Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/385—Haptens or antigens, bound to carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70539—MHC-molecules, e.g. HLA-molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/605—MHC molecules or ligands thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• the present invention relates to therapeutical uses of non-classical human major histocompatibility complex (MHC) molecules (also named MHC class lb molecules) in combination with peptide antigens for the treatment of Parkinson's disease.
• MHC human major histocompatibility complex
• the invention more specifically relates to recombinant polypeptides comprising peptide antigens and one or more domains of a non-classical MHC class lb molecule.
• the invention also relates to methods of producing such recombinant polypeptides, pharmaceutical compositions comprising the same, as well as their uses for treating Parkinson's disease.
• Parkinson's disease is the most common neurodegenerative movement disorder with over 7 million people affected worldwide and still no cure existing. Patients suffer from severe motor and non-motor symptoms such as tremor, bradykinesia, rigidity, postural instability, and depression. Pathologic disease hallmarks are degeneration of dopaminergic neurons in the substantia nigra and formation of o-Synuclein- containing protein aggregates called Lewy-bodies. While the physiological role of o-Synuclein (aSyn) points towards functions in synaptic transmitter release and amplification of immune responses (Alam et al, Cell Reports 38:110090.
• Autopsies of human PD brains are characterized by microglia activation and T cell infiltration in the substantia nigra (Brochard V et al., 2009;McGeer PL et al., 1988).
• Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-o, interferon (IFN)-y and interleukins IL-1 p and IL-6 in the nigrostriatal system further confirm that PD goes along with significant neuroinflammation (Mogi M et al., 1994;Mogi M et al., 1994).
• PD therapy mostly relies on compensating for SN neuron loss by administering L-Dopamine or derivative substances. This intervention initially improves disease symptoms, but does not prevent disease progression.
• T cells in PD have mostly been analyzed in toxin-based models such as the 1 -methyl-4- phenyl-1 ,2,3,6-tetrahydropyridine (MPTP) or the 6-hydroxydopamine (6-OHDA) models.
• MPTP 1 -methyl-4- phenyl-1 ,2,3,6-tetrahydropyridine
• 6-OHDA 6-hydroxydopamine
• insoluble aSyn aggregates that show S129 phosphorylation and Lewy-like neurites and bodies are detectable in this animal model (Ip CW et al., 2017).
• the haSyn PD model thus provides an important tool to advance the preclinical and translational work towards new and better therapies for patients with PD.
• the inventors confirmed a broadly pro-inflammatory profile of brain immune cells in this haSyn PD model, and demonstrated infiltration of CD4 + and CD8 + activated T cell subsets into the brain of haSyn PD mice. These T cells induced neurodegeneration with PD-like symptoms.
• T cell deficiency (RAG-1 7 ) reduced dopaminergic neurodegeneration, while T cell reconstitution aggravated neuronal loss in vivo.
• T cells isolated from brains of haSyn PD mice were cytotoxic towards haSyn-expressing neuronal cells in vitro (Karikari et al., Brain Behav Immun. 2022 Mar; 101 : 194-210.)
• Immunosuppressive MHC class lb molecule such as HLA-G are critical for tolerance induction during pregnancy. They exert immunosuppressive effects on various immune cells via immunosuppressive receptors such as ILT2, ILT4 and Kir2DL4.
• WO 2018/215340 relates to combinations of MHO class lb molecules and peptides for targeted therapeutic immunomodulation but remains silent on the treatment of Parkinson's disease.
• MHO class lb molecules such as HLA-G possess the ability to induce antigen-specific tolerance towards presented peptide antigens.
• MHO class lb molecules can advantageously be used according to the invention to suppress immune responses in an antigen-specific manner.
• molecules other than naturally occurring MHC class lb molecules and in particular polypeptides which only comprise at least one domain of an MHC class lb molecule, preferably at least an [alpha]3 domain of an MHC class lb molecule, can be used:
• the [alpha]1 and [alpha]2 domains of variable class I a molecules can be combined with the [alpha]3 domain of a human MHC class lb molecule in order to suppress immune responses towards peptides presented by these antigens.
• Antigen-loaded HLA-G molecules can be unstable.
• the inventors designed soluble recombinant polypeptides comprising a peptide antigen, an MHC class lb molecule such as HLA-G and p2-microglobulin (b2m), and connected these three components covalently (e.g., via covalent linkers).
• the antigen-binding a1 and a2 domains of an MHC class lb molecule such as HLA-G were exchanged by the respective domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (see, for instance, Figure 2).
• These alternative recombinant polypeptides can be designed with antigen-binding domains of other human HLA molecules.
• constructs comprising the a1 and a2 domains of murine H2-K b can present the ovalbumin-derived peptide SIINFEKL to OT-1 T cells.
• OT-1 T cells express a transgenic T cell receptor that specifically recognizes this antigen) (WO 2018/215340).
• Parkinson's disease is a neurodegenerative disease caused by pathogenic accumulation of alpha-Synuclein.
• the more recent concept that crucial pathogenic events are due to immune responses against alpha-Synuclein has not yet been translated towards therapeutics development.
