Patent Application
20250222083
The present invention relates to therapeutical uses of non-classical human major histocompatibility complex (MHC) molecules (also named MHC class Ib molecules) in combination with peptide antigens for the treatment of multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica. The invention more specifically relates to recombinant polypeptides comprising peptide antigens and one or more domains of a non-classical MHC class Ib molecule. The invention also relates to methods of producing such recombinant polypeptides, pharmaceutical compositions comprising the same, as well as their uses for treating multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica.
Multiple sclerosis (MS) and MOG encephalomyelitis (also known as myelin-oligodendrocyte glycoprotein antibody disease, MOG antibody disease, MOGAD) are autoimmune diseases in which T cells of the immune system attack myelin sheaths in the central nervous system, resulting in gradually progressive neurodegeneration. The relapsing flare-up of disease activity characteristic of MS can be effectively suppressed by immunomodulatory therapies. For the rare MOGAD, which has long been considered a subtype of MS, there are no approved therapeutics, so therapy initially consists of watching and waiting for the disease to progress. In both diseases, slowly progressive neurodegeneration occurs. Some therapeutics effective in MS, such as the antibodies natalizumab or ocrelizumab or the orally bioavailable S1P inhibitor fingolimod, even carry the risk that a persistent and normally harmless intracerebral infection with JC virus can no longer be controlled, resulting in an often fatal progressive multifocal leukoencephalopathy. In this respect, the problem of either insufficient immunomodulation or immunomodulation with too severe side effects remains unsolved in both diseases, especially with regard to slow progression. Common biologics do not cross the blood-brain barrier and thus cannot have an anti-inflammatory effect in situ. In contrast, regulatory cells have been described to cross it well (Schneider-Hohendorf et al., Eur J Immunol. 2010 December; 40(12):3581-90).
Two strategies have been evaluated so far, at least in early clinical studies. Similar to hyposensitization strategies in allergy, large amounts of antigens have been administered by different routes to induce antigen-specific tolerance. However, in autoimmune diseases, these strategies have caused severe side effects and have not been clinically successful. Attempts to induce tolerance by adoptive transfer of antigen-specific regulatory T cells or antigen-loaded tolerogenic dendritic cells appear more promising. However, these strategies are extremely complex and expensive, requiring GMP-compliant production and quality control processes for each individual patient. Therefore, even if small clinical trials have been successful, it is highly questionable whether adoptive transfer therapies will be available for many patients in the foreseeable future. Immunosuppressive MHC class Ib molecules 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 MHC class Ib molecules and peptides for targeted therapeutic immunomodulation.
Taken together, there remains a need for improved drugs for the treatment of multiple sclerosis (MS) and MOG antibody disease. Similarly, there also remains a need for improved drugs for the treatment of MOG antibody positive neuromyelitis optica (NMO).
The inventors have found that human MHC class Ib molecules such as HLA-G possess the ability to induce antigen-specific tolerance towards presented peptide antigens. Thus, albeit being of similar structure and sequence as classical human MHC class Ia molecules which induce antigen peptide-specific immune responses, MHC class Ib molecules can advantageously be used according to the invention to suppress immune responses in an antigen-specific manner. Additionally, the inventors have found that for the suppression of immune responses according to the invention, molecules other than naturally occurring MHC class Ib molecules, and in particular polypeptides which only comprise at least one domain of an MHC class Ib molecule, preferably at least an [alpha]3 domain of an MHC class Ib 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 Ib molecule in order to suppress immune responses towards peptides presented by these antigens.
Antigen-loaded HLA-G molecules can be unstable. Thus, the inventors designed soluble recombinant polypeptides comprising a peptide antigen, an MHC class Ib molecule such as HLA-G and β2-microglobulin (b2m), and connected these three components covalently (e.g., via covalent linkers). Alternatively, the antigen-binding α1 and α2 domains of an MHC class Ib 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,
Surprisingly, the inventors have found that by using the recombinant polypeptides of the invention, immune responses against human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) can be suppressed. Thus, according to the invention, multiple sclerosis (MS), myelin-oligodendrocyte glycoprotein antibody disease (MOG antibody disease) and MOG antibody positive neuromyelitis optica can be treated by the recombinant polypeptides of the invention.
Experimental data of the inventors show that a suitable peptide antigen and the presence of an [alpha]3 domain of an MHC class Ib molecule (e.g. HLA-G) are required to achieve the desired effect. Therefore, this approach goes beyond previously described strategies that either use antigenic peptides in the absence of costimulation (resulting in anergic rather than tolerogenic T cells), or MHC class Ib molecules in an antigen-unspecific setting.
