Vaccine for the treatment of amyloidosis

Abstract

Peptide-based vaccines are provided to treat or prevent amyloid-transthyretin amyloidosis (ATTR).

Description

[Technical Field]

[0001] The present invention relates to a vaccine comprising a transthyretin (TTR)-derived peptide, i.e., a TTR peptide, and to the vaccine for use in methods for treating or preventing amyloid-transthyretin amyloidosis (ATTR). More specifically, the TTR peptide comprises neoepitopes that are selectively presented and available in misfolded, oligomeric, and / or aggregated TTRs, respectively. The present invention further relates to a kit comprising the vaccine. [Background technology]

[0002] Amyloid-transthyretin amyloidosis (ATTR) is a serious age-related disorder that leads to cardiomyopathy and / or sensorimotor polyneuropathy (Non-Patent Literature 1), and includes two subtypes that differ in their pathogenesis: wild-type ATTR (ATTRwt) and mutant ATTR (ATTRv). Their common precursor protein, transthyretin (TTR), physiologically functions as a transporter of thyroxine and retinol-binding proteins. TTR is primarily synthesized in the liver and exists as a tetramer in its native form (Non-Patent Literature 2). ATTRv, formerly known as hereditary / mutant ATTR, is an autosomal dominant disorder. In both wild-type TTR (TTRwt) and mutant / variant TTR (TTRv) proteins, the pathogenic mechanism of ATTR is induced by partial unfolding of the TTR protein and subsequent aggregation into beta-pleated sheets that form amyloid fibrils (Non-Patent Literature 3).

[0003] ATTR is characterized by two main forms of clinical findings. When amyloid fibrils are predominantly accumulated in cardiac tissue, it leads to cardiomyopathy, while fibril deposition in nerve fibers leads to polyneuropathy (Non-Patent Literature 4). The factors that induce amyloid deposition in specific organs are still not understood. Patients generally present with a mix of symptoms, and only a small number of TTR mutations are known to cause purely cardiac or neurological disorders (Non-Patent Literature 5).

[0004] There are currently three established concepts in the treatment of ATTR: (i) After orthotopic liver transplantation, TTRwt synthesis in the donor liver almost completely replaces TTRv in the peripheral blood. In addition to the well-known shortage of organ donors, ATTRv patients who have received liver transplants show a gradual but continuous deterioration of their condition. (ii) Low molecular weight compounds stabilize the TTR tetramer, thereby minimizing the formation of amyloid precursors. Diflunisal, AG10, and tafamidis stabilize the physiological TTR tetramer. Tafamidis has been approved as a treatment for stage 1 ATTR since 2011. (iii) Gene silencers (mRNA inhibitory oligonucleotides) reduce TTRv and TTRwt secreted from the liver. Inotercene is an antisense oligonucleotide administered subcutaneously (sc) once a week. Patisiran acts as an siRNA oligonucleotide administered intravenously (iv) every three weeks in combination with premedication. Both gene silencers were approved in 2018 as treatments for stage 1 and stage 2 ATTR.

[0005] Meanwhile, CRISPR / CAS9, a gene editing protocol that enables targeted in vivo genome editing, is being explored as a novel treatment for ATTR amyloidosis in a Phase I open-label, multicenter trial using an in vivo gene editing agent called NTLA-2001. In addition, monoclonal antibodies (mAbs) are being investigated as an additional potential therapeutic concept for ATTR. Examples include PRX004 (NNC6019-0001), an investigational mAb designed to inhibit fibrillation by specifically targeting and removing misfolded TTR proteins found in ATTR-CM, and NI006, an investigational human mAb that targets TTR amyloid and, most recently, has been shown to be safe and dose-dependent in depleting cardiac ATTR in a Phase I trial (Non-Patent Literature 6). Non-Patent Literature 7 outlines various treatment strategies.

[0006] While each of the current drugs and concepts holds great promise for improving ATTR and related pathologies, alternative treatment strategies remain necessary because patients respond differently to specific treatments, and therefore, having different treatment options is always desirable.

[0007] This technical problem is characterized in the claims, further described below, and solved by embodiments illustrated in the examples and drawings. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Gertz et al., J. Am. Coll. Cardiol. 66 (2015), 2451-24661 [Non-Patent Document 2] Alshehri et al., J. Neuroendocrinol. 27 (2015), 303-3239 [Non-Patent Document 3] Eisele et al., Nat. Rev. Drug Discov. 14 (2015), 759-780

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

[0009] The present invention generally relates to vaccines useful for the treatment and prevention of amyloid-transthyretin amyloidosis (ATTR) and disorders caused by or associated with TTR deposition. TTR tetramer stabilizers, gene silencers, or antibodies are primarily used in the treatment of acute and chronic disorders caused by ATTR, i.e., at the onset or presence of the disease. However, vaccines can be used for disease prevention before the onset of the disease in subjects at risk, for example due to genetic predisposition, or judged to be at risk, for example due to changes in the levels of indicator biomarkers. In addition, since vaccination provides permanent protection due to priming of the immune system, continuous administration of therapeutic agents is usually not necessary. More specifically, the present invention relates to a vaccine comprising an immunogen, which in the context of the present invention is a TTR-derived peptide, i.e., a TTR peptide, which comprises an epitope of TTR, which is selectively presented or available only in misfolded, oligomeric, and / or aggregated TTR, as in the case of a neoepitope, and / or which is not present in physiologically active TTR, for example, even if it is an epitope available in the monomer of wild-type or mutant TTR protein, it is hidden in the physiologically active tetramer and is no longer available for antibody binding. Most preferably, to avoid any cross-reactivity, the epitope is not present on the native TTR monomer. Typically, the peptide-based vaccine of the present invention comprises a TTR peptide and an adjuvant to stimulate an immune response. The vaccine of the present invention is preferably used in the treatment and prevention of ATTR.

[0010] Approximately 20 years ago, Terazaki et al., Lab Invest. 86 (2006) 23-31 described immunizing transgenic mice carrying V30M (a transthyretin mutant in which valine at position 30 is replaced with methionine), the most common FAP-associated TTR mutant, with Y78F (a transthyretin mutant in which tyrosine at position 78 is replaced with phenylalanine), a TTR mutant designed to destabilize the native structure. This mutant has been shown to expose a cryptic epitope that is recognized by a monoclonal antibody that reacts only with amyloid fibrils or with highly amyloid-forming mutants exhibiting amyloid folds. These results suggested that Y78F induces the production of antibodies that specifically react with deposits and lead to an immune response effective in removing / preventing TTR deposition. Therefore, the authors concluded that TTR immunization with selected TTR mutants may have potential applications in FAP immunotherapy.

[0011] However, since then, this approach has not been further pursued toward vaccine development. The reason for this is likely one of the major concerns associated with active immunotherapy: the possibility that the antigen (in this case, the mutant TTR protein) may still trigger an immune response to physiological TTR species. On the other hand, the Y78F mutant protein has been used because it was thought that by using this mutant TTR protein, the desired immune response could be induced by presenting a cryptic epitope.

[0012] The present invention is based on the concept of identifying segments in the TTR amino acid sequence that are available for the immune response of the organism, but are specific to TTR fibers and amyloid, designing corresponding peptides containing at least the immunogenic part of that region, and forcibly changing each of the peptide and the immunogenic part into a 3D conformation similar to the conformation that each segment may have in the amyloid conformation. For this purpose, based on the possible mechanisms of misfolding of the TTR protein, Cryo-EM studies were screened, whereby it was suggested that after the native tetramer decomposes and unfolds and subsequently the polypeptide chains assemble into an initial fibrillar state, a segment of structurally disordered residues Ala36-His56 that is not present on the native TTR tetramer is formed in the solvent-exposed conformation (Schmidt et al., Nat Commun 10 (2019), 5008, see especially Fig. 3 and the legend of that figure. These are incorporated herein by reference). In fact, recent publications have reported the 3D structure of ATTR fibers extracted from patient tissues and determined using cryo-electron microscopy (cryo-EM) (see Schmidt et al. (2019) supra, Iakovleva et al., Nat Commun 12 (2021), 7141, Steinebrei et al., Nat Commun 13 (2022), 6398).

[0013] The amyloid structure was virtually identical in all three cases and was characterized by a rigid core structure interrupted by an undefined segment extending from Lys35 to Gly57. This indicated the presence of loose segments with variable and non-rigid conformations. Nevertheless, according to the present invention, the proximity of Lys35 and Gly57 in the amyloid core indicates that the undefined segment formed a loop, which, as shown in the examples, was used to design the corresponding peptide.

[0014] Examination of the amino acid sequence revealed that this loose segment contains the epitope of NI006, i.e., WEPFA (SEQ ID NO: 1), i.e., a selective anti-ATTR antibody shown to remove cardiac amyloid (see Non-Patent Document 6 supra and Michalon et al., Nat Commun 12 (2021), 3142). Therefore, it is reasonable to assume that NI006 can bind to the loose segment in ATTR and thus a peptide derived from that segment can be used to determine whether it can mimic the 3D conformation in TTR amyloid and be designed to bind substantially with the same selectivity and high affinity as the antibody for TTR amyloid.

[0015] In this context, the peptide, i.e., the immunogen, is typically formulated with an adjuvant or immunogenic enhancer such as a protein carrier to generate an immune response strong enough to protect the subject to be vaccinated from the disease, and it must be taken into account that the presence of, for example, a protein carrier may adversely affect the structure of the peptide and lead to the loss of formation of neoepitopes as seen in the 3D conformation of the loose segment in TTR amyloid.

[0016] Therefore, attempts have been made to stabilize the desired 3D conformation of the peptide by causing cyclization of the corresponding peptide sequence.

[0017] Therefore, experiments conducted within the scope of the present invention have surprisingly revealed that TTR peptides coupled to the carrier bovine serum albumin (BSA) can function as antigens for anti-TTR antibodies, and that cyclization of the peptides promotes antibody binding by more than 100 times (see Examples 1 and 2). In particular, the binding of anti-TTR antibodies, exemplified based on antibody NI-301.37F1, to the linear TTR peptide TTR34-54 and the cyclic peptide TTR34-54cyc (both containing the epitope WEPFA (SEQ ID NO: 1)) was observed in ELISA assays in the nanomolar and picomolar ranges, respectively (see Example 1). Furthermore, antibody binding is still possible even when a carrier protein (BSA is shown here as an example) is coupled to TTR peptides, particularly the cyclic peptides TTR34-54cyc and TTR39-50cyc (see Example 2).

[0018] The antibody NI-301.37F1, which binds to the TTR epitope WEPFA (SEQ ID NO: 1), is described in International Publication No. 2015 / 092077. In addition, the usefulness of TTR peptides as immunogens and, consequently, for vaccination approaches was confirmed in in vivo mouse studies described in Example 3. Specifically, mice were immunized with linear and cyclic BSA-coupled TTR peptides of 12-amino acid and 21-amino acid lengths, and the immune response was characterized by ELISA against amyloid TTR (ATTR, mis.WT-TTR) and tetrameric TTR (TTR). As shown in Figure 3, administration of TTR peptides induced the formation of antibodies against TTR and ATTR after 38 days, where the tested serum showed a much stronger serum reactivity against ATTR than against TTR. Surprisingly, and in contrast to what could have been expected from the initial ELISA assays described in Examples 1 and 2, the linear peptides TTR34-54 and TTR39-50 showed EC 50(ATTR / TTR) The indices are 15.8 and 14.7 respectively, and both are EC 50(ATTR / TTR) The cyclic peptide TTR34-54cyc and EC have an index of 13.4. 50(ATTR / TTR)It showed higher amyloid selectivity than the cyclic peptide TTR39-50cyc, which has an index of 4.3 (see Table 1 and Figure 3). Furthermore, the highest titer of ATTR-specific antibodies was detected in serum obtained from mice immunized with the linear peptide TTR34-54, meaning that mice administered with the TTR34-54 immunogen showed a higher immune response compared to mice administered with other peptides (see Figure 4).

