Patent Application
20250205323
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 type 1 diabetes (T1D). 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 type 1 diabetes (T1D).
As in all autoimmune diseases, an excessive immune reaction against the body's own tissue, which is mistakenly recognized as foreign and attacked, leads to type 1 diabetes (T1D). T cells, which can recognize individual target structures (antigens) very selectively due to their receptors, play a decisive role here. Currently, such diseases are mainly treated with anti-inflammatory drugs or antibodies, which systemically inhibit immune responses and thus dampen symptoms or slow down the progression of diseases. At the same time, however, functioning T cells in particular are also essential for the survival of patients with autoimmune diseases, as they are able to recognize and combat dangerous viruses, bacteria, parasites and mutated cells. Systemic immunosuppression can therefore only be used in a narrow therapeutic window. Thus, in the case of T1D, one attempt is to partially compensate for defects that have developed, by administering insulin in type 1 diabetes. However, blood glucose levels monitoring and accurate dosing of insulin is very difficult. Thus, the unmet medical need is very high, T1D patients have a life expectancy reduced by 11-13 years due to numerous sequelae (Livingstone et al, JAMA. 2015 Jan. 6; 313(1):37-44).
CD8 T cells that attack islet cells play a crucial role in T1D (Tsai S, Shameli A, Santamaria P. CD8+ T cells in type 1 diabetes. Adv Immunol. 2008; 100:79-124.)
However, one of the key diagnostic tools for T1D is testing serum for autoantibodies such as Islet cell cytoplasmic autoantibodies (ICA), Glutamic acid decarboxylase autoantibodies (GADA), Insulinoma-associated-2 autoantibodies (IA-2A) or Insulin autoantibodies (IAA). This way, at risk patients can be identified before a significant destruction of islet cells occurs. Furthermore, inhibition of both CD8 T cell responses and autoantibody formation could significantly improve disease outcome.
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 type 1 diabetes (T1D).
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. The inventors further found that the antigen which is accommodated in the peptide-binding cleft of HLA-G induces selective tolerance in cognate T cells. The inventors observed, inter alia, two mechanisms: induction of apoptosis in highly activated cytotoxic CD8*T cells, and induction of regulatory T cells in cognate naïve T cells. Accordingly, the invention allows to induce selective tolerance induction towards specific antigens without compromising protective immune responses against pathogens.
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 32-microglobulin (b2m), and connected these three components covalently (e.g., via covalent linkers). Alternatively, the antigen-binding a1 and a2 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 human proinsulin/human insulin (INS), human Glutamate decarboxylase 65 (GAD65), human islet amyloid polypeptide (IAPP) and human Zinc transporter 8 (ZNT8) can be suppressed. Thus, according to the invention, type 1 diabetes (T1D) can be treated by the recombinant polypeptides of the invention.
Moreover, according to the invention, the recombinant polypeptides of the invention do not only modulate T-cell responses but also prevent the formation of autoantibodies to human proinsulin and human insulin (INS), human Glutamate decarboxylase 65 (GAD65), human islet amyloid polypeptide (IAPP) and human Zinc transporter 8 (ZNT8). It is expected that this advantage will contribute to a clinical improvement in human patients having type 1 diabetes (T1D), because such autoantibodies are, besides CD8+ T cells, involved in the pathology of type 1 diabetes (T1D).
Accordingly, the invention relates to the following preferred embodiments:
The pharmaceutical compositions or kits for use of the invention can also be used in a treatment for type 1 diabetes in human patients, wherein the treatment is a co-treatment with a stem cell therapy for regenerating the pancreatic tissue. It is expected that such a co-treatment will be beneficial, since the recombinant polypeptides of the invention will, due to their specific immunosuppressive effect, promote regeneration of the pancreatic tissue by the stem cell therapy.
The pharmaceutical compositions or kits for use of the invention can also be used in a treatment for type 1 diabetes in human patients, wherein the treatment is a co-treatment with a stem cell therapy or a human beta cell regenerative drug therapy for regenerating the pancreatic tissue. Human beta cell regenerative drug therapy for diabetes has been reviewed in P Wang, E Karakose, L Choleva, K Kumar, R J DeVita., A Garcia-Ocaha, A F Stewart Andrew. Human Beta Cell Regenerative Drug Therapy for Diabetes: Past Achievements and Future Challenges. Frontiers in Endocrinology 12, 2021. DOI=10.3389/fendo.2021.671946.
The presented peptide antigens 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 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). 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 cleavage site. Furthermore, the antigenic peptide, beta 2-microglobulin and MHC Ib [alpha]chain can be linked in order to increase the stability. The vector map was generated using Snapgene Viewer Software.
(A) experimental design; (B) results
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 alpha 3 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 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 alpha 3 domain and beta-2-microglobulin or just PBS were injected the first day.
The Mog44 peptide containing surrogate molecule significantly reduced EAE symptoms and weight loss.
(A) experimental design; (B) EAE score; (C) body weight
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, ficed 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)
Briefly, murine serum was collected from heart puncture after mice were sacrificed. 10 μg/ml Mog35-55 were used for coating over night, wells were blocked using 1% BSA, and anti-Mog35-55 antibodies were detected using the indicated secondary HRP coupled antibodies.
The recombinant polypeptides referred to in the Figure as follows:
Sterile 96-well white plates were used. Luciferase expressing Panc02 target cells were loaded with 20 μg/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 0 h, 24 h, 48 h.