Furthermore, according to the invention, the recombinant polypeptides of the invention are expected to be highly advantageous in the immunoterapeutic treatment of multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica, because are expected to show an improved safety profile as compared to conventional drugs aganst these diseases, which may cause severe side effects such as progressive multifocal leukoencephalopathy.
Moreover the inventors have surprisingly found that the recombinant polypeptides of the invention do not only modulate T-cell responses but also prevent the formation of MOG-specific autoantibodies in model experiments. It is expected that this finding will translate into a clinical improvement in patients having multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica, because MOG-specific autoantibodies are involved in the pathology of these diseases.
Accordingly, the invention relates to the following preferred embodiments:
The presented peptide antigenis depicted in dotted spheres, the HLA-G alpha1-3 domains are sketched in light-grey, and the beta2microglobulin domain is shown in dark grey. An optional linker connecting the antigenic peptide with the beta2microglobulin molecule is displayed in grey stick style, and an optional disulfide trap is depicted in black spheres. This figure was generated using Pymol and is adapted from structures published in Clements et al., Proc Natl Acad Sci USA. 2005 Mar. 1; 102(9):3360-5 and Hansen et al., Trends Immunol. 2010 October; 31(10):363-9.
HLA-G1 and HLA-G5 each consist of 3 [alpha] domains (here in black), a non-covalently associated beta2-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). As shown here, the [alpha]-3 domain is capable of binding to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci USA. 2003 Jul. 22; 100(15):8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci USA. 2006 Oct. 31; 103(44):16412-7) on immune cells. Physiologically, these sequences form a non-covalently linked MHC class 1 complex. To simplify purification of the complex MHC Ib molecule, one or more protein tags (such as SpotTag, myc tag and/or His(6×) tag) may be introduced. They may be introduced in such a way as to enable their later optional removal via cleavage using an optional Factor Xa or Furin cleavage site. Furthermore, the antigenic peptide, beta2-microglobulin and MHC Ib [alpha]chain can be linked in order to increase the stability. The vector map was generated using Snapgene Viewer Software.
In this experiment, 100 μg of surrogate molecules consisting of a viral (Gp34) or Ovalbumin (Ova) model peptide antigen, murine H2-Kb alpha1 and 2 domains, and human HLA-G alpha3 domain and beta-2-microglobulin were injected i.p. into 12 week old C57BL/6 mice. After 14 days, mice were sacrificed and splenocytes isolated via Ficoll gradient were rechallenged with 5 μg/ml of either Gp34 or Ova peptide in an 48 h standard murine IL-10 ELISpot assay (Mabtech mouse IL-10 HRP ELISpot Kit) (A).
A significant increase in regulatory T cells that secreted IL-10 only in response to a rechallenge with the peptide towards which tolerance was induced via surrogate molecule injection was detected (B).
In this MS mouse model, the model antigen ovalbumin (OVA) is expressed in oligodendrocytes under the control of the myelin basic protein (MBP) promoter (ODC-OVA). This leads to the presentation of the OVA257-264 peptide on H-2Kb MHC molecules on oligodendrocytes. OT-I mice express a T cell receptor (OT-I) on their CD8+ T cells, which recognizes exactly this peptide-MHC combination. When CD8+ T cells from these mice are transferred into 10 day old ODC-OVA mice, these develop an experimental autoimmune encephalomyelitis (EAE) which resembles in many aspects the pathogenesis and symptomatology of MS (Na et al., Brain, Volume 131, Issue 9, September 2008, Pages 2353-2365). In this experiment, 500 μg of surrogate molecules consisting of a viral (Gp34) or Ovalbumin (Ova) model peptide antigen, murine H2-Kb alpha1 and 2 domains, and human HLA-G alpha3 domain and beta-2-microglobulin or just PBS were injected the same day. EAE was scored according to Bittner et al., J Vis Exp. 2014 Apr. 15; (86):51275. Only Ovalbumin-tolerance inducing surrogate molecules almost completely prevented EAE symptoms.
(A) experimental design; (B) results.
In this experiment, it was tested whether the the Mog44 peptide surrogate molecule could induce protective T cells that can inhibit cytotoxic T cells targeting another peptide presented by the same cells. 250 μg per mouse of this molecule described in
With this lower dose of surrogate molecules, EAE symptoms could not be completely prevented, but significantly reduced both with molecules inducing tolerance towards the directly targeted CD8 epitope (Ova_KbG) or towards another epitope (Mog44) expressed on the same cells (Mog44_DbG).
