LU92815B1 - Inhibitor of dj-1 for therapy - Google Patents

Abstract

The present invention relates to an Inhibitor of DJ-1 (PARK7) for use in the treatment or prevention of one or more of autoimmune disease, allergy, infectious disease or cancer, wherein the cancer is not lung cancer. In addition, the present invention relates to pharmaceutical compositions comprising such ihibitorsand kits comprising the inhibitor.

Description

Western blotting analysis, samples were stained with the primary antibodies against DJ-1 (3018257, Abeam) or control GAPDH (sc-25778, Santa Cruz) followed by the secondary antibody goat anti-rabbit IgG-HRP (172-1019, Bio-Rad).

[127] Real-time RT-PCR (qPCR) [128] RNA was isolated with the RNeasy Mini Kit (Qiagen) according to manufacturer's protocol and additionally purified by on-column RNase-free DNase digestion (Qiagen). Purified and concentrated RNA was then reverse-transcribed using Superscript ill reverse transcriptase (Invitrogen). qPCR was conducted on a LightCycier 480 (Roche) as previously described (Pfoertner et al, 2006). The following primers were finally used in our qPCR analysis. Human PARK7/DJ-1 forward (5--3) TTG TAG GCT GAG AAA TCT CTG TG (SEQ ID NO: 3); PARK7 reverse (5-3) ATC CAT TCT CAC TGT GTT CGC (SEQ ID NO: 4); FOXP3 forward ACC TAC GCC ACG CTC ATC (SEQ ID NO: 5); FOXP3 reverse TCATTG AGTGTC CGC TGC T (SEQ ID NO: 6); CTLA4 forward TGC AGO AGT TAG TTC GGG GTT GTT (SEQ ID NO: 7); CTLA4 reverse CTG GCT CTG TTG GGG GCATTT TC (SEQ ID NO; 8), TGFB1 forward CGC AAG GAC CTC GGC TGG AAG TGG (SEQ ID NO: 9); TGFB1 reverse GAG GCG CCC GGG TTA TGC TGG TTG (SEQ ID NO: 10). The primers GARP toward GAT GGG GAA ACT GAG GCT TAG GAA (SEQ ID NO: 11); GARP reverse ACC CCC AAT CTC ACC CCA CAA ATA (SEQ ID NO: 12); LGMN forward CTC GCT CCA GGA CCT TCT TCA CAA (SEQ ID NO: 13); LGMN reverse GCT TCC TGC TCC TCA AAA CTA ACA (SEQ ID NO: 14) and ÏL4 forward CGG CAA CTT TGT CCA CGG A (SEQ ID NO: 15), IL4 reverse TCT GTT ACG GTC AAC TCG GTG (SEQ ID NO: 16) were also from our previous work (Probst-Kepper et al, 2009). We also used qPCR primers from Qiagen for the genes RPS9, GAPDH, PLAU, 1L1R1, S1PR1, IKZF4 (EOS), IKZF2 (Helios), GITR, LGALS3, CD127, IL13, CSF2, 1L5, and IL2. RPS9 or GAPDH was used as an internal standard (Bruder et al, 2004) in our qPCR analysis.

[129] Microarray measurement and data preprocessing [130] The cells were centrifuged, and the cell pellets were lysed in RLT buffer (Qiagen) and immediately stored at -70C. RNAwas isolated with the RNeasy Mini Kit (Qiagen) according to manufacturer's protocol and additionally purified by on-column RNase-free DNase digestion (Qiagen). RNA quality was checked by RNA 6000 Nano assay (2100 Bioanalyzer, Agilent). For oligonucleotide microarray hybridization, RNA was labeled, fragmented, and hybridized to an Affymetrix GeneChip Human Gene 2.0 ST oligonucleotide array at the Genomics Gore facility of the EMBL (Heidelberg), After the arrays were scanned, the expression value for each gene was calculated by using Affymetrix Microarray software 5.0 (MAS5). The average intensity difference values were normalized across the sample set Probe sets that were absent in all samples, according to Affymetrix flags, were removed.