[0019] Therefore, since the exemplary tested TTR peptides are thought to mimic the 3D conformation of loose segments in TTR amyloid and should evoke a neoepitope-specific immune response in mammals, it is reasonable to provide further evidence that the TTR peptides designed according to the present invention, when administered to a target, for example in the form of a vaccine, will induce an immune response including the stimulation of T cells and other reactive immune cells directed towards the TTR peptide, leading to the production of anti-TTR antibodies that exhibit high selectivity against amyloid-forming TTR. As described above, antibodies that bind within the TTR segment ranging from Lys35 to Gly57 have the ability to remove misfolded TTR aggregates by phagocytosis, and therefore, TTR peptides that specifically induce antibodies against that segment, preferably those containing the amino acid sequence WEPFA (SEQ ID NO: 1), can be used as immunogens in vaccines, and it is reasonable to expect that they can induce the removal of misfolded TTR aggregates before they further aggregate into fibrils. This avoids the expression of disease phenotypes, i.e., the accumulation of TTR fibrils. Therefore, it is reasonable to expect that the TTR peptide of the present invention can be used to initiate an immune response against amyloid-forming / misfolded TTRs for immunization, i.e., to prevent the onset of ATTR, or, if the disease has already developed, in the treatment of the disease. The theory underlying the present invention is supported by Example 3, which demonstrates an effective immune response to the immunogen of the present invention, in which serum antibodies preferentially bind to aggregated TTRs. It is thought that preventing the (further) formation of TTR aggregates can prevent further disease progression and / or complete remission of TTR aggregates, thereby halting the disease cascade.

[0020] As described above, the structurally disordered segment of the Ala36-His56 residue of TTR is formed in the solvent-exposed conformation of TTR amyloid and is not present on the native TTR tetramer, i.e., it is a neoepitope and provides a favorable basis for the design of TTR peptides. Antibody binding to such neoepitopes is key to their safety and therapeutic efficacy, and consequently, the induction of such antibodies by immunogens is key to the efficacy of vaccines containing the immunogens. Therefore, the present invention relates to vaccines containing TTR-derived peptides that include an epitope of TTR, wherein this epitope is selectively presented or available for antibody binding only in misfolded, oligomeric, and / or aggregated forms of TTR. In a preferred embodiment, the vaccine of the present invention comprises a peptide containing the amino acid sequence WEPFA (SEQ ID NO: 1), which preferably comprises or consists of the amino acid sequence RKAADDTWEPFASGKTSESGE (SEQ ID NO: 2, TTR34-54) or DTWEPFASGKTS (SEQ ID NO: 3, TTR39-50), and most preferably comprises or consists of the amino acid sequence RKAADDTWEPFASGKTSESGE (SEQ ID NO: 2, TTR34-54). In a preferred embodiment, the peptide is a linear peptide.

[0021] Subunit vaccines, primarily composed of peptides or proteins, may face limitations in terms of immunogenicity, potentially requiring multiple immunizations to achieve a high level of immune response. This has led to the development of various approaches to enhance the response of subunit vaccines, including the presentation of epitopes in multimeric form (e.g., virus-like particles, VLPs, or nanoparticles). This strategy can boost the immune response by increasing the half-life of the epitope by reducing renal clearance and sensitivity to proteolysis (see Malonis et al., Chem Rev. 120 (2020), 3210-3229). While not intended to be theoretically bound, the use of cyclic peptides as vaccines may yield a similar effect, namely enhanced vaccine response, due to the constraint that cyclic peptides adopt a very stable conformation because both ends are linked together. Therefore, in one embodiment, the vaccine of the present invention comprises a TTR peptide capable of forming a cyclic compound. That is, in one embodiment, the vaccine of the present invention comprises a cyclized TTR compound. Cyclization is preferably carried out by linkers coupled to the N-terminal and C-terminal residues of the peptide. In preferred embodiments, the cyclic compound comprises or consists of the amino acid sequence GCGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 4, TTR34-54cyc) or GCGGGDTWEPFASGKTSGGGCG (SEQ ID NO: 5, TTR39-50cyc).

[0022] The observation made in Example 2 that BSA coupling does not substantially hinder antibody binding is important for the successful development of peptide-based vaccines. In particular, in typical peptide vaccination protocols, in addition to or instead of approaches that present epitopes in polymer form, peptides containing the epitope of interest can be conjugated to a carrier protein, thereby boosting the immune response by increasing the half-life of the epitope by reducing renal clearance and sensitivity to proteolysis. Binding to the carrier protein is typically achieved by chemical conjugation. Carriers are generally known to have immunogenic properties, and therefore, simply covalently binding the epitope to an immunogenic species is often sufficient to enhance the immune response (see Malonis et al., Chem Rev. 120 (2020), 3210-3229). BSA is an example of an immunogenic carrier. Therefore, TTR peptides are considered suitable candidates for vaccines. Accordingly, in one embodiment, the vaccine of the present invention comprises a preferably cyclic peptide coupled to a carrier protein, such as BSA.

[0023] As discussed earlier, the accurate presentation of the neoepitope's 3D structure is key to inducing an immune response that preferentially recognizes ATTRs, i.e., aggregated TTRs, particularly wild-type TTR species. Therefore, it was initially thought that peptide cyclization would not only confer serum stability but also mimic the structures on misfolded and aggregated TTRs by aiding peptide folding, thereby stabilizing the conformation against potential harmful interference to accurate folding by immunogenic carriers (here, BSAs, which are much larger polypeptides and are quite likely to adversely affect peptide folding or shield the epitope). However, experiments conducted according to the present invention have surprisingly revealed that cyclization is neither necessary nor advantageous for the efficacy and specificity of TTR peptide immunogens (see Example 3, and Figures 3 and 4).

[0024] Therefore, in a preferred embodiment of the present invention, the TTR peptide-based immunogen is not cyclized and preferably has not undergone any other modifications. Rather, the TTR peptide in the immunogen preferably comprises a minimal epitope / antigen sequence representing a neoepitope specific to pathological variants, oligomers, and / or aggregates of TTR, as determined experimentally and / or by in silico analysis, and optionally containing about 1 to 10 additional amino acids at its N-terminus and / or C-terminus, preferably, where the additional amino acids are also present in the original TTR amino acid sequence (see also Examples).

[0025] As described above, the immunogens for vaccines of the present invention can be chemically synthesized or produced using recombinant DNA technology (see, for example, Wang et al., Sig Transduct. Target Ther. 7 (2022); https: / / doi.org / 10.1038 / s41392-022-00904-4 for a review). Any recombinantly expressed antigenic TTR peptide according to the present invention, whether coupled to an immunogenic carrier or not, also includes nucleic acids encoding the peptide or protein, and an expression vector containing this nucleic acid, as well as host cells containing the expression vector (autonomously or chromosomally inserted), also constitute an embodiment of the present invention.

[0026] Accordingly, in one or more embodiments, the present invention relates to nucleic acids encoding immunogens described herein, expression vectors containing such nucleic acids, and / or host cells containing such nucleic acids or expression vectors. A further embodiment of the present invention is a method for recombinantly producing immunogens by expressing them in host cells and isolating them therefrom.

[0027] The nucleic acids and expression vectors of the present invention may be part of a kit or composition, which optionally further comprises an immunogenicity enhancer such as an immunogenicity carrier or an adjuvant.

[0028] The present invention further relates to a kit comprising the vaccine of the present invention.

[0029] Further embodiments of the present invention will become apparent from the following description and examples. [Brief explanation of the drawing]

[0030] [Figure 1] The results of the ELISA assay demonstrate that the exemplary TTR peptides, upon binding to the ATTR-specific antibody NI-301.37F1, retain the 3D conformation of the loose segment uniquely present in TTR amyloid, and that cyclization promotes antibody binding, indicating that TTR peptides, particularly cyclic TTR peptides, are suitable as antigens and immunogens. (A) In ELISA-1, antibody binding to peptides TTR34-54cyc and mis-WT-TTR was analyzed, and the results showed that antibody binding to the cyclic TTR34-54cyc peptide was much stronger than binding to mis-WT-TTR, i.e., about 10 times stronger; (B) In ELISA-2, antibody binding to peptides TTR34-54_bt and TTR34-54cyc_bt, as well as to the BSA control, was analyzed, and the results showed that both linear and cyclic TTR peptides were bound by ATTR-specific antibodies, but cyclization promoted NI-301.37F1 binding by more than 100 times; (C) In ELISA-3, antibody binding to peptides TTR34-54cyc and TTR40-49 was analyzed, and no antibody binding to TTR40-49 was observed, which indicates that proper folding requires a certain length of the peptide and the antigenic / immunogenic portion of the peptide, respectively. [Figure 2]The results of the ELISA assay showed that the BSA-coupled TTR peptide exhibits a TTR amyloid-specific epitope, as demonstrated by its binding to the antibody NI-301.37F1. (A) In ELISA-1, antibody binding to the peptides TTR34-54cyc_BSA, TTR34-54_BSA, TTR34-54SCRcyc_BSA, and TTR34-54SCR_BSA was analyzed. The results showed that both linear and cyclic TTR peptides coupled to BSA were bound by the antibody, but cyclization promoted NI-301.37F1 binding by more than 100-fold. (B) In ELISA-2, antibody binding to the peptides TTR39-50cyc_BSA, TTR39-50_BSA, TTR39-50SCRcyc_BSA, and TTR39-50SCR_BSA was analyzed. The results showed antibody binding to the cyclic BSA-coupled TTR39-50cyc_BSA peptide, but binding to the corresponding linear peptide was barely detectable. Binding to the antigen control, i.e., the scrambled (SCR) peptide, was not observed in any of the ELISA assays. [Figure 3]The results of the ELISA assay showed that the immune response, as measured by serum antibody titers, increased over time (comparing day 0 (d0) to day 38 (d38)), and that serum obtained from BalbC mice after immunization with TTR peptide showed higher reactivity to ATTR than to TTR. (A) ELISA for TTR and ATTR showed that the reactivity (EC50) of serum obtained from mice after immunization with peptide / immunogen TTR39-50 was 1038 for TTR and 15271 for ATTR, and consequently the amyloid selectivity (EC50 (ATTR / TTR) index) was 14.7; (B) ELISA for TTR and ATTR showed that the reactivity (EC50) of serum obtained from mice after immunization with peptide / immunogen TTR39-50cyc was 1813 for TTR and 24343 for ATTR, and consequently the amyloid selectivity (EC50 (ATTR / TTR) index) was 15.4; (C) ELISA for TTR and ATTR showed that the reactivity (EC50) of serum obtained from mice after immunization with peptide / immunogen TTR34 (D) ELISA for TTR and ATTR showed that serum obtained from mice after immunization with -54 had a reactivity (EC50) of 3400 relative to TTR and 53809 relative to ATTR, and consequently an amyloid selectivity (EC50(ATTR / TTR) index) of 15.8; (D) ELISA for TTR and ATTR showed that serum obtained from mice after immunization with peptide / immunogen TTR34-54cyc had a reactivity (EC50) of 7785 relative to TTR and 33587 relative to ATTR, and consequently an amyloid selectivity (EC50(ATTR / TTR) index) of 4.3; (D) Control ELISA for TTR and ATTR showed the reactivity (EC50) of serum obtained from mice after immunization with control peptide / immunogen PR906. Serum OD450 values ​​were measured and plotted on a logarithmic scale against serum dilution (dilution range 1:100 to 1:590-4900). [Figure 4]The results of the ELISA assay showed that serum obtained from BalbC mice immunized with TTR peptides TTR39-50, TTR39-50cyc, TTR34-54, and TTR34-54cyc showed higher reactivity to ATTR than to TTR, and that the highest antibody titers, particularly those of ATTR-specific antibodies, were detected in serum obtained from mice immunized with the linear peptide TTR34-54. A serum dilution of 1:24300 was used in this assay. [Modes for carrying out the invention]

[0031] Detailed description of the invention The present invention relates to a peptide-based vaccine comprising a transthyretin (TTR)-derived peptide, i.e., a TTR peptide, which comprises an epitope of TTR, which is selectively presented or available for antibody binding only in misfolded, oligomeric, and / or aggregated TTR.

