants based on their HLA haplotype. Adverse effects in the smaller of the two clinical trials led to both trials being halted prematurely, and neither demonstrated any overall effect on the clinical progression of disease over the course of treatment, although in the larger trial, patients receiving weekly 5-mg doses of APL (for a 4–16-week period) reportedly displayed decreasing frequency and volume of gadolinium-enhancing lesions and induction of a Th2 response to the native antigen which persisted for several years in some individuals [117, 118]. In the smaller trial which tested APLs in eight patients, post hoc analysis showed that only one individual carried HLA-DRB1∗ 15:01, the HLA type for which the APL was designed [116]. Relapse of disease in another patient in this trial was attributable to treatment (thus leading to the cessation of the trial), and was due to the expansion of a pool of pathogenic T cells for which both MBP83–99 and APL acted as agonists. Further immunological studies confirmed the high cross-reactivity and wide heterogeneity of T cells reactive to APL and MBP83–99 . Some of the tested APL-reactive clones demonstrated very high antigen avidity to MBP83–99 ; these clones displayed a Th1/Th0 phenotype, and stimulation with MBP83–99 elicited signaling protein phosphorylation patterns associated with full activation. However, in patients whose disease activity scores improved, the frequency of MBP83–99 -reactive T cells in the peripheral blood decreased over the treatment period. Recently, results of a phase 1 clinical trial using an RTL, RTL1000, which comprises the DR2 α1 and β1 domains covalently linked to MOG35–55 , have been reported [119]. In this trial, doses of up to 60 mg of RTL in a single intravenous infusion were found to be safe and well tolerated by patients; higher doses caused hypotension and diarrheoa in patients. A phase II trial of this molecule is reportedly in the planning stage. Another recent phase 1 trial in MS involved the intravenous administration of autologous peripheral blood mononuclear cells (PBMCs) coupled (using ECDI) with seven myelin peptides (MOG1–20 , MOG35–55 , MBP13–32 , MBP83–99 , MBP111–129 , MBP146–170 and PLP139–154 ) to a group of nine MS patients, who were selected based on reactivity to at least one of these peptides [120]. This trial was not set up to show clinical efficacy, but it showed that the method appears safe in humans. Several other peptides of MOG and PLP have also been implicated as targets of autoreactivity in MS patients [121–125], and it may be that expanding the repertoire of peptides coupled to the autologous white blood cells could enhance clinical efficacy. APLs in T1D Altered peptide ligands derived from a range of autoreactive epitopes relating to the pathogenesis of T1D in humans have been tested in vitro and in vivo. The earliest in vitro studies used an APL based on Imogen, a 38 kD islet mitochondrial protein. The APL could antagonize the production of IFN-γ by a single CD4+ T cell clone derived from a patient with T1D when loaded into the same APCs as the native antigen but not when each peptide was loaded into separate populations of APCs (which were subsequently mixed), suggesting that the APL interfered with either antigen presentation or with the formation of immunological synapses [126]. Subsequent studies have tested several APLs produced by the modification of human GAD65 residues 554–570. These APLs were tested against panels of T cell clones, but none of these APLs showed consistent results on all clones, with some having no effects, others antagonizing some clones but not others, and some substitutions making the APLs strong agonists [127–129]. C Informa Healthcare USA, Inc. Copyright


 E. L. Sauer et al. Furthermore, when antagonizable T cell clones were pooled with non-antagonizable clones, IL-2 produced by the latter prevented antagonism of the former, suggesting that these APLs would not be effective on polyclonal populations or in vivo [127]. The APL NBI-6024, which is derived from the insulin B9–23 peptide and had shown promising results in NOD mice [130], was subsequently tested in T1D patients. Most participants in this phase I trial expressed HLA-DQ8 (DQA1∗ 03:01; DQB1∗ 03:02), the restriction element for B9–23 in humans [131]. The APL, which was administered for five times at biweekly intervals, was well tolerated by the patients with recent-onset T1D in phase I clinical trials [132]. However, longitudinal instability in the cytokine responses of participant’s PBMCs to the APL and to the cognate antigen, B9–23 , complicated the analysis of immunomodulatory effects of treatment. Most placebo-treated patients, but very few healthy control participants, demonstrated some reactivity to NBI-6024, suggesting that many patients do have T cells that recognize the insulin B9–23 peptide and that there is a degree of cross-reactivity