[131] Correlation network construction and analysis Network construction [132] In this work, we demonstrated the identification of key genes by constructing and analyzing a Treg-,specific correlation network. The correlation network construction and analysis has been described in detail in our recent work (He et a!., 2012, Molecular Systems Biology). We constructed the correlation network by identifying correlation linkages among pairs of genes. The correlation linkages were identified by employing both the LC and the TC methods. Both methods are capable of discovering potential functional association between genes from time-series data by considering positive/inverted correlation and time shift. A significant correlation with time shift between two genes might indicate a transcription regulatory relationship (Bar-Joseph et al, 2012). But to obtain accurate inference, we need much additional information, a big portion of which are not available. We therefore only simply considered the pairs with this type of correlation as potential function-associated linkages in this work by not distinguishing the specific type of detailed functional association (e.g., signal transduction, transcription regulation or In the same pathways). The LC method has been widely used to identify potential functional association mainly based on a point-to-point comparison of gene expression values from time-series data as recently reviewed (Bar-Joseph et al, 2012). The TG method has also been demonstrated to be useful in identifying potential functional association from time-series data and to be complementary to the LC method based on extracting main features of the change trend and the change level of gene expression between consecutive time points (He and Zeng, 2006). Due to different principles behind the LC and the TC methods, we therefore combined both methods to complement each other in order to identify more complete potential functional associations between genes. For this purpose, we calculated the LC score as defined by the LC method (Qian et al, 2001), the maximal matched change trend (sc) score and the correlation coefficient (cc) score for the maximal matched change trend as defined by the TC method (He and Zeng, 2Q06) for each pair of genes In ail the time-series data sets of Tregs and Teffs. We then calculated PLC for the scores resulting from the LC method as described (Qian et ai, 2001 ).We also calculated PTC1 for the TC method by extraction procedure I, as previously described (He and Zeng, 2006). For the TC method, we here introduced another adapted P-vaiue (PTC2) calculation based on the reordered sc and cc distribution curve. Briefly speaking, the reason to reorder the distribution table (curve) is that we found that some of the pairs, which have a very high cc score but the sc score is not very high, might still have a high chance to be potentially functionally linked. So far, we obtained three different P-values (PTC1, PTC2, and PLC) for each pair of genes in each replicate of the time-series data set generated for a given ceil type (Tregs or Teffs). In the following, we set rules to extract Treg-specific correlation linkages. First, to guarantee the reproducibility of the results, we restricted the significantly correlated pairs to those repeatedly showing significant P-vaiues in both replicates of Tregs constantly by one of the three P-vaiues (PTC1, PTC2, and PLC). A standard P-value cutoff of 0.05, the well established and extensively used threshold was applied. Second, in order to obtain cumulative evidence generated in both replicates and gained by both methods, we employed naive Bayesian integration (combination) (Lu et al, 2005) by multiplying different P-vaiues from both replicates by the LC and the TC methods. This is based on the assumption of the independency of the P-values generated by two different methods due to the utilization of different principles and the independency of the results from each replicate due to the difficulty in obtaining synchronized T-cell stage before activation although ail the T ceils were at the resting stage. Since both PTC1 and PTC2 were caused by the TC method, we chose the minimal P-value generated by one of them. Thus, only four P-values were eventually utilized for the integration purpose. The cutoff for the integrated P-values was chosen at 1e__9 based on a more stringent but also well-established P-value threshold 0.01. The overall threshold 1e 9 was even stricter than the product of the four individual P-values ((0.01 )4.1 e__8) in order to avoid the cases, such as those pairs with all the individual P-vaiues very close to the threshold of 0.01, 'which could be marginal. We therefore set min (PTC1,Treg1*PTC1,Treg2, PTC2,Treg1* PTC2,Treg2) *PLC,Treg1*PLC,Treg2p1e_9, where min () indicates the minimal value of the two products of P values; Treg1/2 indicates the replicate of Treg 1 or 2. In the end, -we only included the pairs satisfying both of the two criteria for the reproducibility and the integrated evidence in Tregs but the same pairs did not even meet the standards of the reproducibility in Teffs, as Treg-specific correlation linkages. The strict criteria of the reproducibility largely excluded those pairs with integrated P-values slightly higher than the integrated P-value threshold in Tregs but slightly lower than the integrated threshold in Teffs. Obviously, the pairs that cannot satisfy the basic prerequisites of the reproducibility (p0.05) has very less chance to pass the integrated P-vaiue threshold (based on 0.01). For example, it has been found that where 99 and 95.5% of ail the Treg-specific correlation linkages show around 3- and 10-fold less P-values, respectively, in average for each of the four individual P-vaiues in Tregs than in Teffs. For the remaining tiny proportion (1%) of the correlation linkages, they showed a very significant correlation only in one of the two replicates of Teffs but had no significance in another one, which still causes a very small final product of P-values Integrated from replicates. Apparently, we cannot consider this tiny fraction as significantly correlated pairs in Teffs without consistent results between replicates. Thus, ail of linkages including the tiny fraction were Treg-specific correlation interactions with high confidence.

