Patent Application
20230355784
The field of present invention relates to the therapy of autoantibody-mediated conditions such as Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), postural orthostatic tachycardia syndrome (POTS), Autoimmune Autonomic Ganglionopathy (AAG), Idiopathic Dilated Cardiomyopathy (IDC), and Chronic Chagas heart disease (cChHD) and other neurological, neuromuscular and neuropsychiatric disorders.
Neuronal receptors represent a special class of targets for disease-causing autoantibodies in neurological autoimmune diseases. In the context with diseases that affect the peripheral autonomic nervous system, these autoepitopes have gained special attention in the context with a variety of neuroimmunological conditions. Comprehensive reviews about autoantibodies against structures of the neuromuscular junction, against peripheral and central neuroreceptors, and against receptors of the autonomic nervous system or against channel proteins, causing channel dysfunction called channelopathies with an autoimmune cause, were published (Vincent 2020; Golden et al, 2019 and Kim, 2014). Importantly, there is growing awareness about the pathogenic significance of this class of disease-causing antibodies against neuroreceptors in the periphery. At the same time, a rich spectrum of disease-associated autoantigens on neuronal surfaces and synapses of the central and the peripheral nerve system is emerging (Zong et al, 2017; Meyer et al, 2018).
Antibodies interfering with the autonomic nervous system are associated with many neuroimmunological conditions including e.g. autoimmune encephalitis, neurodegenerative diseases, multiple sclerosis but also paraneoplastic syndromes or even heart failure. Antibodies and autoantibodies can also target channel proteins (that is, cause channelopathies). Although there is still no complete functional and mechanistic understanding for the role of this type of autoantibodies, a growing body of evidence supports that their therapeutic removal is a useful and promising treatment strategy. Several different types of autoantibodies, typically against components of the autonomic nervous system, were shown to be associated with autonomic dysfunction (or Dysautonomia), which describes a general malfunction of autonomic functions. Dysautonomia is a complex and heterogeneous clinical picture involving several major organ systems such as the heart, intestines, bladder, brain, blood vessels, pupils, glands, and others (Thornton et al, 2017). It is also reviewed by Low & Engstrom, 2017.
Dysautonomia is also found in paraneoplastic syndromes with associated clinical conditions such as autoimmune autonomic ganglionopathy (Nakane et al, 2018), Lambert-Eaton myasthenic syndrome (Vincent 2020), limbic encephalitis or Morvan syndrome (Masood 2021), autonomic neuropathies, encephalitides, and various other manifestations of dysautonomias (reviewed by Golden et al, 2019 and Kaur et al, 2021). McKeon (McKeon et al, 2016) describes the role of autoantibodies and autoimmune autonomic disorders (including autoimmune autonomic ganglionopathy, paraneoplastic autonomic neuropathy, and acute autonomic and sensory neuropathy).
The focus of the present invention is mainly on a subgroup of dysautonomia-related conditions that are in particular associated with autoantibodies against the peripheral autonomic nervous system.
One of the most relevant diseases that involve the peripheral autonomic nervous system is the Chronic fatigue syndrome/Myalgic encephalopathy (CFS/ME, also designated “ME/CFS”); see Sotzny et al, 2018, or Cortes Rivera et al, 2019.
ME/CFS is a complex multisystemic condition where patients typically lose the ability to follow their daily activities because of severe fatigue, sleeping problem and stress intolerance, which has strong impact on their social life and their professional activity. Excessive exhaustibility and severe fatigue are typically combined with cognitive impairment and many other symptoms. It is thought that immunological, genetic, and infectious factors might contribute to a multicausal pathogenesis. To date, neither standardized diagnostics, nor well validated biomarkers, nor appropriate therapies or medications exist. The treatment of ME/CFS is essentially limited to symptomatic therapies. Numerous studies support that autoantibodies against the autonomic nervous system may play a causative role in ME/CFS (reviewed by Sotzny et al, 2018). Remarkably, general removal of antibodies by extracorporeal immunoapheresis could also deplete anti-neuroreceptor antibodies and this could be correlated with clinical improvement of the condition (Scheibenbogen et al, 2018). The association between clinical symptoms and the presence of anti-adrenergic and anti-cholinergic autoantibodies in ME/CFS patients was further corroborated by Bynke et al, 2020.
Importantly, the postural orthostatic tachycardia syndrome (POTS) is a related condition [Zhao et al, 2020, Ruzieh et al, 2017]. As ME/CFS, it is associated, among others, with anti-adrenergic- and muscarinic receptor autoantibodies (Gunning et al, 2019). POTS typically manifests with chronic orthostatic intolerance and a variety of other co-morbidities. The hallmark is typically a strong increase of the heart rate upon standing, often combined with blurred vision, mental clouding, chest discomfort and other heterogenous autonomic abnormalities (see f. ex. Jacob et al, 2020, and citations therein). Mechanistic evidence for the causative role of autoantibodies against neuroreceptors of the autonomic nervous system was provided by in vitro functional blocking of the M3 AChR with patient serum containing autoantibodies against the receptor protein [Palma et al, 2020]. Other examples of a causative role for this type of anti-neuronal autoantibodies were also found in chronic heart failure (Nagatomo et al, 2014).
Complex Regional Pain Syndrome (CRPS) is a pain condition after injury or surgery to a limb and associated with autoantibodies against autonomous neuroreceptors. Again, anti-GPCR antibodies were also functionally assigned to autonomic dysfunction in the autoimmune disease Sjogren's syndrome (reviewed in Shoenfeld et al, 2020). The role of autoantibodies in autoimmune autonomic ganglionopathy was described (Nakane et al, 2018).
Evidence that Ganglionic Acetylcholine Receptor Antibodies play a role in several rheumatic autoimmune diseases (including Sjögren's syndrome, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus) was published (Imamura et al, 2020).
Idiopathic dilated cardiomyopathy (IDC) is typically regarded as a primary myocardial disease characterized by left ventricular or biventricular dilatation and impaired myocardial contractility. Wallukat and Müller (Wallukat et al, 2002; Müller et al, 2000) provided clinical evidence, whereby autoantibodies against beta-1 adrenergic receptor could be non-selectively removed in patients with IDC. Schimke et al, 2005, showed immunoadsorption of anti-beta-1 adrenoreceptor autoantibodies by immunoapheresis in patients with IDC, leading to a reduction in oxidative stress and an improvement in cardiac performance. Matsui et al., 1997, showed that peptides derived from G-protein-coupled receptors can induce morphological cardiomyopathic changes in immunized rabbits. Bornholz et al., 2014, provide a discussion of using beta-1 adrenergic autoantibodies for diagnostic and biomarker purpose.
Chronic Chagas heart disease (cChHD) typically is a chronic manifestation of the Trypanosoma cruzi infection, usually characterized by high antibody levels against the C-terminal region of the ribosomal P proteins. Labovsky et al., 2007, showed autoantibodies against beta-1-adrenergic receptor in patients with cChHD.
Düngen et al., 2020 provide an overview of the relation of beta-1 adrenoreceptor autoantibodies with heart disease.
The pathogenic role for these autoantibodies is supported by results from B-cell depletion or other immunosuppressive therapies including immunoapheresis, where clinical improvement in ME/CFS patients could be observed (Scheibenbogen et al, 2018; Kim et al, 2020). Furthermore, plasma exchange therapy was performed in POTS (Wells et al, 2020), and several Ig depleting approaches, including IVIG therapy, plasma exchange and rituximab treatment supported a causative role for the autoantibodies in these diseases.
However, general immunosuppression or non-selective antibody depletion e.g. by B-cell depletion or immunoapheresis is inconvenient and stressful, and associated with a multitude of undesired side-effects and high cost
It is thus an object of the present invention to provide compounds and methods for the therapy of autoantibody-mediated conditions, preferably selected from ME/CFS, POTS, AAG, IDC, and cChHD, which address one or more of the shortcomings of existing therapies described above and/or which lead to improved treatment outcome.
The present invention provides a compound (typically for the sequestration, or depletion, of antibodies, in particular antibodies associated with autoantibody-mediated conditions, preferably selected from ME/CFS, POTS, AAG, IDC, and cChHD or other conditions mentioned herein, present in a human individual) comprising a biopolymer scaffold and at least two peptides with a sequence length of 6-13 amino acids, wherein each of the peptides independently comprises a 6-amino-acid fragment, preferably a 7-, more preferably an 8-, even more preferably a 9-, even more preferably a 10-, even more preferably an 11-, yet even more preferably a 12-, most preferably a 13-amino-acid fragment, of an amino-acid sequence (preferably of a (preferably human) neuroreceptor), identified by a UniProt accession code selected from the group consisting of:
P02708, P07510, P07550, P08172, P08173, P08588, P08908, P08912, P08913, P11229, P11230, P13945, P17787, P18089, P18825, P20309, P25098, P25100, P30532, P30926, P32297, P35348, P35368, P35626, P36544, P43681, Q04844, Q05901, Q07001, Q15822, Q15825, Q9GZZ6, Q9UGM1, A0A0G2JKS1, A5X5Y0, A6NL88, A8MPY1, B4DS77, B8ZZ34, O00222, O00591, O14490, O14764, O15303, O15399, O43424, 043653, O60359, O60391, O60403, O60404, O60936, O75311, O75916, 076027, O94772, O95264, O95502, O95868, O95886, P01579, P05026, P05067, P06850, P07196, P07384, POC7T3, POC8F1, PODP57, PODP58, P12931, P13500, P14416, P14867, P15382, P16066, P17342, P18505, P18507, P19634, P20594, P21452, P21728, P21917, P21918, P23415, P23416, P24046, P24387, P25021, P25101, P28221, P28222, P28223, P28335, P28472, P28476, P28566, P29274, P29275, P29323, P30411, P30542, P30556, P30939, P31644, P32418, P34903, P34969, P35367, P35372, P35462, P35609, P37288, P39086, P41594, P41595, P41597, P42261, P42262, P42263, P43119, P46098, P47869, P47870, P47898, P47901, P47972, P48058, P48067, P48167, P48169, P48549, P49354, P50052, P50406, P53355, P55000, P62955, P63252, P78334, P78352, P78509, Q00535, Q05586, Q06413, Q07699, Q12879, Q12959, Q13002, Q13003, Q13224, Q13255, Q13387, Q13639, Q13702, Q13936, Q13972, Q14289, Q14416, Q14500, Q14571, Q14573, Q14643, Q14831, Q14832, Q14833, Q14957, Q15700, Q15818, Q16099, Q16445, Q16478, Q16553, Q16602, Q401N2, Q494W8, Q5SQ64, Q6PI25, Q6TFL4, Q6UXU4, Q6ZSJ9, Q70Z44, Q86Y78, Q86YM7, Q8N1C3, Q8N2G4, Q8N2Q7, Q8N4C8, Q8NC67, Q8NFZ4, Q8NG75, Q8NGA5, Q8NGA6, Q8NGC8, Q8NGC9, Q8NGG2, Q8NGG3, Q8NGH5, Q8NGH8, Q8NGN1, Q8NGS4, Q8NGY7, Q8NHC4, Q8NI32, Q8TBE1, Q8TCU5, Q8TDF5, Q8WXA2, Q8WXA8, Q8WXS5, Q92736, Q92796, Q96G91, Q96NW7, Q96P66, Q99928, Q99996, Q9BUH8, Q9BXM7, Q9BYB0, Q9GZV3, Q9H3N8, Q9NPA1, Q9NZ94, Q9P1A6, Q9UBK2, Q9UBN1, Q9UBS5, Q9UF02, Q9ULKO, Q9UN88, Q9UPX8, Q9Y2H0, Q9Y4A9, Q9Y566, Q9Y5N1, Q9Y691, Q9Y698, P37088, P51168, P51170, P51172, O94759, Q16515, O60741, Q9NZQ8, P78348, Q8TDD5, Q9NY37, Q13002, P39086, P48664, A6NGN9, 000305, O00555, O15146, O43448, O43497, O43525, O43526, O60840, 075096, O95180, O95259, O95970, P06213, P16389, P16473, P17658, P22001, P22459, P22460, P24530, P42658, P43146, P48547, P49418, P51787, P54284, P54289, P56696, Q00975, Q01668, Q02246, Q02641, Q03721, Q05329, Q06432, Q08289, Q09470, Q12809, Q13018, Q13303, Q13698, Q14003, Q14721, Q14722, Q15878, Q6PIL6, Q6PIU1, Q6X4W1, Q7Z3S7, Q7Z429, Q8IZS8, Q8NCM2, Q8TAE7, Q8TDN1, Q8TDN2, Q8WWG9, Q92953, Q96KK3, Q96L42, Q96PR1, Q96RP8, Q9BQ31, Q9BXT2, Q9H252, Q9H3M0, Q9NR82, Q9NS40, Q9NS61, Q9NSA2, Q9NY47, Q9NZI2, Q9NZV8, Q9POX4, Q9UHC6, Q9UIX4, Q9UJ90, Q9UJ96, Q9UK17, Q9ULD8, Q9ULS6, Q9UQ05, Q9Y2W7, Q9Y6H6, Q9Y6J6, P48058, P55087, Q9BPU6, P52799, P15328, Q05329, Q16653, Q9Y4C0, Q5F0I5, Q99719, P17600, Q13148, P01266, P07202, and Q9Y6A1, (preferably identified by an UniProt accession code selected from Table 1, Table 2 or Table 3 below, in particular Table 1 or Table 3), optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
Furthermore, the present invention provides a pharmaceutical composition comprising the compound according to the invention and at least one pharmaceutically acceptable excipient.
In an aspect, this pharmaceutical composition is for use in prevention or treatment of autoantibody-mediated conditions, preferably selected from ME/CFS, POTS, AAG, IDC, cChHD, encephalitis such as limbic encephalitis or paraneoplastic striatal encephalitis or Anti-mGluR1 encephalitis or Anti-mGluR5 encephalitis or acute disseminated encephalomyelitis (ADEM) or NMDAR encephalitis, paraneoplastic syndrome, stiff man syndrome, autoimmune channelopathies, neuromyelitis optica, neuromyotonia, Morvan's syndrome, neuropathic pain, myelitis, optic neuritis, retinitis, parkinsonism, chorea, psychosis, dystonia, mutism, movement disorders, confusion, hallucinations, prodromal diarrhoea, memory loss, hyperexcitability, encephalitis psychiatric syndrome, narcolepsy, autism spectrum disorders, seizures, status epilepticus, chronic epilepsy, myoclonus, encephalomyelitis, myoclonus, parasomnia, sleep apnoea, cognitive impairment, gait abnormalities, faciobrachial dystonic seizures, paraneoplastic syndrome, cerebellar ataxia, dysautonomia, Tourette, ADHD, cerebellar ataxia, oscillopsia, amyotrophic lateral sclerosis (ALS), thyroid disorder and headache with neurological deficits and lymphocytosis (HaNDL), in an individual, preferably a human individual.
As described herein above, there are numerous studies supporting that autoantibodies against neuroreceptors or membrane channel proteins play a causative role in ME/CFS, POTS, AAG, IDC, and cChHD. For instance, Bynke et al. found that there exists a general pattern of increased antibody levels to adrenergic and muscarinic receptors in ME/CFS patients (Bynke et al., 2020). In particular, significant increases in autoantibody levels directed against beta-1 and beta-2 adrenergic receptors as well as M3 and M4 muscarinic acetylcholine receptors were observed. Scheibenbogen et al. also observed elevated autoantibodies, in particular against beta-2 adrenergic receptors, and M3 and M4 muscarinic acetylcholine receptors in ME/CFS patients (Scheibenbogen et al., 2020). General antibody depletion by immunoadsorption was shown to be effective in removing autoantibodies and lead to clinical improvement in ME/CFS patients.
However, prior to the present invention, there were no selective approaches to specifically target disease-causing antibodies. Non-specific antibody depletion or immunosuppression are highly inconvenient and come with a multitude of undesired side effects.
In the course of the present invention, a compound was developed which is able to deplete (or sequester) such antibodies against neuroreceptors in vivo and is therefore suitable for use in the prevention or treatment of autoantibody-mediated conditions, such as ME/CFS, POTS, AAG, IDC, and cChHD and other conditions mentioned herein.
Further, it was surprisingly found that the approach which is also used in the invention is particularly effective in reducing titres of undesired antibodies in an individual. In particular, the compound achieved especially good results with regard to selectivity, duration of titre reduction and/or level of titre reduction in an in vivo model (see experimental examples). Moreover it was found that the approach allowed antibody sequestration within less than 24 hours.
The detailed description given below relates to all of the above aspects of the invention unless explicitly excluded.
In general, antibodies are essential components of the humoral immune system, offering protection from infections by foreign organisms including bacteria, viruses, fungi or parasites. However, under certain circumstances—including autoimmune diseases, organ transplantation, blood transfusion or upon administration of biomolecular drugs or gene delivery vectors—antibodies can target the patient's own body (or the foreign tissue or cells or the biomolecular drug or vector just administered), thereby turning into harmful or disease-causing entities. Certain antibodies can also interfere with probes for diagnostic imaging. In the following, such antibodies are generally referred to as “undesired antibodies” or “undesirable antibodies”.