[0032] Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. The present invention can be carried out or tested using methods and materials similar to or equivalent to those described herein, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references referenced herein constitute part of this specification by citation. Materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope of this invention.

[0033] Further embodiments of the present invention will become apparent from the following description, examples, and claims. Those skilled in the art will understand that all provisions of the general features of the general embodiments below can and preferably be combined with provisions of one or more other features of such general embodiments. To avoid doubt, it is emphasized that expressions such as “in some embodiments,” “in a particular embodiment,” “in a particular case,” “in some cases,” “in further embodiments,” and “in one embodiment” should be read with in mind that any of the embodiments described herein can be combined with the respective features of those embodiments, and that this disclosure should be treated as if such combinations of features of those embodiments were described in detail in one embodiment. The same applies to any combination of embodiments and features described in the appended claims and shown in the examples, which are also intended to be combined with features of the corresponding embodiments disclosed herein, where, for the sake of consistency and brevity, embodiments are characterized by dependencies; however, in practice, each combination of embodiments and features that can be interpreted as arising out of (numerous) dependencies should be seen as literally disclosed and not as a choice from different options.

[0034] Unless otherwise specified, terms used herein are defined as those provided in the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford University Press, published 1997, revised 2000, and reprinted 2003, ISBN 0-19-850673-2, second edition published 2006, ISBN 0-19-852917-1 978-0-19852917-0).

[0035] In this specification, the term "cyclic peptide" may refer to a completely proteinaceous compound, such as a compound whose linker has two, three, four, five, six, seven, or eight amino acids, or a compound that does not have a linker. For example, an amino acid stretch containing a native protein sequence, i.e., an antibody epitope, allows for cyclization without the addition of extra amino acids, for example, due to the presence of two cysteines at an appropriate distance. It is understood that the properties described for cyclic peptides determined in the examples may be incorporated into other compounds, such as cyclic compounds containing non-amino acid linker molecules. When a cyclic compound is composed of amino acids, the terms "cyclic peptide" and "cyclic compound" can be used interchangeably.

[0036] The term "immunogenic" refers to a substance that induces antibody production and activates lymphocytes or other reactive immune cells directed towards the antigenic portion of the immunogen (in this case, the TTR peptide).

[0037] As used herein, “immunogen” or “immunogenic compound” means a substance that elicits an immune response and results in antibody production, and may include, for example, TTR peptides, particularly cyclic peptides as described herein, conjugated as multiantigenic peptides and / or fused with immunogenicity enhancers such as carrier proteins like BSA. In addition to the conjugates described herein, immunogenic peptide mimes that induce cross-reactive antibodies against epitopes referred to herein also constitute immunogens.

[0038] The term "corresponding linear compound" in relation to cyclic compounds refers to compounds that contain or consist of the same sequence or chemical parts as the cyclic compound, but are in a linear (acyclic) form, or optionally, peptides.

[0039] As used herein, the term “linker” means a chemical moiety, preferably low or non-immunogenic, that can be covalently bound directly or indirectly to a protein fragment or peptide as defined herein. The ends of a linker can, for example, bind to form a cyclic compound. Linkers can be located at the N-terminus and C-terminus. Alternatively, linkers may be located internally at a “certain distance” from the ends. The ends of a linker can, for example, bind to form a cyclic compound. A linker may contain one or more functionalizable moieties, such as one or more cysteine ​​(C) residues. A linker can also bind to other proteins or components via its functionalizable moieties. A linker can bind to a carrier protein or immunogen enhancer, such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), via its functionalizable moieties. Cyclic compounds containing linkers have a length longer than the peptide or protein fragment itself. The linker may, but is not limited to, a non-immunogenic moiety, such as the amino acids glycine (G) and alanine (A), or polyethylene glycol (PEG) repeats.

[0040] As used herein, the term “functionalizable moiety” refers to a chemical entity having a “functional group,” and as used herein, “functional group” refers to a group of atoms or a single atom that reacts with another group of atoms or a single atom (a so-called “complementary functional group”) to form a chemical interaction between the two groups or atoms. In the case of cysteine ​​(C), the functional group may be -SH, which can react to form a disulfide bond. The reaction with another group of atoms may be covalent or non-covalent, for example, the biotin-streptavidin bond (which may have a dissociation constant (Kd) of about 1e-14). As used herein, a strong non-covalent bond means an interaction with a Kd of at least 1e-9, at least 1e-10, at least 1e-11, at least 1e-12, at least 1e-13, or at least 1e-14.

[0041] For example, to enhance immunogenicity, proteins and / or other active ingredients may be coupled to a cyclic compound. For this purpose, any functionalizable moiety capable of reacting (e.g., forming a covalent or non-covalent but strong bond) can be used. In one specific embodiment, the functionalizable moiety is a cysteine ​​residue which is reacted to form a disulfide bond with an unpaired cysteine ​​on the protein of interest (which may be an immunogenicity enhancer such as a carrier protein like bovine serum albumin (BSA), or a T helper cell epitope). As used herein, the term “reacts with” generally means that there is a flow of electrons or a transfer of static charge, resulting in the formation of a chemical interaction.

[0042] As used herein, the term "effective dose" refers to the amount of antigenic / immunogenic composition that, when administered to a human or animal, elicits an immune response. The effective dose is readily determined by those skilled in the art according to common procedures.

[0043] As used herein, the term “carrier protein” refers to an immunogenic protein used to enhance the immune system’s response to compounds that are not inherently immunogenic, such as another protein / peptide. Carrier proteins are particularly useful for small compounds or compounds with very low immunogenicity, where the number of potential target epitopes for the immune response is very limited. Preferred carrier proteins have a high compound-to-carrier ratio by allowing multiple compounds to be coupled to a single carrier protein in order to enhance the immunogenicity of the carrier protein-antigen complex.

[0044] The term "adjuvant" refers to a compound that, when administered in combination with an antigen (in this case, a TTR peptide), increases, stimulates, activates, enhances, or modulates the immune response to the antigen, but does not produce an immune response to the antigen when administered alone. Adjuvants can enhance the immune response through several mechanisms, including lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of macrophages. Adjuvants may be natural compounds, modified or derivative compounds of natural compounds, or synthetic compounds. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate, or aluminum hydroxide such as Alhydrogel®, often also called alum), organic adjuvants (e.g., saponins or squalene), oily adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microcrosspheries), visomes, bacterial adjuvants (e.g., monophosphoryl lipid A (MPL), or muramyl peptide), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or polynucleotide adjuvants (e.g., CpG oligodeoxynucleotide). QS-21 is a saponin extracted from Quillaya saponaria. MF59 is an oil-in-water emulsion containing squalene, polysorbate 80, and sorbitan trioleate. AS03 is an oil-in-water emulsion containing squalene, polysorbate 80, and α-tocopherol. AS01 is a combination of liposomes, MPL, and QS21. AS02 is a combination of oil-in-water emulsion, MPL, and QS21. AS04 is a complex of MPL with aluminum hydroxide or aluminum phosphate. IC31 is a combination of KLK peptide and oligodeoxynucleotide ODN1.Hiltonol (Poly-ICLC) is a synthetic complex of carboxymethylcellulose, polyinosinate-polycytidylic acid, and poly-L-lysine double-stranded RNA.

[0045] The term "immunization" typically refers to the process of activating, strengthening, or boosting an individual's immune system against a substance ("vaccine") that causes or induces a disease or disorder. Therefore, immunizing a healthy individual against such a substance can prevent the onset of disease or disorder. Immunizing a patient suffering from a disease or disorder can treat the disease or disorder or prevent its further progression. Immunization can be achieved through various techniques, but most commonly, it is achieved by vaccinating a healthy individual or a patient suffering from a disease or disorder. The underlying principle of active immunity mediated by vaccines (in this case, vaccines containing TTR peptides) is the generation of immunological "memory." In the context of ATTRs, when cellular antigens, i.e., amyloid-forming TTRs, are continuously present, the goal of active immunity is to maintain high antibody levels through regular immunization throughout life. For example, challenging an individual's immune system with a vaccine containing a disease-specific immunogen induces the formation and / or proliferation of immune cells that specifically recognize the immunogen contained in the vaccine. At least some of the immune cells mentioned above survive for a certain period, which may extend to 10, 20, or even 30 years after vaccination. If an individual's immune system encounters the immunogen again within the aforementioned period, the immune cells generated by vaccination are reactivated, enhancing the immune response to the immunogen compared to the immune response of an individual that has not been challenged by the vaccine and is encountering the immunogen for the first time. In many cases, a single dose of vaccine is not sufficient to generate the number of persistent immune cells necessary for effective protection against the disease or disorder. As a result, repeated challenges with disease-specific biological agents are required to establish persistent and protective immunity against the disease or to cure the disease.

[0046] As described in detail above, the present invention is based on the concept of identifying segments within the TTR amino acid sequence that are available for use in the immune response of living organisms but are specific to TTR fibers and amyloid. Analysis of the amino acid sequence revealed that such segments include the NI006 epitope, namely WEPFA (SEQ ID NO: 1). As shown in the examples, several different TTR peptides, including the epitope WEPFA (SEQ ID NO: 1), which is contained in loose segments and is hidden in the naturally folded conformation of the TTR protein but is a neoepitope in the sense that it becomes available for antibody binding after unfolding and aggregation, maintain an amyloid-specific conformation and are therefore suitable as immunogens. As described in more detail above, it is reasonable to expect that such peptides, when administered once to a subject, for example in the form of a vaccine, will induce an immune response specific to TTR aggregates, as observed in passive immunotherapy using NI006, resulting in the removal of TTR amyloid and thus preventing ATTR expression. Therefore, the TTR peptides described herein are useful as immunogens in vaccines.

[0047] Accordingly, the present invention relates to a vaccine comprising a TTR-derived peptide, i.e., a TTR peptide, comprising a TTR epitope, wherein the epitope is selectively presented or available only in misfolded, oligomeric, and / or aggregated TTR, and to the use of the TTR peptide as an immunogen, i.e., an immunogenic compound. In other words, the present invention relates to a TTR-derived peptide, i.e., a TTR peptide, comprising a TTR epitope, for use as a vaccine, wherein the epitope is selectively presented or available only in misfolded, oligomeric, and / or aggregated TTR.