[133] EXAMPLE 2: DJ-1 Is highly expressed in Tregs and Treg-important genes are enriched in DJ-1 correlation network [134] Starting from our own published datasets, we were able to find that DJ-1 was highly expressed in both unstimulated and stimulated human Tregs and Teffs according to the high-time-resolutson time-series transcriptome data in the first 6 hrs following TCR stimulation [19], Quantitative Realtime PCR results from other healthy donors have also confirmed that its expression level is as high as some highly-expressed reference genes such as RPS9 (Figure 1). As expected, DJ-1 protein is also highly expressed in both human Tregs and Teffs (Figure 1, western blot and FACS figure).

[135] Furthermore, by utilizing a network strategy which has a potential to Infer novel key genes for Tregs from Treg-specific correlation network [19], we analysed the first-degree neighbourhoods in the DJ-1 subnetwork. We cannot find a significant enrichment for the collection of 400 T-cell function related genes. However, we noticed that almost ail of its first-degree neighbours were significantly enriched for the collection of the 400 genes. Since the second-degree neighbours in the subnetwork might also influence the activity or function of the given gene/node [20], we further analysed the subnetwork composed of second-order neighbours of DJ-1. Intriguingiy, we found that in the second-degree gene network DJ-1 was highly connected with well-known key players for Treg function (4.3E-12), such as FOXP3, CTLA4, ÎCOS, GATAS, CD44 and others [13, 21]. This indicates that DJ-1 is probably involved In Treg suppressor function (Figure 2) according to our network analysis strategy.

[136] EXAMPLE 3: Inhibiting DJ~1 promotes Treg suppressor function in order to test whether DJ-1 plays a rale in the suppressor function of Tregs, we knocked-down DJ-1 in human natural Tregs (nTregs) with DJ-1 specific siRNA (si-DJ-T). After co-culturing nTregs with carboxyfluorescein succinimidyi ester (CFSE)-iabelied Teffs at various ratios, Epstein-Barr virus (EBV)-transiormed B ceils (representing antigen presenting celis), unexpectedly, we observed that knockdown of DJ-fsignificantly enhanced the Treg suppressor function in the cases of higher (eg. 1:4 and 1:8) but not lower ratios (Figure 3).

[137] Since the control Tregs (with non-specific siRNA, si__NS) have already quite high suppressive function at lower ratios, we expect that it is almost impossible to further increase their suppressor function. For this reason, we proposed to Improve the suppressive function for the so-called ‘defective’ nTregs, i.e,, with impaired suppressor function, which is often the case in autoimmune diseases. As F0XP3 is the so-called master regulator of Tregs and its deficiency impairs Treg suppressor function, we here tested the effects of DJ-1 on nTregs with F0XP3 deficiency. As expected, knockîng-down F0XP3 indeed led to an impairment of human Treg suppressor function (Figure 3). Remarkably, silencing of DJ-1 rescued the impaired Treg suppressive function (Figure 3). Recently, Dj-1 -knockout mice have shown reduced inflammation in white adipose tissue [22] although one has no clue whether Treg function has been enhanced or not Here, we further asked whether Dj-1-knockout murine CD4+CD25+Tregs demonstrated enhanced suppressive function by co-culturing sorted Dj-1~'~, Dj-Tl+ or Dj-1+I* Tregs with CFSE-iabelled WT Tbs and feeder celis.