With few exceptions, selective removal of undesired antibodies has not reached clinical practice. It is presently restricted to very few indications: One of the known techniques for selective antibody removal (although not widely established) is immunoapheresis. In contrast to immunoapheresis (which removes immunoglobulin), selective immunoapheresis involves the filtration of plasma through an extracorporeal, selective antibody-adsorber cartridge that will deplete the undesired antibody based on selective binding to its antigen binding site. Selective immunoapheresis has for instance been used for removing anti-A or anti-B antibodies from the blood prior to ABO-incompatible transplantation or with respect to indications in transfusion medicine (Teschner et al). Selective apheresis was also experimentally applied in other indications, such as neuroimmunological indications (Tetala et al) or myasthenia gravis (Lazaridis et al), but is not yet established in the clinical routine. One reason that selective immunoapheresis is only hesitantly applied is the fact that it is a cost intensive and cumbersome intervention procedure that requires specialized medical care. Moreover, it is not known in the prior art how to deplete undesired antibodies rapidly and efficiently.
Unrelated to apheresis, Morimoto et al. discloses dextran as a generally applicable multivalent scaffold for improving immunoglobulin-binding affinities of peptide and peptidomimetic ligands such as the FLAG peptide. WO 2011/130324 A1 relates to compounds for prevention of cell injury. EP 3 059 244 A1 relates to a C-met protein agonist.
As mentioned, apheresis is applied extracorporeally. By contrast, also several approaches to deplete undesirable antibodies intracorporeally were proposed in the prior art, mostly in connection with certain autoimmune diseases involving autoantibodies or anti-drug antibodies:
Lorentz et al discloses a technique whereby erythrocytes are charged in situ with a tolerogenic payload driving the deletion of antigen-specific T cells. This is supposed to ultimately lead to reduction of the undesired humoral response against a model antigen. A similar approach is proposed in Pishesha et al. In this approach, erythrocytes are loaded ex vivo with a peptide-antigen construct that is covalently bound to the surface and reinjected into the animal model for general immunotolerance induction.
WO 92/13558 A1 relates to conjugates of stable nonimmunogenic polymers and analogs of immunogens that possess the specific B cell binding ability of the immunogen and which, when introduced into individuals, induce humoral anergy to the immunogen. Accordingly, these conjugates are disclosed to be useful for treating antibody-mediated pathologies that are caused by foreign- or self-immunogens. In this connection, see also EP 0 498 658 A2.
Taddeo et al discloses selectively depleting antibody producing plasma cells using anti-CD138 antibody derivatives fused to an ovalbumin model antigen thereby inducing receptor crosslinking and cell suicide in vitro selectively in those cells that express the antibody against the model antigen.
Apitope International NV (Belgium) is presently developing soluble tolerogenic T-cell epitope peptides which may lead to expression of low levels of co-stimulatory molecules from antigen presenting cells inducing tolerance, thereby suppressing antibody response (see e.g. Jansson et al). These products are currently under preclinical and early clinical evaluation, e.g. in multiple sclerosis, Grave's disease, intermediate uveitis, and other autoimmune conditions as well as Factor VIII intolerance.
Similarly, Selecta Biosciences, Inc. (USA) is currently pursuing strategies of tolerance induction by so-called Synthetic Vaccine Particles (SVPs). SVP-Rapamycin is supposed to induce tolerance by preventing undesired antibody production via selectively inducing regulatory T cells (see Mazor et al).
Mingozzi et al discloses decoy adeno-associated virus (AAV) capsids that adsorb antibodies but cannot enter a target cell.
WO 2015/136027 A1 discloses carbohydrate ligands presenting the minimal Human Natural Killer-1 (HNK-1) epitope that bind to anti-MAG (myelin-associated glycoprotein) IgM antibodies, and their use in diagnosis as well as for the treatment of anti-MAG neuropathy. WO 2017/046172 A1 discloses further carbohydrate ligands and moieties, respectively, mimicking glycoepitopes comprised by glycosphingolipids of the nervous system which are bound by anti-glycan antibodies associated with neurological diseases. The document further relates to the use of these carbohydrate ligands/moieties in diagnosis as well as for the treatment of neurological diseases associated with anti-glycan antibodies.
US 2004/0258683 A1 discloses methods for treating systemic lupus erythematosus (SLE) including renal SLE and methods of reducing risk of renal flare in individuals with SLE, and methods of monitoring such treatment. One disclosed method of treating SLE including renal SLE and reducing risk of renal flare in an individual with SLE involves the administration of an effective amount of an agent for reducing the level of anti-double-stranded DNA (dsDNA) antibody, such as a dsDNA epitope as in the form of an epitope-presenting carrier or an epitope-presenting valency platform molecule, to the individual.
U.S. Pat. No. 5,637,454 relates to assays and treatments of autoimmune diseases. Agents used for treatment might include peptides homologous to the identified antigenic, molecular mimicry sequences. It is disclosed that these peptides could be delivered to a patient in order to decrease the amount of circulating antibody with a particular specificity.
US 2007/0026396 A1 relates to peptides directed against antibodies, which cause cold-intolerance, and the use thereof. It is taught that by using the disclosed peptides, in vivo or ex vivo neutralization of undesired autoantibodies is possible. A comparable approach is disclosed in WO 1992/014150 A1 or in WO 1998/030586 A2.
WO 2018/102668 A1 discloses a fusion protein for selective degradation of disease-causing or otherwise undesired antibodies. The fusion protein (termed “Seldeg”) includes a targeting component that specifically binds to a cell surface receptor or other cell surface molecule at near-neutral pH, and an antigen component fused directly or indirectly to the targeting component. Also disclosed is a method of depleting a target antigen-specific antibody from a patient by administering to the patient a Seldeg having an antigen component configured to specifically bind the target antigen-specific antibody.
WO 2015/181393 A1 concerns peptides grafted into sunflower-trypsin-inhibitor-(SFTI-) and cyclotide-based scaffolds. These peptides are disclosed to be effective in autoimmune disease, for instance citrullinated fibrinogen sequences that are grafted into the SFTI scaffold have been shown to block autoantibodies in rheumatoid arthritis and inhibit inflammation and pain. These scaffolds are disclosed to be non-immunogenic.
Erlandsson et al discloses in vivo clearing of idiotypic antibodies with anti-idiotypic antibodies and their derivatives.
Berlin Cures Holding AG (Germany) has proposed an intravenous broad spectrum neutralizer DNA aptamer (see e.g. WO 2016/020377 A1 and WO 2012/000889 A1) for the treatment of dilated cardiomyopathy and other GPCR-autoantibody related diseases that in high dosage is supposed to block autoantibodies by competitive binding to the antigen binding regions of autoantibodies. In general, aptamers did not yet achieve a breakthrough and are still in a preliminary stage of clinical development. The major concerns are still biostability and bioavailability, constraints such as nuclease sensitivity, toxicity, small size and renal clearance. A particular problem with respect to their use as selective antibody antagonists are their propensity to stimulate the innate immune response.
WO 00/33887 A2 discloses methods for reducing circulating levels of antibodies, particularly disease-associated antibodies. The methods entail administering effective amounts of epitope-presenting carriers to an individual. In addition, ex vivo methods for reducing circulating levels of antibodies are disclosed which employ epitope-presenting carriers.
U.S. Pat. No. 6,022,544 A relates to a method for reducing an undesired antibody response in a mammal by administering to the mammal a non-immunogenic construct which is free of high molecular weight immunostimulatory molecules. The construct is disclosed to contain at least two copies of a B cell membrane immunoglobulin receptor epitope bound to a pharmaceutically acceptable non-immunogenic carrier.
However, the approaches to deplete undesirable antibodies intracorporeally disclosed in the prior art have many shortcomings. In particular, neither of them has been approved for regular clinical use.
With respect to the compound of the present invention, it is preferred that said neurotransmitter is a neuroreceptor of the autonomic nervous system, more preferably a neuroreceptor selected from the group consisting of muscarinic, and nicotinic cholinergic receptors, alpha- and beta-adrenergic receptors, serotonin receptors, angiotensin- and endothelin receptors; most preferably a neuroreceptor selected from the group consisting of beta-1 adrenergic receptor, beta-2 adrenergic receptor, M3 muscarinic acetylcholine receptor, and M4 muscarinic acetylcholine receptor. In all instances, it is preferred that the neuroreceptor is a human neuroreceptor.
In a preference, each of the at least two peptides (comprised by the inventive compound), independently comprises a 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-amino acid fragment (in increasing order of preference) of an amino acid sequence (preferably of a neuroreceptor of the autonomic nervous system) identified by a UniProt accession code selected from the group consisting of: P02708, P07510, P07550, P08172, P08173, P08588, P08908, P08912, P08913, P11229, P11230, P13945, P17787, P18089, P18825, P20309, P25098, P25100, P30532, P30926, P32297, P35348, P35368, P35626, P36544, P43681, Q04844, Q05901, Q07001, Q15822, Q15825, Q9GZZ6, Q9UGM1; P37088, P51168, P51170, P51172, 094759, Q16515, 060741, Q9NZQ8, P78348, Q8TDD5, Q9NY37, Q13002, P39086, and P48664.
It is even more preferred, if said amino acid sequence is an amino acid sequence of a neuroreceptor selected from the group consisting of muscarinic, and nicotinic cholinergic receptors, alpha- and beta-adrenergic receptors, serotonin receptors, angiotensin- and endothelin receptors.
In a particular preference, said amino acid sequence is an amino acid sequence (preferably of a neuroreceptor selected from the group consisting of beta-1 adrenergic receptor, beta-2 adrenergic receptor, M3 muscarinic acetylcholine receptor, and M4 muscarinic acetylcholine receptor) identified by a UniProt accession code selected from the group consisting of: P08588, P07550, P20309, and P08173.
The definitions of preferred amino acid sequences (with respect to neurotransmitters and/or UniProt accession numbers) disclosed in the preceding paragraphs, as well as in the summary of the invention disclosed herein above, equally apply as preferred embodiments to all definitions of peptides comprised in the inventive compound (designated herein below e.g. as P, P1, P2, Pa-J).
It is well established that many neurological, neuromuscular and neuropsychiatric disorders are associated with or caused by autoantibodies. Since the discovery of the disease-causing effect of autoantibodies that target the neuromuscular junction (e.g. antibodies to the nicotinic acetylcholine receptor in myasthenia gravis), several paradigms with similar pathogenetic features were described. In many cases correlations and associations were shown in the clinic, for some of these cases functional proof of concepts was established by using animal models in which the pathogenic effects of human autoantibodies (either from autoimmune sera, or from cloned antibodies, or directly induced by active immunization) were demonstrated, as reviewed for example by Giannoccaro et al., 2020. In the course of the present invention, sera from human donors (including ME/CFS patients) were screened for peptides that are able to bind to the paratopes of autoantibodies against human proteins, in particular receptors and ion channels or other membrane channels, involved in neurological, neuromuscular and neuropsychiatric disorders (see in particular Example 12 and Table 1 below, as well as e.g. Table 2). The found peptides or fragments thereof are suitable to deplete disease-causing autoantibodies in a patient when administered in the form of the inventive compound.
Several neurological or neuropsychiatric disease conditions or syndromes can be associated with one or several common autoantibody targets (i.e. autoantigens). Autoantigens do not necessarily need to be located in the extracellular space, such as is the case with neuroreceptors and membrane channels (related to autoimmune channelopathies)—many autoantibodies are in fact associated with intracellular antigens, as listed below. Importantly, the association of neuroimmunological symptoms is found in a variety of conditions such as tumors, neurodegenerative diseases or autoimmune diseases. The present invention provides a solution of removing such autoantibodies regardless of whether the corresponding autoantigens are located in the extracellular or intracellular space. The peptides derived from neuroreceptors and other proteins disclosed herein provide binding moieties for autoantibodies regardless of whether the peptides have been derived from an extra- or intracellular portion of a protein chain. Furthermore, the peptide identification strategy provided in the present invention may yield peptide hits which only represent a partial epitope structure, and not an entire, “natural” epitope structure—it is not required that the linear or cyclic peptides of the present invention should mimic an entire epitope per se (in fact, representing only a partial epitope is preferred in order to further reduce any potential immunogenicity of the compounds of the present invention). In other words, a purpose of the peptides of the present invention is to bind to undesired and disease-causing antibodies such as the type of autoantibodies involved in neurological or neuropsychiatric diseases (see in particular Tables 1-3 below).
An extensive list of top autoantigens involved in neurological or neuropsychiatric conditions is provided in Table 2 below. See e.g. Prüss, 2021; Garza et al., 2021; Giannoccaro et al. 2018; Gardoni et al 2021. In addition, Hansen & TimAus, 2021 provide a review with a special focus on autoantibodies in psychiatric conditions, most importantly autoimmune encephalitis with psychiatric syndromes and related diseases. Galli et al, 2020, provide a review about the role of autoantibodies in paraneoplastic diseases. 2020). Also relevant are intracellular antigens such as Ma2[Ta], Hu, Ri, Yo, CV2/CRMP5, amphiphysin, GAD65, and antinuclear antigens (ANAs), or thyroid tissue antigens such as TG, TPO or TRAK in the context of neurological diseases.
Table 3 below is a selection from Table 1 and lists peptides based on the top autoantigens of Table 2 found in the autoantibody screen performed in the course of the present invention.
In light of the findings described in Tables 1-3 above as well as in Example 12, a particularly preferred embodiment is directed to the inventive compound wherein, for at least one of the peptides (preferably for each of the peptides), said amino-acid fragment comprises at least 4, preferably at least 5 or even at least 6, more preferably at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of a sequence identified by any one of SEQ ID NOs: 45-3536, preferably any one of SEQ ID NOs: 45-863, especially any one of SEQ ID NOs: 45-201, optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
According to yet another preference, the peptides used in the compound of the present invention (e.g. peptide P or Pa or Pb or P1 or P2) comprise at least 4, preferably at least 5 or even at least 6, more preferably at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of a sequence identified by any one of SEQ ID NOs: 45-3536, preferably any one of SEQ ID NOs: 45-863, especially any one of SEQ ID NOs: 45-201, optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
According to another preferred embodiment, the respective amino acid sequences of the at least two peptides of the inventive compound are the same. In other words, the at least two peptides are identical.
Narcolepsy type 1 is another autoantibody-associated disease (see e.g. Vuorela et al, 2021). The involved autoantigen turned out to be protein-O-mannosyltransferase 1 (POMT1), UniProt accession number Q9Y6A1. More specifically, one autoepitope discovered was located in residues 697-711 of UniProt accession number Q9Y6A1. Accordingly, POMT1 is a particularly preferred target of the present invention. Even more preferably, said amino-acid fragment comprises at least 4, preferably at least 5 or even at least 6, more preferably at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of residues 697-711 of UniProt accession number Q9Y6A1, optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
Preferably, in the entire context of the present invention, the at least two peptides comprise a peptide P1 and a peptide P2, wherein P1 and P2 independently comprise a 6-, preferably a 7-, more preferably an 8-, even more preferably a 9-, even more preferably a 10-, yet even more preferably an 11-, especially a 12-, most preferably a 13-amino-acid fragment of an amino-acid sequence as disclosed hereinabove (by the indicated neuroreceptors and/or UniProt accession numbers), wherein P1 and P2 are present in form of a peptide dimer P1-S-P2, wherein S is a non-peptide spacer, wherein the peptide dimer is covalently bound to the biopolymer scaffold, preferably via a linker.
A preferred embodiment of the inventive compound relates to a compound comprising
P(-S-P)(n-1) and
P(-S-P)(n-1);
The biopolymer scaffold used in the present invention may be a mammalian biopolymer such as a human biopolymer, a non-human primate biopolymer, a sheep biopolymer, a pig biopolymer, a dog biopolymer or a rodent biopolymer. In particular the biopolymer scaffold is a protein, especially a (non-modified or non-modified with respect to its amino-acid sequence) plasma protein. Preferably, the biopolymer scaffold is a mammalian protein such as a human protein, a non-human primate protein, a sheep protein, a pig protein, a dog protein or a rodent protein. Typically, the biopolymer scaffold is a non-immunogenic and/or non-toxic protein that preferably circulates in the plasma of healthy (human) individuals and can e.g. be efficiently scavenged or recycled by scavenging receptors, such as e.g. present on myeloid cells or on liver sinusoidal endothelial cells (reviewed by Sorensen et al 2015).
According to a particular preference, the biopolymer scaffold is a (preferably human) globulin, preferably selected from the group consisting of immunoglobulins, alpha1-globulins, alpha2-globulins and beta-globulins, in particular immunoglobulin G, haptoglobin and transferrin. Haptoglobin in particular has several advantageous properties, as shown in Examples 5-9, especially an advantageous safety profile.
The biopolymer scaffold may also be (preferably human) albumin, hemopexin, alpha-1-antitrypsin, C1 esterase inhibitor, lactoferrin or non-immunogenic (i.e. non-immunogenic in the individual to be treated) fragments of all of the aforementioned proteins, including the globulins.