• the inventors have found that by suppressing immune responses against alpha-Synuclein using surrogates of the recombinant polypeptides of the invention in a mouse model which faithfully mimics human Parkinson's disease, a therapeutic effect in the treatment of Parkinson's disease can be achieved.
• Parkinson's disease can be treated by the recombinant polypeptides of the invention.
• the recombinant polypeptides of the invention are expected to be highly advantageous in the immunoterapeutic treatment of Parkinson's disease, because they cause hardly any systemic immunosuppression.
• early-stage patients suffering from Parkinson's disease should not be exposed to the strong side effects of systemic immunosuppression, as this would likely result in opportunistic and potentially deadly infections.
• late-stage patients however, neurodegeneration is often irreversible.
• a highly specific tolerance induction achieved by presenting alpha-Synuclein antigens on the recombinant polypeptides of the invention would, however, be already tolerable at early stages of disease such as prodromal Parkinson's disease, which will open new therapeutic avenues.
• the invention relates to the following preferred embodiments:
• a recombinant polypeptide capable of presenting a peptide antigen comprising, in an N- to C-terminal order,
• a peptide antigen presented by said recombinant polypeptide wherein the peptide antigen is a peptide of human alpha-Synuclein; ii) optionally a linker sequence; ill) optionally a sequence of a human polypeptide domain comprising a sequence of a human p2 microglobulin, or an amino acid sequence at least 90% identical to the amino acid sequence of human p2 microglobulin represented by SEQ ID NO: 5; iv) optionally a linker sequence; v) optionally an [alpha] 1 domain of an MHO molecule; vi) optionally an [alpha] 2 domain of an MHO molecule; vii) an [alpha] 3 domain of an MHO class lb molecule or a derivative of an [alpha] 3 domain of an MHO class lb molecule, said derivative being capable of binding to ILT2 or ILT4; viii) optionally a protease cleavage site; lx) optionally a
• peptide antigen consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.
• the recombinant polypeptide according to item 3 wherein said peptide antigen consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, and SEQ ID NO: 33.
• the recombinant polypeptide according to item 3 wherein said peptide antigen consists of the amino acid sequence of SEQ ID NO: 20.
• the recombinant polypeptide according to item 3, wherein said peptide antigen consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, and SEQ ID NO: 33.
• the recombinant polypeptide according to item 8 wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human MHC class la molecule.
• the recombinant polypeptide according to item 10 wherein said [alpha] 1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human HLA-A2 molecule.
• the recombinant polypeptide according to item 10 wherein said [alpha]1 domain according to (v) and said [alpha]2 domain according to (vi) are from a human HLA-A11 molecule.
• the recombinant polypeptide according to item 16 wherein the [alpha]3 domain according to (vii) is identical to the [alpha]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.
• the recombinant polypeptide according to item 23 wherein the linker sequence according to (ii) comprises the amino acid sequence (GGGGS)n, and wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and is preferably selected from the group consisting of 2, 3, 4 and 5.
• the recombinant polypeptide according to any one of the preceding items wherein said sequence of a human polypeptide domain according to (ill) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5 and more preferably identical to the amino acid sequence of SEQ ID NO: 5.
• a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46; and
• a pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1-33.
• composition or kit according to item 37, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1-33, and wherein each of the different polypeptides comprises a different peptide antigen as defined in any one of items 3 to 7.
• a recombinant host cell comprising a nucleic acid or a vector according to item 34 or 35 and expressing the recombinant polypeptide according to any one of items 1-33.
• a method for obtaining pharmaceutical composition comprising a polypeptide according to any one of items 1-33, the method comprising the steps of (a) culturing the recombinant host cell of item 43 under conditions allowing expression of the recombinant polypeptide from the nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition.
• Figure 1 Depiction of a peptide-loaded soluble MHO lb molecule suitable to achieve therapeutic antigenspecific immunomodulation.
• HLA-G1 and HLA-G5 each consist of 3 [alpha] domains (here in black), a non-covalently associated beta 2- microglobulin subunit (here in dark grey) and the antigenic peptide presented on HLA-G (short black arrow).
• HLA-G1 further contains a transmembrane domain and a short intracellular chain (not shown here).
• the [alpha]-3 domain is capable of binding to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci U S A. 2003 July 22; 100(15):8856-8861 ) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci U S A.
• MHC class 1 2006 Oct 31; 103(44): 16412-7
• these sequences form a non-covalently linked MHC class 1 complex.
• one or more protein tags such as SpotTag, myc tag and/or His(6x) tag
• they may be introduced in such a way as to enable their later optional removal via cleavage using an optional Factor Xa cleavage site.
• the antigenic peptide, beta 2-microglobulin and MHC lb [alpha]chain can be linked in order to increase the stability.
• the vector map was generated using Snapgene Viewer Software.
• Splenocytes from two initially non-responsive haSyn mice and two controls were cultured for 10 days in the presence of all 10 peptides (1 pig/ml each) in RPMI with 10% FCS and 20 ng/ml IL-2.
• Multiscreen PVDF plates (MSIPS4510, Millipore) were activated with 35% ethanol, washed with sterile PBS and coated overnight with 2 pig/ml filtered anti-mouse IFN-y antibody (AN18, Mabtech) at 4 °C.