[138] EXAMPLE 4: DJ-1 mediates Treg suppressor function via FOXP3 and CTLA4 [139] To figure out the mechanism underlying DJ-7-medidated Treg suppressor function, we further assessed the effects of DJ-1 on the expression of known Treg key genes. Remarkably, knocking-down DJ-1 followed by anti-CD3/-CD28/IL2 stimulation significantly up-regulated several reported Treg important genes in FOXR3-deficient human Tregs, such as F0XP3, GARP, EOS (JKZF4) and others (Figure 4). The regulation of some gene expression was FOXP3-dependent but the expression of others was not (Figure 4). Concordantty, knockdown of DJ-1 increased P0XP3 protein expression in FOXFU-silenced Tregs (Figure 4). As FOXP3 normally inhibits the expression of cytokines, knocking-down FOXP3 indeed increased their expression (Figure 4, right), interestingly, silencing of DJ-1 significantly inhibited the expression of these increased cytokines in FOXP3-silenced Tregs possibly by rescuing F0XP3 expression (Figure 4, right).

[140] Reactive oxygen species (RQS) are regulated by DJ-1 in other cellular types and conditions [6, 7] and play an important role in signaling transduction [5, 23-25]. For this reason, we further investigated whether RQS were involved in DJ-1-mediated nTreg suppressor function. More recently, RQS were demonstrated to be involved in antigen-specific T cell activation when the authors used total CD4+ or CDS-*- T ceils as subjects [26]. Qur results show that the RQS burst level was significantly Increased in DJ-f-knockdown nTregs (Figure 5).

[141] Notably, inhibiting the RQS burst with a specific ROS inhibitor (Diphenyleneiodonium, DPI) significantly decreased the expression of some Treg key genes (FOXP3 and CTLA4) in a dose-dependent manner, which were upregulated after silencing DJ-1 in FOXP3-deficiency Tregs (Figure 5, right).

[142] Moreover, ROS inhibitor did not result in significant cell death of Tregs. However, we were unable to observe any significant effect on several tested potential Treg-relevant signalling pathways, such as STAT5, ERK, AKT and others following DJ-1 knockdown In FOXP3-deficient Tregs. These lines of evidence support our hypothesis that DJ-1 might play an unexpected inhibitory role in Treg suppressor function in contrast to its classical 'positive’ role in neurons and tumor cells.

[143] EXAMPLE 5: DJ-1 mediates Treg suppressor function via regulating ceil cycle and TCR signaling pathway.

[144] To systematically investigate which genes and pathways/subnetworks were involved In DJ-1 -mediated Treg suppressor function, we measured genome-scale transcription of si_NS treated nTregs, FOXP3-knockdown Tregs, FQXP3 and DJ-1-dual-knockdown Tregs. Since stimulation is required for Treg suppressor function, before transcriptome measurement we first knocked-down FOXP3 and/or DJ-1 by specific siRNA and then stimulated Tregs for 1 day. Overall, in two Independent microarray sets from different human donors, we repeatedly observed that most of genes were downregulated (494) while only a very small fraction of genes (62) were upregulated following DJ-1 inhibition (Figure 6).