In another preference, the biopolymer scaffold is an anti-CD163 antibody (i.e. an antibody specific for a CD163 protein) or CD163-binding fragment thereof.
Human CD163 (Cluster of Differentiation 163) is a 130 kDa membrane glycoprotein (formerly called M130) and prototypic class I scavenger receptor with an extracellular portion consisting of nine scavenger receptor cysteine-rich (SRCR) domains that are responsible for ligand binding. CD163 is an endocytic receptor present on macrophages and monocytes, it removes hemoglobin/haptoglobin complexes from the blood but it also plays a role in anti-inflammatory processes and wound healing. Highest expression levels of CD163 are found on tissue macrophages (e.g. Kupffer cells in the liver) and on certain macrophages in spleen and bone marrow. Because of its tissue- and cell-specific expression and entirely unrelated to depletion of undesirable antibodies, CD163 is regarded as a macrophage target for drug delivery of e.g. immunotoxins, liposomes or other therapeutic compound classes (Skytthe et al., 2020).
Monoclonal anti-CD163 antibodies and the SRCR domains they are binding are for instance disclosed in Madsen et al., 2004, in particular
Entirely unrelated to depletion of undesirable antibodies, CD163 was proposed as a target for cell-specific drug delivery because of its physiological properties. Tumor-associated macrophages represent one of the main targets where the potential benefit of CD163-targeting is currently explored. Remarkably, numerous tumors and malignancies were shown to correlate with CD163 expression levels, supporting the use of this target for tumor therapy. Other proposed applications include CD163 targeting by anti-drug conjugates (ADCs) in chronic inflammation and neuroinflammation (reviewed in Skytthe et al., 2020). Therefore, CD163-targeting by ADCs notably with dexamethasone or stealth liposome conjugates represents therapeutic principle which is currently studied (Graversen et al., 2012; Etzerodt et al., 2012).
In that context, there are references indicating that anti-CD163 antibodies can be rapidly internalized by endocytosis when applied in vivo. This was shown for example for mAB Ed-2 (Dijkstra et al., 1985; Graversen et al., 2012) or for mAB Mac2-158/KN2/NRY (Granfeldt et al., 2013). Based on those observations in combination with observations made in the course of the present invention (see in particular example section), anti-CD163 antibodies and CD163-binding turned out to be highly suitable biopolymer scaffolds for depletion/sequestration of undesirable antibodies.
Numerous anti-CD163 antibodies and CD163-binding fragments thereof are known in the art (see e.g. above). These are suitable to be used as a biopolymer scaffold for the present invention. For instance, any anti-CD163 antibody or fragment thereof mentioned herein or in WO 2011/039510 A2 (which is included herein by reference) may be used as a biopolymer scaffold in the invention. Preferably, the biopolymer scaffold of the inventive compound is antibody Mac2-48, Mac2-158, 5C6-FAT, BerMac3, or E10B10 as disclosed in WO 2011/039510, in particular humanised Mac2-48 or Mac2-158 as disclosed in WO 2011/039510 A2.
In a preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a heavy-chain variable (VH) region comprising one or more complementarity-determining region (CDR) sequences selected from the group consisting of SEQ ID NOs: 11-13 of WO 2011/039510 A2.
In addition, or alternatively thereto, in a preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a light-chain variable (VL) region comprising one or more CDR sequences selected from the group consisting of SEQ ID NOs: 14-16 of WO 2011/039510 A2 or selected from the group consisting of SEQ ID NOs:17-19 of WO 2011/039510 A2.
In a further preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a heavy-chain variable (VH) region comprising or consisting of the amino acid sequence of SEQ ID NO: 20 of WO 2011/039510 A2.
In addition, or alternatively thereto, in a preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a light-chain variable (VL) region comprising or consisting of the amino acid sequence of SEQ ID NO: 21 of WO 2011/039510 A2.
In a further preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a heavy-chain variable (VH) region comprising or consisting of the amino acid sequence of SEQ ID NO: 22 of WO 2011/039510 A2.
In addition, or alternatively thereto, in a preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a light-chain variable (VL) region comprising or consisting of the amino acid sequence of SEQ ID NO: 23 of WO 2011/039510 A2.
In a further preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a heavy-chain variable (VH) region comprising or consisting of the amino acid sequence of SEQ ID NO: 24 of WO 2011/039510 A2.
In addition, or alternatively thereto, in a preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof comprises a light-chain variable (VL) region comprising or consisting of the amino acid sequence of SEQ ID NO: 25 of WO 2011/039510 A2.
In the context of the present invention, the anti-CD163 antibody may be a mammalian antibody such as a humanized or human antibody, a non-human primate antibody, a sheep antibody, a pig antibody, a dog antibody or a rodent antibody. In embodiments, the anti-CD163 antibody may monoclonal.
According to a preference, the anti-CD163 antibody is selected from IgG, IgA, IgD, IgE and IgM.
According to a further preference, the CD163-binding fragment is selected from a Fab, a Fab′, a F(ab)2, a Fv, a single-chain antibody, a nanobody and an antigen-binding domain.
CD163 amino acid sequences are for instance disclosed in WO 2011/039510 A2 (which is included here by reference). In the context of the present invention, the anti-CD163 antibody or CD163-binding fragment thereof is preferably specific for a human CD163, especially with the amino acid sequence of any one of SEQ ID NOs: 28-31 of WO 2011/039510 A2.
In a further preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof is specific for the extracellular region of CD163 (e.g. for human CD163: amino acids 42-1050 of UniProt Q86VB7, sequence version 2), preferably for an SRCR domain of CD163, more preferably for any one of SRCR domains 1-9 of CD163 (e.g. for human CD163: amino acids 51-152, 159-259, 266-366, 373-473, 478-578, 583-683, 719-819, 824-926 and 929-1029, respectively, of UniProt Q86VB7, sequence version 2), even more preferably for any one of SRCR domains 1-3 of CD163 (e.g. for human CD163: amino acids 51-152, 159-259, 266-366, and 373-473, respectively, of UniProt Q86VB7, sequence version 2), especially for SRCR domain 1 of CD163 (in particular with the amino acid sequence of any one of SEQ ID NOs: 1-8 of WO 2011/039510 A2, especially SEQ ID NO: 1 of WO 2011/039510 A2).
In a particular preference, the anti-CD163 antibody or CD163-binding fragment thereof is capable of competing for binding to (preferably human) CD163 with a (preferably human) hemoglobin-haptoglobin complex (e.g. in an ELISA).
In another particular preference, the anti-CD163 antibody or CD163-binding fragment thereof is capable of competing for binding to human CD163 with any of the anti-human CD163 mAbs disclosed herein, in particular Mac2-48 or Mac2-158 as disclosed in WO 2011/039510 A2.
In yet another particular preference, the anti-CD163 antibody or CD163-binding fragment thereof is capable of competing for binding to human CD163 with an antibody having a heavy chain variable (VH) region consisting of the amino acid sequence
Details on competitive binding experiments are known to the person of skilled in the art (e.g. based on ELISA) and are for instance disclosed in WO 2011/039510 A2 (which is included herein by reference).
In the course of the present invention, the epitopes of antibodies E10B10 and Mac2-158 as disclosed in WO 2011/039510 were mapped by fine mapping using circular peptide arrays, whereby the peptides were derived from CD163. These epitopes are particularly suitable for binding of the anti-CD163 antibody (or CD163-binding fragment thereof) of the inventive compound.
Accordingly, in particularly preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof is specific for peptide consisting of 7-25, preferably 8-20, even more preferably 9-15, especially 10-13 amino acids, wherein the peptide comprises the amino acid sequence CSGRVEVKVQEEWGTVCNNGWSMEA (SEQ ID NO: 3) or a 7-24 amino-acid fragment thereof. Preferably, this peptide comprises the amino acid sequence GRVEVKVQEEW (SEQ ID NO: 4), WGTVCNNGWS (SEQ ID NO: 5) or WGTVCNNGW (SEQ ID NO: 6). More preferably, the peptide comprises an amino acid sequence selected from EWGTVCNNGWSME (SEQ ID NO: 7), QEEWGTVCNNGWS (SEQ ID NO: 8), WGTVCNNGWSMEA (SEQ ID NO: 9), EEWGTVCNNGWSM (SEQ ID NO: 10), VQEEWGTVCNNGW (SEQ ID NO: 11), EWGTVCNNGW (SEQ ID NO: 12) and WGTVCNNGWS (SEQ ID NO: 5). Even more preferably, the peptide consists of an amino acid sequence selected from EWGTVCNNGWSME (SEQ ID NO: 7), QEEWGTVCNNGWS (SEQ ID NO: 8), WGTVCNNGWSMEA (SEQ ID NO: 9), EEWGTVCNNGWSM (SEQ ID NO: 10), VQEEWGTVCNNGW (SEQ ID NO: 11), EWGTVCNNGW (SEQ ID NO: 12) and WGTVCNNGWS (SEQ ID NO: 5), optionally with an N-terminal and/or C-terminal cysteine residue.
Accordingly, in another particularly preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof is specific for a peptide consisting of 7-25, preferably 8-20, even more preferably 9-15, especially 10-13 amino acids, wherein the peptide comprises the amino acid sequence DHVSCRGNESALWDCKHDGWG (SEQ ID NO: 13) or a 7-20 amino-acid fragment thereof. Preferably, this peptide comprises the amino acid sequence ESALW (SEQ ID NO: 14) or ALW. More preferably, the peptide comprises an amino acid sequence selected from ESALWDC (SEQ ID NO: 15), RGNESALWDC (SEQ ID NO: 16), SCRGNESALW (SEQ ID NO: 17), VSCRGNESALWDC (SEQ ID NO: 18), ALWDCKHDGW (SEQ ID NO: 19), DHVSCRGNESALW (SEQ ID NO: 20), CRGNESALWD (SEQ ID NO: 21), NESALWDCKHDGW (SEQ ID NO: 22) and ESALWDCKHDGWG (SEQ ID NO: 23). Even more preferably, the peptide consists of an amino acid sequence selected from ESALWDC (SEQ ID NO: 15), RGNESALWDC (SEQ ID NO: 16), SCRGNESALW (SEQ ID NO: 17), VSCRGNESALWDC (SEQ ID NO: 18), ALWDCKHDGW (SEQ ID NO: 19), DHVSCRGNESALW (SEQ ID NO: 20), CRGNESALWD (SEQ ID NO: 21), NESALWDCKHDGW (SEQ ID NO: 22) and ESALWDCKHDGWG (SEQ ID NO: 23), optionally with an N-terminal and/or C-terminal cysteine residue.
Accordingly, in another particularly preferred embodiment, the anti-CD163 antibody or CD163-binding fragment thereof is specific for a peptide consisting of 7-25, preferably 8-20, even more preferably 9-15, especially 10-13 amino acids, wherein the peptide comprises the amino acid sequence SSLGGTDKELRLVDGENKCS (SEQ ID NO: 24) or a 7-19 amino-acid fragment thereof. Preferably, this peptide comprises the amino acid sequence SSLGGTDKELR (SEQ ID NO: 25) or SSLGG (SEQ ID NO: 26). More preferably, the peptide comprises an amino acid sequence selected from SSLGGTDKELR (SEQ ID NO: 25), SSLGGTDKEL (SEQ ID NO: 28), SSLGGTDKE (SEQ ID NO: 29), SSLGGTDK (SEQ ID NO: 30), SSLGGTD (SEQ ID NO: 31), SSLGGT (SEQ ID NO: 32) and SSLGG (SEQ ID NO: 26). Even more preferably, the peptide consists of an amino acid sequence selected from SSLGGTDKELR (SEQ ID NO: 25), SSLGGTDKEL (SEQ ID NO: 28), SSLGGTDKE (SEQ ID NO: 29), SSLGGTDK (SEQ ID NO: 30), SSLGGTD (SEQ ID NO: 31), SSLGGT (SEQ ID NO: 32) and SSLGG (SEQ ID NO: 26), optionally with an N-terminal and/or C-terminal cysteine residue.
The peptides (or peptide n-mers) are preferably covalently conjugated (or covalently bound) to the biopolymer scaffold via a (non-immunogenic) linker known in the art such as for example amine-to-sulfhydryl linkers and bifunctional NHS-PEG-maleimide linkers or other linkers known in the art. Alternatively, the peptides (or peptide n-mers) can be bound to the epitope carrier scaffold e.g. by formation of a disulfide bond between the protein and the peptide (which is also referred to as “linker” herein), or using non-covalent assembly techniques, spontaneous isopeptide bond formation or unnatural amino acids for bio-orthogonal chemistry via genetic code expansion techniques (reviewed by Howarth et al 2018 and Lim et al 2016).
The compound of the present invention may comprise e.g. at least two, preferably between 3 and 40 copies of one or several different peptides (which may be present in different forms of peptide n-mers as disclosed herein). The compound may comprise one type of epitopic peptide (in other words: antibody-binding peptide or paratope-binding peptide), however the diversity of epitopic peptides bound to one biopolymer scaffold molecule can be a mixture of e.g. up to 8 different epitopic peptides.
Typically, since the peptides present in the inventive compound specifically bind to selected undesired antibodies, their sequence is usually selected and optimized such that they provide specific binding in order to guarantee selectivity of undesired antibody depletion from the blood. For this purpose, the peptide sequence of the peptides typically corresponds to the entire epitope sequence or portions of the undesired antibody epitope. The peptides used in the present invention can be further optimized by exchanging one, two or up to three amino-acid positions, allowing e.g. for modulating the binding affinity to the undesired antibody that needs to be depleted. Such single or multiple amino-acid substitution strategies that can provide “mimotopes” with increased binding affinity and are known in the field and were previously developed using phage display strategies or peptide microarrays. In other words, the peptides used in the present invention do not have to be completely identical to the native epitope sequences of the undesired antibodies.
Typically, the peptides used in the compound of the present invention (e.g. peptide P or Pa or Pb or P1 or P2) are composed of one or more of the 20 amino acids commonly present in mammalian proteins. In addition, the amino acid repertoire used in the peptides may be expanded to post-translationally modified amino acids e.g. affecting antigenicity of proteins such as post translational modifications, in particular oxidative post translational modifications (see e.g. Ryan 2014) or modifications to the peptide backbone (see e.g. Müller 2018), or to non-natural amino acids (see e.g. Meister et al, 2018). These modifications may also be used in the peptides e.g. to adapt the binding interaction and specificity between the peptide and the variable region of an undesired antibody. In particular, epitopes (and therefore the peptides used in the compound of the present invention) can also contain citrulline as for example in autoimmune diseases. Furthermore, by introducing modifications into the peptide sequence the propensity of binding to an HLA molecule may be reduced, the stability and the physicochemical characteristics may be improved or the affinity to the undesired antibody may be increased.
In many cases, the undesired antibody that is to be depleted is oligo- or polyclonal (e.g. autoantibodies, ADAs or alloantibodies are typically poly- or oligoclonal), implying that undesired (polyclonal) antibody epitope covers a larger epitopic region of a target molecule. To adapt to this situation, the compound of the present invention may comprise a mixture of two or several epitopic peptides (in other words: antibody-binding peptides or paratope-binding peptides), thereby allowing to adapt to the polyclonality or oligoclonality of an undesired antibody.
Such poly-epitopic compounds of the present invention can effectively deplete undesired antibodies and are more often effective than mono-epitopic compounds in case the epitope of the undesired antibody extends to larger amino acid sequence stretches.
It is advantageous if the peptides used for the inventive compound are designed such that they will be specifically recognized by the variable region of the undesired antibodies to be depleted. The sequences of peptides used in the present invention may e.g. be selected by applying fine epitope mapping techniques (i.e. epitope walks, peptide deletion mapping, amino acid substitution scanning using peptide arrays such as described in Carter et al 2004, and Hansen et al 2013) on the undesired antibodies.
It is highly preferred that the peptides used for the inventive compound do not bind to any HLA Class I or HLA Class II molecule (i.e. of the individual to be treated, e.g. human), in order to prevent presentation and stimulation via a T-cell receptor in vivo and thereby induce an immune reaction. It is generally not desired to involve any suppressive (or stimulatory) T-cell reaction in contrast to antigen-specific immunologic tolerization approaches. Therefore, to avoid T-cell epitope activity as much as possible, the peptides of the compound of the present invention (e.g. peptide P or Pa or Pb or P1 or P2) preferably fulfil one or more of the following characteristics:
For stronger reduction of the titre of the undesired antibodies, it is preferred that the peptides used in the present invention are circularized (see also Example 4). Accordingly, in a preferred embodiment, at least one occurrence of P is a circularized peptide. Preferably at least 10% of all occurrences of P are circularized peptides, more preferably at least 25% of all occurrences of P are circularized peptides, yet more preferably at least 50% of all occurrences of P are circularized peptides, even more preferably at least 75% of all occurrences of P are circularized peptides, yet even more preferably at least 90% of all occurrences of P are circularized peptides or even at least 95% of all occurrences of P are circularized peptides, especially all of the occurrences of P are circularized peptides. Several common techniques are available for circularization of peptides, see e.g. Ong et al 2017. It goes without saying that “circularized peptide” as used herein shall be understood as the peptide itself being circularized, as e.g. disclosed in Ong et al. (and not e.g. grafted on a circular scaffold with a sequence length that is longer than 13 amino acids). Such peptides may also be referred to as cyclopeptides herein.