• Isolated single cell suspensions from cervical lymph nodes and spleen were plated with or without 5 pig/ml synthetic peptide in IMDM with 7.5% FCS.
• IFN-y spots were then detected with 2 pig/ml biotinylated anti-mouse IFN-y antibody (R4-6A2, Mabtech), followed by horseradish peroxidase-conjugated streptavidin (Cell Signaling) in PBS-T supplemented with 0.05% BSA.
• the assay was developed with filtered "ready to use” TMB substrate (Mabtech) and analyzed using an Immunopost S6 Core Analyzer Figure 4: aSyn-loaded AIM Bio surrogate molecules prevent neuron death in PD model and may improve endurance.
• Polypeptides comprising a peptide, here an aSyn peptide, MHC class 1 antigen presenting domains and HLA- G alpha3 domain (AIM Bio) treatment once a week for 9 cycles resulted in a significant, dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice compared to vehicle-treated haSyn PD mice with significantly higher numbers of tyrosine hydroxylase (TH) + neuron numbers in the substantia nigra of the treated groups.
• Immunohistochemical stainings were performed using 40 pm PFA-fixed cryosections processed for unbiased stereology (TH) covering the whole SN.
• C Latencies relative to pre-op, assessed by the Rotarod test.
• Figure 5 aSyn-loaded AIM Bio surrogate molecules prevent infiltration of CD11b + myeloid cells (microglia) but induce infiltration of CD8 + T cells into the striatum in PD model.
• Immunohistochemical stainings of mouse tissue for CD11 b + myeloid cells and CD8 + T cells were performed using 10 pirn fresh coronal cryo-sections of the striatum. After 4% PFA fixation or aceton fixation, respectively, sections were incubated with either rat anti-mouse CD11b (1 :100, Serotec) or rat anti CD8 MCA609G 1 :500 antibodies, respectively, followed by biotinylated rabbit anti-rat secondary antibodies (Vector Labs). Development was done using (DAB)-HCI-peroxidase (Vector Labs). Microglia were quantified at a magnification of 200x in the region of the SN and striatum as depicted by consecutive sections stained for TH on a BH2 light microscope (Olympus).
• B quantification.
• D quantification.
• Figure 7 aSyn-loaded AIM Bio protect Tyrosine Hydroxylase - fibers in the striatum and substantia nigra neurons
• B quantification.
• C Repeat experiment of Figure 4 using an alternative Nissl staining to show neurons.
• FIG. 8 aSyn-loaded AIM Bio induce CD8 + CD122 + regulatory T cells
• CD8 + CD122 + regulatory T cells both in the spleen and cervical lymph nodes was observed in response to aSyn-loaded AIM Bio treatment.
• Frozen sections were fixed with aceton for 10min at -20 °C, washed and blocked with 5% BSA; 5% NGS, 0.2% Triton-X100 in PBS for 1 h.
• CD122 was stained with antiCD122i rabbit polyclonal antibody (MyBiosource, 1 :100) and anti-CD8i clone YTS191.1 rat (Biorad, 1:200) in 1% BSA; 1% NGS, 0.2% Triton-X100 over night.
• Secondary andibody staining used anti-rabbit-Cy3 (Dianova, 1 :300) and anti-rat-AF488 (Invitrogen, 1 :300) for 1 h.
• Figure 9 Stability of purified single-chain MHC lb molecules. After purification of the single chain MHC lb molecules, their stability was analysed after 1 and 3 freeze-thawing cycles, storage for 5 days at room temperature and heating up to a temperature of 50°C for 30 min. For this, A) a Coomassie gel staining of a 12% polyacrylamide gel using 2 pig AIM Bio and B) an aHLA-G Western blot using the 2A12aHLA-G antibody (1 :1000) blot using 1 pig protein was performed under non-reducing conditions. Both monomers and dimers are detectable.
• FIG. 10 Single-chain MHC lb molecules are thermally stable.
• TSA Thermal Shift Assay
• 3 pig of the respective single chain MHC lb molecule or Motavizumab as control molecule were diluted with PBS and 5x SYPRO Orange dye (stock 5000x, final concentration: 5x) to a volume of 25 pil.
• a melting curve program was set up on a StepOnePlus Instrument using the StepOnePlus Software 2.3. The start temperature was 25°C for one minute followed by a temperature increase of 1 °C per minute to a final temperature of 95°C for 2 min, thereby measuring the autofluorescence as arbitrary unit. Data were exported and graphs were drawn in Prism V7.04. For determination of the melting temperature (Tm), the Boltzman sigmoidal function was used.
• FIG. 11 Single-chain MHC lb molecules induce Treg in a dose-dependent manner.
• OT-I mice were injected i.p. with indicated amounts of single-chain H2_K b alphal +2 and HLA-G alpha3 domain constructs with human beta-2-microglobulin and the indicated peptide or carrier (PBS).
• Ova is the cognate peptide for the OT-I TOR in these mice, Gp34 is an irrelevant, virus derived control peptide.