[145] This mainstream downregulation is very much consistent with the essential known function of DJ-1 as a coactivator [3]. As a snapshot, the genes (277) upreguiated by FOXP3 knockdown but then downreguiated by DJ-1 knockdown, referred to as FOXP3-dependent downreguiated genes, were mainly enriched for the processes of nucleotide binding (P-va!ue=8.5E-11), ribonucleotide binding (2.6E-8), microtubule organizing center (2.9E-6), cel! cycle (1.3E-6), small GTPase regulator activity (8.2E-3) and ubiquitin-mediated proteolysis (9.6E-3). Among these processes, DJ-1 is known io bind to RNA even at a very low concentration [4], Since DJ-1 interacts with a number of E3 ligases [27, 28], it is not shocking to observe its downstream regulatory effects on the gene expression involved in ubiquitin-mediated proteolysis. Furthermore, a recent quantitative interaction proteomics analysis shows that proteins binding to several neurodegenerative disease proteins are significantly enriched for the genes related to proteolysis [29]. The common theme on ubiquitin-mediated proteolysis of DJ-1 action observed by us and others although using different techniques, definitely signifies the proteolysis function of DJ-1. Surprisingly, the TCR signaling pathway (1.5E-2) that was regulated by FOXP3 [30] was also significantly affected by DJ-1 (Figure 6), again indicating the important role of DJ-1 in Treg suppressor function. Out of the 217 FOXP3-independent gene downreguiated by DJ-1 expression reduction, we could observe that the processes of ribonucleotide binding, GTPase regulator activity and cell cycle were still significantly enriched. This in fact validates the aforementioned pathway results of FOXP3-dependent downreguiated genes although the affected genes of the same pathways are different. A positive transcription effect of DJ-1 on genes forming endoplasmic reticulum membrane was also observed among FGXP3-independent downreguiated genes.

[146] For the 29 downreguiated genes by FOXP3 but upreguiated by DJ-1 downregulation, we could not find a significant enrichment for any known biological process or pathway, again exhibiting the non-essentiaiity of the down-regulatory activity of DJ-1. DJ-1 knockdown enhances only 33 FOXP3-independent genes, inhibiting DJ-1 not only significantly downreguiated E1 ubiquitin-activating enzyme (UBE1) and E2 ubiquitin-conjugating enzymes UBE2D3 and UBE2G, but also enhances some of E3 ubiquitin ligases (e.g., UBE4B, ÏRIP12 and SMURF2), which might compensate each other to modulate ubiquitin-mediated proteolysis.

[147] More specifically, decrease of SKP2 (S-phase kinase-associated protein 2) was observed In £>J-7-FOXP3-siRNA Tregs relative to FOXP3-siRNA Tregs.

Interestingly, knockdown of SKP2 converts diabetogenic T cells to FOXP3+ regulatory T cells [31], In addition to SKP2, several other genes regulating S-phase arrest from the ÎPA database were also downregulated In DJ-1 and FQXP3 dual knockdown Tregs compared with FQXPS-siRNA Tregs. The downregulation of those genes together following DJ-1 knockdown might inhibit Treg proliferation, reminiscent characteristics of Tregs. Although in the peripheral immunocytes, we observed that many genes which inhibits congenital malformation of brain were downregulated following DJ-1 knockdown, again demonstrating that DJ-1 does not only play a privilege role in neurons. To further understand the hierarchal structure through which DJ-1 mediates Treg suppressor function, we further mapped the down- or up-regulated genes in £?J-f-FOXP3-siRNA vs FQXP3-SÎRNA Tregs Into known transcription regulatory networks. The ingenuity Pathway Analysis (!PA) of the transcriptomics data shows that DJ-1 controlled the down-regulated genes via NUPR1, PIAS1, TCR and EP400 in Tregs (Figure 7). It is worthy to notice that EP400 modulates cell fate decisions by the regulation of RQS homeostasis [32] and DJ-1 directly binds to the androgen receptor-binding region of PÎAS1 (protein inhibitor of activated STAT) [33].

[148] EXAMPLE 6: DJ-1 binds to GZMB„ RNA processing factors and MARK signaling proteins preferentially In stimulated Tregs but LGALS3BP In unstimulated Tregs.

[149] Since DJ-1 was highly expressed in both unstimuiated and stimulated Tregs, next, we examined whether and how DJ-1 mediated Treg suppressor function via preferentially binding to certain binding partners in different statuses of Tregs by co-immuno-precipitation (IP) proteomic analysis. Following the Co-IP mass spectrum analysis with strict criteria (see Methods), we compared the abundancy of DJ-1 binding partners between unstimulated and stimulated Tregs,'Teffs. First of all, our results were able to confirm the 12 known partners (Figure 8) of DJ-1 measured in other ceiiular types, including DAXX and others, showing the reliability of our techniques.