Further, for stronger reduction of the titre of the undesired antibodies relative to the amount of scaffold used, in a preferred embodiment of the compound of the present invention, independently for each of the peptide n-mers, n is at least 2, more preferably at least 3, especially at least 4. Usually, in order to avoid complexities in the manufacturing process, independently for each of the peptide n-mers, n is less than 10, preferably less than 9, more preferably less than 8, even more preferably less than 7, yet even more preferably less than 6, especially less than 5. To benefit from higher avidity through divalent binding of the undesired antibody, it is highly preferred that, for each of the peptide n-mers, n is 2.
For multivalent binding of the undesired antibodies, it is advantageous that the peptide dimers or n-mers are spaced by a hydrophilic, structurally flexible, immunologically inert, non-toxic and clinically approved spacer such as (hetero-)bifunctional and -trifunctional polyethylene glycol (PEG) spacers (e.g. NHS-PEG-Maleimide)—a wide range of PEG chains is available and PEG is approved by the FDA. Alternatives to PEG linkers such as immunologically inert and non-toxic synthetic polymers or glycans are also suitable. Accordingly, in the context of the present invention, the spacer (e.g. spacer S) is preferably selected from PEG molecules or glycans. For instance, the spacer such as PEG can be introduced during peptide synthesis. Such spacers (e.g. PEG spacers) may have a molecular weight of e.g. 10000 Dalton. Evidently, within the context of the present invention, the covalent binding of the peptide n-mers to the biopolymer scaffold via a linker each may for example also be achieved by binding of the linker directly to a spacer of the peptide n-mer (instead of, e.g., to a peptide of the peptide n-mer).
Preferably, each of the peptide n-mers is covalently bound to the biopolymer scaffold, preferably via a linker each.
As used herein, the linker may e.g. be selected from disulphide bridges and PEG molecules.
According to a further preferred embodiment of the inventive compound, at least one occurrence of P is Pa and/or at least one occurrence of P is Pb (wherein Pa and Pb each independently is a peptide as defined above for P and/or P1 and P2). Preferably, independently for each occurrence, P is Pa or Pb.
Furthermore, it is preferred when in the first peptide n-mer, each occurrence of P is Pa and, in the second peptide n-mer, each occurrence of P is Pb. Alternatively, or in addition thereto, Pa and/or Pb is circularized.
Divalent binding is particularly suitable to reduce antibody titres. According, in a preferred embodiment,
For increasing effectivity, in particular in autoimmune disease (which is usually based on polyclonal antibodies, see above), in a preferred embodiment the first peptide n-mer is different from the second peptide n-mer. For similar reasons, preferably, the peptide Pa is different from the peptide Pb, preferably wherein the peptide Pa and the peptide Pb are two different epitopes of the same antigen or two different epitope parts of the same epitope.
Especially for better targeting of polyclonal antibodies, it is advantageous when the peptide Pa and the peptide Pb comprise the same amino-acid sequence fragment, wherein the amino-acid sequence fragment has a length of at least 2 amino acids, preferably at least 3 amino acids, more preferably at least 4 amino acids, yet more preferably at least 5 amino acids, even more preferably at least 6 amino acids, yet even more preferably at least 7 amino acids, especially at least 8 amino acids or even at least 9 amino acids.
Further, for stronger reduction of the titre of the undesired antibodies relative to the amount of scaffold used, the compound comprises a plurality of said first peptide n-mer (e.g. up to 10 or 20 or 30) and/or a plurality of said second peptide n-mer (e.g. up to 10 or 20 or 30). For stronger reduction of the titre of the undesired antibodies relative to the amount of scaffold used, the compound may also comprise at least
P(-S-P)(n-1),
P(-S-P)(n-1),
P(-S-P)(n-1),
P(-S-P)(n-1),
P(-S-P)(n-1),
P(-S-P)(n-1),
P(-S-P)(n-1),
P(-S-P)(n-1),
Peptides Pc-Pj may have one or more of same features (e.g. sequence) as disclosed herein for peptides Pa and Pb (and/or for peptides P, P1, P2). All preferred features disclosed herein for P, P1, and P2, are also preferred features of the peptides Pa-Pj. As also illustrated above, it is highly preferred when the compound of the present invention is non-immunogenic in a mammal, preferably in a human, in a non-human primate, in a sheep, in a pig, in a dog or in a rodent.
In the context of the present invention, a non-immunogenic compound preferably is a compound wherein the biopolymer scaffold (if it is a protein) and/or the peptides (of the peptide n-mers) have an IC50 higher than 100 nM, preferably higher than 500 nM, even more preferably higher than 1000 nM, especially higher than 2000 nM, against HLA-DRB1_0101 as predicted by the NetMHCII-2.3 algorithm. The NetMHCII-2.3 algorithm is described in detail in Jensen et al, which is incorporated herein by reference. The algorithm is publicly available under http://www.cbs.dtu.dk/services/NetMHCII-2.3/. Even more preferably, a non-immunogenic compound (or pharmaceutical composition) does not bind to any HLA and/or MHC molecule (e.g. in a mammal, preferably in a human, in a non-human primate, in a sheep, in a pig, in a dog or in a rodent; or of the individual to be treated) in vivo.
According to a further preference, the compound is for intracorporeal sequestration (or intracorporeal depletion) of at least one antibody in an individual, preferably in the bloodstream of the individual and/or for reduction of the titre of at least one antibody in the individual, preferably in the bloodstream of the individual. Preferably the antibody is an antibody specific for a (human) neuroreceptor, preferably a (human) neuroreceptor of the autonomic nervous system, more preferably a (human) neuroreceptor selected from the group consisting of muscarinic, and nicotinic cholinergic receptors, alpha- and beta-adrenergic receptors, serotonin receptors, angiotensin- and endothelin receptors; most preferably a (human) neuroreceptor selected from the group consisting of beta-1 adrenergic receptor, beta-2 adrenergic receptor, M3 muscarinic acetylcholine receptor, and M4 muscarinic acetylcholine receptor; preferably defined by a UniProt accession number disclosed herein above (in the context of the peptides comprised in the inventive compound).
In an aspect, the present invention relates to a pharmaceutical composition comprising the inventive compound and at least one pharmaceutically acceptable excipient.
In embodiments, the composition is prepared for intraperitoneal, subcutaneous, intramuscular and/or intravenous administration. In particular, the composition is for repeated administration (since it is typically non-immunogenic).
In a preference, the molar ratio of peptides (e.g. P or Pa or Pb) to biopolymer scaffold in the composition is from 2:1 to 100:1, preferably from 3:1 to 90:1, more preferably from 4:1 to 80:1, even more preferably from 5:1 to 70:1, yet even more preferably from 6:1 to 60:1, especially from 7:1 to 50:1 or even from 8:10 to 40:1.
In another aspect, the compound and/or the pharmaceutical composition of the present invention is for use in therapy.
Preferably, the compound and/or the pharmaceutical composition is for use in prevention or treatment of ME/CFS in an individual.
In a further preference, the compound and/or the pharmaceutical composition is for use in prevention or treatment of POTS in an individual.
In yet a further preference, the compound and/or the pharmaceutical composition is for use in prevention or treatment of AAG in an individual.
In yet a further preference, the compound and/or the pharmaceutical composition is for use in prevention or treatment of IDC in an individual.
In yet a further preference, the compound and/or the pharmaceutical composition is for use in prevention or treatment of cChHD in an individual.
In yet a further preference, the compound and/or the pharmaceutical composition is for use in prevention or treatment of encephalitis such as limbic encephalitis or paraneoplastic striatal encephalitis or Anti-mGluR1 encephalitis or Anti-mGluR5 encephalitis or acute disseminated encephalomyelitis (ADEM) or NMDAR encephalitis, paraneoplastic syndrome, stiff man syndrome, autoimmune channelopathies, neuromyelitis optica, neuromyotonia, Morvan's syndrome, neuropathic pain, myelitis, optic neuritis, retinitis, parkinsonism, chorea, psychosis, dystonia, mutism, movement disorders, confusion, hallucinations, prodromal diarrhoea, memory loss, hyperexcitability, encephalitis psychiatric syndrome, narcolepsy, autism spectrum disorders, seizures, status epilepticus, chronic epilepsy, myoclonus, encephalomyelitis, myoclonus, parasomnia, sleep apnoea, cognitive impairment, gait abnormalities, faciobrachial dystonic seizures, paraneoplastic syndrome, cerebellar ataxia, dysautonomia, Tourette, ADHD, cerebellar ataxia, oscillopsia, amyotrophic lateral sclerosis (ALS), thyroid disorder and headache with neurological deficits or lymphocytosis (HaNDL) in an individual.
In the course of the present invention, it turned out that the in vivo kinetics of undesirable-antibody lowering by the inventive compound is typically very fast, sometimes followed by a mild rebound of the undesirable antibody. It is thus particularly preferred when the compound (or the pharmaceutical composition comprising the compound) is administered at least twice within a 96-hour window, preferably within a 72-hour window, more preferably within a 48-hour window, even more preferably within a 36-hour window, yet even more preferably within a 24-hour window, especially within a 18-hour window or even within a 12-hour window.
In embodiments, one or more antibodies are present in the individual which are specific for at least one occurrence of the peptide of the inventive compound (e.g. the peptide P, P1, P2, or for peptide Pa and/or peptide Pb), preferably wherein said antibodies are specific for a neuroreceptor as defined herein above.
It is highly preferred that the composition is non-immunogenic in the individual (e.g. it does not comprise an adjuvant or an immunostimulatory substance that stimulates the innate or the adaptive immune system, e.g. such as an adjuvant or a T-cell epitope).
The composition of the present invention may be administered at a dose of 1-1000 mg, preferably 2-500 mg, more preferably 3-250 mg, even more preferably 4-100 mg, especially 5-50 mg, compound per kg body weight of the individual, preferably wherein the composition is administered repeatedly. Such administration may be intraperitoneally, subcutaneously, intramuscularly or intravenously.
In an aspect, the present invention relates to a method of ameliorating or treating an autoantibody-mediated condition, preferably selected from CFS/ME, POTS, AAG, IDC, and cChHD and encephalitis such as limbic encephalitis or paraneoplastic striatal encephalitis or Anti-mGluR1 encephalitis or Anti-mGluR5 encephalitis or acute disseminated encephalomyelitis (ADEM) or NMDAR encephalitis, paraneoplastic syndrome, stiff man syndrome, autoimmune channelopathies, neuromyelitis optica, neuromyotonia, Morvan's syndrome, neuropathic pain, myelitis, optic neuritis, retinitis, parkinsonism, chorea, psychosis, dystonia, mutism, movement disorders, confusion, hallucinations, prodromal diarrhoea, memory loss, hyperexcitability, encephalitis psychiatric syndrome, narcolepsy, autism spectrum disorders, seizures, status epilepticus, chronic epilepsy, myoclonus, encephalomyelitis, myoclonus, parasomnia, sleep apnoea, cognitive impairment, gait abnormalities, faciobrachial dystonic seizures, paraneoplastic syndrome, cerebellar ataxia, dysautonomia, Tourette, ADHD, cerebellar ataxia, oscillopsia, amyotrophic lateral sclerosis (ALS), thyroid disorder and headache with neurological deficits and lymphocytosis (HaNDL), in an individual in need thereof, comprising
In a further aspect, the present invention relates to a method of sequestering (or depleting) one or more antibodies present in an individual, comprising
In a preference, the one or more antibodies are specific for a neuroreceptor, preferably a neuroreceptor as defined herein above.
Preferably, the biopolymer scaffold is autologous with respect to the individual, preferably wherein the biopolymer scaffold is an autologous protein (i.e. murine albumin is used when the individual is a mouse).
In a further aspect, the present invention relates to a peptide, wherein the peptide is defined as disclosed herein for any one of the at least two peptides of the inventive compound, P, P1, P2, Pa, or Pb. Preferably, the peptide comprises a 6-amino-acid fragment, preferably a 7-, more preferably an 8-, even more preferably a 9-, even more preferably a 10-, even more preferably an 11-, yet even more preferably a 12-, most preferably a 13-amino-acid fragment, of an amino-acid sequence, identified by a UniProt accession code selected from the group consisting of: P02708, P07510, P07550, P08172, P08173, P08588, P08908, P08912, P08913, P11229, P11230, P13945, P17787, P18089, P18825, P20309, P25098, P25100, P30532, P30926, P32297, P35348, P35368, P35626, P36544, P43681, Q04844, Q05901, Q07001, Q15822, Q15825, Q9GZZ6, Q9UGM1, A0A0G2JKS1, A5X5Y0, A6NL88, A8MPY1, B4DS77, B8ZZ34, 000222, 000591, 014490, 014764, 015303, 015399, 043424, 043653, 060359, 060391, 060403, 060404, 060936, 075311, 075916, 076027, 094772, 095264, 095502, 095868, 095886, P01579, P05026, P05067, P06850, P07196, P07384, POC7T3, POC8F1, PODP57, PODP58, P12931, P13500, P14416, P14867, P15382, P16066, P17342, P18505, P18507, P19634, P20594, P21452, P21728, P21917, P21918, P23415, P23416, P24046, P24387, P25021, P25101, P28221, P28222, P28223, P28335, P28472, P28476, P28566, P29274, P29275, P29323, P30411, P30542, P30556, P30939, P31644, P32418, P34903, P34969, P35367, P35372, P35462, P35609, P37288, P39086, P41594, P41595, P41597, P42261, P42262, P42263, P43119, P46098, P47869, P47870, P47898, P47901, P47972, P48058, P48067, P48167, P48169, P48549, P49354, P50052, P50406, P53355, P55000, P62955, P63252, P78334, P78352, P78509, Q00535, Q05586, Q06413, Q07699, Q12879, Q12959, Q13002, Q13003, Q13224, Q13255, Q13387, Q13639, Q13702, Q13936, Q13972, Q14289, Q14416, Q14500, Q14571, Q14573, Q14643, Q14831, Q14832, Q14833, Q14957, Q15700, Q15818, Q16099, Q16445, Q16478, Q16553, Q16602, Q401N2, Q494W8, Q5SQ64, Q6PI25, Q6TFL4, Q6UXU4, Q6ZSJ9, Q70Z44, Q86Y78, Q86YM7, Q8N1C3, Q8N2G4, Q8N2Q7, Q8N4C8, Q8NC67, Q8NFZ4, Q8NG75, Q8NGA5, Q8NGA6, Q8NGC8, Q8NGC9, Q8NGG2, Q8NGG3, Q8NGH5, Q8NGH8, Q8NGN1, Q8NGS4, Q8NGY7, Q8NHC4, Q8NI32, Q8TBE1, Q8TCU5, Q8TDF5, Q8WXA2, Q8WXA8, Q8WXS5, Q92736, Q92796, Q96G91, Q96NW7, Q96P66, Q99928, Q99996, Q9BUH8, Q9BXM7, Q9BYB0, Q9GZV3, Q9H3N8, Q9NPA1, Q9NZ94, Q9P1A6, Q9UBK2, Q9UBN1, Q9UBS5, Q9UF02, Q9ULKO, Q9UN88, Q9UPX8, Q9Y2H0, Q9Y4A9, Q9Y566, Q9Y5N1, Q9Y691, Q9Y698, P37088, P51168, P51170, P51172, 094759, Q16515, 060741, Q9NZQ8, P78348, Q8TDD5, Q9NY37, Q13002, P39086, P48664, A6NGN9, 000305, 000555, 015146, 043448, 043497, 043525, 043526, 060840, 075096, 095180, 095259, 095970, P06213, P16389, P16473, P17658, P22001, P22459, P22460, P24530, P42658, P43146, P48547, P49418, P51787, P54284, P54289, P56696, Q00975, Q01668, Q02246, Q02641, Q03721, Q05329, Q06432, Q08289, Q09470, Q12809, Q13018, Q13303, Q13698, Q14003, Q14721, Q14722, Q15878, Q6PIL6, Q6PIU1, Q6X4W1, Q7Z3S7, Q7Z429, Q8IZS8, Q8NCM2, Q8TAE7, Q8TDN1, Q8TDN2, Q8WWG9, Q92953, Q96KK3, Q96L42, Q96PR1, Q96RP8, Q9BQ31, Q9BXT2, Q9H252, Q9H3M0, Q9NR82, Q9NS40, Q9NS61, Q9NSA2, Q9NY47, Q9NZI2, Q9NZV8, Q9POX4, Q9UHC6, Q9UIX4, Q9UJ90, Q9UJ96, Q9UK17, Q9ULD8, Q9ULS6, Q9UQ05, Q9Y2W7, Q9Y6H6, Q9Y6J6, P48058, P55087, Q9BPU6, P52799, P15328, Q05329, Q16653, Q9Y4C0, Q5F0I5, Q99719, P17600, Q13148, P01266, Q9Y6A1, Q9Y6A1, and P07202, optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid. Even more preferably, said amino-acid fragment comprises at least 4, preferably at least 5 or even at least 6, more preferably at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of a sequence identified by any one of SEQ ID NOs: 45-3536 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of the SEQ ID NO given in Table 1 is the same), preferably any one of SEQ ID NOs: 45-863 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of the SEQ ID NO given in Table 1 is the same), especially any one of SEQ ID NOs: 45-201 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of the SEQ ID NO given in Table 1 is the same), optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
In certain embodiments, such peptides may be used as probes for the diagnostic typing and analysis of autoantibody-mediated conditions such as disclosed herein. The peptides can e.g. be used as part of a diagnostic autoantibody-mediated condition typing or screening device or kit or procedure, as a companion diagnostic, for patient stratification or for monitoring autoantibody levels in the course of therapeutic treatments.