[0048] In a particularly preferred embodiment, the TTR peptide is derived from a loose segment of TTR ranging from Lys35 to Gly57. Therefore, it is reasonable to expect that further TTR peptides, which are present in or overlap with the above loose fragments and which include epitopes exposed in misfolded variants of TTR, as well as on aggregates, fibrous bodies, and / or oligomers of TTR, are also suitable for the vaccine of the present invention. Accordingly, in one embodiment, the TTR peptide contained in the vaccine of the present invention comprises at least four amino acid residues, preferably all amino acids, of the amino acid sequence present in or overlapping with the loose TTR fragment and exposed in the misfolded variant of TTR, and on the aggregates, fibrous bodies and / or oligomers of TTR, respectively, including, for example, 54-ELXGLTXE-61 (SEQ ID NO: 13), a peptide recognized by antibody NI-301.35G11 disclosed in International Publication No. 2015 / 092077 (where X can be any amino acid); WEPFASG (SEQ ID NO: 14), a peptide recognized by antibody NI-301.12D3 disclosed in International Publication No. 2015 / 092077; and 30-VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE-66 (SEQ ID NO: 20), a peptide recognized by antibody described in International Publication No. 2014 / 124334. Furthermore, the amino acid sequence used for the TTR peptide may be modified, for example, by amino acid substitution / deletion / addition, compared to the original amino acid sequence in the loose segment, as long as the 3D conformation is not inherently affected. For example, the TTR peptide may contain an epitope comprising the amino acid sequence WXPFA (SEQ ID NO: 11) (where X can be any native amino acid), which is a peptide recognized by the antibody NI-301.28B3, for example, disclosed in International Publication No. 2015 / 092077.

[0049] Furthermore, it is reasonable to expect that misfolded variants of TTR, as well as additional TTR peptides containing epitopes exposed on TTR aggregates, fibrous bodies, and / or oligomers, can generally promote the induction of antibodies that can eliminate pathological TTR aggregates when administered to a subject, and thus be useful as immunogens for vaccines.Therefore, in one embodiment, the TTR peptide contained in the vaccine of the present invention comprises at least four amino acid residues, preferably all amino acids, of the amino acid sequence exposed in the misfolded variant of TTR and on the aggregates, fibrous bodies, and / or oligomers of TTR, for example, EEFGEGIY (SEQ ID NO: 12), which is a peptide recognized by the antibody NI-301.59F1 disclosed in International Publication No. 2015 / 092077 (where X is any amino acid). It may be an acid; for example, TTAVVTNPKE (SEQ ID NO 15), a peptide recognized by antibody NI-301.18C4 disclosed in International Publication No. 2015 / 092077; KCPLMVK and VFRK (SEQ ID NO 16 and SEQ ID NO 17), representing peptides containing conformational epitopes requiring at least C in the first sequence and V and F in the second sequence, and recognized by antibody NI-301.44E4 in International Publication No. 2015 / 092077; for example, among others, Higaki et al., Amyloid 23 (2016), EHAEVVFTA (SEQ ID NO: 18), a peptide recognized by antibody 14G8 / PRX004 / NN-6019 disclosed in 86-97; GPRRYTIAA (SEQ ID NO: 19), a peptide recognized by antibody 18C5, for example, described in International Publication No. 2019 / 071205; ALLSPYSYSTTAV (SEQ ID NO: 21), a peptide recognized by antibody that binds to TTR109-121, as described in International Publication No. 2014 / 124334; for example, International Publication No. 2 This includes WKALGISPFHE (SEQ ID NO: 22), a peptide recognized by antibody 371M as described in International Publication No. 015 / 115332; SYSTTAVVTN (SEQ ID NO: 23), a peptide recognized by antibody 313M (RT24) as described in International Publication No. 2015 / 115331; or LLSPYSYSTTAVVTNPKE (SEQ ID NO: 24), a peptide recognized by antibody that binds to TTR100-127 as described in International Publication No. 2014 / 124334.

[0050] The amino acid sequence contained in the loose segment of TTR is derived from the wild-type TTR amino acid sequence and is not specific to any variant type TTR (TTRv). Therefore, in one embodiment, the TTR peptide contained in the vaccine of the present invention contains an epitope consisting of the wild-type TTR amino acid sequence, and consequently the vaccine of the present invention is useful in preventing the occurrence of sporadic wild-type ATTR caused by misfolded wild-type TTR.

[0051] As described above, the exemplary TTR epitope WEPFA (SEQ ID NO: 1) is a neoepitope that is not only selectively presented and usable in misfolded, oligomeric, and / or aggregated TTRs, but is also absent in the tetramer of bioactive TTR and is not present on the native TTR monomer. Therefore, according to the present invention, the TTR peptide comprises an epitope that is selectively presented or usable in misfolded, oligomeric, and / or aggregated TTRs. In addition, or alternatively, the TTR peptide comprises an epitope that is absent in the tetramer of bioactive TTR (but may be present on the native and / or mutant TTR monomers), preferably such epitope is also absent on the native TTR monomer. Further exemplary epitopes not present on the natural TTR monomer are known from the applicant's prior research disclosed in International Publication No. 2015 / 092077, and include, for example, the amino acid sequence EEFZEGIY (SEQ ID NO: 12) (where X can be any amino acid), which is an epitope recognized by antibody NI-301.59F1, or the peptide ELXGLTXE (SEQ ID NO: 13) (where X can be any amino acid), which is recognized by antibody NI-301.35G11. Thus, in one embodiment, the TTR peptide contained in the vaccine of the present invention contains an epitope comprising or consisting of the amino acid sequence WEPFA (SEQ ID NO: 1), EEFZEGIY (SEQ ID NO: 12), or ELXGLTXE (SEQ ID NO: 13). Most preferably, the TTR peptide comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

[0052] As further shown in the examples, particularly Example 2, the tested anti-TTR antibodies were shown to bind to TTR39-50 in addition to TTR34-54.

[0053] Accordingly, in one embodiment of the present invention, the TTR peptide comprises or consists of 5 to 40 amino acids of the TTR protein, preferably 10 to 30 amino acids, more preferably 12 to 25 amino acids, more preferably 12 to 21 amino acids, more preferably 12 or 21 amino acids, and most preferably 21 amino acids. In one embodiment, the TTR peptide comprises or consists of at least 5 amino acid residues of the TTR protein, preferably at least 10, more preferably at least 12 amino acids, more preferably at least 15, most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues. As described above, these amino acid residues comprise epitopes presented or available in misfolded, oligomeric, and / or aggregated TTRs. More specifically, as is known to those skilled in the art, there should be at least an epitope consisting of as few as 4 amino acids, to which a sufficient and necessary number of amino acids and / or other parts, such as a linker part necessary for cyclization, may be supplemented to provide a stable peptide.

[0054] However, in principle, there is no limit to the length of a peptide as long as it is stable and immunogenic, that is, as long as it induces antibodies when administered to a target and can be selectively cyclized. Therefore, the TTR peptide contained in the vaccine of the present invention contains all four to all amino acids of the TTR protein. Preferably, the TTR peptide contains 4 to 100 amino acids, more preferably 4 to 90 amino acids, more preferably 4 to 80 amino acids, more preferably 4 to 70 amino acids, more preferably 4 to 60 amino acids, more preferably 4 to 50 amino acids, more preferably 4 to 45 amino acids, more preferably 4 to 40 amino acids, more preferably 4 to 35 amino acids, more preferably 4 to 30 amino acids, more preferably 4 to 25 amino acids, or 4 to 24 amino acids, or 4 to 23 amino acids, or 4 to 22 amino acids, or 4 to 21 amino acids, or 4 to 20 amino acids, preferably 5 to 25 amino acids, or 5 to 24 amino acids, or 5 to 23 amino acids, or 5 to 22 amino acids, or 5 to 21 amino acids, or 5 to 20 amino acids.

[0055] The amino acids represent either only the epitopes present in the TTR protein, or the epitopes and adjacent amino acids. In a preferred embodiment, the TTR peptide comprises amino acid residues of the TTR protein, where these amino acid residues include epitopes and adjacent amino acids.

[0056] Most preferably, the peptide contained in the vaccine of the present invention contains the amino acid sequence shown in SEQ ID NO: 2 (TTR34-54) or SEQ ID NO: 3 (TTR39-50).

[0057] Relatively short immunogenic peptides, i.e., TTR peptides (less than approximately 50 amino acids), are typically synthesized using standard chemical peptide synthesis techniques, such as solid-phase synthesis, in which the C-terminal amino acids of the sequence are attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence. The techniques of solid-phase synthesis are known to those skilled in the art. Alternatively, TTR peptides can also be synthesized using recombinant nucleic acid methods. Generally, this involves creating a nucleic acid sequence encoding the peptide, placing the nucleic acid in an expression cassette under the control of a specific promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide, and regenerating the peptide as needed. Sufficient techniques to guide those skilled in the art in performing such procedures can be found in the literature. Once expressed, recombinant peptides can be purified using standard procedures including ammonium sulfate precipitation, affinity columns, column chromatography, and gel electrophoresis.

[0058] In Examples 1 and 2, the cyclic peptide showed superior performance to the corresponding linear peptide in ELISA assays. In particular, the binding affinity of the TTR antibody to the cyclized TTR peptide was approximately 10 to 100 times higher than that to the corresponding linear peptide. Therefore, in one embodiment, the peptide contained in the vaccine of the present invention forms a cyclic compound. In other words, in one embodiment, the present invention relates to a vaccine containing a cyclic compound containing a peptide containing an epitope of the TTR protein, wherein the epitope is preferably available for binding only by misfolded and / or aggregated proteins, as in the case of a neoepitope, and / or the epitope is not present in the physiologically active protein, for example, even if it is an epitope available in the monomer of the TTR protein, it is hidden in the physiologically active tetramer and is no longer available for antibody binding. In particular, in one embodiment, the vaccine of the present invention contains the TTR peptide as defined above in a cyclizable form. In a preferred embodiment, the TTR peptide contains a linker that is covalently bonded to the N-terminal and C-terminal residues of the peptide to form a cyclic compound.

[0059] As described in Example 3 and shown in Figures 3 and 4, immunization with the cyclic peptides TTR34-54cyc and TTR39-50cyc, and, remarkably, with the linear peptides TTR34-54 and TTR39-50 respectively, resulted in a selective immune response, as measured by serum reactivity and amyloid selectivity, in that antibodies specific to ATTR were generated during immunization with these peptides.

[0060] Therefore, the present invention also relates to the use of TTR peptide forms as immunogens, i.e., immunogenic compounds. The vaccine of the present invention may comprise one immunogenic compound, i.e., one type of TTR peptide, or two or more types of TTR peptides, for example, two, three or four different types of TTR peptides.

[0061] The linear TTR peptides used in the examples consist of the amino acid sequence RKAADDTWEPFASGKTSESGE (TTR34-54; SEQ ID NO: 2), which has a total of 21 amino acids in the amyloid-forming protein TTR containing 5 amino acid epitopes WEPFA, and the amino acid sequence DTWEPFASGKTS (TTR394-50; SEQ ID NO: 3), which has a total of 12 amino acids in the amyloid-forming protein TTR containing 5 amino acid epitopes WEPFA. Therefore, in preferred embodiments, the linear peptide consists of a total of 5 to 30 amino acids, preferably 10 to 30, more preferably 10 to 25, even more preferably 20 ± 1, 2, 3, or 4 amino acids, or 10 ± 1, 2, 3, or 4 amino acids, but most preferably 20 ± 1, 2, 3, or 4 amino acids.