[150] In order to obtain more robust results, we determined those binding partners which showed similar change trends between unstimuiated and stimulated Tregs, Teffs as weii as Tregs treated without the addition of IL2. Very interestingly, six proteins regulating RNA splicing preferentially bound in the stimulated rather than resting Tregs/Teffs (Figure 8). Furthermore, UPF1 (the regulator of nonsense- mediated mRNA decay) also mainly bound in stimulated Tregs/Teff. Another RNA processing protein, a pre-mRNA cleavage and poiyadenyiation specificity factor (PPP1CB) also mainly bound in stimulated Tregs. These Co-IP proteomic results are in concordance with our observation on the RNA processing pathways that were significantly affected in transcription levels by DJ-1 expression. A protein ATP5D (a subunit of mitochondrial ATP synthase) mainly slicked to DJ-1 in stimulated Tregs/Teffs, reminding the essential role of DJ-1 in mitochondria! function. Interestingly, two translation initiation factors (EÎF2A and EIF5B) also preferentially bound to DJ-1 in stimulated Tregs/Teffs. Satisfyingly, we also found that some proteins preferentially-bound in stimulated Tregs, such as MAP4K1 and ANXA4, which regulate JNK-STAT, MARK and NFKB signalling pathways, indicating a potent role of DJ-1 in Treg suppressor function since these pathways provide clear clues linking DJ-1 and downstream T cell functions/celiular phenotypes.

[151] Unexpectedly, Granzyme B (GZMB), a known Important Treg gene [34], preferentially bound to both stimulated Tregs and Teffs. But this preference was much higher in Tregs than in Teffs (>8 vs ~2, Figure 8), indicating a specific role of GZMB in DJ-1 -mediated Treg suppressor function, in contrast, we noticed that GaIetin-3-binding protein (LGALS3BP) bound with DJ-1 preferentially in unstimulated Tregs than in stimulated Tregs with a factor of up to 10 for both Treg sample pairs. Since Galectin 3 regulates Treg function [35], this observation was already able to partially illustrate how DJ-1 mediated Treg suppressor function in this work.

[152] Furthermore, we determined 8 proteins, which bound to DJ-1 preferentially in unstimulated Tregs/Teffs. it is worthy to mention that a RNA binding factor MOV10 (RiSC complex RNA helicase) bound wifh DJ-1 at least 10 folds higher in unstimuiated Tregs/Teffs than in the stimulated ones, again showing that RNA processing pathways were one of the main binding sources of DJ-1. DJ-1 regulates cellular metabolic homeostasis via modulating ROS levels as demonstrated in murine skeletal muscles [36], Interestingly, in human Tregs/Teffs, we also observed that PDHB (pyruvate dehydrogenase (lipoamide) beta), the essential component of glucose catabolism, preferentially bound in unstimuiated Tregs/Teffs, not only confirming the reports about the role of DJ-1 in metabolic homeostasis, but also discovering a molecular pathway underlying the metabolic function.

[153] Discussion

This is the first work to demonstrate that a well-known Parkinson’s disease gene DJ- 1 unexpectedly mediates the suppressive function of a peripheral immunocyte, ie. Tregs, provoking reconsideration that disease genes in multiple cellular types instead of single ceiiular types might contribute to the pathogenesis of a complex neurodegenerative disease. We are the first to show that inhibiting a gene (DJ-1) can significantly rescue FOXP3-deficient Treg suppressor function, which will provide a promising alternative strategy for targeting autoimmune diseases since inhibition of a target, e.g., by a promising siRNA in vivo treatment [40], is generally easier than activation from a drug-development point of view.

[154] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[155] All publications and patents cited in this disclosure are incorporated by reference In their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[156] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[157] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, 'will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. 'When used herein the term “comprising” can be substituted with the term “containing” or sometimes when used herein with the term “having”.

[168] 'When used herein “consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[159] In each instance herein any of the terms ''comprising", "consisting essentially of' and "consisting of may be replaced with either of the other two terms.