In a further aspect, the invention relates to a method for detecting and/or quantifying autoantibodies in a biological sample comprising the steps of
The skilled person is familiar with methods for detecting and/or quantifying antibodies in biological samples. The method can e.g. be a sandwich assay, preferably an enzyme-linked immunosorbent assay (ELISA), or a surface plasmon resonance (SPR) assay.
In a preference, the peptide (especially at least 10, more preferably at least 100, even more preferably at least 1000, especially at least 10000 different peptides of the invention) are immobilized on a solid support, preferably an ELISA plate or an SPR chip or a biosensor-based diagnostic device with an electrochemical, fluorescent, magnetic, electronic, gravimetric or optical biotransducer. Alternatively, or in addition thereto, the peptide (especially at least 10, more preferably at least 100, even more preferably at least 1000, especially at least 10000 different peptides of the invention) may be coupled to a reporter or reporter fragment, such as a reporter fragment suitable for a protein-fragment complementation assay (PCA); see e.g. Li et al, 2019, or Kanulainen et al, 2021.
Preferably, the sample is obtained from a mammal, preferably a human. Preferably the sample is a blood sample, preferably a whole blood, serum, or plasma sample.
The invention further relates to the use of a peptide defined as disclosed herein (e.g. for P, P1, P2, Pa, or Pb) in a diagnostic assay, preferably ELISA, preferably as disclosed herein above.
A further aspect of the invention relates to a diagnostic device comprising the peptide defined as disclosed herein (e.g. for P, P1, P2, Pa, or Pb), preferably immobilized on a solid support. In a preference, the solid support is an ELISA plate or a surface plasmon resonance chip. In another preference, the diagnostic device is a biosensor-based diagnostic device with an electrochemical, fluorescent, magnetic, electronic, gravimetric or optical biotransducer.
In another preferred embodiment, the diagnostic device is a lateral flow assay.
The invention further relates to a diagnostic kit comprising a peptide defined as disclosed herein (e.g. for P, P1, P2, Pa, or Pb), preferably a diagnostic device as defined herein. Preferably the diagnostic kit further comprises one or more selected from the group of a buffer, a reagent, instructions. Preferably the diagnostic kit is an ELISA kit.
A further aspect relates to an apheresis device comprising the peptide defined as disclosed herein (e.g. for P, P1, P2, Pa, or Pb). Preferably the peptide is immobilized on a solid carrier. It is especially preferred if the apheresis device comprises at least two, preferably at least three, more preferably at least four different peptides defined as disclosed herein (e.g. for P, P1, P2, Pa, or Pb). In a preferred embodiment the solid carrier comprises the inventive compound.
Preferably, the solid carrier is capable of being contacted with blood or plasma flow. Preferably, the solid carrier is a sterile and pyrogen-free column.
In the context of the present invention, for improved bioavailability, it is preferred that the inventive compound has a solubility in water at 25° C. of at least 0.1 μg/ml, preferably at least 1 μg/ml, more preferably at least 10 μg/ml, even more preferably at least 100 μg/ml, especially at least 1000 μg/ml.
The term “preventing” or “prevention” as used herein means to stop a disease state or condition from occurring in a patient or subject completely or almost completely or at least to a (preferably significant) extent, especially when the patient or subject or individual is predisposed to such a risk of contracting a disease state or condition.
The pharmaceutical composition of the present invention is preferably provided as a (typically aqueous) solution, (typically aqueous) suspension or (typically aqueous) emulsion. Excipients suitable for the pharmaceutical composition of the present invention are known to the person skilled in the art, upon having read the present specification, for example water (especially water for injection), saline, Ringer's solution, dextrose solution, buffers, Hank solution, vesicle forming compounds (e.g. lipids), fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable excipients include any compound that does not itself induce the production of antibodies in the patient (or individual) that are harmful for the patient (or individual). Examples are well tolerable proteins, polysaccharides, polylactic acids, polyglycolic acid, polymeric amino acids and amino acid copolymers. This pharmaceutical composition can (as a drug) be administered via appropriate procedures known to the skilled person (upon having read the present specification) to a patient or individual in need thereof (i.e. a patient or individual having or having the risk of developing the diseases or conditions mentioned herein). The preferred route of administration of said pharmaceutical composition is parenteral administration, in particular through intraperitoneal, subcutaneous, intramuscular and/or intravenous administration. For parenteral administration, the pharmaceutical composition of the present invention is preferably provided in injectable dosage unit form, e.g. as a solution (typically as an aqueous solution), suspension or emulsion, formulated in conjunction with the above-defined pharmaceutically acceptable excipients. The dosage and method of administration, however, depends on the individual patient or individual to be treated. Said pharmaceutical composition can be administered in any suitable dosage known from other biological dosage regimens or specifically evaluated and optimised for a given individual. For example, the active agent may be present in the pharmaceutical composition in an amount from 1 mg to 10 g, preferably 50 mg to 2 g, in particular 100 mg to 1 g. Usual dosages can also be determined on the basis of kg body weight of the patient, for example preferred dosages are in the range of 0.1 mg to 100 mg/kg body weight, especially 1 to 10 mg/kg body weight (per administration session). The administration may occur e.g. once daily, once every other day, once per week or once every two weeks. As the preferred mode of administration of the inventive pharmaceutical composition is parenteral administration, the pharmaceutical composition according to the present invention is preferably liquid or ready to be dissolved in liquid such sterile, de-ionised or distilled water or sterile isotonic phosphate-buffered saline (PBS). Preferably, 1000 μg (dry-weight) of such a composition comprises or consists of 0.1-990 μg, preferably 1-900 μg, more preferably 10-200 μg compound, and option-ally 1-500 μg, preferably 1-100 μg, more preferably 5-15 μg (buffer) salts (preferably to yield an isotonic buffer in the final volume), and optionally 0.1-999.9 μg, preferably 100-999.9 μg, more preferably 200-999 μg other excipients. Preferably, 100 mg of such a dry composition is dissolved in sterile, de-ionised/distilled water or sterile isotonic phosphate-buffered saline (PBS) to yield a final volume of 0.1-100 ml, preferably 0.5-20 ml, more preferably 1-10 ml.
It is evident to the skilled person that active agents and drugs described herein can also be administered in salt-form (i.e. as a pharmaceutically acceptable salt of the active agent). Accordingly, any mention of an active agent herein shall also include any pharmaceutically acceptable salt forms thereof.
Methods for chemical synthesis of peptides used for the compound of the present invention are well-known in the art. Of course, it is also possible to produce the peptides using recombinant methods. The peptides can be produced in microorganisms such as bacteria, yeast or fungi, in eukaryotic cells such as mammalian or insect cells, or in a recombinant virus vector such as adenovirus, poxvirus, herpesvirus, Simliki forest virus, baculovirus, bacteriophage, sindbis virus or sendai virus. Suitable bacteria for producing the peptides include E. coli, B. subtilis or any other bacterium that is capable of expressing such peptides. Suitable yeast cells for expressing the peptides of the present invention include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida, Pichiapastoris or any other yeast capable of expressing peptides. Corresponding means and methods are well known in the art. Also, methods for isolating and purifying recombinantly produced peptides are well known in the art and include e.g. gel filtration, affinity chromatography, ion exchange chromatography etc.
Beneficially, cysteine residues are added to the peptides at the N- and/or C-terminus to facilitate coupling to the biopolymer scaffold, especially.
To facilitate isolation of said peptides, fusion polypeptides may be made wherein the peptides are translationally fused (covalently linked) to a heterologous polypeptide which enables isolation by affinity chromatography. Typical heterologous polypeptides are His-Tag (e.g. His6; 6 histidine residues), GST-Tag (Glutathione-S-transferase) etc. The fusion polypeptide facilitates not only the purification of the peptides but can also prevent the degradation of the peptides during the purification steps. If it is desired to remove the heterologous polypeptide after purification, the fusion polypeptide may comprise a cleavage site at the junction between the peptide and the heterologous polypeptide. The cleavage site may consist of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site (e.g. proteases).
The coupling/conjugation chemistry used to link the peptides/peptide n-mers to the biopolymer scaffold (e.g. via heterobifunctional compounds such as GMBS and of course also others as described in “Bioconjugate Techniques”, Greg T. Hermanson) or used to conjugate the spacer to the peptides in the context of the present invention can also be selected from reactions known to the skilled in the art. The biopolymer scaffold itself may be recombinantly produced or obtained from natural sources.
Herein, the term “specific for”—as in “molecule A specific for molecule B”—means that molecule A has a binding preference for molecule B compared to other molecules in an individual's body. Typically, this entails that molecule A (such as an antibody) has a dissociation constant (also called “affinity”) in regard to molecule B (such as the antigen, specifically the binding epitope thereof) that is lower than (i.e. “stronger than”) 1000 nM, preferably lower than 100 nM, more preferably lower than 50 nM, even more preferably lower than 10 nM, especially lower than 5 nM.
Herein, “UniProt” refers to the Universal Protein Resource. UniProt is a comprehensive resource for protein sequence and annotation data. UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics and the Protein Information Resource (PIR). Across the three institutes more than 100 people are involved through different tasks such as database curation, software development and support. Website: https://www.uniprot.org/
Entries in the UniProt databases are identified by their accession codes (referred to herein e.g. as “UniProt accession code” or briefly as “UniProt” followed by the accession code), usually a code of six alphanumeric letters (e.g. “Q1HVF7”). If not specified otherwise, the accession codes used herein refer to entries in the Protein Knowledgebase (UniProtKB) of UniProt. If not stated otherwise, the UniProt database state for all entries referenced herein is of 22 Sep. 2020 (UniProt/UniProtKB Release 2020_04).
In the context of the present application, sequence variants (designated as “natural variant” in UniProt) are expressly included when referring to a UniProt database entry.
“Percent (%) amino acid sequence identity” or “X % identical” (such as “70% identical”) with respect to a reference polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR) or the “needle” pairwise sequence alignment application of the EMBOSS software package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are calculated using the sequence alignment of the computer programme “needle” of the EMBOSS software package (publicly available from European Molecular Biology Laboratory; Rice et al., 2000).
The needle programme can be accessed under the web site http://www.ebi.ac.uk/Tools/psa/emboss_needle/ or downloaded for local installation as part of the EMBOSS package from http://emboss.sourceforge.net/. It runs on many widely-used UNIX operating systems, such as Linux.
To align two protein sequences, the needle programme is preferably run with the following parameters:
Commandline: needle-auto-stdout-asequence SEQUENCE_FILE_A-bsequence SEQUENCE_FILE_B-datafile EBLOSUM62-gapopen 10.0-gapextend 0.5-endopen 10.0-endextend 0.5-aformat3 pair-sprotein1-sprotein2 (Align_format: pair Report_file: stdout)
The % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
The present invention further relates to the following embodiments:
Embodiment 1. A compound comprising a biopolymer scaffold and at least two peptides with a sequence length of 6-13 amino acids,
Embodiment 2. The compound of embodiment 1, wherein said amino-acid sequence is an amino acid sequence, preferably of a (preferably human) neuroreceptor of the autonomic nervous system, identified by a UniProt accession code selected from the group consisting of: P02708, P07510, P07550, P08172, P08173, P08588, P08908, P08912, P08913, P11229, P11230, P13945, P17787, P18089, P18825, P20309, P25098, P25100, P30532, P30926, P32297, P35348, P35368, P35626, P36544, P43681, Q04844, Q05901, Q07001, Q15822, Q15825, Q9GZZ6, Q9UGM1; P37088, P51168, P51170, P51172, O94759, Q16515, 060741, Q9NZQ8, P78348, Q8TDD5, Q9NY37, Q13002, P39086, and P48664.
Embodiment 3. The compound of embodiment 1 or 2, wherein said amino acid sequence is an amino acid sequence of a (preferably human) neuroreceptor selected from the group consisting of muscarinic, and nicotinic cholinergic receptors, alpha- and beta-adrenergic receptors, serotonin receptors, angiotensin- and endothelin receptors.
Embodiment 4. The compound of any one of embodiments 1 to 3, wherein said amino-acid sequence is an amino acid sequence, preferably of a (preferably human) neuroreceptor selected from the group consisting of beta-1 adrenergic receptor, beta-2 adrenergic receptor, M3 muscarinic acetylcholine receptor, and M4 muscarinic acetylcholine receptor, identified by a UniProt accession code selected from the group consisting of: P08588, P07550, P20309, and P08173.
Embodiment 5. The compound of any one of embodiments 1 to 4, wherein said amino-acid sequence is an amino acid sequence identified by a UniProt accession code selected from the group consisting of: P02708, P07510, P07550, P08172, P08173, P08588, P08908, P08912, P08913, P11229, P11230, P13945, P17787, P18089, P18825, P20309, P25098, P25100, P30532, P30926, P32297, P35348, P35368, P35626, P36544, P43681, Q04844, Q05901, Q07001, Q15822, Q15825, Q9GZZ6, Q9UGM1, A0A0G2JKS1, A5X5Y0, A6NL88, A8MPY1, B4DS77, B8ZZ34, O00222, O00591, O14490, O14764, O15303, O15399, O43424, O43653, O60359, O60391, O60403, O60404, O60936, O75311, O75916, O76027, O94772, O95264, O95502, O95868, O95886, P01579, P05026, P05067, P06850, P07196, P07384, POC7T3, POC8F1, PODP57, PODP58, P12931, P13500, P14416, P14867, P15382, P16066, P17342, P18505, P18507, P19634, P20594, P21452, P21728, P21917, P21918, P23415, P23416, P24046, P24387, P25021, P25101, P28221, P28222, P28223, P28335, P28472, P28476, P28566, P29274, P29275, P29323, P30411, P30542, P30556, P30939, P31644, P32418, P34903, P34969, P35367, P35372, P35462, P35609, P37288, P39086, P41594, P41595, P41597, P42261, P42262, P42263, P43119, P46098, P47869, P47870, P47898, P47901, P47972, P48058, P48067, P48167, P48169, P48549, P49354, P50052, P50406, P53355, P55000, P62955, P63252, P78334, P78352, P78509, Q00535, Q05586, Q06413, Q07699, Q12879, Q12959, Q13002, Q13003, Q13224, Q13255, Q13387, Q13639, Q13702, Q13936, Q13972, Q14289, Q14416, Q14500, Q14571, Q14573, Q14643, Q14831, Q14832, Q14833, Q14957, Q15700, Q15818, Q16099, Q16445, Q16478, Q16553, Q16602, Q401N2, Q494W8, Q5SQ64, Q6PI25, Q6TFL4, Q6UXU4, Q6ZSJ9, Q70Z44, Q86Y78, Q86YM7, Q8N1C3, Q8N2G4, Q8N2Q7, Q8N4C8, Q8NC67, Q8NFZ4, Q8NG75, Q8NGA5, Q8NGA6, Q8NGC8, Q8NGC9, Q8NGG2, Q8NGG3, Q8NGH5, Q8NGH8, Q8NGN1, Q8NGS4, Q8NGY7, Q8NHC4, Q8NI32, Q8TBE1, Q8TCU5, Q8TDF5, Q8WXA2, Q8WXA8, Q8WXS5, Q92736, Q92796, Q96G91, Q96NW7, Q96P66, Q99928, Q99996, Q9BUH8, Q9BXM7, Q9BYB0, Q9GZV3, Q9H3N8, Q9NPA1, Q9NZ94, Q9P1A6, Q9UBK2, Q9UBN1, Q9UBS5, Q9UF02, Q9ULKO, Q9UN88, Q9UPX8, Q9Y2H0, Q9Y4A9, Q9Y566, Q9Y5N1, Q9Y691, Q9Y698, P37088, P51168, P51170, P51172, O94759, Q16515, O60741, Q9NZQ8, P78348, Q8TDD5, Q9NY37, Q13002, P39086 and P48664.