• mice were sacrificed and splenocytes tested for IL10 secreting cells in a recall mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 pig/ml of the indicated peptide or only PBS were added, 48h).
• a recall mouse IL-10 ELISpot 200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 pig/ml of the indicated peptide or only PBS were added, 48h.
• a clear induction of IL-10 secreting cells reactive to Ova peptide was observed when 50 and 500 pig mouse adapted Ova_KbG were injected.
• FIG. 12 Single-chain MHC lb molecules inhibit T cell lysis in a dose-dependent manner.
• OT1/BL6 Mice were sacrificed and splenocytes were collected and washed once in RPMI 5% FCS. Red blood cells were removed with 2ml 1x sterile RBC lysis buffer for 3 min.
• Cells were cultured in high density culture (10mio cells/ml) for 72h in RPMI 10% FCS medium with GMCSF 20 ng/mL, IL-2 20ng/ml and IL-4 10 ng/ml and increasing doses of Ova_KbG. Cells are then scraped from the plates, CD8+ cells are then purified via magnetic beads.
• Luciferase expressing Panc02 target cells were loaded with 20pig/ml Ova peptide (SIINFEKL) for 60 min at 37°C with 500 rpm shaking.
• CD8+ effector T cells were added in a 50:1 ratio, as well as luciferin. Luminescence was measured after Oh, 24h, 48h.
• FIG. 13 Single-chain MHC lb molecules induce expression of IL-10 in EAE-ODC Ova mice.
• Serum cytokines from EAE-ODC Ova mice were measured with Th1/Th2 10plex Flowcytomix Kit (eBioscience) according to the manufacturer's instruction. The kit was used for the simultaneous detection of mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1 alpha (IL-1 a), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), t interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-a) in a single sample.
• GMCSF mouse granulocyte-macrophage colony-stimulating factor
• IL-1 a interleukin-1 a
• IL-2 interleukin-2
• IL-4 interleukin-4
• IL-6 interleukin-6
• FIG. 15 Thermal Shift Assay.
• TSA Thermal Shift Assay
• 3 pig of the respective single chain MHC lb molecule were diluted with PBS and 5x SYPRO Orange dye (stock 5000x, final concentration: 5x) to a volume of 25 pil.
• a melting curve program was set up on a StepOnePlus Instrument using the StepOnePlus Software 2.3. The start temperature was 25°C for one minute followed by a temperature increase of 1 °C per minute to a final temperature of 95°C for 2 min with autofluorescence being measured throughout the process. Data were exported and graphs were drawn in Prism V7.04.
• the melting temperature (Tm) was determined via the Boltzmann sigmoidal function. The high melting temperatures indicate good protein stability for therapeutic use.
• Figure 16 Stability of polypeptide constructs of the invention (A, B).
• sc HLA-A2 or A11 alpha1-2 HLA-G aplha3 constructs with indicated peptides were purified via spot-cap purification according to the manufacturers protocol, followed by ON dialysis. Constriucts then underwent 3 freeze-thaw cycles, were heated to 50 °C for 30min or kept for 6 days at room temperature. Degradation was then analyzed on a Coomassie genl (12% gel, 4% stacking gel, 1 pig purified protein, non-reducing conditions, 15 pil sample +5 pil 4x Laemmli.
• Roti Mark TRICOLOR XTRA (Roth, 2244) was used for coomassie staining for 5-6 h. Destaining was done with 12.5 ml Methanol + 37.5 ml H2O dest for 5 min, another round of destaining with H2O dest
• FIG. 17 ELISpot results from healthy donor PBMCs. Induction of regulatory, IL-10 secreting cells through indicated constructs of the invention over a period of 14 days in PBMCs from healthy donors is shown
• Figure 18 IL-10 ELISpot results from parkinson patients (A) and healthy/age matched individuals (B). Induction of regulatory, IL-10 secreting cells through indicated constructs of the invention and age-matched healthy donors (01-03) or normal healthy donors (B10, B12, B13) over a period of 14 days is shown.
• All proteins in accordance with the invention including the recombinant polypeptides of the invention, can be obtained by methods known in the art. Such methods include methods for the production of recombinant polypeptides.
• the recombinant polypeptides of the invention can be expressed in recombinant host cells according to the invention.
• Recombinant host cells of the invention are preferably mammalian cells such as CHO and HEK cells.
• the recombinant polypeptides of the invention are meant to optionally include a secretion signal peptide sequence.
• the recombinant polypeptides of the invention are meant to also optionally include affinity tags, e.g. in order to facilitate purification, and optional protease cleavage sites between the tag and the polypeptide, e.g. in order to facilitate removal of the tags by protease cleavage.
• any reference to amino acid sequences referred to herein is meant to encompass not only the unmodified amino acid sequence but also typical posttranslational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, the clipping of particular amino acids or other posttranslational modifications) occurring in cellular expression systems known in the art, including mammalian cells such as CHO and HEK cells.
• polypeptides of the invention are meant to optionally include the respective pro-peptides.
• the recombinant polypeptides of the invention can be in form of their soluble or their membrane-bound form. Whether a recombinant polypeptide is "soluble” under these conditions can be determined by methods known in the art, e.g., by measuring the turbidity of the recombinant polypeptide under the above-indicated reference conditions. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.