[0062] The cyclic TTR peptides used in the examples consist of two amino acid sequences: GCGGGRKAADDTWEPFASGKTSESGEGGGCG (TTR34-54cyc; SEQ ID NO: 4), which has a total of 31 amino acids, comprising 21 amino acids from the amyloid-forming protein TTR containing 5 amino acid epitopes WEPFA, and a linker sequence of 10 amino acids consisting of 5 amino acids each at the N-terminus and C-terminus of a 21-amino acid stretch from TTR; and GCGGGDTWEPFASGKTSGGGCG (TTR394-50cyc; SEQ ID NO: 5), which has a total of 22 amino acids, comprising 12 amino acids from the amyloid-forming protein TTR containing 5 amino acid epitopes WEPFA, and a linker sequence of 10 amino acids consisting of 5 amino acids each at the N-terminus and C-terminus of a 12-amino acid stretch from TTR. Therefore, in a preferred embodiment, the cyclic compound consists of a total of 15 to 40 amino acids, preferably 20 to 40, more preferably 20 to 35, even more preferably 30 ± 1, 2, 3, or 4 amino acids, or 20 ± 1, 2, 3, or 4 amino acids, but most preferably consists of 30 ± 1, 2, 3, or 4 amino acids, or, if non-amino acid residues are incorporated, for example as linkers, the structure is configured to be similar to the corresponding peptide. In this embodiment, the amino acid sequence derived from the TTR protein present in the cyclic compound may consist of 10 to 40 amino acids, preferably 10 to 25, more preferably 20 ± 1, 2, 3, or 4, or 10 ± 1, 2, 3, or 4, most preferably 20 ± 1, 2, 3, or 4, and optionally supplemented with linkers of preferably 5 to 20 amino acids in length, more preferably 5 to 15 amino acids, most preferably 10 ± 1, 2, 3, or 4 amino acids, which are distributed at both ends of the N-terminus and C-terminus, or at only one end. Furthermore, for example, if the epitope of the target-binding molecule is a conformational epitope or a discontinuous epitope, the linker sequence or "filling" sequence may be located within the amino acid sequence derived from the amyloid-forming protein.

[0063] Therefore, the cyclic compounds present in the vaccine of the present invention may consist of or comprise a TTR neoepitope or a TTR peptide containing an epitope, which means that additional amino acids or other chemical entities used for cyclization of the peptide or protein fragment may be present in the protein fragment or peptide forming the cyclic compound, as will be further explained below, for example.

[0064] The additional amino acids may be amino acids naturally located adjacent to the epitope sequence, i.e., amino acids located laterally to the epitope sequence and present in the TTR protein sequence from which the peptide originates. In other words, the TTR peptide forming a cyclic compound includes the TTR epitope, the additional amino acids adjacent to the epitope, and the additional amino acids located laterally to the epitope. The number of these adjacent / lateral amino acids may vary, for example, from 1, 2, or 3 amino acids to 50 amino acids, preferably 1, 2, or 3 amino acids to 40 amino acids, more preferably 1, 2, or 3 amino acids to 30 amino acids, more preferably 1, 2, or 3 amino acids to 20 amino acids, and more preferably 10 to 20 amino acids, where the amino acids are evenly distributed on the N-terminal and C-terminal sides of the epitope sequence, or unevenly distributed, for example, with 7 additional amino acids on the N-terminal side and 9 amino acids on the C-terminal side of the epitope.

[0065] In addition, or alternatively, the TTR peptide may, in one embodiment, include a linker, i.e., the protein fragment or peptide may include an epitope and a linker without any adjacent amino acids, or it may include the epitope and adjacent amino acids as defined above and a linker. In a preferred embodiment, the TTR peptide forming the cyclic compound contained in the vaccine of the present invention includes a neoepitope, an amino acid adjacent to the epitope, and a linker. Preferably, the linker is directly or indirectly covalently bonded to the N-terminal residue and the C-terminal residue of the TTR peptide.

[0066] In one embodiment, the linker amino acid is selected from non-immunogenic or low-immunogenic amino acid residues such as G or A, for example, the linker may be GG, GGG, GAG, G(PEG)G, PEG-PEG (also called PEG2)-GG, etc. For example, to couple the compound to an immunogenicity enhancer such as a carrier such as BSA, it may contain one or more functionally modifiable moieties, such as amino acids having a functional group.

[0067] Methods for cyclizing peptides are generally known in the art. For example, cyclization can be carried out by chemical crosslinking using a chemical skeleton. Crosslinking requires functional groups, and a small number of protein chemical targets, such as primary amine groups (-NH2) (where this group is present at the N-terminus of each polypeptide chain and in the side chains of lysine residues); carboxyl groups (-COOH) (where this group is present at the C-terminus of each polypeptide chain and in the side chains of aspartic acid and glutamic acid); and sulfhydryl groups (-SH) (where this group is present in the side chain of cysteine), account for the majority of crosslinking techniques.

[0068] Backbone-based cyclization is one of the most frequently used methods because it can be applied to chemically or biologically synthesized peptides. Generally, backbone compounds such as organic halides (most frequently organic bromides) selectively react with the sulfhydryl group of cysteine. Non-sulfhydryl groups, such as primary amines of lysine or the N-terminal amino group in peptides, can also be used in cyclization, for example, using N-hydroxysuccinimide (NHS)-containing chemicals. Specially designed non-natural amino acids can also be used in peptide cyclization via bioorthogonal reactions. For example, if azide-containing amino acids such as azidohomoalanine or azidophenylalanine are present in the peptide, cyclization can occur via a copper-mediated click reaction using an alkyne-containing backbone.

[0069] Furthermore, it is possible to link cysteine ​​molecules together via disulfide bonds (-SS-) between their side chains, or to perform amide cyclization (head-to-tail, or main chain cyclization) without using any skeleton.

[0070] For example, peptides having "C" residues at the N-terminus and C-terminus, such as the cyclic TTR compounds GCGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 4) and GCGGGDTWEPFASGKTSGGGCG (SEQ ID NO: 5) used in Examples 1 and 2, can be reacted by SS cyclization to produce cyclic peptides. These cyclic compounds can be synthesized as linear molecules having a linker covalently bonded to the N-terminus or vicinity of the TTR peptide or a peptide containing the relevant epitope as referred herein, or to the C-terminus or vicinity, prior to cyclization. Alternatively, before cyclization, part of the linker can be covalently bonded to the N-terminus or vicinity, and part to the C-terminus or vicinity. In either case, the linear compound is cyclized, for example, by SS bond cyclization. Therefore, a compound can be cyclized by 1) forming a peptide bond by covalent bonding at the N-terminus or its vicinity and the C-terminus or its vicinity of the peptide + linker (e.g., cyclizing the main chain), 2) bonding to a side chain in the peptide + linker at the N-terminus or its vicinity, or at the C-terminus or its vicinity, or 3) bonding to two side chains in the peptide + linker. In this context, "nearby" is defined as being within one, two, or three amino acid residues from the N-terminus or C-terminus. Preferably, the linker is coupled to the N-terminus or C-terminus.

[0071] As described above, peptides can be cyclized by oxidation of thiol or mercaptan group-containing residues located at or near the N-terminus, C-terminus, or within the peptide, including, for example, cysteine ​​and homocysteine. For example, two cysteine ​​residues located laterally on the peptide can be oxidized to form a disulfide bond. Possible oxidizing agents include, for example, oxygen (air), dimethyl sulfoxide, oxidized glutathione, cystine, copper(II) chloride, potassium ferricyanide, thallium(III) trifluoroacetate, or other oxidizing agents known to those skilled in the art and that can be used in methods known to those skilled in the art. Crosslinking agents are also known in the art and can be selected, for example, based on the functional group used for crosslinking (see, for example, the Crosslinker Selection Tool provided by Thermo Fisher Scientific).

[0072] Therefore, in one embodiment, the linker includes a functionally modifiable portion, for example, an amino acid having one of the above-mentioned functional groups such as lysine, aspartic acid, glutamic acid, or cysteine, a non-natural amino acid such as azidohomoalanine or azidophenylalanine, or a molecule with equivalent function such as polyethylene glycol (PEG).

[0073] When the functionalizable portion is a natural amino acid such as lysine, aspartic acid, glutamic acid, serine, threonine, or cysteine, the functionalizable portion does not necessarily have to be in the linker, but can also be in the protein fragment forming the cyclic peptide, or in an epitope or adjacent amino acid within the peptide. Therefore, cyclization of the TTR peptide can also be carried out without a linker. Thus, in one embodiment, the TTR peptide forms the cyclic compound present in the vaccine of the present invention without a linker. The bond may occur via the side chain of one or more amino acids, for example, the sulfhydryl moiety of a cysteine ​​residue, the carboxylic acid moiety of an aspartic acid or glutamic acid residue, the hydroxyl group of a serine or threonine residue, or the amine of a lysine or arginine residue.

[0074] In a preferred embodiment, at least one functionalizable moiety is present in the linker, i.e., the linker comprises one or more functionalizable moieties. The linker may contain or consist of any amino acid, including non-natural amino acids, but preferably comprises at least one of the above-mentioned functionalizable moieties, i.e., lysine, aspartic acid, glutamic acid, or non-natural amino acids such as cysteine, azidohomoalanine, or azidophenylalanine, or a molecule with equivalent function such as polyethylene glycol (PEG). In one embodiment, the linker comprises one or more PEG molecules as functionalizable moieties. In a preferred embodiment, the linker comprises cysteine ​​as a functionalizable moiety.

[0075] Therefore, in a preferred embodiment, a linker of any length and sequence can be described by the following sequence: X-nX-1FX1-Xn (where F is any functionalizable moiety, preferably C (cysteine), and X is any amino acid, including non-natural amino acids). In a further preferred embodiment, the linker amino acid is selected from alanine (A), glycine (G), or serine (S), or from alanine (A) and glycine (G), or from glycine (G) and serine (S), but is preferably glycine (G).

[0076] More preferably, the linker amino acid is selected from alanine (A), glycine (G), or serine (S), or from alanine (A) and glycine (G), or from glycine (G) and serine (S), preferably glycine (G), and the functionalizable portion is cysteine ​​(C). Therefore, preferably, cyclization is carried out using a skeletal compound such as an organic halide, preferably an organic bromide, that selectively reacts with the sulfhydryl group of cysteine, or via a disulfide bridge. Most preferably, cyclization is carried out via a disulfide bridge.

[0077] In a preferred embodiment, the linker comprises 1 to 40 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 30 amino acids, more preferably 1 to 25 amino acids, more preferably 1 to 20 amino acids, more preferably 1 to 10 amino acids, more preferably 1 to 9 amino acids, most preferably 1 to 8 amino acids, particularly 1, 2, 3, 4, 5, 6, 7, or 8 amino acids, and / or molecules having equivalent functions, and / or combinations thereof, where, if the linker comprises only amino acids, it is preferable that at least one amino acid having any of the above-described functional groups, preferably cysteine, is present among the amino acids. Other amino acids included in the linker may be selected from any known amino acids, including non-natural amino acids, but are preferably alanine (A) and / or glycine (G), and more preferably glycine (G).

[0078] As described above, the linker length can vary and may consist of, for example, 9 amino acids, e.g., GGGGCGGGG (SEQ ID NO: 27), or 8 amino acids, e.g., GGGCGGGG (SEQ ID NO: 28), GGCGGGGG (SEQ ID NO: 29), or GCGGGGGG (SEQ ID NO: 30), or 7 amino acids, e.g., GGGGCGG (SEQ ID NO: 31), GGGGCGGGG (SEQ ID NO: 32), GGCGGGG (SEQ ID NO: 33), or GCGGGGG (SEQ ID NO: 34), or 6 amino acids, e.g., GGGCGG (SEQ ID NO: 35), GGCGGG (SEQ ID NO: 36), or GCGGGG (SEQ ID NO: 37), or 5 amino acids, e.g., GCGGG (SEQ ID NO: 25), or GGGCG (SEQ ID NO: 26), or 4 amino acids, e.g., GCGG (SEQ ID NO: 38), or GGCG (SEQ ID NO: 39), or 3 amino acids, e.g., GCG. Most preferably, the linker in the cyclic compound contains or consists of GCGGG (SEQ ID NO: 25) or GGGCG (SEQ ID NO: 26).