Embodiment 6. The compound of any one of embodiments 1 to 4, wherein said amino-acid sequence is an amino acid sequence identified by a UniProt accession code selected from the group consisting of: P02708, P07510, P07550, P08172, P08173, P08588, P08908, P08912, P08913, P11229, P11230, P13945, P17787, P18089, P18825, P20309, P25098, P25100, P30532, P30926, P32297, P35348, P35368, P35626, P36544, P43681, Q04844, Q05901, Q07001, Q15822, Q15825, Q9GZZ6, Q9UGM1, A0A0G2JKS1, A5X5Y0, A6NL88, A8MPY1, B4DS77, B8ZZ34, O00222, O00591, O14490, O14764, O15303, O15399, O43424, O43653, O60359, O60391, O60403, O60404, O60936, O75311, O75916, O76027, O94772, O95264, O95502, O95868, O95886, P01579, P05026, P05067, P06850, P07196, P07384, POC7T3, POC8F1, PODP57, PODP58, P12931, P13500, P14416, P14867, P15382, P16066, P17342, P18505, P18507, P19634, P20594, P21452, P21728, P21917, P21918, P23415, P23416, P24046, P24387, P25021, P25101, P28221, P28222, P28223, P28335, P28472, P28476, P28566, P29274, P29275, P29323, P30411, P30542, P30556, P30939, P31644, P32418, P34903, P34969, P35367, P35372, P35462, P35609, P37288, P39086, P41594, P41595, P41597, P42261, P42262, P42263, P43119, P46098, P47869, P47870, P47898, P47901, P47972, P48058, P48067, P48167, P48169, P48549, P49354, P50052, P50406, P53355, P55000, P62955, P63252, P78334, P78352, P78509, Q00535, Q05586, Q06413, Q07699, Q12879, Q12959, Q13002, Q13003, Q13224, Q13255, Q13387, Q13639, Q13702, Q13936, Q13972, Q14289, Q14416, Q14500, Q14571, Q14573, Q14643, Q14831, Q14832, Q14833, Q14957, Q15700, Q15818, Q16099, Q16445, Q16478, Q16553, Q16602, Q401N2, Q494W8, Q5SQ64, Q6PI25, Q6TFL4, Q6UXU4, Q6ZSJ9, Q70Z44, Q86Y78, Q86YM7, Q8N1C3, Q8N2G4, Q8N2Q7, Q8N4C8, Q8NC67, Q8NFZ4, Q8NG75, Q8NGA5, Q8NGA6, Q8NGC8, Q8NGC9, Q8NGG2, Q8NGG3, Q8NGH5, Q8NGH8, Q8NGN1, Q8NGS4, Q8NGY7, Q8NHC4, Q8NI32, Q8TBE1, Q8TCU5, Q8TDF5, Q8WXA2, Q8WXA8, Q8WXS5, Q92736, Q92796, Q96G91, Q96NW7, Q96P66, Q99928, Q99996, Q9BUH8, Q9BXM7, Q9BYB0, Q9GZV3, Q9H3N8, Q9NPA1, Q9NZ94, Q9P1A6, Q9UBK2, Q9UBN1, Q9UBS5, Q9UF02, Q9ULKO, Q9UN88, Q9UPX8, Q9Y2H0, Q9Y4A9, Q9Y566, Q9Y5N1, Q9Y691, Q9Y698, P37088, P51168, P51170, P51172, O94759, Q16515, O60741, Q9NZQ8, P78348, Q8TDD5, Q9NY37, Q13002, P39086, P48664, A6NGN9, O00305, O00555, O15146, O43448, O43497, O43525, O43526, O60840, O75096, O95180, O95259, O95970, P06213, P16389, P16473, P17658, P22001, P22459, P22460, P24530, P42658, P43146, P48547, P49418, P51787, P54284, P54289, P56696, Q00975, Q01668, Q02246, Q02641, Q03721, Q05329, Q06432, Q08289, Q09470, Q12809, Q13018, Q13303, Q13698, Q14003, Q14721, Q14722, Q15878, Q6PIL6, Q6PIU1, Q6X4W1, Q7Z3S7, Q7Z429, Q8IZS8, Q8NCM2, Q8TAE7, Q8TDN1, Q8TDN2, Q8WWG9, Q92953, Q96KK3, Q96L42, Q96PR1, Q96RP8, Q9BQ31, Q9BXT2, Q9H252, Q9H3M0, Q9NR82, Q9NS40, Q9NS61, Q9NSA2, Q9NY47, Q9NZI2, Q9NZV8, Q9POX4, Q9UHC6, Q9UIX4, Q9UJ90, Q9UJ96, Q9UK17, Q9ULD8, Q9ULS6, Q9UQ05, Q9Y2W7, Q9Y6H6 and Q9Y6J6.
Embodiment 7. The compound of any one of embodiments 1 to 4, wherein said amino-acid sequence is an amino acid sequence identified by a UniProt accession code selected from the group consisting of: O00555, O43497, O95180, P02708, P18505, P31644, P41594, P42263, Q00975, Q01668, Q05586, Q13224, Q13936, Q14957, Q15878, Q16445, Q8TCU5, Q9POX4, A6NGN9, O15399, O60840, P14416, P16473, P23415, P34903, P42261, P42262, P42658, P47869, Q09470, Q12879, Q13255, Q9UHC6, O15146, O95970, P14867, P28472, P47870, P48169, and P49418.
Embodiment 8. The compound of any one of embodiments 1 to 4, wherein said amino-acid sequence is an amino acid sequence identified by a UniProt accession code selected from the group consisting of: P02708, P18505, P31644, P41594, P42263, Q05586, Q13224, Q13936, Q14957, Q16445, Q8TCU5, O15399, P14416, P23415, P34903, P42261, P42262, P47869, Q12879, Q13255, P14867, P28472, P47870, and P48169.
Embodiment 9. The compound of any one of embodiments 1 to 8, wherein, for at least one of the peptides (preferably for each of the peptides), said amino-acid fragment comprises at least 4, preferably at least 5 or even at least 6, more preferably at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of a sequence identified by any one of SEQ ID NOs: 45-3536 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of the SEQ ID NO given in Table 1 is the same), preferably any one of SEQ ID NOs: 45-863 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of the SEQ ID NO given in Table 1 is the same), especially any one of SEQ ID NOs: 45-201 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of the SEQ ID NO given in Table 1 is the same), optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid. Embodiment 10. The compound of any one of embodiments 1 to 9, wherein, for at least one of the peptides (preferably for each of the peptides), said amino-acid fragment comprises at least 4, preferably at least 5 or even at least 6, more preferably at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of a sequence listed in Table 3 (with the proviso that the UniProt accession code of said amino-acid sequence and the UniProt accession code of said sequence listed in Table 3 is the same), optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
Embodiment 11. The compound of any one of embodiments 1 to 10, wherein at most three, preferably at most two, more preferably at most one amino acid of said fragment is independently substituted by any other amino acid.
Embodiment 12. The compound of any one of embodiments 1 to 10, wherein three amino acids of said fragment are independently substituted by any other amino acid.
Embodiment 13. The compound of any one of embodiments 1 to 10, wherein two amino acids of said fragment are independently substituted by any other amino acid.
Embodiment 14. The compound of any one of embodiments 1 to 10, wherein one amino acid of said fragment is substituted by any other amino acid.
Embodiment 15. The compound of any one of embodiments 1 to 14, wherein the biopolymer scaffold is a human protein.
Embodiment 16. The compound of any one of embodiments 1 to 15, wherein the at least two peptides comprise a peptide P1 and a peptide P2, wherein P1 and P2 independently comprise a 6-amino-acid fragment, preferably a 7-, more preferably an 8-, more preferably a 9-, even more preferably a 10-, yet even more preferably an 11-, especially a 12-, most preferably a 13-amino-acid fragment, of an amino acid sequence as defined in any one of embodiments 1 to 14, wherein P1 and P2 are present in form of a peptide dimer Pi-S-P2, wherein S is a non-peptide spacer, wherein the peptide dimer is covalently bound to the biopolymer scaffold, preferably via a linker.
Embodiment 17. The compound of any one of embodiments 1 to 16, wherein the biopolymer scaffold is selected from human globulins and human albumin.
Embodiment 18. The compound of any one of embodiments 1 to 17, wherein at least one of the at least two peptides is circularized.
Embodiment 19. The compound of any one of embodiments 1 to 18, wherein each of the at least two peptides is circularized.
Embodiment 20. The compound of any one of embodiments 1 to 19, wherein the compound is non-immunogenic in humans.
Embodiment 21. The compound of any one of embodiments 1 to 20, wherein the biopolymer scaffold is selected from human transferrin and human albumin.
Embodiment 22. A compound, preferably the compound of any one of embodiments 1 to 21, comprising
P(-S-P)(n-1) and
Embodiment 23. The compound of embodiment 22, wherein at least one occurrence of P is a circularized peptide, preferably wherein at least 10% of all occurrences of P are circularized peptides, more preferably wherein at least 25% of all occurrences of P are circularized peptides, yet more preferably wherein at least 50% of all occurrences of P are circularized peptides, even more preferably wherein at least 75% of all occurrences of P are circularized peptides, yet even more preferably wherein at least 90% of all occurrences of P are circularized peptides or even wherein at least 95% of all occurrences of P are circularized peptides, especially wherein all of the occurrences of P are circularized peptides.
Embodiment 24. The compound of embodiment 22 or 23, wherein, independently for each of the peptide n-mers, n is at least 2, more preferably at least 3, especially at least 4.
Embodiment 25. The compound of any one of embodiments 22 to 24, wherein, independently for each of the peptide n-mers, n is less than 10, preferably less than 9, more preferably less than 8, even more preferably less than 7, yet even more preferably less than 6, especially less than 5.
Embodiment 26. The compound of any one of embodiments 22 to 25, wherein, for each of the peptide n-mers, n is 2.
Embodiment 27. The compound of any one of embodiments 22 to 26, wherein at least one occurrence of P is Pa and/or at least one occurrence of P is Pb,
Embodiment 28. The compound of any one of embodiments 22 to 27, wherein, independently for each occurrence, P is Pa or Pb.
Embodiment 29. The compound of any one of embodiments 22 to 28, wherein, in the first peptide n-mer, each occurrence of P is Pa and, in the second peptide n-mer, each occurrence of P is Pb.
Embodiment 30. The compound of any one of embodiments 22 to 29, wherein
Embodiment 31. A compound comprising
Embodiment 32. The compound of embodiment 31, further comprising a second peptide n-mer which is a peptide dimer of the formula Pb-S-Pb or Pa-S-Pb,
Embodiment 33. The compound of any one of embodiments 22 to 30 and 32, wherein the first peptide n-mer is different from the second peptide n-mer.
Embodiment 34. The compound of any one of embodiments 27 to 33, wherein the peptide Pa is different from the peptide Pb, preferably wherein the peptide Pa and the peptide Pb are two different epitopes of the same antigen or two different epitope parts of the same epitope.
Embodiment 35. The compound of any one of embodiments 27 to 34, wherein the peptide Pa and the peptide Pb comprise the same amino-acid sequence fragment, wherein the amino-acid sequence fragment has a length of at least 2 amino acids, preferably at least 3 amino acids, more preferably at least 4 amino acids, yet more preferably at least 5 amino acids, even more preferably at least 6 amino acids, yet even more preferably at least 7 amino acids, especially at least 8 amino acids or even at least 9 amino acids.
Embodiment 36. The compound of any one of embodiments 27 to 35, wherein Pa and/or Pb is circularized.
Embodiment 37. The compound of any one of embodiments 22 to 36, wherein the compound comprises a plurality of said first peptide n-mer and/or a plurality of said second peptide n-mer.
Embodiment 38. The compound of any one of embodiments 1 to 37, wherein the biopolymer scaffold is a protein, preferably a mammalian protein such as a human protein, a non-human primate protein, a sheep protein, a pig protein, a dog protein or a rodent protein.
Embodiment 39. The compound of any one of embodiments 1 to 38, wherein the biopolymer scaffold is a globulin.
Embodiment 40. The compound of any one of embodiments 1 to 39, wherein the biopolymer scaffold is selected from the group consisting of immunoglobulins, alpha1-globulins, alpha2-globulins and beta-globulins.
Embodiment 41. The compound of any one of embodiments 1 to 40, wherein the biopolymer scaffold is selected from the group consisting of immunoglobulin G, haptoglobin and transferrin.
Embodiment 42. The compound of any one of embodiments 1 to 41, wherein the biopolymer scaffold is haptoglobin.
Embodiment 43. The compound of any one of embodiments 1 to 38, wherein the biopolymer scaffold is an albumin.
Embodiment 44. The compound of embodiment 38, wherein the biopolymer scaffold is an anti-CD163 antibody (i.e. an antibody specific for a CD163 protein) or CD163-binding fragment thereof.
Embodiment 45. The compound of embodiment 44, wherein the anti-CD163 antibody or CD163-binding fragment thereof is specific for human CD163 and/or is specific for the extracellular region of CD163, preferably for an SRCR domain of CD163, more preferably for any one of SRCR domains 1-9 of CD163, even more preferably for any one of SRCR domains 1-3 of CD163, especially for SRCR domain 1 of CD163.
Embodiment 46. The compound of embodiment 44 or 45, wherein the anti-CD163 antibody or CD163-binding fragment thereof is specific for one of the following peptides:
Embodiment 47. The compound of embodiment 44 or 45, wherein the anti-CD163 antibody or CD163-binding fragment thereof is specific for a peptide comprising the amino acid sequence ESALW (SEQ ID NO: 14) or ALW.
Embodiment 48. The compound of embodiment 44 or 45, wherein the anti-CD163 antibody or CD163-binding fragment thereof is specific for a peptide comprising the amino acid sequence GRVEVKVQEEW (SEQ ID NO: 4), WGTVCNNGWS (SEQ ID NO: 5) or WGTVCNNGW (SEQ ID NO: 6).
Embodiment 49. The compound of embodiment 44 or 45, wherein the anti-CD163 antibody or CD163-binding fragment thereof is specific for a peptide comprising the amino acid sequence SSLGGTDKELR (SEQ ID NO: 25) or SSLGG (SEQ ID NO: 26).
Embodiment 50. The compound of any one of embodiments 1 to 49, wherein the compound is non-immunogenic in a mammal, preferably in a human, in a non-human primate, in a sheep, in a pig, in a dog or in a rodent.
Embodiment 51. The compound of any one of embodiments 1 to 50, wherein the compound is for intracorporeal sequestration (or intracorporeal depletion) of at least one antibody in an individual, preferably in the bloodstream of the individual and/or for reduction of the titre of at least one antibody in the individual, preferably in the bloodstream of the individual.
Embodiment 52. The compound of any one of embodiments 1 to 51, wherein the compound further comprises at least
P(-S-P)(n-1),
Embodiment 53. The compound of embodiment 52, wherein the compound further comprises at least
P(-S-P)(n-1),
P(-S-P)(n-1),
Embodiment 55. The compound of embodiment 54, wherein the compound further comprises at least
P(-S-P)(n-1),
P(-S-P)(n-1),
Embodiment 57. The compound of embodiment 56, wherein the compound further comprises at least
P(-S-P)(n-1),
Embodiment 58. The compound of embodiment 57, wherein the compound further comprises at least
P(-S-P)(n-1),
Embodiment 59. The compound of embodiment 58, wherein the compound further comprises at least
P(-S-P)(n-1),
Embodiment 60. The compound of any one of embodiments 22 to 59, wherein each of the peptide n-mers is covalently bound to the biopolymer scaffold, preferably via a linker each.
Embodiment 61. The compound of any one of embodiments 1 to 60, wherein at least one of said linkers is selected from disulphide bridges and PEG molecules.
Embodiment 62. The compound of any one of embodiments 1 to 61, wherein at least one of the spacers S is selected from PEG molecules or glycans.
Embodiment 63. The compound of any one of embodiments 1 to 62, wherein the first peptide n-mer is Pa-S-Pb and the second peptide n-mer is Pa-S-Pb.
Embodiment 64. The compound of any one of embodiments 1 to 63, wherein the peptide Pa and the peptide Pb comprise the same amino-acid sequence fragment, wherein the amino-acid sequence fragment has a length of at least 5 amino acids, even more preferably at least 6 amino acids, yet even more preferably at least 7 amino acids, especially at least 8 amino acids or even at least 9 amino acids.
Embodiment 65. The compound of any one of embodiments 1 to 64, wherein the compounds is for the sequestration (or depletion) of an antibody specific for a (human) neuroreceptor, preferably wherein the neuroreceptor is defined as in any one of embodiments 1 to 8.
Embodiment 66. A pharmaceutical composition comprising the compound of any one of embodiments 1 to 65 and at least one pharmaceutically acceptable excipient.
Embodiment 67. The pharmaceutical composition of embodiment 66, wherein the molar ratio of the peptides to scaffold in the composition is from 2:1 to 100:1, preferably from 3:1 to 90:1, more preferably from 4:1 to 80:1, even more preferably from 5:1 to 70:1, yet even more preferably from 6:1 to 60:1, especially from 7:1 to 50:1 or even from 8:10 to 40:1.
Embodiment 68. The pharmaceutical composition of embodiment 66 or 67, wherein the composition is prepared for intraperitoneal, subcutaneous, intramuscular and/or intravenous administration and/or wherein the composition is for repeated administration.
Embodiment 69. The pharmaceutical composition of any one of embodiments 66 to 68, or the compound of any one of embodiments 22 to 65, wherein the molar ratio of peptide P to biopolymer scaffold in the composition is from 2:1 to 100:1, preferably from 3:1 to 90:1, more preferably from 4:1 to 80:1, even more preferably from 5:1 to 70:1, yet even more preferably from 6:1 to 60:1, especially from 7:1 to 50:1 or even from 8:10 to 40:1.