• Single chain MHC molecules can be stored, for instance, in PBS at -80°C (with or without 0.1% human albumin as carrier, depending on the protein concentration) or in 50% glycerol at -20°C.
• MHC molecules are preferably human MHC molecules.
• the recombinant polypeptides of the invention are preferably isolated recombinant polypeptides. It will be understood how a recombinant polypeptide capable of binding and presenting an peptide antigen according to the invention can be prepared.
• peptide antigen-binding domains such as [alpha] 1 and [alpha]2 domains are well-known, and modifications of these domains can be made.
• a peptide antigen to bind to the polypeptides and MHC molecules according to the invention can be determined by techniques known in the art, including but not limited to explorative methods such as MHC peptide elution followed by Mass spectrometry and bio-informatic prediction in silico, and confirmative methods such as MHC peptide multimere binding methods and stimulation assays.
• the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in a human patient.
• the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in the treatment of Parkinson's disease in a human patient.
• the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immunological tolerance against human alpha-Synuclein, e.g., in a human patient.
• any lenghts of these peptide antigens referred to herein are meant to refer to the length of the peptide antigens themselves.
• the lenghts of peptide antigens referred to herein do not include the length conferred by additional amino acids which are not part of the peptide antigens such as additional amino acids from possible linker sequences etc.
• each occurrence of the term “comprising” may optionally be substituted with the term “consisting of'.
• the methods used in the present invention are performed in accordance with procedures known in the art, e.g. the procedures described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.”, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology.” Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane (“Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.
• Protein-protein binding such as binding of antibodies to their respective target proteins, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.
• binding of MHC class lb molecules or recombinant polypeptides according to the invention to their receptors, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy measurements. More preferably, binding of MHC class lb molecules or recombinant polypeptides according to the invention to their receptors is assessed by surface plasmon resonance measurements at 25°C. Appropriate conditions for such surface plasmon resonance measurements have been described by Shiroishi et al., Proc Natl Acad Sci U S A. 2003 July 22; 100(15):8856-8861 .
• Sequence Alignments of sequences according to the invention are performed by using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool.” Journal of Molecular Biology 215. p. 403-410.; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.).
• Appropriate parameters for sequence alignments of short peptides by the BLAST algorithm which are suitable for peptide antigens in accordance with the invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignments for a short input sequence.
• the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment.
• identity or “identical” preferably refer to the identity value obtained by using the BLAST algorithm.
• compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions.
• compositions are prepared in a way that they can be stored and administered appropriately.
• the pharmaceutical compositions of the invention may therefore comprise pharmaceutically acceptable components such as carriers, excipients and/or stabilizers.
• Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition to a human patient.
• the pharmaceutical acceptable components added to the pharmaceutical compositions may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical compositions and the route of administration.
• compositions comprising the nucleic acids of the invention may also be formulated in accordance with knowledge available in the art, e.g. using liposomal formulations targeting dendritic cells.
• peptide antigens which can be used in accordance with the invention are not particularly limited other than by their ability to be presented on MHC molecules. It is understood that a "peptide antigen presented by said recombinant polypeptide” as referred to in relation to the invention is a peptide antigen that is presented by said recombinant polypeptide to human T cells, if such T cells are present, in a way that it binds to a T cell receptor on the human T-cells.
• MHC molecules which are able to be presented on MHC molecules can be generated as known in the art (see, for instance, Rammensee, Bachmann, Emmerich, Bachor, Stevanovic. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9; Pearson et al. MHC class l-associated peptides derive from selective regions of the human genome. J Clin Invest. 2016 Dec 1 ; 126(12):4690-4701 ; and Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov;37(11)724-737).
• Peptide antigens are generally known in the art.
• the peptide antigens in accordance with the invention are capable of binding to MHC class I proteins. It will be understood by a person skilled in the art that for each MHC class lb molecule or polypeptide capable of presenting peptides in accordance with the invention, peptide antigens which are capable of binding to said MHC class lb molecule or recombinant polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.
• Binding of peptide antigens to MHC class lb molecules or to polypeptides capable of peptide antigen binding in accordance with the invention can be assessed by methods known in the art, e.g. the methods of:
• Such methods include experimental methods and methods for the prediction of peptide antigen binding.
• Anchor residues which serve to anchor the peptide antigen on the MHC class I molecule and to ensure binding of the peptide antigen to the MHC class I molecule are known in the art.
• the peptide antigen used in accordance with the invention contain any of the anchor or preferred amino acid residues in the positions as predicted for MHC class I molecules.
• the peptide antigen is from human alpha-Synuclein.
• non-anchor amino acid residues of the peptide antigen of the invention may or may not contain conservative substitutions, preferably not more than two conservative substitutions, more preferably one conservative subsitution with respect to the corresponding amino acid sequence of a peptide antigen from human alpha-Synuclein.
• Peptide antigens of the invention preferably consist of naturally occurring amino acids. However, non-naturally occurring amino acids such as modified amino acids can also be used.
• a peptide antigen of the invention encompasses the peptidomimetic of the indicated peptide antigen amino acid sequence of human alpha-Synuclein.