of those cells with the APL. Encouragingly, adolescent T1D patients treated with APL showed a dose-dependent shift in cytokine production towards a Th2 response. In spite of this promising start, a subsequent phase II trial of NBI-6024, in which monthly doses of APL were administered for two years, failed to show any effect on the rate of deterioration of endogenous insulin production, or on anti-islet antibodies or T cell numbers [133]. In the phase II trial, participants were not selected on the basis of HLA haplotype or immunoreactivity towards B9–23 . In addition, although the positive effects seen in the phase I trial occurred at a dose of 5 mg of APL biweekly, the maximum dose tested in the phase II trial was 1 mg monthly. It was speculated that the frequency, timing or dose of NBI-6024 may have been insufficient to generate an effect. Modified Ligands in Rheumatoid Arthritis Rheumatoid arthritis is closely associated with HLA-DRB1 alleles that encode the “shared epitope”, which include HLA-DRB1∗ 01:01, HLA-DRB1∗ 04:01, HLADRB1∗ 04:04, HLA-DRB1∗ 04:05 and HLA-DRB1∗ 09:01 [134]. In many patients, there is generation of autoreactive antibodies, including rheumatoid factor, directed against Ig Fc, and anti-citrullinated peptide antibodies (ACPAs), which recognize citrullinated epitopes within matrix proteins. HLA-DR-restricted T cell reactivity against CII263–272 is also detected in many patients with RA. This epitope can bind both HLA-DR1 and HLA-DR4 via residues F263 and K264, and, to a lesser extent, E266 [96, 135]. MVPs generated by amino acid substitutions at positions 263 and 266 altered cytokine production by CII-sensitized T cells from HLA-DR4-transgenic mice from a Th1 to a Th2 response in vitro and in vivo, but were effective in suppressing CIA only when used at a 480-fold molar excess with the cognate antigen [97]. Computer modeling suggests that residues 267 and 270 project from the binding groove of both DR1 and DR4 so that these are available to contact TCRs [136], but binding studies revealed heterogeneity among T cell clones, with differing sensitivities to substitutions at almost every position between 262 and 270 [96, 135]. Consequently, antagonistic APLs have been effective only against limited numbers of T cell clones [137, 138]. Some APLs, however, particularly those carrying substitutions at two or three positions, have been able to suppress the responses to the cognate antigen among PBMCs and peripheral blood lymphocytes from people with RA [139, 140]. WHY HAVE MODIFIED PEPTIDE THERAPIES BEEN SUCCESSFUL IN ANIMAL MODELS BUT NOT IN HUMANS? A number of factors underlie the failure to translate success in preventing or ameliorating animal models of autoimmune diseases into successful clinical trials of International Reviews of Immunology





modified peptides. Addressing these factors will be critical if progress is to be made. Firstly, modified peptides have often been tested on monoclonal T cell populations in vitro or in TCR transgenic mice in vivo, yet physiological T cell responses to antigens are polyclonal, involving a spectrum of affinities for an antigen and variation in the antigenic residues that contribute to ligation. Consequently, as has been described, modified peptides may elicit different responses from sub-populations of T cells in a polyclonal response: antagonizing some, while agonizing others, for example. Sampling many clonal T cell responses does not guarantee that a representative picture can be built up, as it cannot account for the paracrine effects of cytokine production on T cell activation. The sensitivity of partially agonistic responses, such as anergy and antagonism to cytokines, may mean that an APL that antagonizes a monoclonal T cell line may fail to antagonize a polyclonal T cell population, if IL-2 production by activated T cells overcomes the dominant-negative antagonistic signal. The second issue is that most human trials have not screened patients to ensure that they carry the appropriate HLA molecules to allow presentation of the peptide in the format required to downregulate the response. The take-home message is that if you design a therapeutic agent that only works in the required manner when it is presented by specific HLA molecules, it should only be used in patients who carry those HLA molecules. Thirdly, clinical outcome measures in human autoimmune diseases are not as clear-cut as in the animal models, making it more difficult to interpret and accurately measure beneficial effects of therapeutic agents, particularly within a relatively short timeframe. This is especially important to recognize in autoimmune diseases where the clinical course is variable, with attacks and remissions interspersed at irregular intervals, or where symptoms are due primarily to events that would be unlikely to be changed by therapies that target the immune system (e.g. symptoms caused