Embodiment 70. The pharmaceutical composition of any one of embodiments 66 to 69, or the compound of any one of embodiments 27 to 65 wherein the molar ratio of peptide Pa to biopolymer scaffold in the composition is from 2:1 to 100:1, preferably from 3:1 to 90:1, more preferably from 4:1 to 80:1, even more preferably from 5:1 to 70:1, yet even more preferably from 6:1 to 60:1, especially from 7:1 to 50:1 or even from 8:10 to 40:1.
Embodiment 71. The pharmaceutical composition of any one of embodiments 66 to 70, or the compound of any one of embodiments 27 to 65, wherein the molar ratio of peptide Pb to biopolymer scaffold in the composition is from 2:1 to 100:1, preferably from 3:1 to 90:1, more preferably from 4:1 to 80:1, even more preferably from 5:1 to 70:1, yet even more preferably from 6:1 to 60:1, especially from 7:1 to 50:1 or even from 8:10 to 40:1.
Embodiment 72. The pharmaceutical composition of any one of embodiments 66 to 71 for use in therapy.
Embodiment 73. The pharmaceutical composition of any one of embodiments 66 to 71 for use in prevention or treatment of an autoantibody-mediated condition, preferably selected from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), postural orthostatic tachycardia syndrome (POTS), Autoimmune Autonomic Ganglionopathy (AAG), Idiopathic Dilated Cardiomyopathy (IDC), and Chronic Chagas heart disease (cChHD), or from encephalitis such as limbic encephalitis or paraneoplastic striatal encephalitis or Anti-mGluR1 encephalitis or Anti-mGluR5 encephalitis or acute disseminated encephalomyelitis (ADEM) or NMDAR encephalitis, paraneoplastic syndrome, stiff man syndrome, autoimmune channelopathies, neuromyelitis optica, neuromyotonia, Morvan's syndrome, neuropathic pain, myelitis, optic neuritis, retinitis, parkinsonism, chorea, psychosis, dystonia, mutism, movement disorders, confusion, hallucinations, prodromal diarrhoea, memory loss, hyperexcitability, encephalitis psychiatric syndrome, narcolepsy, autism spectrum disorders, seizures, status epilepticus, chronic epilepsy, myoclonus, encephalomyelitis, myoclonus, parasomnia, sleep apnoea, cognitive impairment, gait abnormalities, faciobrachial dystonic seizures, paraneoplastic syndrome, cerebellar ataxia, dysautonomia, Tourette, ADHD, cerebellar ataxia, oscillopsia, amyotrophic lateral sclerosis (ALS), thyroid disorder and headache with neurological deficits and lymphocytosis (HaNDL), in an individual.
Embodiment 74. The pharmaceutical composition for use according to embodiment 72 or 73, wherein the pharmaceutical composition is administered at least twice within a 96-hour window, preferably within a 72-hour window, more preferably within a 48-hour window, even more preferably within a 36-hour window, yet even more preferably within a 24-hour window, especially within a 18-hour window or even within a 12-hour window.
Embodiment 75. The pharmaceutical composition for use according to any one of embodiments 72 to 74, wherein the composition is administered at a dose of 1-1000 mg, preferably 2-500 mg, more preferably 3-250 mg, even more preferably 4-100 mg, especially 5-50 mg, compound per kg body weight of the individual.
Embodiment 76. The pharmaceutical composition for use according to any one of embodiments 72 to 75, wherein the composition is administered intraperitoneally, subcutaneously, intramuscularly or intravenously.
Embodiment 77. The pharmaceutical composition for use according to any one of embodiments 72 to 76, wherein one or more antibodies are present in the individual which are specific for at least one occurrence of peptide P, or for peptide Pa and/or peptide Pb.
Embodiment 78. The pharmaceutical composition for use according to any one of embodiments 72 to 77, wherein one or more antibodies are present in the individual which are specific for a neuroreceptor, preferably wherein the neuroreceptor is defined as in any one of embodiments 1 to 8.
Embodiment 79. The pharmaceutical composition for use according to any one of embodiments 72 to 78, wherein the composition is non-immunogenic in the individual.
Embodiment 80. The pharmaceutical composition for use according to any one of embodiments 72 to 79, wherein the composition is administered at a dose of 1-1000 mg, preferably 2-500 mg, more preferably 3-250 mg, even more preferably 4-100 mg, especially 5-50 mg, compound per kg body weight of the individual.
Embodiment 81. A method of ameliorating or treating an autoantibody-mediated condition, selected from CFS/ME, POTS, AAG, IDC, and cChHD, in an individual in need thereof, comprising
Embodiment 82. The method according to embodiment 81, wherein the method is defined as in any one of embodiments 72 to 80.
Embodiment 83. A method of sequestering (or depleting) one or more antibodies present in an individual, comprising
Embodiment 84. The method of embodiment 83, wherein the one or more antibodies are specific for a neuroreceptor, preferably wherein the neuroreceptor is defined as in any one of embodiments 1 to 8.
Embodiment 85. The method of embodiment 83 or 84, wherein the individual is a non-human animal, preferably a non-human primate, a sheep, a pig, a dog or a rodent, in particular a mouse.
Embodiment 86. The method of any one of embodiments 83 to 85, wherein the biopolymer scaffold is autologous with respect to the individual, preferably wherein the biopolymer scaffold is an autologous protein.
Embodiment 87. The method of any one of embodiments 83 to 86, wherein the composition is administered intraperitoneally, subcutaneously, intramuscularly or intravenously.
Embodiment 88. A peptide (preferably with a sequence length of 6-13 amino acids), wherein the peptide comprises a 6-amino-acid fragment, preferably a 7-, more preferably an 8-, even more preferably a 9-, even more preferably a 10-, even more preferably an 11-, yet even more preferably a 12-, most preferably a 13-amino-acid fragment, of an amino-acid sequence identified by a UniProt accession code selected from the group consisting of:
Embodiment 89. The peptide of embodiment 88, wherein the peptide is further defined as in any one of embodiments 1 to 14.
Embodiment 90. A peptide, preferably with a sequence length of 7-14 amino-acids, comprising, preferably consisting of, at least 7 or even at least 8, yet more preferably at least 9, even more preferably at least 10, yet even more preferably at least 11, especially at least 12 or even 13 consecutive amino acids of a sequence identified by any one of SEQ ID NOs: 45-3536, preferably any one of SEQ ID NOs: 45-863, especially any one of SEQ ID NOs: 45-201, optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
Embodiment 91. A peptide, preferably with a sequence length of 7-14 amino-acids, comprising, preferably consisting of, the sequence identified by any one of SEQ ID NOs: 45-3536, preferably any one of SEQ ID NOs: 45-863, especially any one of SEQ ID NOs: 45-201, optionally wherein at most three, preferably at most two, more preferably at most one amino acid is independently substituted by any other amino acid.
Embodiment 92. The peptide of any one of embodiments 88 to 91, wherein the peptide is linear or circularized.
Embodiment 93. A method for detecting and/or quantifying autoantibodies in a biological sample comprising the steps of
Embodiment 94. The method of embodiment 93, wherein the peptide is immobilized on a solid support, in particular a biosensor-based diagnostic device with an electrochemical, fluorescent, magnetic, electronic, gravimetric or optical biotransducer and/or wherein the peptide is coupled to a reporter or reporter fragment, such as a reporter fragment suitable for a PCA.
Embodiment 95. The method of embodiment 93 or 94, wherein the method is a sandwich assay, preferably an enzyme-linked immunosorbent assay (ELISA).
Embodiment 96. The method of any one of embodiments 93 to 95, wherein the sample is obtained from a mammal, preferably a human.
Embodiment 97. The method of any one of embodiments 93 to 96, wherein the sample is a blood sample, preferably whole blood, serum, or plasma.
Embodiment 98. Use of the peptide according to any one of embodiments 88 to 92 in an enzyme-linked immunosorbent assay (ELISA), preferably for a method as defined in any one of embodiments 93 to 97.
Embodiment 99. A diagnostic device comprising the peptide according to any one of embodiments 88 to 92, wherein the peptide is immobilized on a solid support and/or wherein the peptide is coupled to a reporter or reporter fragment, such as a reporter fragment suitable for a PCA.
Embodiment 100. The diagnostic device according to embodiment 99, wherein the solid support is an ELISA plate or a surface plasmon resonance chip.
Embodiment 101. The diagnostic device according to embodiment 99, wherein the diagnostic device is a lateral flow assay device or a biosensor-based diagnostic device with an electrochemical, fluorescent, magnetic, electronic, gravimetric or optical biotransducer.
Embodiment 102. A diagnostic kit comprising a peptide according to any one of embodiments 88 to 92, preferably a diagnostic device according to any one of embodiment 99 to 101, and preferably one or more selected from the group of a buffer, a reagent, and instructions.
Embodiment 103. An apheresis device comprising the peptide according to any one of embodiments 88 to 92, preferably immobilized on a solid carrier.
Embodiment 104. The apheresis device according to embodiment 103, wherein the solid carrier is capable of being contacted with blood or plasma flow.
Embodiment 105. The apheresis device according to embodiment 103 or 104, wherein the solid carrier comprises the compound according to any one of embodiments 1 to 65.
Embodiment 106. The apheresis device according to any one of embodiment 103 to 105, wherein the solid carrier is a sterile and pyrogen-free column.
Embodiment 107. The apheresis device according to any one of embodiments 103 to 106, wherein the apheresis device comprises at least two, preferably at least three, more preferably at least four different peptides according to any one of embodiments 88 to 92.
The present invention is further illustrated by the following figures and examples, without being restricted thereto.
In the context of the following figures and examples the compound on which the inventive approach is based is also referred to as “Selective Antibody Depletion Compound” (SADC).
Examples 1-10 relate to the general working principle of SADCs, demonstrating the selective removal of antibodies. Example 11 relates to the specific application of this therapeutic concept to CFS/ME, POTS, AAG, IDC, and cChHD.
Animal Models:
In order to provide in vivo models with measurable titers of prototypic undesired antibodies in human indications, BALB/c mice were immunized using standard experimental vaccination with KLH-conjugated peptide vaccines derived from established human autoantigens or anti-drug antibodies. After titer evaluation by standard peptide ELISA, immunized animals were treated with the corresponding test SADCs to demonstrate selective antibody lowering by SADC treatment. All experiments were performed in compliance with the guidelines by the corresponding animal ethics authorities.
Immunization of Mice with Model Antigens:
Female BALB/c mice (aged 8-10 weeks) were supplied by Janvier (France), maintained under a 12h light/12h dark cycle and given free access to food and water. Immunizations were performed by s.c. application of KLH carrier-conjugated peptide vaccines injected 3 times in biweekly intervals. KLH conjugates were generated with peptide T3-2 (SEQ ID NO. 33: CGRPQKRPSCIGCKG), which represents an example for molecular mimicry between a viral antigen (EBNA-1) and an endogenous human receptor antigen, namely the placental GPR50 protein, that was shown to be relevant to preeclampsia (Elliott et al.). In order to confirm the generality of this approach, a larger antigenic peptide derived from the autoimmune condition myasthenia gravis was used for immunization of mice with a human autoepitope. In analogy to peptide T3-2, animals were immunized with peptide T1-1 (SEQ ID NO. 34: LKWNPDDYGGVKKIHIPSEKGC), derived from the MIR (main immunogenic region) of the human AChR protein which plays a fundamental role in pathogenesis of the disease (Luo et al.). The T1-1 peptide was used for immunizing mice with a surrogate partial model epitope of the human AChR autoantigen. The peptide T8-1 (SEQ ID NO. 35: DHTLYTPYHTHPG) was used to immunize control mice to provide a control titer for proof of selectivity of the system. For vaccine conjugate preparation, KLH carrier (Sigma) was activated with sulfo-GMBS (Cat. Nr. 22324 Thermo), according to the manufacturer's instructions, followed by addition of either N- or C-terminally cysteinylated peptides T3-2 and T1-1 and final addition of Alhydrogel® before injection into the flank of the animals. The doses for vaccines T3-2 and T1-1 were 15 μg of conjugate in a volume of 100 ul per injection containing Alhydrogel® (InvivoGen VAC-Alu-250) at a final concentration of 1% per dose.
Generation of Prototypic SADCs:
For testing selective antibody lowering activity by SADCs of T3-2 and T1-1 immunized mice, SADCs were prepared with mouse serum albumin (MSA) or mouse immunoglobulin (mouse-Ig) as biopolymer scaffold in order to provide an autologous biopolymer scaffold, that will not induce any immune reaction in mice, or non-autologuous human haptoglobin as biopolymer scaffold (that did not induce an allogenic reaction after one-time injection within 72 hours). N-terminally cysteinylated SADC peptide E049 (SEQ ID NO. 36: GRPQKRPSCIG) and/or C-terminally cysteinylated SADC peptide E006 (SEQ ID NO. 37: VKKIHIPSEKG) were linked to the scaffold using sulfo-GMBS (Cat. Nr. 22324 Thermo)-activated MSA (Sigma; Cat. Nr. A3559) or -mouse-Ig (Sigma, 15381) or -human haptoglobin (Sigma H0138) according to the instructions of the manufacturer, thereby providing MSA-, Ig- and haptoglobin-based SADCs with the corresponding cysteinylated peptides, that were covalently attached to the lysines of the corresponding biopolymer scaffold. Beside conjugation of the cysteinylated peptides to the lysines via a bifunctional amine-to-sulfhydryl crosslinker, a portion of the added cysteinylated SADC peptides directly reacted with sulfhydryl groups of cysteins of the albumin scaffold protein, which can be detected by treating the conjugates with DTT followed by subsequent detection of free peptides using mass spectrometry or any other analytical method that detects free peptide. Finally, these SADC conjugates were dialysed against water using Pur-A-Lyzer™ (Sigma) and subsequently lyophilized. The lyophilized material was resuspended in PBS before injection into animals.
In Vivo Functional Testing of SADCs:
Prototypic SADCs, SADC-E049 and SADC-E006 were injected intraperitoneally (i.p.; as a surrogate for an intended intravenous application in humans and larger animals) into the mice that had previously been immunized with peptide vaccine T3-2 (carrying the EBNA-1 model epitope) and peptide vaccine T1-1 (carrying the AChR MIR model epitope). The applied dose was 30 μg SADC conjugate in a volume of 50 μl PBS. Blood takes were performed by submandibular vein puncture, before (−48 h, −24 h) and after (+24 h, +48 h, +72 h, etc.) i.p. SADC injections, respectively, using capillary micro-hematocrit tubes. Using ELISA analysis (see below), it was found that both prototypic SADCs were able to clearly reduce the titers over a period of at least 72 hrs in the present animal model. It could therefore be concluded that SADCs can be used to effectively reduce titers in vivo.
Titer Analysis:
Peptide ELISAs were performed according to standard procedures using 96-well plates (Nunc Medisorp plates; Thermofisher, Cat Nr 467320) coated for 1 h at RT with BSA-coupled peptides (30 nM, dissolved in PBS) and incubated with the appropriate buffers while shaking (blocking buffer, 1% BSA, 1×PBS; washing buffer, 1×PBS/0.1% Tween; dilution buffer, 1×PBS/0.1% BSA/0.1% Tween). After serum incubation (dilutions starting at 1:50 in PBS; typically in 1:3 or 1:2 titration steps), bound antibodies were detected using Horseradish Peroxidase-conjugated goat anti-mouse IgG (Fc) from Jackson immunoresearch (115-035-008). After stopping the reaction, plates were measured at 450 nm for 20 min using TMB. EC50 were calculated from readout values using curve fitting with a 4-parameter logistic regression model (GraphPad Prism) according to the procedures recommended by the manufacturer. Constraining parameters for ceiling and floor values were set accordingly, providing curve fitting quality levels of R2>0.98.
A similar example is shown in
The haptoglobin-based SADC was generated using human Haptoglobin as a surrogate although the autologuous scaffold protein would be preferred. In order to avoid formation of anti-human-haptoglobin antibodies, only one single SADC injection per mouse of the non-autologuous scaffold haptoglobin was used for the present experimental conditions. As expected, under the present experimental conditions (i.e. one-time application), no antibody reactivity was observed against the present surrogate haptoglobin homologue.
In order to exclude immunogenicity of SADCs, prototypic candidate SADCs were tested for their propensity to induce antibodies upon repeated injection. Peptides T3-1 and T9-1 were used for this test. T3-1 is a 10-amino acid peptide derived from a reference epitope of the Angiotensin receptor, against which agonistic autoantibodies are formed in a pre-eclampsia animal model (Zhou et al.); T9-1 is a 12-amino acid peptide derived from a reference anti-drug antibody epitope of human IFN gamma (Lin et al.). These control SADC conjugates were injected 8× every two weeks i.p. into naïve, non-immunized female BALB/c mice starting at an age of 8-10 weeks.