• Sequences Preferred amino acid sequences referred to in the present application can be independently selected from the following sequences.
• the sequences are represented in an N-terminal to C-terminal order; and they are represented in the one-letter amino acid code.
• Leader Peptide (absent from the recombinant polypeptide due to processing during cellular expression): e.g. MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1)
• Peptide antigen any MHO class I peptide corresponding to MHO class I [alpha] 1&2 domains, e.g. GAPQEGIL (SEQ ID NO: 20)
• First linker For instance GGGGSGGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4) beta 2 Microglobulin, for instance:
• Second Linker for instance:
• [Alpha] 1 & 2 domain derived either from human HLA-G or from any other MHC class I [alpha]1&2 domain suitable to present the selected antigenic peptide, Y84 may be C in DT variant e.g. [Alpha] 1 & 2 domain derived from human HLA-G: E.g.: GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAA QISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 7)
• Human HLA-A2 [alpha]1 & 2 domain E.g.:
• Human HLA-G [alpha]3 domain (or any MHC lb [alpha]3 domain, such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for instance:
• a shorter form of a human HLA-G [alpha]3 domain may be used which lacks the optional C- terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47), i.e. :
• IEGRTGTKLGP SEQ ID NO: 10.
• Spacer sequence e.g. NSAVD (SEQ ID NO: 14) or GS
• exemplary peptide antigens which can be part of the recombinant polypeptides of the invention are as follows:
• GAPQEGIL (SEQ ID NO: 20), preferably used in recombinant polypeptides containing human HLA-G [alpha] 1 & 2 domain
• KTKEGVLYV SEQ ID NO: 28
• KTKEGVLYV SEQ ID NO: 28
• AWTGVTAV (SEQ ID NO: 33) , preferably used in recombinant polypeptides containing human HLA-A2 [alpha] 1 & 2 domain
• GWHGVTTV (SEQ ID NO: 31), preferably used in recombinant polypeptides containing human HLA-A2
• MDVFMKGLSK (SEQ ID NO: 22), preferably used in recombinant polypeptides containing human HLA-A11 [alpha] 1 & 2 domain
• GWAAAEKTK (SEQ ID NO: 25), preferably used in recombinant polypeptides containing human HLA-A11 [alpha] 1 & 2 domain
• MPVDPDNEAY (SEQ ID NO: 21), preferably used in recombinant polypeptides containing human HLA-A1 [alpha] 1 & 2 domain
• PVDPDNEAY (SEQ ID NO: 44), preferably used in recombinant polypeptides containing human HLA-A1 [alpha] 1 & 2 domain SIAAATGFV (SEQ ID NO: 37), preferably used in recombinant polypeptides containing human HLA-A2 [alpha] 1 & 2 domain
• WTGVTAVA (SEQ ID NO: 34), preferably used in recombinant polypeptides containing human HLA-A2 [alpha] 1 & 2 domain
• WAAAEKTK (SEQ ID NO: 26), preferably used in recombinant polypeptides containing human HLA-A11 [alpha] 1 & 2 domain
• VFMKGLSKAK (SEQ ID NO: 24), preferably used in recombinant polypeptides containing human HLA-A11 [alpha] 1 & 2 domain
• AAATGFVKKD (SEQ ID NO: 42), preferably used in recombinant polypeptides containing human HLA-A11 [alpha] 1 & 2 domain
• AATGFVKK (SEQ ID NO: 43), preferably used in recombinant polypeptides containing human HLA-A11 [alpha] 1 & 2 domain
• sequence of the peptide antigen (here: GAPQEGIL) of the above full length protein can be substituted by any peptide antigen sequence in accordance with the invention, i.e. by any peptide antigen presented by said recombinant polypeptide, wherein the peptide antigen is a peptide of human alpha- Synuclein.
• recombinant polypeptides of the invention may consist of a sequence consisting of a peptide antigen which is a peptide of human alpha-Synuclein (e.g., any one of the peptide antigens of SEQ ID NOs: 20-46), followed by the sequence of
• the receptors ILT2 also known as LILRB1 and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of these receptors in accordance with the invention are as follows:
• HNLSSE WSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKE
• PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH SEQ ID NO: 18
• the sequence of human alpha-Synuclein is known in the art.
• a perferred human alpha-Synuclein is as follows:
• Parkinson's disease including prodromal Parkinson's disease (prodromal-PD), and the classification thereof, are known in the art.
• Prodromal-PD refers to the stage at which individuals do not fulfill diagnostic criteria for PD (ie, bradykinesia and at least 1 other motor sign) but do exhibit signs and symptoms that indicate a higher than average risk of developing motor symptoms and a diagnosis of PD in the future. Most prodromal symptoms are nonmotor and have a major impact on quality of life both for patients with prodromal-PD and for those whose disease stage has progressed to motor-PD. Thus, early detection and treatment of these prodromal symptoms is essential for high-quality care.
• the best-characterized symptoms of prodromal-PD include hyposmia, constipation, mood disorders, and REM sleep behavior disorder (RBD).
