Anti-Metallothionein Therapeutics opportunities for the treatment of inflammatory bowel diseases

1. Anti-MetallothioneinTherapeuticsopportunities for the treatment of inflammatory bowel diseases Martine De Vos, Debby Laukens and Lindsey Devisscher (University of Gent, Ghent, Belgium) and Michael Lynes (University of Connecticut, Storrs, CT, USA)

2. Outline • Metallothionein (MT) overview • The role of mammalian MT on immune functions • Focus on extracellular MT • The presence of MT in inflammatory bowel disease and the consequences of MT manipulations • Future directions and opportunities

3. Stressors initiate homeostatic responses, and can induce a spectrum of proteins • Heat shock proteins • glucose regulated proteins • FKBP • cyclophilins • acute phase proteins • some cytokines • histone 2B • ubiquitin • glucocorticoids metallothionein

4. CC C cysteine motifs CXC CX3C Metallothionein: an unusual biochemistry • Small (6-7 kDa), heat stable molecule • About 61 amino acids • 20/61 are cysteines • 4-11 molecules of heavy metal divalent cation per molecule of MT • no aromatic or histidine residues, no disulfide linkages • No signal peptide MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA Crystal structure of Cd5, Zn2-MT2 (based on Robbins, A.H, et al. PDB structure 4MT2)

5. Highly homologous isoforms of Mammalian MT Palacios O, Atrian S, Capdevila M. Zn- and Cu-thioneins:a functional classification for metallothioneins. J BiolInorgChem 2011;16:991-1009 Expression profiles: MT1 and MT2 are ubiquitous MT3 predominantly expressed in the brain MT4 predominantly expressed in squamous cell epithelium

6. inflammatory agents Ca TNF IL-6 IL-1 iono- H O phore phorbal GC 2 2 metal IFN ester cations [Ca] cAMP DAG ROS Calmodulin- PKA PK PKC GC-R MBP AP2 SP1 AP1 GRE ISRE GRE BLE MRE TRE GC MRE TATA -800 -300 +1 1000 Induction of MT Gene Transcription • Structural MT gene: three exons interrupted by two introns • Chromosome 8 (mouse) and Chromosome 16 (human) All of these inducers are immunomodulatory

7. Syntenic relationships between metallothionein gene clusters in humans and mice mouse human

8. A summary of metallothionein functions • Intracellular functions • decreases toxic effects of heavy metals • acts as a free radical scavenger, regulates cellular redox potential • serves as a reservoir for essential heavy metals • regulates NF-kB, Sp-1 transcription factor activity • Extracellular functions • Redistribution of metal cations within body • Interactions with membrane bound receptors • Reports of an astrocyte receptor • Interactions with megalin (surface molecule on kidney cells) • Hypothesis: Metallothionein that is synthesized as a result of stress can alter the capacity of the immune system, and manipulation of metallothionein can influence adaptive and innate immune activities and immune-related diseases.

9. Metallothionein: an extracellular pool Secretome P analysis http://www.cbs.dtu.dk/services/SecretomeP/ MT1A_HUMAN” predictions NN-score Odds Weighted 0.835 4.229 0.008 Non-classically secreted proteins should obtain an NN-score exceeding the normal threshold of 0.5. MT has been found in serum, urine, pancreatic acini, liver sinusoids, glomeruli, etc.

10. Targeted disruption of Mt1 and Mt2 genes decreasesPtpn6me-v lifespan 50% survival Ptpn6me-v or “viable motheaten” is a mutation in a cytosolic protein tyrosine phosphatase negative regulator of immune function that causes congenital inflammation Wild type and congenic mutant pups Mutant adult

11. Metallothionein is detectable on the surface of viable motheaten splenocytes UC1MT-FITC binding to splenocytes from mev/mev and +/mev mice

12. Divalent heavy metal cations (Zn or Cd) induce Metallothionein in T cells Jurkat-T cells (1x106 cells/ml) were cultured in 24-well plates in RPMI-1640 supplemented with 20 µM Cd, 100 µM Zn, or vehicle control for 6 hours. After incubation, cells were fixed. Cells were then treated with UC1MT (IgG1) or isotype-matched MOPC21 and then stained with goat-anti-mouse IgG-FITC. Cells were mounted using InvitrogenProLong Gold and analyzed using a Leica SP2 spectral confocal microscope.

13. 80 60 40 ova ova/mt 20 10 12 14 16 18 20 22 Exogenous extracellular metallothionein-mediatedhumoral immunosuppressionin vivo mOD/min 0 days Collect serum Mice were injected with 200 ug OVA with or without the addition of 120 ug MT on day 0 and day 10. Samples obtained on the days indicated were used in ELISA to determine the anti-OVA activity. Results are representative of three independent experiments and are reported as the average of triplicates + s.d.