[0079] As described above, the cyclic compound comprises a peptide containing a TTR epitope, most preferably an epitope containing the amino acid sequence WEPFA (SEQ ID NO: 1) and adjacent amino acids, and linkers at the N-terminus and C-terminus of the peptide, where the linker may, in principle, contain any of the linker sequences described above, preferably containing the amino acid sequence GCGGG (SEQ ID NO: 25) or GGGCG (SEQ ID NO: 26). Therefore, in a preferred embodiment, the cyclic compound contains or consists of the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 4) or H-GCGGGDTWEPFASGKTSGGGCG-OH (TTR39-50cyc; SEQ ID NO: 5), which were shown as preferred target antigens in Examples 1 and 2.

[0080] In some cases, the immunogenicity of peptides is limited. Techniques for conferring immunogenicity to peptides are known in the art and include, for example, conjugation to immunogenicity enhancers such as carriers or T helper cell epitopes, or administration in the presence of adjuvants.

[0081] In one embodiment, the TTR peptide contained in the vaccine of the present invention is formulated together with an immunogenicity enhancer, i.e., an active substance that helps induce an immune response to the peptide.

[0082] In one embodiment, the TTR peptide contained in the vaccine of the present invention further comprises a carrier such as BSA used in Example 2. For example, proteins such as BSA and / or other active substances may be coupled to the TTR peptide to enhance immunogenicity. Suitable carriers for this purpose are known in the art and include, but are not limited to, maltose-binding protein "MBP", bovine serum albumin (BSA), KLH (keyhole limpet hemocyanin), ovalbumin, flagellin, serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, polymers of D-amino acids and / or L-amino acids, tetanus toxoid (TT), diphtheria toxoid (DT), CRM197, a genetically modified cross-reactive substance (CRM) of diphtheria toxin, meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), and Pseudomonas aeruginosa exotoxin A. Various methods for chemically crosslinking peptides to carrier proteins are known in the art, typically involving reactive sulfhydryl and / or amino groups. Many of these systems are commercially available (e.g., ThermoFisher Scientific's Imject®). The most common chemical crosslinking is performed using sulfhydryl groups in the antigen, which can be done by adding cysteine ​​residues to the antigenic peptide, e.g., C-(GA)10, or via primary amino groups (e.g., ThermoFisher Scientific's Imject® Maleimide and Imject® EDC products). Furthermore, methods for conjugating peptides to immunogenicity enhancers such as KLH or carriers such as BSA are described in Lateef et al., Journal of Biomolecular Techniques 18 (2007), 173-176, which are incorporated herein by reference.

[0083] In one embodiment, the carrier coupled to the TTR peptide is BSA or KLH, i.e., the TTR peptide comprises a carrier such as BSA or KLH and an immunogenicity enhancer. In a preferred embodiment, the carrier coupled to the TTR peptide is BSA, i.e., the TTR peptide comprises a carrier such as BSA and an immunogenicity enhancer. The immunogenicity enhancer / carrier can be coupled to the peptide directly by amide bonds or disulfide bonds, or indirectly via a chemical linker. In particular, BSA is covalently bonded using a free amine reaction that links the free amine on the peptide to the free amine on the BSA protein.

[0084] Furthermore, the use of KLH is known to potentially lead to the production of large amounts of antibodies directed against KLH, which is an undesirable side effect and should therefore be avoided. Accordingly, in one embodiment, the vaccine does not contain KLH as an immunogenicity enhancer.

[0085] In one embodiment, the immunogen containing the TTR peptide and the TTR peptide each contain a T helper cell epitope. More specifically, the TTR peptide can be linked with a heterologous T helper cell epitope peptide to form a peptide immunogen construct. Optionally, linking can be carried out via a heterologous spacer. As used herein, the term “heterologous” refers to an amino acid sequence derived from an amino acid sequence that is not part of or homologous to the wild-type sequence of TTR. A heterologous spacer can be any molecule or chemical structure capable of linking two amino acids and / or peptides together, and may include compounds, native amino acids, non-native amino acids, or any combination thereof. A heterologous T helper cell epitope can be any T helper cell epitope capable of enhancing the immune response to the TTR epitope. The T helper cell epitope may also contain promiscuous binding motifs to multiple MHC class II molecules, or may contain multiple promiscuous MHC class II binding motifs to enable maximal activation of T helper cells leading to the initiation and regulation of the immune response. T helper cell epitopes are preferably immunologically silent in themselves; that is, antibodies generated by TTR peptide immunogen constructs, if present, are unlikely to be directed towards the T helper cell epitopes, thereby enabling a highly concentrated immune response directed towards targeted TTR epitope peptides. T helper cell epitopes may contain amino acid sequences derived from exogenous pathogens. In the art, several universal T helper cell epitopes available herein are known, such as tetanus toxin and diphtheria toxin-derived universal T helper cell epitopes like P30 (Deithelm-Okita et al., The Journal of Infectious Diseases 181 (2000), 1001-1009; Swartz et al., npj Vaccines 6 (2021), 12).

[0086] Therefore, in one embodiment, the peptide is formulated with a carrier molecule, preferably a carrier protein such as BSA, or an immunogenicity enhancer linked to a T helper cell epitope, to form a conjugate that helps induce an immune response to the peptide, and preferably the antibody thereby induced specifically binds to amyloid-forming TTRs, i.e., TTR aggregates, in the target and removes them, thereby inhibiting the formation of TTR deposits and resulting in the treatment or prevention of the disease.

[0087] Alternatively, the immunogen may be a multi-antigenic peptide (MAP) containing a TTR peptide. To avoid the adverse effects associated with conventional vaccines (i.e., attenuated pathogens, dead pathogens, or inactivated pathogens), carrier proteins, and cytotoxic adjuvants, multi-antigenic peptide vaccine systems have been developed. Two main approaches have been used in the development of multi-antigenic peptide vaccine systems: (1) the addition of functional components, e.g., T cell epitopes, cell-permeable peptides, and lipophilic moieties; and (2) a synthetic approach using size-defined nanomaterials, e.g., self-assembling peptides, non-peptide dendrimers, and gold nanoparticles as antigen-presenting platforms. The use of multi-antigenic peptide (MAP) systems can improve upon the problem of sometimes low immunogenicity of subunit peptide vaccines. In the MAP system, multiple copies of an antigenic peptide are simultaneously bound to the α-amino and ε-amino groups of a non-immunogenic Lys-based dendrimer skeleton, thereby conferring stability against degradation, and thus enhancing molecular recognition by immune cells, inducing a more potent immune response compared to a small antigenic peptide alone. In some compositions, MAP comprises one or more of the following: a Lys-based dendrimer skeleton, a helper T cell epitope, an immunostimulatory lipophilic moiety, a cell-permeable peptide, radical-induced polymerization, self-assembling nanoparticles as an antigen-presenting platform, and gold nanoparticles. Thus, in one embodiment, the TTR peptide contained in the vaccine of the present invention is prepared as a MAP.

[0088] This invention does not include a MAP in which eight copies of the sequence GGEHAEVVFTAGGGK are synthesized on a MAP dendrimer core ([fluorenylmethyloxycarbonyl(Fmoc)Fmoc-Lys(Fmoc)]4-Lys2-Lys-bAla) bonded to a Wang resin. This MAP is described in Higaki et al., Amyloid, 23:2 (2016), 86-97, and is also shown in Bugyei-Twum 2012 (Antoinette Bugyei-Twum, "Inhibition of Transthyretin Fibrillogenesis Using a Conformation Specific Antibody," a dissertation submitted as a requirement for obtaining a Master of Science degree at the University of Toronto's School of Chemistry).

[0089] In one embodiment, the vaccine of the present invention comprises an adjuvant in addition to the TTR peptide. Most vaccines are injected with an adjuvant to stimulate an immune response. The incorporation of adjuvants into vaccine formulations is intended to enhance, accelerate, and prolong a specific immune response toward a desired response to the vaccine antigen. The benefits of adjuvants include enhancing the immunogenicity of the antigen, modifying the nature of the immune response, reducing the amount of antigen required for successful immunization, reducing the frequency of required booster immunizations, and improving the immune response in elderly and immunocompromised patients. Adjuvants that can be used to carry out the present invention generally include any adjuvant known in the art that boosts the immune response to the vaccine and / or provides more sustained immunity without giving rise to an immune response by itself. Preferred adjuvants allow for a reduction in the immunogenic composition applied to obtain a sufficient immune response. However, the properties of adjuvants can vary widely. For example, conformationally designed epitopes may require adjuvants that do not denature or emulsify the antigen. Therefore, in one embodiment, the vaccine of the present invention comprises an adjuvant, preferably an adjuvant that does not affect the structure of the peptide, such as the cyclization structure.

[0090] Suitable adjuvants may be selected and evaluated by those skilled in the art, for example, based on the European Medicines Agency's "Guideline on adjuvants in vaccine for human use" (January 20, 2005, last updated February 16, 2023 (EMEA / CHMP / VEG / 134716 / 2004)). The adjuvant may be administered as a single composition with the immunogen. Alternatively, the adjuvant may be administered before, concurrently with, and / or after administration of the immunogen. Exemplary adjuvants are listed above and below: The group of usable adjuvants includes different types, namely (i) delivery systems, e.g., mineral salts, e.g., aluminum salts (aluminum hydroxide, aluminum sulfate, and aluminum phosphate), emulsions, e.g., Freund's adjuvant, MF59, or AS03, and particulate matter, e.g., virus-like particles. (ii) Virosom, PLA / PLGA, (ii) Immunostimulants, e.g., TLR1 / 2 agonists, e.g., L-pampo, MALP-2, Pam2CSK4 and Pam3CSK4, TLR3 agonists, e.g., poly(I:C)(polyinosinic acid:polycytidylic acid), poly-ICLC, TLR4 agonists, e.g., monophosphoryl lipid A (MPL), TLR5 agonists (iii) complex adjuvants, such as flagellin, TLR7 / 8 agonists, such as imiquimod (R837; 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine) and reximod (R848, 4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol), and TLR9 agonists, such as CpG ODN; (iii) complex adjuvants, such as AS01 and AS02, AS04, and (iv) mucosal adjuvants, such as cholera toxin (CT), heat-unstable enterotoxins (LTK3 and LTR72), and chitosan (see Facciola et al., Vaccines 10 (2022), 819 for a review).Some adjuvants that are preferred include incomplete Freund's adjuvant (IFA) (Montanide ISA-51) or CpG oligodeoxynucleotide (ODN).

[0091] According to the present invention, the immunogen for the vaccine of the present invention can be chemically synthesized or produced using recombinant DNA technology, wherein the TTR peptide disclosed herein may or may not be coupled to an immunogenic carrier. Corresponding means and methods are known in the art; see, for example, Hou et al., Trans. Tianjin Univ. 23 (2017), 401-41, https: / / doi.org / 10.1007 / s12209-017-0068-8, Molecular Biotechnology: Principles and Applications of Recombinant DNA, 6th Edition, Eds. Glick and Patten; ISBN: 978-1-683-67366-8 February 2022, and Textbook on Cloning, Expression and Purification of Recombinant Proteins, 2022 Ed. Kakoli Bose; ISBN: 978-981-16-4986-8.