• mice injected with haSyn or control virus were sacrificed. Immune cells were isolated from brain, peripheral lymph nodes and spleen. Then, these cells were incubated with A53T- aSyn peptides predicted in silico (e.g. via NetMHC).
• A53T- aSyn peptides predicted in silico e.g. via NetMHC.
• MHC I- and MHC Il-restricted haSyn peptides that induce IFN-y release from T cells of diseased mice (Fig. 3). T cell reactivity towards haSyn peptides was also shown for cervical lymph node and brain derived T cells.
• Example 1 Prevention of Neuron Death in a PD in vivo Model
• this haSyn PD mouse model which closely resembles the human disease, is ideally suited to evaluate the efficacy of immune modulation on the background of a PD- like aSyn-based pathology, and to evaluate the suitability of recombinant polypeptides of the invention for the treatment of PD.
• the inventors further reasoned that induction of immune tolerance towards aSyn will induce a tissue-wide protection via local antigen-specific activation of Treg.
• the inventors developed a recombinant polypeptide, which corresponds to the recombinant polypeptides of the invention but carries this peptide antigen which is immunogenic in mice and murine H2-D b alphal and 2 domains (this recombinant polypeptide is hereinafter also referred to as "mouse AIM Bio surrogate molecule” or "AIM Bio a syn”).
• haSyn PD mice were injected weekly with either 0.5 pig/g AIM Bio a s y n or 2 pig/g AIM Bio a s y n or an off-target- irrelevant control peptide (gp34), starting 3 days after AAV injection.
• the Gp34 peptide is a well-characterized T cell epitope derived from Lymphocytic Choriomeningitis virus (LCMV) Glycoprotein. While this antigen was traditionally named Gp33, the epitope presented on H2-K b was later found to comprise just amino acids 34-41.
• LCMV Lymphocytic Choriomeningitis virus
• H2-K b epitope Gp34 (An epitope beginning at amino acid 33 is, in contrast, presented on H2-K d .) Therefore, we call the H2-K b epitope Gp34, which is in line with the most recent recommendations. Still, there is an ambiguous use of the Gp33 and Gp34 nomenclature in the literature.
• the sequence of the peptide used here is AVYNFATM (SEQ ID NO: 48).
• Control mice were injected with carrier (PBS) only. 10 weeks after AAV delivery the inventors found that PBS-injected or Gp34_KbG injected haSyn PD mice had lost about 45% of substantia nigra neurons compared to animals injected with a non-pathogenic empty vector control (EV).
• PBS carrier
• this neuron loss was attenuated by both low-dose (0.5pig/g) and high dose (2 pig/g) AIM Bio a s y n in a dose-dependent manner (Fig. 4). Furthermore, the inventors found that aSyn-loaded AIM Bio surrogate molecules prevent infiltration of CD11 b + myeloid cells into the striatum in PD model (Fig. 5), aSyn-loaded AIM Bio surrogate molecules reduce aSyn accumulation in Substantia Nigra in PD model (Fig. 6), and aSyn-loaded AIM Bio protect Tyrosine Hydroxylase - fibers in the striatum (Fig. 7).
• Example 2 aSyn-specific MHC class l-based Therapeutics for the Treatment of PD in Human Patients
• the inventors' findings show that single-chain proteins containing an aSyn peptide antigen and an HLA-G alpha 3 domain can largely prevent neuron death in an animal model for Parkinson's disease, i.e. that they can be used for the treatment of PD. Based on these findings, the inventors have generated aSyn-peptide and MHC class I fusion molecules for therapeutic use in human PD patients.
• Target sequence human alpha-Synuclein
• GAPQEGIL SEQ ID NO: 20
• VFMKGLSKAK (SEQ ID NO: 24)
• WAAAEKTK (SEQ ID NO: 26)
• VLYVGSKTK (SEQ ID NO: 30)
• GVTTVAEKTK (SEQ ID NO: 32) (based on A53T mutant)
• VTGVTAVAQK (SEQ ID NO: 35)
• GSIAAATGF SEQ ID NO: 36
• GSIAAATGFVK SEQ ID NO: 40
• AAATGFVKK (SEQ ID NO: 41)
• AAATGFVKKD (SEQ ID NO: 42)
• AATGFVKK (SEQ ID NO: 43)
• PVDPDNEAY (SEQ ID NO: 44)
• SIAAATGFV SEQ ID NO: 37 +HLA-A2 presenting domains
• KTKEGVLYV SEQ ID NO: 28 +HLA-A2 presenting domains
• AWTGVTAV SEQ ID NO: 33
• HLA-A2 presenting domain
• GWHGVTTV SEQ ID NO: 31
• GSIAAATGFVK SEQ ID NO: 40 +HLA-A11 presenting domains
• AAATGFVKK (SEQ ID NO: 41) +HLA-A11 presenting domains
• VLYVGSKTK (SEQ ID NO: 30) +HLA-A11 presenting domains
• PVDPDNEAY SEQ ID NO: 44 +HLA-A1 presenting domains
• WAAAEKTK SEQ ID NO: 26 +HLA-A11 presenting domains
• VFMKGLSKAK (SEQ ID NO: 24) +HLA-A11 presenting domains
• AAATGFVKKD (SEQ ID NO: 42) +HLA-A11 presenting domains
• AATGFVKK (SEQ ID NO: 43) +HLA-A11 presenting domains
• GWAAAEKTK (SEQ ID NO: 25) +HLA-A11 presenting domains
• GAPQEGIL SEQ ID NO: 20 +HLA-G presenting domains
• Expi-293F cells (Thermo Fisher), grown in Expi-293TM expression medium (Thermo Fisher): transfection of 1 pig DNA into 2.5x10 6 cells/ml using the ExpifectamineTM 293 Transfection kit (Thermo Fisher) using Opti-MEM (Thermo Fisher) for complexation of DNA with Expifectamine, after 18-20 h, addition of enhancer according to the protocol, harvesting of the supernatant after 4-6 days (37°C, 8% CO2, humidified incubator), 19 mm 2 orbital shaker 125 rpm Spot-tag protein purification: equilibration of Spot-Cap resin: transfer of desired slurry amount into an appropriate tube, sediment beads by centrifugation (4°C, 4 min, 2500 g), remove & discard supernatant, add 10 bed volumes PBS (cold) to beads, invert to mix, sediment beads by centrifugation (4°C, 4 min, 2500 g), remove & discard supernatant, repeat 2
• PBMC peripheral blood mononuclear cells
• PBMCs were thawed 1 day prior to PBMC pulsing (d-1) and kept over night in 5 ml X-VIVO 15 medium containing 5% human AB serum in a well of a 6 well plate at 37°C.