by irreversible axonal loss in patients with progressive MS). It will be important to use modified peptide therapeutics in patients who are actively making an immune response. As the majority of modified peptides appear to act through indirect bystander suppression, inducing a new sub-population of anti-inflammatory or regulatory cells, outcome measures should incorporate assays to determine cross-reactivity and cytokine profiles of both auto-reactive cells and treatment-specific cells within the T cell repertoire. The last issue that needs to be considered relates to the form and dosage of the modified peptide. Peptides by themselves are typically not very immunogenic; in the animal models, modified peptide therapeutics have typically been administered in adjuvants such as Freund’s Complete Adjuvant (CFA) and/or at extremely high doses, relative to the size of the animal (milligram quantity in a 20-g mouse). As the use of adjuvants in humans often leads to granuloma formation and many unwanted side effects, modified peptides have typically been administered in water or saline. Some human trials of APLs have reported injection site reactions in some patients following subcutaneous administration, even though the dosages used have not been comparable to those used in the animal models on a weight:weight basis (milligram quantity in a 60-kg human). In addition, the half-life of peptides in serum is typically very short, as they are targeted by serum proteases. Results from the animal models suggest that many of the beneficial effects of modified peptides derive from their ability to themself induce a strong regulatory response, but this is unlikely to develop if the half-life of the peptide is short and the amount of peptide that could be presented to the immune system is low. Several strategies have been tested to enhance the stability and immunogenicity of modified peptides. One strategy is the substitution of amino acids that are susceptible to proteolysis with D-amino acids: this approach was shown to enhance the in vitro C Informa Healthcare USA, Inc. Copyright


 E. L. Sauer et al. activity of APLs on T cell clones from patients with T1D [141, 142], and was also used in the APLs used in the MS human clinical trials [116, 117]. Replacing amino acids with non-natural amino acids has also been tested. An MVP created from an epitope of human cartilage glycoprotein-39 (HC gp-39), but with the non-natural amino acids diphenylalanine or 1,2,3,4-tetrahydroisoquinoline-3-carbonyl (3Tic) used in place of a phenylalanine at the P1 MHC-binding residue, did not affect the affinity of the peptide for HLA-DR4 but induced stronger proliferation and changed the cytokine and chemokine profiles of the cells, compared with the cognate antigen [143]. Another method that has been tested to increase resistance to proteolysis is to create cyclical peptides [144–146]. A cyclical form of an antagonistic APL (derived from MBP) had similar clinical efficacy as the linear form in an EAE model, and induced similar responses to the linear APL in MBP-specific human T cell clones, but was more resistant to degradation in serum [144, 147]. Another approach to improve immunogenicity has been to use dendrimers, prepared by chemical ligation (thioester, oxime, hydrazone) of tetravalent or octavalent polylysine scaffolds with multiple peptides [148]. A dendrimer linking two different APLs (designed to treat Celiac disease) increased the ability of the APLs to competitively block T cell activation by native peptides at significantly lower doses than those at which they could act individually [149, 150]. Finally, it has recently been shown that thioacylation of APL by attachment of a palmitic acid chain via a thioester bond to a cysteine residue can markedly enhance the immunogenic and therapeutic properties of APL in an EAE model [151]. Of note, thioacylation of APL markedly enhances the half-life of the APL in serum, and drives uptake of the peptide into the MHC class II presentation pathway [152]. CONCLUSIONS Therapeutic strategies using modified peptides have been shown to be an effective means of controlling autoimmune disease in multiple animal models. Thus far, such therapeutic agents have not been successfully applied to human disease, although the reasons underlying this failure are evident and addressable. Translation of the efficacy seen in animal models into humans will require consideration of (i) the polyclonal nature of physiological T cell responses; (ii) use of peptide-based therapeutics in patients of the correct HLA type; (iii) the choice of relevant outcome measures; and (iv) applying strategies to increase bioavailability of peptides. Declaration of Interest The authors report no declaration of interest. The authors alone are responsible for the content and writing of this paper. REFERENCES [1]

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