Animals C1-C4 were treated i.p. (as described in example 1) with SADC T3-1. Animals C5-C8 were treated i.p. with an SADC carrying the peptide T9-1. As a reference signal for ELISA analysis, plasma from a control animal that was vaccinated 3 times with KLH-peptide T1-1 (derived from the AChR-MIR, explained in Example 1) was used. Using BSA-conjugated peptide probes T3-1, T9-1 and E005 (SEQ ID NO. 38: GGVKKIHIPSEK), respectively, for antibody titer detection by standard ELISA at a dilution of 1:100, it could be demonstrated that antibody induction was absent in SADC-treated animals, when compared to the vaccine-treated control animal C (see
Plasma of E006-KLH (VKKIHIPSEKG (SEQ ID NO: 37) with C-terminal cysteine, conjugated to KLH) vaccinated mice was diluted 1:3200 in dilution buffer (PBS+0.1% w/v BSA+0.1% Tween20) and incubated (100 μl, room temperature) sequentially (10 min/well) four times on single wells of a microtiter plate that was coated with 2.5 μg/ml (250 ng/well) of SADC or 5 μg/ml (500 ng/well) albumin as negative control.
In order to determine the amount of free, unbound antibody present before and after incubation on SADC coated wells, 50 μl of the diluted serum were taken before and after the depletion and quantified by standard ELISA using E006-BSA coated plates (10 nM peptide) and detection by goat anti mouse IgG bio (Southern Biotech, diluted 1:2000). Subsequently, the biotinylated antibody was detected with Streptavidin-HRP (Thermo Scientific, diluted 1:5000) using TMB as substrate. Development of the signal was stopped with 0.5 M sulfuric acid.
ELISA was measured at OD450 nm (y-axis). As a result, the antibody was efficiently adsorbed by either coated mono- or divalent SADCs containing peptide E006 with C-terminal cysteine (sequence VKKIHIPSEKGC, SEQ ID NO: 39) (before=non-depleted starting material; mono-divalent corresponds to peptides displayed on the SADC surface; neg. control was albumin; indicated on the x-axis). See
This demonstrates that SADCs with mono- or divalent peptides are very suitable to adsorb antibodies and thereby deplete them.
Linear and circular peptides derived from wild-type or modified peptide amino acid sequences can be used for the construction of specific SADCs for the selective removal of harmful, disease-causing or otherwise unwanted antibodies directed against a particular epitope. In case of a particular epitope, linear peptides or constrained peptides such as cyclopeptides containing portions of an epitope or variants thereof, where for example, one or several amino acids have been substituted or chemically modified in order to improve affinity to an antibody (mimotopes), can be used for constructing SADCs. A peptide screen can be performed with the aim of identifying peptides with optimized affinity to a disease-inducing autoantibody. The flexibility of structural or chemical peptide modification provided a solution to minimize the risk of immunogenicity, in particular of binding of the peptide to HLA and thus the risk of unwanted immune stimulation.
Therefore, wild-type as well as modified linear and circular peptide sequences were derived from a known epitope associated with an autoimmune disease. Peptides of various length and positions were systematically permutated by amino acid substitutions and synthesized on a peptide array. This allowed screening of 60000 circular and linear wild-type and mimotope peptides derived from these sequences. The peptide arrays were incubated with an autoantibody known to be involved in the autoimmune disease. This autoantibody was therefore used to screen the 60000 peptides and 100 circular and 100 linear peptide hits were selected based on their relative binding strength to the autoantibody. Of these 200 peptides, 51 sequences were identical between the circular and the linear peptide group. All of the best peptides identified had at least one amino acid substitution when aligned to the original sequences, respectively and are therefore regarded as mimotopes. It also turned out that higher binding strengths can be achieved with circularized peptides.
These newly identified peptides, preferentially those with high relative binding values, are used to generate SADCs that are able to remove autoantibodies directed against this particular epitope or to develop further mimotopes and derivatives based on their sequences.
10 μg of model undesired antibody mAB anti V5 (Thermo Scientific) was injected i.p. into female Balb/c mice (5 animals per treatment group; aged 9-11 weeks) followed by intravenous injection of 50 μg SADC (different biopolymer scaffolds with tagged V5 peptides bound, see below) 48 hrs after the initial antibody administration. Blood was collected at 24 hrs intervals from the submandibular vein. Blood samples for time point 0 hrs were taken just before SADC administration.
Blood was collected every 24 hrs until time point 120 hrs after the SADC administration (x-axis). The decay and reduction of plasma anti-V5 IgG levels after SADC administration was determined by anti V5 titer readout using standard ELISA procedures in combination with coated V5-peptide-BSA (peptide sequence IPNPLLGLDC—SEQ ID NO: 40) and detection by goat anti mouse IgG bio (Southern Biotech, diluted 1:2000) as shown in
EC50[OD450] values were determined using 4 parameter logistic curve fitting and relative signal decay between the initial level (set to 1 at time point 0) and the following time points (x-axis) was calculated as ratio of the EC50 values (y-axis, fold signal reduction EC50). All SADC peptides contained tags for direct detection of SADC and immunocomplexes from plasma samples; peptide sequences used for SADCs were: IPNPLLGLDGGSGDYKDDDDKGK(SEQ ID NO: 41)-(BiotinAca)GC (SADC with albumin scaffold—SADC-ALB, SADC with immunoglobulin scaffold—SADC-IG, SADC with haptoglobin scaffold—SADC-HP, and SADC with transferrin scaffold—SADC-TF) and unrelated peptide VKKIHIPSEKGGSGDYKDDDDKGK(SEQ ID NO: 42)-(BiotinAca)GC as negative control SADC (SADC-CTR).
The SADC scaffolds for the different treatment groups of 5 animals are displayed in black/grey shades (see inset of
Treated groups exhibited rapid and pronounced antibody reduction already at 24 hrs (in particular SADC-TF) when compared to the mock treated control group SADC-CTL. SADC-CTR was used as reference for a normal antibody decay since it has no antibody lowering activity because its peptide sequence is not recognized by the administered anti V5 antibody. The decay of SADC-CTR is thus marked with a trend line, emphasizing the antibody level differences between treated and mock treated animals.
In order to determine the effectivity of selective antibody lowering under these experimental conditions, a two-way ANOVA test was performed using a Dunnett's multiple comparison test. 48 hrs after SADC administration, the antibody EC50 was highly significantly reduced in all SADC groups (p<0.0001) compared to the SADC-CTR reference group (trend line). At 120 hrs after SADC administration, antibody decrease was highly significant in the SADC-ALB and SADC-TF groups (both p<0.0001) and significant in the SADC-HP group (p=0.0292), whereas the SADC-IG group showed a trend towards an EC50 reduction(p=0.0722) 120 hrs after SADC administration. Of note, selective antibody reduction was highly significant (p<0.0001) in the SADC-ALB and SADC-TF groups at all tested time-points after SADC administration.
It is concluded that all SADC biopolymer scaffolds were able to selectively reduce antibody levels. Titer reduction was most pronounced with SADC-ALB and SADC-TF and no rebound or recycling of antibody levels was detected towards the last time points suggesting that undesired antibodies are degraded as intended.
Plasma levels of different SADC variants at 24 hrs after i.v. injection into Balb/c mice. Determination of Plasma levels (y-axis) of SADC-ALB, -IG, —HP, -TF and the negative control SADC-CTR (x-axis), were detected in the plasmas from the animals already described in example 5. Injected plasma SADC levels were detected by standard ELISA whereby SADCs were captured via their biotin moieties of their peptides in combination with streptavidin coated plates (Thermo Scientific). Captured SADCs were detected by mouse anti Flag-HRP antibody (Thermo Scientific, 1:2,000 diluted) detecting the Flag-tagged peptides (see also example 7):
Assuming a theoretical amount in the order of 25 μg/ml in blood after injecting 50 μg SADC i.v., the detectable amount of SADC ranged between 799 and 623 ng/ml for SADC-ALB or SADC-IG and up to approximately 5000 ng/ml for SADC-TF, 24 hrs after SADC injection. However surprisingly and in contrast, SADC-HP and control SADC-CTR (which is also a SADC-HP variant, however carrying the in this case unrelated negative control peptide E006, see previous examples), had completely disappeared from circulation 24 hrs after injection, and were not detectable anymore. See
This demonstrates that both Haptoglobin scaffold-based SADCs tested in the present example ((namely SADC-HP and SADC-CTR) exhibit a relatively shorter plasma half-life which represents an advantage over SADCs such as SADC-ALB, SADC-IG oder SADC-TF in regard of their potential role in complement-dependent vascular and renal damage due to the in vivo risk of immunocomplex formation. Another advantage of SADC-HP is the accelerated clearance rate of their unwanted target antibody from blood in cases where a rapid therapeutic effect is needed. The present results demonstrate that Haptoglobin-based SADC scaffolds (as represented by SADC-HP and SADC-CTR) are subject to rapid clearance from the blood, regardless of whether SADC-binding antibodies are present in the blood, thereby minimizing undesirable immunocomplex formation and showing rapid and efficient clearance. Haptoglobin-based SADCs such as SADC-HP in the present example thus provide a therapeutically relevant advantage over other SADC biopolymer scaffolds, such as demonstrated by SADC-TF or SADC-ALB, both of which are still detectable 24 hrs after injection under the described conditions, in contrast to SADC-HP or SADC-CTR which both are completely cleared 24 hrs after injection.
In order to determine the amount IgG bound to SADCs in vivo, after i.v. injection of 10 μg anti V5 IgG (Thermo Scientific) followed by injection of SADC-ALB, —HP, -TF and -CTR (50 μg) administered i.v. 48h after antibody injection, plasma was collected from the submandibular vein, 24 hrs after SADC injection, and incubated on streptavidin plates for capturing SADCs from plasma via their biotinylated SADC-V5-peptide [IPNPLLGLDGGSGDYKDDDDKGK(SEQ ID NO: 41) (BiotinAca)GC or in case of SADC-CTR the negative control peptide VKKIHIPSEKGGSGDYKDDDDKGK(SEQ ID NO: 42) (BiotinAca)GC]. IgG bound to the streptavidin-captured SADCs was detected by ELISA using a goat anti mouse IgG HRP antibody (Jackson Immuno Research, diluted 1:2,000) for detection of the SADC-antibody complexes present in plasma 24 hrs after SADC injection. OD450 nm values (y-axis) obtained for a negative control serum from untreated animals were subtracted from the OD450 nm values of the test groups (x-axis) for background correction.
As shown in
SADC-antibody complex formation was analyzed by pre-incubating 1 μg/ml of human anti V5 antibody (anti V5 epitope tag [SV5-P-K], human IgG3, Absolute Antibody) with increasing concentrations of SADC-ALB, -IG, —HP, -TF and -CTR (displayed on the x-axis) in PBS+0.1% w/v BSA+0.1% v/v Tween20 for 2 hours at room temperature in order to allow for immunocomplex formation in vitro. After complex formation, samples were incubated on ELISA plates that had previously been coated with 10 μg/ml of human C1q (CompTech) for 1 h at room temperature, in order to allow capturing of in vitro formed immunocomplexes. Complexes were subsequently detected by ELISA using anti human IgG (Fab specific)-Peroxidase (Sigma, diluted 1:1,000). Measured signals at OD450 nm (y-axis) reflect Antibody-SADC complex formation in vitro.
As shown in
Together with the in vivo data (previous examples), these findings corroborate the finding that haptoglobin scaffolds are advantageous over other SADC biopolymer scaffolds because of the reduced propensity to activate the complement system. In contrast, SADC-TF or SADC-ALB show higher complexation, and thereby carry a certain risk of activating the C1 complex with initiation of the classical complement pathway (a risk which may be tolerable in some settings, however).
Immunocomplexes were allowed to form in vitro, similar to the previous example, using 1 μg/ml mouse anti V5 antibody (Thermo Scientific) in combination with increasing amounts of SADCs (displayed on the x-axis). SADC-antibody complexes were captured on a streptavidin coated ELISA plate via the biotinylated SADC-peptides (see previous examples), followed by detection of bound anti-V5 using anti mouse IgG-HRP (Jackson Immuno Research, diluted 1:2,000).
Under these assay conditions, SADC-HP showed markedly less antibody binding capacity in vitro when compared to SADC-TF or SADC-ALB (see
This in vitro finding is consistent with the observation (see previous examples) that SADC-HP has a lower immunocomplex formation capacity when compared to SADC-TF or SADC-ALB which is regarded as a safety advantage with respect to its therapeutic use for the depletion of unwanted antibodies.
Rapid in vivo blood clearance of anti-mouse-CD163 mAB E10B10 (as disclosed in WO 2011/039510 A2). mAB E10B10 was resynthesized with a mouse IgG2a backbone. 50 μg mAb E10B10 and Mac2-158 (human-specific anti-CD163 mAb as disclosed in WO 2011/039510 A2, used as negative control in this example since it does not bind to mouse CD163) were injected i.v. into mice and measured after 12, 24, 36, 48, 72, 96 hours in an ELISA to determine the blood clearance.
mAb E10B10 was much more rapidly cleared from circulation than control mAb Mac2-158 was, as shown in
In conclusion, anti-CD163 antibodies are highly suitable as SADC scaffold because of their clearance profile. SADCs with such scaffolds will rapidly clear undesirable antibodies from circulation.
Detailed Methods:
50 ug of biotinylated monoclonal antibodies E10B10 and biotinylated Mac2-158 were injected i.v. into mice and measured after 12, 24, 36, 48, 72, 96 hours to determine the clearance by ELISA: Streptavidin plates were incubated with plasma samples diluted in PBS+0.1% BSA+0.1% Tween20 for 1 h at room temperature (50 μl/well). After washing (3× with PBS+0.1% Tween20), bound biotinylated antibodies were detected with anti-mouse IgG+IgM-HRP antibody at a 1:1000 dilution. After washing, TMB substrate was added and development of the substrate was stopped with TMB Stop Solution. The signal at OD450 nm was read. The EC50 values were calculated by non-linear regression using 4 parametric curve fitting with constrained curves and least squares regression. EC50 values at time-point T12 (this was the first measured time-point after antibody injection) was set at 100%, all other EC50 values were compared to the levels at T12.
SADCs are prepared essentially as described in Example 1, using human transferrin as biopolymer scaffold.
N-terminally cysteinylated peptides RATHQEAINCYA (SEQ ID NO: 43) and YANETC (SEQ ID NO: 44), both derived from the second extra-cellular loop of human beta-2 adrenergic receptor (UniProt accession code P07550; cf. Magnusson et al., 1989), are linked to the scaffold using sulfo-GMBS-activated human transferrin, thereby providing transferrin-based SADCs with the corresponding cysteinylated peptides, that are thereby covalently attached to the lysines of the corresponding biopolymer scaffold. These SADC conjugates are purified and resuspended in PBS.
To three ME/CFS patients 150 mg, 250 mg, and 500 mg, respectively, of resuspended SADC conjugate is administered intravenously, in order to reduce autoantibodies against beta-2 adrenergic receptors in the plasma of the patients and thereby ameliorate the symptoms of ME/CFS. The same procedure is carried out for three POTS patients, three AAG patients, three IDC patients, and three cChHD patients.
A screening for autoantibodies against peptides on microarrays containing 72886 cyclic (and, to a lesser extent, linear) peptides (derived from 184 human neuroreceptors as well as proteins involved in neurological or neuropsychiatric conditions) with a sequence length between 7 and 14 amino-acids, was performed to identify peptide stretches from antigenic protein sequences that are recognized by autoantibodies. IgG was prepared from blood obtained from 30 human donors (including ME/CFS patients) by protein G purification. Each IgG sample was incubated with peptide microarrays and Ig binding signals were detected by fluorescence. All antibody binding signals to the peptides on the arrays were background subtracted and ranked for each sample and a deduplicated aggregate of the respective top 250 peptide hits for each donor with the corresponding protein sequence of origin (as obtained from UniProt) was compiled (designated as group III). Further, the deduplicated aggregate of the respective top 50 peptide hits for each donor was compiled and designated as group II. Finally, the deduplicated aggregate of the respective top 50 peptide hits for each donor was compiled and designated as group I.
Altogether, group I contains 157 distinct peptide hits, group II contains 819 distinct peptide hits and group III contains 3492 distinct peptide hits. Evidently, group I is a subset of group II which in turn is a subset of group III. Groups I-III correspond to 0.2%, 1.1% and 4.8%, respectively, of all peptides screened.
The peptide hits belonging to groups I-III are listed in Table 1, in the general description above.
Thus, all listed peptides, preferably peptides belonging to group II, even more preferably belonging to group I, provide sequences from which (optionally shorter) peptide sequences can be derived for antibody depletion according to the present invention. Furthermore, also other peptide sequences (or fragments) from the proteins from which the peptides of Table 1 were derived (preferably from group II, more preferably however from group I), are well suited to be used for SADCs according to the present invention. These peptides and fragments thereof are also highly suitable for autoantibody profiling for diagnostic or predictive purposes.
In a manner similar to example 12, blood samples from human donors were screened with a peptide microarray based solely on a selection of 62 linear peptides from human neuroreceptors listed herein. This screen provided 52 positive IgG binding hits, i.e. confirmed autoantigenic hits.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20197934.1 | Sep 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/076176 | 9/23/2021 | WO |