• X-VIVO 15 complete medium X-VIVO 15 medium + 2% human AB serum supplemented with cytokine cocktail: 10 ng/ml TGF-b1 , 10 ng/ml IL-4, 20 ng/ml IL-2, 20 ng/ml GM-CSF
• ELISPOT plates were coated using anti-hlLW (clone 9D-7, 1 :500 dilution in PBS, sterile filtered) and alL10 (10G8-biotin) and on day 14, 200,000 cells were seeded per well on the ELISPOT plates in duplicates, including negative controls (cells plus PBS) and a positive control (e.g. LPS).
• anti-hlLW clone 9D-7, 1 :500 dilution in PBS, sterile filtered
• alL10 10G8-biotin
• the PFDF membrane was activated with 50 pl/well EtOH (35% v/v) for 1 min followed by 5x washing with 200 pl distilled sterile water. Plate was coated with 100 pl/well antibody solution at 4°C over night. On the next day, unbound coating antibody was removed, 5 washing steps were performed with 200 pl PBS and 200 pl blocking buffer (X-VIVO 15 5% hAB serum) was added and the plate incubated for 30 min - 2 h at room temperature.
• the respective antigenic peptide in DMSO or DMSO as a control were prepared, and a final amount of 5 pg peptide/ml was added to the final volume of 100 pl/well.
• 150,000 cells were seeded per well in X-VIVO 15 medium with 5% human AB serum.
• Blocking buffer (X VIVO 15 medum + 5% hAB serum) was carefully removed, and medium with PBS as negative control and stimulants (5 pg/ml total volume in each well) were added to the other wells and incubated at 37°C over night.
• Secondary antibody was prepared: 1 pg/ml alL-10-biotinylated antibody in 0.5% BSA/1x PBS (1 :1000 dilution) and horseradish peroxidase-conjugated streptavidin (1 :750 in 0.5% BSA/PBS), tetramethylbenzidine solution was filtered using a 0.45 pm filter and stored at 4°C till use.
• Capture antibodies anti-hlLW (Clone: 9D-7, Mabtech #3430-3-250; 1 :500 dilution), anti-hlL10-biotinylated (Mabtech, #3430-6-250) 1x PBS (sterile) 35% EtOH (v/v)
• Blocking buffer X-vivo 5% hAB serum (sterile) [blocking is done in the same medium as cell culture]
• Example 4 Further proof-of-principle of stability and effects of the recombinant polypeptides of the invention. Additionally, the inventors set out to obtain and test recombinant polypeptidies having the general structure of the recombinant polypeptides of the invention but containing various different peptide antigens, in order to obtain further proof-of-principle that recombinant polypeptidies of the invention and surrogates thereof are stable and efficacious. As shown in Figures 9 and 10, respectively, the tested recombinant polypeptidies are stable during freeze-thawing and storage and are thermally stable. Further, they induce Treg in a dosedependent manner (Figure 11) and inhibit T cell lysis in a dose-dependent manner ( Figure 12).
• Example 5 ELISpot results from healthy donor PBMCs (Figure 17), and IL-10 ELISpot results from parkinson patients (Figure 18 A) and healthy/age matched individuals ( Figure 18 B). These experiments show that a significant induction of immunosuppressive cells in response to the constructs of the invention can be observed in vitro already after 14 days both in healthy donor PBMCs and patient PBMCs. References:
• Tumor necrosis factor-alpha increases both in the brain and in the cerebrospinal fluid from parkinsonian patients.
• compositions, polypeptides, nucleic acids, cells, and products for use in the invention are industrially applicable. For example, they can be used in the manufacture of, or as, pharmaceutical products.

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