(A) EAE score; (B): body weight.
(A) experimental design In this model, a strong, myelin-specific autoimmune response is triggered by administration of MOG 35-55 peptide in combination with Complete Freund's adjuvant, which activates CD4+ Th17 cells, and pertussis toxin, which makes the blood-brain barrier more permeable (Protocol: Bittner et al., J Vis Exp. 2014 Apr. 15; (86): 51275). Here, CD4+ cells as well as antibodies play a crucial role in the development of EAE (Tigno-Aranjuez et al., J Immunol Nov. 1, 2009, 183 (9) 5654-5661). In addition, 100 μg/mouse of surrogate molecules consisting of a viral (Gp34) or two Mog peptide antigens (Mog37 or Mog44), murine H2-Db alpha1 and 2 domains, and human HLA-G alpha3 domain and beta-2-microglobulin or just PBS were injected the first day.
The Mog44 peptide containing surrogate molecule significantly reduced EAE symptoms (B) and weight loss (C).
10 μm fresh frozen sections were stained with commercial Toluidine 1× staining reagent for 1 h at room temperature. A strong infiltration of immune cells was detected in EAE, but prevented by Mog44_Db_G. 10 μm fresh frozen sections were briefly dried at room temperature, fixed with acetone, blocked with 5% BSA 10% normal goat serum in PBS, stained with 1:100 anti-CD8 antibody, secondary antibody coupled to HRP and DAB solution (detailed methods: Karikari et al., Brain Behav Immun. 2022 Jan. 12; 101:194-210). Mog35-55 induced EAE lead to a strong infiltration of CD8+ cells into the spinal cord which was prevented by MOG44_Db_G surrogate molecule.
Murine serum was collected from heart puncture after mice were sacrificed and diluted 1:50 in PBS. 10 μg/ml Mog35-55 in PBS were used for coating over night, wells were then blocked using 1% BSA for 2 h, before diluted sera were added for 1 h. Anti-Mog35-55 antibodies were detected using the indicated secondary HRP coupled antibodies (dilution 1:5000 in PBS). Mog35-55 induced EAE correlated with high levels of Mog35-55 specific IgG autoantibodies, which were not detectable in animals treated with 100 μg MOG44_Db_G surrogate molecule.
The myelin (MAG, MBP, MOG, PLP) peptide and MHC class I presenting molecules are as follows:
It is further shown which combinations of myelin peptides and antigen-presenting MHC class I alpha1 and 2 domains (HLAG=HLA-G, A2G=HLA-A2 presenting domains+HLA-G alpha3 domain) lead to good results with regards to expression/production, ELISpot based prioritization in healthy blood donors (described in
In vitro Treg induction mediated by AIM Biologicals was carried out as follows:
OT1/BL6 Mice were sacrificed and splenocytes were collected and washed once in RPMI 5% FCS. Red blood cells were removed with 2 ml 1× sterile RBC lysis buffer for 3 min. Cells were cultured in high density culture (10mio cells/ml) for 72 h in RPMI 10% FCS medium with GMCSF 20 ng/ml, IL-2 20 ng/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.
Use sterile 96-well white plate. Load Panc02 fluc+ target cells with 20 μg/ml Ova peptide (SIINFEKL) for 60 min at 37° C. with 500 rpm shaking. Mix effector CD8+ T cells 50:1 with PancO2 target cells (5000 target cells), add luciferin. Measure luminescence at 0 h, 24 h, 48 h.
Unless otherwise defined below, the terms used in the present invention shall be understood in accordance with their common meaning known to the person skilled in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited either by specifying the full literature reference in the text.
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.
It will be understood that the recombinant polypeptides of the invention are meant to optionally include a secretion signal peptide sequence. Similarly, 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.
It is also understood that 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.
Likewise, it will be understood that the recombinant polypeptides of the invention are meant to optionally include the respective pro-peptides.
It will also be understood that 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.
According to the invention, 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. For example, peptide antigen-binding domains such as [alpha]1 and [alpha]2 domains are well-known, and modifications of these domains can be made. The capability of 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.
In accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in a human patient.
In accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in the treatment of multiple sclerosis (MS), MOG antibody disease or MOG antibody positive neuromyelitis optica in a human patient.
In accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immunological tolerance against human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1), e.g., in a human patient.
It is understood that in accordance with the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are stable.
It will be understood that in connection with the peptide antigens used in accordance with the invention, any lengths of these peptide antigens referred to herein (e.g. “7 to 11 amino acids in length”) are meant to refer to the length of the peptide antigens themselves. Thus, the lengths 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.
In accordance with the present invention, each occurrence of the term “comprising” may optionally be substituted with the term “consisting of”.
Generally, unless otherwise defined herein, the methods used in the present invention (e.g. cloning methods or methods relating to antibodies) are performed in accordance with procedures known in the art, e.g. the procedures described in Sambrook et al. (“Molecular Cloning: A Laboratory Manual.”, 2nd 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.
For instance, binding of MHC class Ib 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 Ib 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 USA. 2003 Jul. 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. In one embodiment, 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. Thus, when used in connection with sequences, terms such as “identity” or “identical” preferably refer to the identity value obtained by using the BLAST algorithm.
Pharmaceutical compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions.
For instance, the pharmaceutical 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. In general, the pharmaceutically acceptable components used in connection with the present invention are used in accordance with knowledge available in the art, e.g. from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising the nucleic acids of the invention (e.g., RNAs) may also be formulated in accordance with knowledge available in the art, e.g. using liposomal formulations targeting dendritic cells.
Peptide Antigens in Accordance with the Invention
The peptide antigens which can be used in accordance with the invention, including the peptide antigens as defined above, 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.
Peptides which are able to be presented on MHC molecules can be generated as known in the art (see, for instance, Rammensee, Bachmann, Emmerich, Bachor, Stevanović. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 November; 50(3-4):213-9; Pearson et al. MHC class I-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 November; 37(11):724-737).
Peptide antigens are generally known in the art. Generally, 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 Ib molecule or polypeptide capable of presenting peptides in accordance with the invention, peptide antigens which are capable of binding to said MHC class Ib 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 Ib 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:
Rammensee, Bachmann, Emmerich, Bachor, Stevanović. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 November; 50(3-4):213-9;
Pearson et al. MHC class I-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 November; 37(11):724-737.
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.
In a preferred embodiment in accordance with all embodiments of the invention, 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.
Such predictions can preferably be made in as described in any one of the following publications:
In the invention, the peptide antigen is from human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).
It is understood that the 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 substitution with respect to the corresponding amino acid sequence of a peptide antigen from human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG) or human myelin proteolipid protein (PLP1).
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. For instance, in one embodiment, a peptide antigen of the invention encompasses the peptidomimetic of the indicated peptide antigen amino acid sequence of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).
Methods for the synthesis of peptide antigens, including peptide antigens in accordance with the invention, are well known in the art.
The recombinant polypeptides of the invention can be used for the treatment of multiple sclerosis, MOG antibody disease and MOG antibody positive neuromyelitis optica.
The treatment can be a treatment by inducing myelin-specific regulatory T cells. As such regulatory T cells (e.g., CD8-positive regulatory T cells) are activated in myelinated structures, they confer target cell protection against cytotoxic T cells recognizing the same or another myelin antigen. Regulatury T-cells (e.g., CD8-positive regulatory T cells) are known in the art and can be detected, for instance, by their secretion of IL-10.
While CD8-positive regulatory T cells are not as well known as CD4CD25 regulatory T cells, they have even been described to be more potent. See, for instance:
While these are characterized by expression of CD122 and CD8 in mice, their human counterparts have been described to be CD8 and CXCR3 positive. See, for instance:
The treatment can be a treatment for reducing plasma or cerebrospinal fluid levels of autoantibodies against human myelin-oligodendrocyte glycoprotein. The human patient can be a patient who had plasma or cerebrospinal fluid autoantibodies against myelin-oligodendrocyte glycoprotein prior to the start of the treatment.