14. Monoclonal anti-metallothionein Ab (clone UC1MT) enhances the humoral response to OVA immunization 300 OVA OVA w/ UC1MT OVA w/ Ig Control 250 200 150 anti OVA response (mOD/min) 100 50 0 0 14 18 21 25 32 35 43 days BALB/cByJ mice were challenged with 200 ug OVA in the presence or absence of UC1MT or isotype control on day 0 and day 10. (similar results were observed whenthe immunogen used was synthetic peptide conjugated to carrier protein)

15. How might this work? • Intracellular MT is critical both as a metal reservoir, as an antioxidant and as a transcription factor regulator • Extracellular MT may interact with membrane receptors and alter immune cell behaviors (e.g. proliferation and cellular trafficking) • The extracellular pool is amenable to manipulation with antibody

16. Sequence comparison of MT with a chemotactic factor, Ccl17 Amino acids compared at a threshold of “85% similarity” are colored grey, boxed amino acids are identical. CCL17 or TARC (thymus and activation regulated chemokine), belongs to the IL8-like chemokine family, and maps close to the MT gene cluster. It induces chemotaxis in T cells and binds CCR4 receptor

17. Measuring chemotaxis: ECIS/taxis electrode design ~ Cell well Contact Pads Target electrode (~5x10-4 cm2) Wiring Chamber Large Chemoattractant Electrode (~0.12 cm2) Well Circuit: 1 volt AC with 1Mohm resistor applied to each well sequentially every x sec. Resistance at the small electrode dominates the circuit due to its small size relative to the large electrode.

18. Single ECIS chamber: side view Diffusing chemoattractant from well Cell Well Agarose matrix Migrating cells L a r g e T a r g e t E l e c t r o d e E l e c t r o d e To ECIS Instrumentation

19. ECIS/taxis- automated measurement of dictyosteliumfolate chemotaxis Migrating cells Impedance measurements Target electrode Diffusing chemoattractant

20. Metallothionein induces leukocyte chemotaxis Metallothionein and SDF-1a evoke a chemotactic response in Jurkat T cells Both cholera toxin and pertussis toxin block the MT-mediated chemotactic response (suggesting a GCPR-type receptor target)

21. Summary thus far: • Chronic inflammation can be associated with MT expression • MT can bind to lymphocyte surfaces, and lymphocytes can also make MT • MT has structural features that are shared with chemokines (chemotactic cytokines) • Metallothionein can act as a chemotactic agent and may act through G protein coupled receptor(s) • Manipulation of MT in mouse models of congenital inflammation changes the course of disease

22. Chromosome 16 (IBD1 locus): 55.156 K 55.180 K 55.200 K MT1C MT3 MT1L MT1E MT1K MT1J MT1A MTM MT1B MT1F MT1G MT1H MT1I MT1X MT4 MT2A MT1D 2000 bases How might MT relate to inflammatory bowel disease? • The MT gene cluster is located at an important locus associatedwith IBD (this is the most replicated locus ever found associated with IBD and also contains NOD2).

23. MT functions relevant in IBD. IBD is characterized by the presence of an increased level of ROS in the mucosal intestinal tissue as well as oxidative DNA and protein damage, defective host-microbe interactions, immune cell infiltration, and a disturbed T cell apoptosis. On all of these elements, MTs can have effects. In addition, MTs can have a dual role in enzyme activation through the release or sequestration of zinc. Finally, MTs are reported to regulate the activation of the transcription factor NF- B, which has a key role in inflammatory responses. AnoukWaeytens, Martine De Vos, and Debby Laukens http://dx.doi.org/10.1155/2009/729172

24. Metallothioneins in clinical samples of IBD: Crohn’s Disease/Ulcerative Colitis AnoukWaeytens, Martine De Vos, and Debby Laukens http://dx.doi.org/10.1155/2009/729172

25. Mouse ModelsAvailable Congenic strains of C57BL/6J • Wild Type Control (MT-WT) – C57BL/6J • MT transgenic (MT-TgN) - Tg(Mt1)174Bri / 174Bri • MT transgenic (MT-TgN het) - Tg(Mt1)174Bri / - • MT knockout (MT-KO) - Mt1tm1Bri Mt2tm1Bri

26. What is the role of endogenous MT in experimental colitis? 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 …. Dextran sulphate sodium-induced colitis - ACUTE Dextran sulphate sodium-induced colitis - CHRONIC MT knockoutand wild type mice in DSS-colitis 4% DSS 4% DSS H2O H2O 4% DSS x3

27. MT knockout mice are favored during DSS-colitis ACUTE COLITIS

28. MT knockout mice show reduced leukocyte infiltration P=0.06

29. MT knockout mice develop a less severe phenotype during DSS-colitis CHRONIC COLITIS

30. Anti-MT antibodytherapy in DSS- and TNBS-colitis Days DSS-colitis Days 100 mg UC1MT or IgG i.p. 0 1 2 3 4% DSS H2O 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 100 mg UC1MT or IgG i.p. samples Randomize TNBS IR randomize samples TNBS-colitis