% increase in IL-10 secreting T cells in response to treatment with the indicated single chain MHC Ib molecule is shown. Black lines indicate an HLA-A2 positive, grey a negative donor. Response a significant increase of Treg is observed bot in HLA-A2 positive and negative donors (response rate indicated in legend)
For the Thermal Shift Assay (TSA), 3 μg of the respective single chain MHC Ib molecule were diluted with PBS and 5×SYPRO Orange dye (stock 5000×, final concentration: 5×) to a volume of 25 μl. 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. The high melting temperatures indicate good protein stability for therapeutic use.
A, C: Western Blots of the indicated recombinant polypeptides. B, D: Coomassie Gels of the indicated recombinant polypeptides (using the same methods).
The data indicate that T1D single chain MHC Ib molecules (recombinant polypeptides of the invention) can be purified and stored and are resistant to freeze-thaw cycles
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. As used herein, the term “soluble” means that the recombinant polypeptide is soluble under the following reference conditions: 5 μg/ml to 5 mg/ml in PBS, optionally with 0.1% human serum, or optionally in 50% glycerol. 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 type 1 diabetes 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 proinsulin or human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide or human Zinc transporter 8, 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.
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:
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 proinsulin or human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide or human Zinc transporter 8.
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 proinsulin or human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide or human Zinc transporter 8.
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 proinsulin or human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide or human Zinc transporter 8.
Methods for the synthesis of peptide antigens, including peptide antigens in accordance with the invention, are well known in the art.
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 the recombinant polypeptides of the invention: Optional leader Peptide (absent from the recombinant polypeptide due to processing during cellular expression): e.g. MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1)
Note that the following underlined amino acids of this sequence are relevant for ILT2 or ILT4 receptor interaction:
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.:
Most preferred exemplary peptide antigens which can be part of the recombinant polypeptides of the invention are as follows:
Further preferred 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 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 proinsulin or human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide or human Zinc transporter 8. That is, recombinant polypeptides of the invention may consist of a sequence consisting of a peptide antigen which is a peptide of human proinsulin or human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide or human Zinc transporter 8 (e.g., any one of the peptide antigens of SEQ ID NOs: 2, 22, 23, 24, 25, 26 and 27), followed by the sequence of
These recombinant polypeptides of the invention may also contain the optional leader peptide as exemplified above.
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 proinsulin and human insulin, human Glutamate decarboxylase 65, human islet amyloid polypeptide and human Zinc transporter 8 are known in the art. Preferred amino acid sequences of these proteins 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 Sep 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
5 μg/ml of an AIM Bio molecule or the respective controls.
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 aIL10 (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. MOG 157) 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.
25 μl diluted HRP-streptavidin (1:750) was added per well and incubated for 1 h at room temperature in the dark followed by 5 washing steps using sterile 1×PBS.
100 μl of filtered TMB substrate was added per well for 15-25 min till blue sports developed. Reaction was stopped by washing the wells thoroughly with tapped water.
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.
Wild type black 6 mice were injected with 100 μg recombinant polypeptides (also referred to as “AIMBio”) having the following sequences,
for inducing tolerance towards an OVA peptide or an viral Gp34 peptide, respectively. 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-Kb was later found to comprise just amino acids 34-41. (An epitope beginning at amino acid 33 is, in contrast, presented on H2-Kd.) Therefore, we call the H2-Kb 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 first 8 amino acids of SEQ ID NO: 35 show the correct sequence (AVYNFA™; SEQ ID NO: 58). After 2 weeks, mice were sacrificed, and splenocytes re-challenged either with the matching or a mismatching peptide. IL-10 secreting cells were quantified by ELIspot. The results are shown in
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 late stage NMO 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 (
At the day of the 33 μg or 100 μg recombinant polypeptide of the invention surrogate molecule (“AIM Bio”) i.p. injection, 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_Kb_G) 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.
T1D autoantigens in accordance with the invention include (pro-)insulin (INS), Glutamate decarboxylase 65 (GAD65), islet amyloid polypeptide (IAPP) or Zinc transporter 8 (ZNT8).
The inventors' findings show that single-chain proteins containing an INS, GAD65, IAPP or ZNT8 peptide antigen 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 (
In correlation with the in vivo experiment shown here, it is plausible that these constructs suppress CD8 T cell mediated and antibody mediated responses directed against islet cell antigens in patients.
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
Further, the high melting temperatures shown in
In vitro Treg induction mediated by peptide-HLA-G containing constructs (AIM Biologicals) was carried out as follows: PBMCs from healthy donors were purified via density centrifugation performed on white blood cells from a leukocyte reduction chamber using Ficoll. 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 5 μg/ml of an AIM Bio molecule or the respective controls.
On day 3, 1 ml complete medium (with cytokines) was added, on day 6, a second pulse with 5 μg/ml 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 plate PVDF 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 anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtered) 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. 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) for 48 h. 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. 25 μl diluted HRP-streptavidin (1:750) was added per well and incubated for 1 h at room temperature in the dark followed by 5 washing steps using sterile 1×PBS. 100 μl of filtered TMB substrate was added per well for 15-25 min till blue sports developed. Reaction was stopped by washing the wells thoroughly with water. Plastic underdrains of the plates were removed and the bottom and sides of the plates were also washed with tap water and dried.
Some recombinant polypeptides of the invention induced at least 30% more IL-10 secreting T reg in PBMCs of ˜75% of all healthy blood donors.
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 |
|---|---|---|---|
| 22164163.2 | Mar 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/057681 | 3/24/2023 | WO |