In accordance with the invention, the autoantibodies can be detected by various methods known in the art. A preferred approach are cell-based assays (CBAs) where the suspected target antigen of the autoantibodies (e.g., myelin-oligodendrocyte glycoprotein) is overexpressed in HEK293 or CHO cells which are then incubated with serum or cerebrospinal fluid, typically for 1 h at room temperature. Mock-transfected sister cells serve as controls. Autoantibodies that bind to the cells are detected with different fluorescently labeled anti-human specific secondary antibodies that recognize total human IgG (heavy and light chain), IgG-Fc (constant chain) or IgG1. Binding is quantified by either flow cytometry (CBA-FACS) or visual scoring by microscopic evaluation of the immunofluorescence (CBA-IF), which is often titrated. In the majority of established cell-based assays for detection of MOG-antibodies, the 218 amino acid α1 isoform of MOG is used, although an alternative in which a truncated version of MOG that contains only the extracellular immunoglobulin and the transmembrane domain has also been tested. Other approaches like enzyme-linked immunosorbent assays (ELISAs) or Western Blots are also possible, but often less sensitive, as conformation-sensitive antibodies may not be detected by these methods. Suitable approaches have been described in Waters, P., Pettingill, P. & Lang, B. Detection methods for neural autoantibodies. Handb. Clin. Neurol. 133, 147-163 (2016).
A detailed overview on Myelin oligodendrocyte glycoprotein (MOG) antibodies can be found in Reindl M, Waters P. Myelin oligodendrocyte glycoprotein antibodies in neurological disease. Nat Rev Neurol. 2019 February; 15 (2): 89-102. doi: 10.1038/s41582-018-0112-x. PMID: 30559466.
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.
Exemplary sequences which are part of of the recombinant polypeptides of the invention:
Alternatively, 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: 20), i.e.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGE EQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 21),
Further (alternative) exemplary peptide antigens which can be part of the recombinant polypeptides of the invention are as follows:
Example for a recombinant polypeptide of the invention (with the optional leader peptide):
Note that the sequence of the peptide antigen (here: VLLAVLPVL) of the above full length recombinant polypeptide 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 myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1). That is, recombinant polypeptides of the invention may consist of a sequence consisting of a peptide antigen which is a peptide of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) (e.g., any one of the peptide antigens of SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33), 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:
The sequences of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) are known in the art. Preferred amino acid sequences of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) are as follows:
The present invention is further illustrated by the following non-limiting examples:
Sediment beads by centrifugation. Use Amicon Ultra-4 centrifugal filters (15 kDa cutoff) for Protein concentration and spot-peptide removal with 15 kDa Amicon cutoff columns
Rinse the Amicon Ultra-4 centrifugal filters (15 kDa cutoff) with PBS followed by 0.1 N NaOH (centrifugation at 4000 g, 4° C.) to remove trace amounts of glycerine.
To isolate peripheral blood mononuclear cells (PBMC), a density centrifugation was performed with white blood cells from a leukocyte reduction chamber and density gradient medium (e.g. Ficoll, or ROTI September 1077). Cells were centrifuged for 20 min at 1200×g without brake followed by collection of the interphase ring that was washed with 1×PBS (5 min, 300×g). PBMC were frozen till further use.
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.
On the next day (d0) cells were counted and resuspended in X-VIVO 15 complete medium (5% hAB serum & cytokine cocktail: 20 ng/ml hIL-2, 20 ng/ml hGM-CSF, 10 ng/ml hIL-4 & 10 ng/ml hTGF-b1) at a cell density of 3×106 cells/ml.
For experiments, 3×106 cells were seeded in the respective wells of a 12-well plate with a final volume of 1000 μl X-VIVO complete medium with cytokine cocktail and
On day 3, 1 ml complete medium (with cytokines) was added, on day 6, a second pulse with 5 μg/ml of a recombinant polypeptide of the invention or a surrogate thereof (collectively referred to as “AIM Bio” molecule) was performed (after removing medium). On days 7, 10 & 12, 1 ml complete medium (with cytokines) was added.
On day 13, ELISPOT plates were coated using anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtered) and all 10 (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).
The PFDF membrane was activated with 50 μl/well EtOH (35% v/v) for 1 min followed by 5× washing with 200 μl distilled sterile water. Plate was coated with 100 μl/well antibody solution at 4° C. over night. On the next day, unbound coating antibody was removed, 5 washing steps were performed with 200 μl PBS and 200 μl 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 (e.g. MOG157) in DMSO or DMSO as a control were prepared, and a final amount of 5 μg peptide/ml was added to the final volume of 100 μl/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 μg/ml total volume in each well) were added to the other wells and incubated at 37° C. over night.
Secondary antibody was prepared: 1 μg/ml aIL-10-biotinylated antibody in 0.5% BSA/1×PBS (1:1000 dilution) and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA/PBS), tetramethylbenzidine solution was filtered using a 0.45 μm filter and stored at 4° C. till use.
Cell supernatant was removed and 5× washed using 100 μl PBS. Last excess buffer was removed using paper towels.