[0092] Accordingly, the present invention also relates to nucleic acids encoding immunogens as described herein, preferably in combination with immunogenicity enhancers such as immunogenic carriers or adjuvants as described above.

[0093] Typically, the nucleic acids of the present invention are in an expressible form, that is, when applied to cells, the nucleic acids express an immunogen or a portion thereof. In one embodiment, this can be achieved, for example, by using a translatable nucleic acid such as mRNA for use in in vitro translation, or by incorporating the nucleic acid into an expression vector capable of expressing the claimed nucleic acid, and thus this also constitutes part of the present invention.

[0094] In further embodiments, the present invention relates to host cells comprising the nucleic acid or expression vector of the present invention, and to the use of said nucleic acid and expression vector for producing immunogens within the cell. The cell may be, for example, a bacterial, yeast, or mammalian cell.

[0095] In one embodiment, the immunogen may be purified from a cell culture and formulated as a vaccine, which may optionally include the step of coupling the immunogen, such as a TTR peptide, with an immunogenicity enhancer, such as an immunogenicity carrier and / or adjuvant. Thus, a method of recombinantly producing the immunogen by expressing it in a host cell and isolating it therefrom is a further embodiment of the present invention. Any suitable host cell, for example, a prokaryote or a eukaryote, may be used, and in certain embodiments, the host cell is selected from the group consisting of bacterial, fungal (including yeast), and mammalian cells.

[0096] The present invention also relates to a kit and composition comprising a nucleic acid encoding the immunogen of the present invention (whether or not it includes an immunogenicity enhancer such as an immunogenicity carrier), an expression vector comprising the nucleic acid, a host cell comprising the nucleic acid or the expression vector, and / or the immunogen of the present invention, and optionally (where applicable) at least one immunogenicity enhancer such as an immunogenicity carrier, and / or an adjuvant.

[0097] The present invention further relates to a kit comprising the vaccine of the present invention, the kit optionally comprising means for administering the vaccine and / or instructions, such as a recommended dose and / or a syringe for administering the vaccine. In a preferred embodiment, the kit comprises the vaccine of the present invention in a pharmaceutical container such as a pre-filled vial or syringe.

[0098] Furthermore, a method for preparing a vaccine composition for inducing an immune response in organisms, particularly animals or humans suffering from ATTR, or healthy organisms at risk of developing ATTR and therefore requiring treatment to prevent, treat, or mitigate the effects of ATTR, is disclosed, the method comprising formulating the TTR peptide according to the present invention in a pharmaceutically acceptable form. Pharmaceutically acceptable carriers for vaccine formulation are known in the art.

[0099] As described above, the vaccine of the present invention can be used to prevent ATTR in subjects by inducing an immune response, for example, by inducing anti-TRR antibodies that can bind to and remove amyloid-forming TTR, i.e., TTR aggregates. Anti-TTR antibodies have been shown to be useful in the treatment of ATTR, such as ATTR-CM (see Non-Patent Literature 6 cited above). The results described in Non-Patent Literature 6, as shown in Figure 2 of Non-Patent Literature 6, further indicate the usefulness of anti-TTR antibodies in the treatment of musculoskeletal disorders, as TTR deposits in the shoulder joint are removed during treatment with antibody NI006. Therefore, the vaccine of the present invention may also be useful in the treatment of ATTR in subjects, particularly in the early stages of ATTR. Accordingly, the present invention also relates to vaccines and kits of the present invention for use in the prevention or treatment of ATTR in subjects, such as ATTR cardiomyopathy (ATTR-CM), ATTR polyneuropathy (ATTR-PN), particularly ATTR-CM, and musculoskeletal disorders or conditions, wherein the latter musculoskeletal disorders or conditions are preferably related to TTR deposition in joints, most preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, arthralgia, shoulder pain, amyloid arthropathy, lumbar spinal stenosis, biceps tendon rupture, trigger finger, and rotator cuff disorders, wherein the musculoskeletal disorder or condition is osteoarthritis or amyloid arthropathy, preferably amyloid arthropathy.

[0100] The progression of immunization with the vaccine of the present invention can be monitored by detecting antibody titers in plasma or serum. Antibody levels can be evaluated using standard ELISA or other immunoassay procedures using the immunogen as the antigen. After immunization, antiserum can be obtained, and polyclonal antibodies can be isolated from the serum if desired (see also Example 3).

[0101] ATTR includes two subtypes: wild-type ATTR (ATTRwt) and variant ATTR (ATTRv). Cardiac TTR deposits attributable to ATTRwt are found in 10% to 15% of individuals aged 65 years or older. An epitope containing the amino acid sequence WEPFA (SEQ ID NO: 1) can be found in TTRwt, and therefore, in one embodiment, the vaccine of the present invention is particularly suitable for the treatment or prevention of subjects with sporadic wild-type ATTR, preferably wild-type ATTR-CM. The vaccine of the present invention is also particularly suitable for the treatment or prevention of subjects who are negative for TTR mutations in genetic testing.

[0102] The present invention further relates to a method for treating or preventing ATTR, e.g., ATTR-PN or ATTR-CM, particularly ATTR-CM, and the aforementioned musculoskeletal disorders, preferably those associated with the accumulation of wild-type TTR, in a subject, comprising administering the vaccine of the present invention in an effective dose. Administration can be carried out by commonly known techniques, e.g., via intravenous, intramuscular, intradermal, or subcutaneous administration routes.

[0103] The vaccine of the present invention may be administered to patient populations at risk of developing ATTR, such as elderly populations, or to known populations of individuals “at risk” who carry known ATTR-promoting mutations. More specifically, suitable subjects for treatment include patients who are currently showing symptoms, as well as individuals who are at risk of the disease but are asymptomatic (including treatment-naïve subjects who have never been treated for the disease in the past). Subjects at risk of the disease include individuals belonging to the elderly population and asymptomatic subjects with known genetic disease risk. Such individuals include those with relatives who have experienced the disease and those whose risk has been determined by genetic analysis or analysis of biochemical markers.

[0104] In prophylactic applications, the vaccine of the present invention can be administered to subjects susceptible to ATTR or at risk of ATTR for other reasons in a regime (dosage, frequency of administration, and route of administration) that is effective in reducing the risk, mitigating the severity, or delaying the onset of at least one sign or symptom of the disease. In particular, such regimes are effective in inhibiting or delaying the formation of amyloid-forming TTRs.

[0105] In therapeutic applications, the vaccine of the present invention can be administered to subjects suspected of having ATTR or patients already suffering from ATTR in a regimen (dosage, frequency of administration, and route of administration) that is effective in improving at least one sign or symptom of the disease, or at least inhibiting further exacerbation. In particular, it is preferable that the regimen is effective in reducing the level of amyloid-forming TTR or at least inhibiting further increase.

[0106] A regimen is considered therapeutically or prophylactically effective if the treated individual achieves a better outcome than the average outcome in a control population of equivalent subjects not treated by the method of the present invention, or if, in a controlled clinical trial, the treated subject showed a better outcome than the control subject. The effective dose varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether other drugs are being administered, and whether the treatment is prophylactic or therapeutic.

[0107] Throughout the text of this specification, several documents are cited. The contents of all cited documents (including references cited throughout this application, including the background art section, issued patents, published patent applications, and manufacturer specifications, instructions, etc.) are expressly part of this specification by reference. However, none of the cited documents are considered to be prior art relating to the present invention.

[0108] A more complete understanding can be obtained by referring to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0109] Example 1: TTR peptide presents a neoepitope of TTR amyloid. To test the immunogenicity potential of the TTR peptide, i.e., its ability to induce protective antibodies (which requires the binding of the peptide to the antibody), we first conducted corresponding binding studies.

[0110] In particular, the ability of anti-TTR antibodies to bind to TTR peptides was exemplary evaluated by an ELISA assay using antibody NI-301.37F1 as the anti-TTR antibody, with cyclic peptides containing amino acid residues 34-54 of wild-type TTR (biotinized and non-biotinized forms of TTR34-54cyc) and the corresponding biotinized linear peptide (TTR34-54) as target antigens. Furthermore, in addition to TTR peptides TTR40-49, misfolded wild-type TTR (mis.WT-TTR) was used as an antigen control.

[0111] The cyclic peptide TTR34-54cyc (1.36 mg / mL) was manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. In particular, the peptide containing the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 4) was synthesized by solid-phase peptide synthesis and cyclized via a disulfide bridge between two cysteine ​​residues in the polyglycine stretch. The TTR peptides containing the amino acid sequences H-RKAADDTWEPFASGKTSESGE-OH (SEQ ID NO: 2, TTR34-54) and H-TWEPFASGKT-OH (SEQ ID NO: 6, TTR40-49, 1.25 mg / mL) were also manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. The biotinylated peptides TTR34-54cyc_bt and TTR34-54_bt contain an aminohexanoic acid (Ahx) spacer between their N-terminus and the biotin residue, namely TTR34-54cyc_bt(biotin-(Ahx)GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 4), 680 μg / mL) and TTR34-54_bt(biotin-(Ahx)RKAADDTWEPFASGKTSESGE-OH (SEQ ID NO: 2)).

[0112] Wild-type TTR protein purified from human plasma was obtained from Bio-Rad Laboratories, Inc. (California, USA; 7600-0604) and subjected to custom purification by protein A / G chromatography followed by lectin column chromatography to remove residual immunoglobulins. Plasma-purified WT-TTR was provided as a solution at a concentration of 1 mg / ml in PBS buffer. Misfolded WT-TTR aggregates (mis.WT-TTR) were prepared in vitro by diluting the WT-TTR stock solution to a concentration of 200 μg / ml in agglutination buffer (50 mM acetic acid-HCl, 100 mM KCl, 1 mM EDTA, pH 3.0) and then incubating at 37°C for 4 hours with shaking at 1000 rpm. The mis.WT-TTR was aliquoted and stored at -20°C until use. The quality of the mis.WT-TTR was confirmed by ELISA and biolayer interferometry (BLI).

[0113] Three ELISA assays were performed. In the first ELISA assay (ELISA-1), antibody binding to the peptides TTR34-54cyc and mis-WT-TTR was analyzed. In the second ELISA assay (ELISA-2), antibody binding to the peptides TTR34-54_bt and TTR34-54cyc_bt, as well as BSA control, was analyzed. In the third ELISA assay (ELISA-3), antibody binding to the peptides TTR34-54cyc and TTR40-49 was analyzed.

[0114] Specifically, 96-well microplates were coated at 37°C for 1 hour with TTR34-54cyc and mis-WT-TTR (ELISA-1), TTR34-54_bt, TTR34-54cyc_bt and BSA (ELISA-2), and TTR34-54cyc and TTR40-49 (ELISA-3), respectively. Here, each target antigen was diluted to a concentration of 10 μg / ml in PBS buffer (pH 7.4). Nonspecific binding sites were blocked at room temperature (RT) for 1 hour using a blocking buffer containing 2% (w / v) bovine serum albumin (BSA) and 0.1% polysorbate 20 in 1× PBS buffer (pH 7.4). NI-301.37F1 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted in blocking buffer to the indicated concentration (dilution series from 400 nM to 4 pM and 0) and incubated overnight at 4°C. Binding was determined using anti-human IgG antibody conjugated with horseradish peroxidase (HRP), followed by measurement of HRP activity using a standard colorimetric assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA). Data were analyzed using GraphPad's Prism software. EC 50 The values ​​were estimated by nonlinear regression of individual data points using a log(agonist) versus response model (with variable slope). Data fitting was performed using the least squares regression method.