[160] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including ail patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[161] When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value. For example, if a formulation comprises about 5 mg/mi of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/mi, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/mi and even more preferably between 4.75 and 5.25 mg/mi, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as “(from) X to Y” equates with an interval which Is defined as “between X and Y”. Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of “5 mg/ml to 10 mg/mi” or "between 5 mg/ml and 10 mg/mi” includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value.

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Claims (15)

1. Inhibitor von DJ-1 (PARK7) zur Verwendung bei der Behandlung oder Prävention von einem oder mehreren aus Äütoimmunerkrankung, Allergie, Infektionskrankheit oder Krebs, wobei der Krebs kein Lungenkrebs ist.An inhibitor of DJ-1 (PARK7) for use in the treatment or prevention of one or more of autoimmune disease, allergy, infectious disease or cancer, wherein the cancer is not a lung cancer. 2. Inhibitor zur Verwendung nach Anspruch 1, wobei der Inhibitor eine siRNA, eine miRNA, ein Bindeprotein, ein kleines Molekül oder eine Verbindung ist.2. An inhibitor for use according to claim 1, wherein the inhibitor is an siRNA, a miRNA, a binding protein, a small molecule or a compound. 3. Inhibitor zur Verwendung nach Anspruch 2, wobei das Bindeprotein ausgewählt ist aus der Gruppe bestehend aus einem Antikörper, einem divalenten Antikörperfragment, einem monovalenten Antikörperfragment oder einem proteinartigen Bindemolekul mit antikörperähnlichen Bindungseigenschaften.The inhibitor for use according to claim 2, wherein the binding protein is selected from the group consisting of an antibody, a divalent antibody fragment, a monovalent antibody fragment or a proteinaceous binding molecule having antibody-like binding properties. 4. Inhibitor zur Verwendung nach Anspruch 2, wobei die siRNA eine Sequenzidentität von mindestens 50%, 60%, 70%, 80%, 90%, 95%, 98% 99% oder 100% zu SEQ ID NO: 1 hat.The inhibitor for use according to claim 2, wherein the siRNA has a sequence identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% 99% or 100% to SEQ ID NO: 1. 5. Inhibitor zur Verwendung nach Anspruch 2, wobei die siRNA oder die miRNA innerhalb eines Plasmidvektors zur Verfügung gestellt werden und/oder durch synthetische oder natürliche Nanopartikel modifiziert oder eingekapselt sind.5. An inhibitor for use according to claim 2, wherein the siRNA or miRNA are provided within a plasmid vector and / or modified or encapsulated by synthetic or natural nanoparticles. 6. Inhibitor zur Verwendung nach Anspruch 5, wobei der Nanopartikel ein liposomaler Nanopartikel ist.The inhibitor for use according to claim 5, wherein the nanoparticle is a liposomal nanoparticle. 7. Inhibitor zur Verwendung nach einem der Ansprüche 1-6, wobei der Inhibitor die Suppressor-Funktion einer regulatorischen T Zelle (Treg) gegenüber der Treg, bevor sie mit dem Inhibitor kontaktiert wurde, erhöht.7. An inhibitor for use according to any one of claims 1-6, wherein the inhibitor increases the suppressor function of a regulatory T cell (Treg) to the Treg before it has been contacted with the inhibitor. 8. Inhibitor zur Verwendung nach Anspruch 7, wobei die Treg eine natürliche menschliche Treg (nTreg) ist und/oder eine Treg mit eingeschränkter Suppressor-Funktion ist.The inhibitor for use according to claim 7, wherein the Treg is a natural human Treg (nTreg) and / or a Treg with restricted suppressor function. 