Plastic underdrains of the plates was removed and the bottom and sides of the plates were washed with tap water and dried.
Plates were read out using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analysed in Excel and graphs/statistics were done in Graphad Prism.
Required: Capture antibodies: anti-hIL10 (Clone: 9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10-biotinylated (Mabtech, #3430-6-250), 1×PBS (sterile), 35% EtOH (v/v), Blocking buffer: X-vivo 5% hAB serum (sterile) [blocking is done in the same medium as cell culture], Dilution buffer: 0.5% BAS in PBS, Washing buffer: 1×PBS, Medium: for T cells, X-VIVO 15 medium (Lonza), Filter syringe: Millex GV, ELISPOT PVDF plate (#MSIP4510, Millipore), TMB substrate
Wild type black 6 mice were injected with 100 μg recombinant polypeptides (also referred to as “AIMBio”) having the following sequences,
As described in (Na et al, Brain. 2008 September; 131 (Pt 9): 2353-65.), the adoptive transfer of CD8+OT-I T cells that recognize an ovalbumin epitope in the context of H2-Kb into mice which express ovalbumin in oligodendrocytes leads to experimental autoimmune encephalomyelitis which recapitulates many MS and MOGAD symptoms. In this animal model, a single injection of 500 μg of recombinant polypeptides surrogate molecules (also referred to as “AIMBio”) that induce tolerance towards the targeted ovalbumin epitope almost completely prevented EAE symptoms, while a surrogate molecule presenting a control peptide hat no significant protective effects (
In the same model, the inventors were further able to show that using an antigen peptide also presented of the target tissue or cell can result in effective bystander immunosuppression. Here, 250 μg of recombinant polypeptide surrogate molecules (also referred to as “AIMBio”) were injected per mouse (
At the day zero, 33 μg or 100 μg recombinant polypeptide of the invention surrogate molecule (“AIM Bio”) was injected i.p., 100 μl MOG35-55 peptide/CFA (Complete Freund's Adjuvance; final concentration Mycobacterium tuberculosis H37RA and peptide each 1 mg/ml) emulsion were injected each left and right s.c. into the flank and 250 ng pertussis toxin (in 200 μl PBS) intraperitoneally. A second pertussis toxin injection was given 3 days later. In this animal model, a single injection AIM Bio surrogate molecules that induce tolerance towards the a Mog epitope (Mog44_DbG) significantly reduced EAE symptoms, while a surrogate molecule presenting a control peptide (Gp34) or a non-functional Mog peptide (Mog37) hat no significant protective effects (
In this model Mog44 AIM Bio also completely prevented the formation of MOG-specific autoantibodies in the serum as tested by ELISA (
Mog-reactive antibodies in sera of AIM Bio (33 or 100 μg) treated mice were detected via standard ELISA protocol, with 3 washes in between each step. Briefly, ELISA plates were coated with 10 μg/ml Mog35-55 peptide, blocked with PBS 1% BSA, before mouse sera diluted 1:25 in PBS 1% BSA were added for 1 h. Anti-mouse IgG-HRP or anti-mouse heavy and light chain-HRP antibodies diluted 1:5000 were used for detection.
The recombinant polypeptides of the invention are newly developed protein complexes derived from the pregnancy-associated immunosuppressive MHC molecule HLA-G. It is likely that HLA-G enables an embryo to influence the maternal immune system to tolerate embryonic antigens but further antagonize antigens from pathogens. The recombinant polypeptides of the invention containing variable peptides were able to selectively eliminate peptide-specific cytotoxic effector T cells as well as induce peptide-specific regulatory T cells in the test tube.
The inventors' findings show that single-chain proteins containing myelin peptide antigens and a HLA-G alpha 3 domain can induce tolerogenic T cells in healthy donors. Thus, CD8 Treg were upregulated by at least 30% in 75% of all healthy blood donors by a recombinant polypeptide of the invention containing the VLLAVLPVL antigen (also referred to as “Mog157_A2G”; see
Additionally, the inventors set out to obtain and test recombinant polypeptides 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 polypeptides of the invention and surrogates thereof are stable and efficacious. As shown in
MOG-tolerance inducing recombinant polypeptides of the invention prevent apoptosis (cleaved caspase 3), CD3+ immune cell infiltration and myelin lesions in the spinal cord. The mouse model is described in
Immunofluorescence analyses of a spinal cord of a MOG-induced EAE model.
The pharmaceutical 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22164122.8 | Mar 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/057679 | 3/24/2023 | WO |