31. UC1MT in acute DSS-colitis

32. UC1MT in acute TNBS-colitis

33. What is the site of action of the UC1MT antibody? Approach: small animal imaging

34. Small animal imaging - µSPECT-CT Indium 111 DOTA Monoclonal UC1MT injection 4 control mice 4 colitis mice, day 7 4 colitis mice, day 14 µSPECT-CT andautoradiography 2 days later

35. Healthy Inflammation Healing SPECT/CT data: Colon kidney Quantifyingradioactivityin the colon Autoradiography of colon section Intensityscale Prox Mid Dist

36. Genetic deletion of MT and antibody-mediated MT inhibition dampens experimental colitis, characterized by reduced leukocyte infiltration UC1MT antibody binds the inflamed colon during colitis Cellular release of MT?

37. MT release from stressed/damaged HT29 cells HT29 cells • CELL DEATH • TNF/IFN • Staurosporine • Freeze/thawing • PRO-INFLAMMATORY STIMULI • LPS • H2O2 • TNF APOPTOSIS NECROSIS Does the supernatantcontainbioactive MT?

38. Metallothioneins are released from necrotic HT29 cells 6 kDa LPS H2O2 TNF 2µM stauro 10µM stauro INF Freeze/thawing

39. Will endogenous, released MT attract leukocytes? Boyden chamber migration assay 500.000 blood isolated leukocytes + anti-MT antibody (100 μg/ml UC1MT) MT containing conditioned medium

40. Endogenous released MT acts as potent chemokine

41. MTs are released from necrotic intestinal epithelial cells • Released MTs acts as potent chemokine in vitro • This chemotactic function can be blocked in vitro by monoclonal therapy

42. Metallothioneins act as dangersignals in the gut • ‘Find-me’ signals • Dimer of ribosomal protein S19 • Endothelial monocyte-activating polypeptide II • Fragments of human tyrosyl tRNA synthetase • Thrombospondin 1 • Soluble IL-6 receptor • Fractalkine • Lysophosphatidylcholine • Sphingosine-1-phosphate • Nucleotides • Lactoferrin • Apoptotic micro-blebs • DAMPs • High mobility group box 1 protein • Hepatoma-derived growth factor • Calgranulin proteins • Heat-shock proteins • ATP • IL-6 • Uric acid • Metallothioneins Kono and Rock 2008, Nature reviews; Peter et al. 2010, Apoptosis

43. Metallothioneins function as chemotactic danger signals and represent a novel target to dampen inflammation by reducing leukocyte infiltration in mice models for inflammatory bowel diseases Pending patent: P10/099: The use of antagonists targeting metallothionein to treat intestinal inflammation

44. MT expression in human IBD? Ileal MT expression Paneth cell

45. Colonic MT expression Healthy control Ulcerative Colitis Colonic Crohn’s Disease

46. MTs are mainlyexpressed in the colonic epithelium MT immunoreactivityshiftsfrommainlyepithelialto the inflammatoryinfiltrateduring colitis Positivecorrelationbetween the severity of colitis andlamina propria MT immunoreactivity No correlationbetweenepithelial MT immunoreactivityand the grade of colitis but MT expression is absent in highlynecroticregions

47. Ongoing studies/UGent: Inductionand release of MT frommacrophages Effect of MT on macrophagepolarization LPS response of BM-derivedmacrophagesfrom MT-KO and WT mice Anti-MT antibodytreatment in T cell transfer – inducedcolitis Effect of anti-MT treatment on lymphocyteproliferation

48. Ongoing studies/Uconn: Role of MT in management of the intracellular Zn pool and immune activity Influences of MT in Cd-mediated immunomodulation Bacterial MT analog (SmtA, Pseudomonas aeruginosa) anditsrole as virulence factor Collaboration: UC1MT influences on Epidermolysis BullosaAquisita Grating-coupledSurface Plasmon Resonance (GCSPR) andGrating-coupledSurface Plasmon CoupledFluorescence (GCSPCE) microarrays and the detection of (a) toxinsandtoxicants, (b) polymicrobialinfections, (c) functional T cellphenotypes in T1D and (d) biomarkersignatures of post traumatic stress disorders

49. Next steps: I. in animal model(s) 1. identification of MT-specific or MT-selective receptors (presumptive G-protein coupled receptors for chemotaxis response) 2. determine cellular signaling cascades altered by MT 3. determine if MT effects influences the microbiome of IBD mice II. in human patients 1. determine if MT expression levels (promoter occupancy, propensity to synthesize MT, etc) correlates with disease severity 2. map the distribution of MT within the IBD wound sites (hypothesis that MT levels in the most severely damaged tissue is down due to the ROS-mediated destruction of MT antigenicity) 3. characterize the effect of extracellular MT on released cytokines and leukocyte proliferation in situ