9. Inhibitor zur Verwendung nach Anspruch 8, wobei die Treg mit eingeschränkter Suppressor-Funktion eine Treg ist, die von einem Subjekt erhalten wurde, das eine Autoimmunerkrankung, eine Allergie, eine Infektionskrankheit oder einen Krebs hat.The inhibitor for use according to claim 8, wherein the Treg with restricted suppressor function is a Treg obtained from a subject having an autoimmune disease, an allergy, an infectious disease or a cancer. 10. Pharmazeutische Zusammensetzung, umfassend den Inhibitor, wie in einem der Ansprüche 1-9 definiert.A pharmaceutical composition comprising the inhibitor as defined in any of claims 1-9. 11. Verfahren zum Durchmustern eines Inhibitor oder Aktivators von DJ-1, umfassend (a) Kontaktieren von Tregs mit einer siRNA, einer miRNA, einem Bindeprotein, einem kleinem Molekül oder einer Verbindung von Interesse; (b) Messen von Suppressor-Funktionen der Tregs, wobei ein Anstieg der Suppressor-Funktion der Tregs, verglichen mit den Tregs vor dem Kontaktieren, anzeigt, dass die siRNA, die miRNA, das Bindeprotein, das kleine Molekül oder die Verbindung von Interesse als Inhibitor von DJ-1 dient oder wobei eine Abnahme der Suppressor-Funktion der Tregs, verglichen mit den Tregs vor dem Kontaktieren, anzeigt, dass die siRNA, die miRNA, das kleine Molekül, das Bindeprotein oder die Verbindung von Interesse als Aktivator von DJ-1 dient.11. A method of screening an inhibitor or activator of DJ-1, comprising (a) contacting Tregs with a siRNA, a miRNA, a binding protein, a small molecule or a compound of interest; (b) measuring suppressor functions of the Tregs, wherein an increase in the suppressor function of the Tregs, as compared to the Tregs prior to contacting, indicates that the siRNA, miRNA, binding protein, small molecule or compound of interest Inhibitor of DJ-1, or wherein a decrease in the suppressor function of the Tregs, as compared to the Tregs prior to contacting, indicates that the siRNA, miRNA, small molecule, binding protein or compound of interest acts as an activator of DJ 1 serves. 12. Verfahren zur Bestimmung, ob eine Zelle empfänglich für die Behandlung mit einem Inhibitor, wie in einem der Ansprüche 1-9 definiert, ist, umfassend Bestimmen, ob die Zelle DJ-1exprimiert oder nicht.12. A method for determining whether a cell is susceptible to treatment with an inhibitor as defined in any one of claims 1-9, comprising determining whether or not the cell expresses DJ-1. 13. Kit, umfassend einen Inhibitor für DJ-1, wie in einem der Ansprüche 1-9 definiert.A kit comprising an inhibitor of DJ-1 as defined in any of claims 1-9. 14. Inhibitor von DJ-1 (PARK7) zur Venvendung bei der Behandlung oder Prävention von einer oder mehreren aus Autoimmunerkrankung, Allergie, Infektionskrankheit oder Krebs, wobei der Krebs ein nicht Lungenkrebs ist, wobei die Behandlung oder Prävention umfasst (i) Kontaktieren von Tregs und/oderT Effektor Zellen (Teffs) mit dem Inhibitor.14. An inhibitor of DJ-1 (PARK7) for use in the treatment or prevention of one or more of autoimmune disease, allergy, infectious disease or cancer, wherein the cancer is a non-lung cancer, wherein the treatment or prevention comprises (i) contacting Tregs and / or T effector cells (Teffs) with the inhibitor. 15. Inhibitor von DJ-1 (PARK7) zur Verwendung bei der Behandlung oder Prävention von einem oder mehreren aus Autoimmunerkrankung, Allergie, Infektionskrankheit oder Krebs, wobei der Krebs kein Lungenkrebs ist, wobei die Behandlung oder Prävention umfasst (i) Erhalten von Tregs und/oder Teffs von einem Subjekt; (ii) Kontaktieren der Tregs und/oder Teffs mit dem Inhibitor, und (iii) Wiedereinfuhren der Tregs und/oder Teffs in das Subjekt.An inhibitor of DJ-1 (PARK7) for use in the treatment or prevention of one or more of autoimmune disease, allergy, infectious disease or cancer, wherein the cancer is not a lung cancer, wherein the treatment or prevention comprises (i) obtaining Tregs and / or teffs of a subject; (ii) contacting the Tregs and / or Teffs with the inhibitor; and (iii) reimporting the Tregs and / or Teffs into the subject.