[0115] ELISA results showed that the antibody NI-301.37F1 binds to the TTR peptide. The ELISA-1 assay showed that the binding of NI-301.37F1 to the cyclic TTR34-54cyc peptide was much stronger than the binding to mis.WT-TTR, i.e., approximately 10 times stronger. In particular, in ELISA-1, the binding of NI-301.37F1 to the cyclic TTR34-54cyc peptide was significant. 50 It is 0.022 nM, and NI-301.37F1 coupled EC to mis.WT-TTR 50The concentration was 0.19 nM (see Figure 1A). In the ELISA-2 assay, both linear and cyclic TTR peptides were conjugated by NI-301.37F1, but cyclization was further shown to promote NI-301.37F1 binding by more than 100-fold. In particular, in the ELISA-2 assay, NI-301.37F1-conjugated EC1 to cyclic TTR34-54cyc_bt peptide was observed. 50 It is 0.11 nM, and NI-301.37F1 coupled EC to linear TTR34-54_bt 50 The concentration was 19.5 nM (see Figure 1B). In the control ELISA assay, i.e., ELISA-3, no binding of NI-301.37F1 to TTR40-49 was observed (see Figure 1C).

[0116] Example 2: BSA-coupled TTR peptide harbors a neoepitope of TTR amyloid-specific antibody. In typical peptide vaccination protocols, a peptide containing the target epitope is conjugated to a carrier protein to boost the immune response by increasing the epitope's half-life, for example, by reducing renal clearance and sensitivity to proteolysis. Accordingly, further ELISA assays were performed to evaluate the ability of anti-TTR antibodies (exemplarily tested here using antibody NI-301.37F1) to bind to BSA-coupled TTR peptides.

[0117] In particular, the ability of anti-TTR antibodies to bind to BSA-coupled TTR peptides was exemplary evaluated by an ELISA assay using antibody NI-301.37F1 as the anti-TTR antibody, with cyclic peptides containing amino acid residues 34-54 of wild-type TTR coupled to BSA (TTR34-54cyc_BSA), cyclic peptides containing amino acid residues 39-50 of wild-type TTR coupled to BSA (TTR39-50cyc_BSA), and corresponding linear peptides coupled to BSA (TTR34-54_BSA and TTR39-50_BSA) as target antigens. Furthermore, scrambled cyclic and linear peptides coupled to BSA (TTR34-54SCRcyc_BSA, TTR39-50SCRcyc_BSA, TTR34-54SCR_BSA, and TTR39-50SCR_BSA) were used as antigen controls.

[0118] The cyclic peptides coupled to BSA, namely TTR34-54cyc_BSA, TTR39-50cyc_BSA, TTR34-54SCRcyc_BSA, and TTR39-50SCRcyc_BSA, were manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. In particular, peptides containing the amino acid sequences H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 4, TTR34-54cyc_BSA), H-GCGGGDTWEPFASGKTSGGGCG-OH (SEQ ID NO: 5, TTR39-50cyc_BSA), H-GCGGGERDDPFKTAWATASGKESESGGGGCG-OH (SEQ ID NO: 9, TTR34-54SCRcyc_BSA), and H-GCGGGEWSDTPTFKGSAGGGCG-OH (SEQ ID NO: 10, TTR39-50SCRcyc_BSA) were synthesized by solid-phase peptide synthesis and cyclized via disulfide crosslinking between two cysteine ​​residues within a polyglycine stretch. Furthermore, BSA was coupled to the cyclic peptides using a divalent free amine coupling reagent. The TTR peptides containing the amino acid sequences H-RKAADDTWEPFASGKTSESGE-OH (SEQ ID NO: 2, TTR34-54_BSA), H-DTWEPFASGKTS-OH (SEQ ID NO: 3, TTR39-50_BSA), H-ERDDPFKTAWATASGKESESG-OH (SEQ ID NO: 7, TTR34-54SCR_BSA), and H-EWSDTPTFKGSA-OH (SEQ ID NO: 8, TTR39-50SCR_BSA) were also manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C.

[0119] Misfolded WT-TTR aggregates were prepared as described in Example 1.

[0120] Two ELISA assays were performed. In the first ELISA assay (ELISA-1), antibody binding to peptide TTR34-54cyc_BSA, TTR34-54_BSA, TTR34-54SCRcyc_BSA, and TTR34-54SCR_BSA was analyzed. In the second ELISA assay (ELISA-2), antibody binding to peptide TTR39-50cyc_BSA, TTR39-50_BSA, TTR39-50SCRcyc_BSA, and TTR39-50SCR_BSA was analyzed. The ELISA assays were performed as described in Example 1.

[0121] The results of the ELISA showed that antibody NI-301.37F1 binds to the BSA-coupled TTR peptides. The ELISA-1 assay showed high binding affinity of antibody NI-301.37F1 to the cyclic BSA-coupled TTR34-54cyc_BSA peptide (EC 50 value: 3.8 nM), and also showed binding of the above antibody to the corresponding BSA-coupled linear peptide (TTR34-54_BSA) (EC 50 value: > 400 nM). Cyclization promoted NI-301.37F1 binding by more than 100-fold. Binding to the antigen control, i.e., the scrambled peptide, was not observed (see Figure 2A). The ELISA-2 assay showed binding to the cyclic BSA-coupled TTR39-50cyc_BSA peptide (EC 50 value: > 400 nM), but binding to the corresponding linear peptide was hardly detectable. Binding to the antigen control, i.e., the scrambled peptide, was not observed. Thus, TTR39-50 is the approximate minimum peptide length for NI-301.37F1 binding (see Figure 2B).

[0122] Example 3: Immunogenic Potential of TTR Peptides in Mammals The immunogenicity potential of TTR peptides, specifically the ability of immunization with TTR peptides to induce a highly selective immune response against amyloid-forming TTRs, was investigated in in vivo mouse studies. In particular, each group of experimental animals was immunized with a target peptide coupled to the carrier protein BSA (coupling to BSA was performed using an amine-reactive crosslinker). Here, the peptide adopted a conformation similar to that present in TTR amyloid fibrils. After repeated injection of the immunogenic peptide, the immune response was monitored by serum titration to characterize the ability to specifically bind to target proteins adopting amyloid conformations.

[0123] immunization Five groups of BalbC mice (30 mice in total), consisting of six mice each, were injected with BSA-coupled TTR peptides in linear or cyclic conformations. To induce a specific immune response to ATTR, peptides of 12-amino acid and 21-amino acid lengths in cyclic and linear conformations were used (Groups 1-4). To characterize response selectivity, an unrelated peptide, referred to herein as peptide PR906, was used as a control.

[0124] The peptide was administered subcutaneously (sc) to mice three times consecutively at approximately two-week intervals. For the first and second injections, the antigen was mixed with RIBI adjuvant. Pre-collected injection solutions were administered without adjuvant. Blood samples were collected before the first injection (day 0) and 38 days later, i.e., after the third injection. Antigen-specific titers for the peptide antigen and ATTR were determined by ELISA.

[0125] List of antigens and immune groups Group 1 TTR34-54_BSA Group 2 TTR34-54cyc_BSA 3rd group TTR39-50_BSA Group 4 TTR39-50cyc_BSA 5th group PR906

[0126] Characterization of the immune response Immune response monitoring (serum titer monitoring) and immune response characterization were performed by ELISA against WT-TTR and mis.WT-TTR (also called ATTR) as follows: ELISA plates were coated with WT-TTR and mis.WT-TTR diluted to 10 μg / mL in PBS at 37°C for 1 hour. The plates were then blocked at room temperature for 1 hour using BSA-free blocking buffer. Serum samples were diluted in PBS in a dilution series from 1:100 to 1:590-4900, transferred to their respective ELISA plates, and incubated at room temperature for 1 hour. After washing, immunogenically bound antibodies were detected with a 1:20000 dilution of HRP-coupled anti-mouse IgG secondary antibody.

[0127] Immune response monitoring was performed on day 0 and day 38. Afterward, the mice were euthanized, and final blood samples were collected. Each serum sample was tested individually to determine the immune response rate for each immunogen.

[0128] As shown in Figure 3, all administered TTR peptides induced the formation of antibodies against ATTR and TTR, and the highest OD was observed in serum samples collected from mice after a 38-day incubation period. 450 Values ​​were detected. Furthermore, it is clear that the serum of all mice contained significantly higher levels of ATTR-specific antibodies than TTR-specific antibodies. Therefore, after immunization of BalbC mice with each TTR peptide, serum antibody titers increased over time (comparing day 0 to day 38), and serum reactivity was higher against mis.WT-TTR / ATTR than against WT-TTR. In particular, serum obtained from mice after immunization with peptide / immunogen TTR39-50 showed a serum reactivity of 1038 (EC) against TTR. 50 ), 15271 serologically reactive (EC) to ATTR 50 ) shows, and therefore amyloid selectivity (EC) of 14.7 50(ATTR / TTR)The index was shown (see Figure 3A and Table 1). Serum obtained from mice after immunization with peptide / immunogen TTR39-50cyc showed a serum reactivity of 1813 (EC) to TTR. 50 ), 24343 serum reactivity to ATTR (EC 50 ) shows, and therefore amyloid selectivity (EC) of 15.4 50(ATTR / TTR) The index was shown (see Figure 3B and Table 1). Serum obtained from mice after immunization with peptide / immunogen TTR34-54 showed a serum reactivity (EC) of 3400 to TTR. 50 ), 53809 serological reactivity to ATTR (EC 50 ) shows, and therefore amyloid selectivity (EC) of 15.8 50(ATTR / TTR) The index was shown (see Figure 3C and Table 1). Serum obtained from mice after immunization with peptide / immunogen TTR34-54cyc showed a serum reactivity (EC) of 7785 to TTR. 50 ), 33587 serological reactivity to ATTR (EC 50 ) demonstrates, and therefore the amyloid selectivity (EC) of 4.3 50(ATTR / TTR) The indices are shown (see Figure 3D and Table 1).

[0129] [Table 1]

[0130] This was also confirmed by Figure 4, which visualizes the results of an ELISA assay using a 1:23300 serum dilution. In particular, the serum reactivity of serum obtained from BalbC mice after immunization with TTR peptides TTR39-50, TTR39-50cyc, TTR34-54, and TTR34-54cyc was (each OD 450 The titers were shown to be significantly higher against ATTR than against TTR (as indicated by the values). Furthermore, the highest antibody titers were detected in serum obtained from mice immunized with the linear peptide TTR34-54.

[0131] In summary, immunization of mice with the TTR peptides described above, which included a carrier protein (in this case, BSA), induced an immune response in both linear and cyclic TTR peptides, leading to the production of anti-TTR antibodies that exhibited high selectivity for amyloid-forming TTR.

Claims

1. A vaccine comprising an immunogen containing a peptide derived from transthyretin (TTR), wherein the peptide comprises a neoepitope that is selectively presented or available in misfolded, oligomeric, and / or aggregated TTR.

2. The vaccine according to claim 1, wherein the TTR peptide is formulated together with an immunogenicity enhancer.

3. The vaccine according to claim 1 or 2, wherein the TTR peptide and the neoepitope each consist of the wild-type (wt) amino acid sequence of TTR.

4. The vaccine according to any one of claims 1 to 3, wherein the TTR peptide comprises at least four amino acid residues of the TTR amino acid sequence from Lys35 to Gly57.

5. The vaccine according to any one of claims 1 to 4, wherein the TTR peptide comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

6. The vaccine according to any one of claims 1 to 5, wherein the TTR peptide comprises or consists of at least 5, preferably at least 10, more preferably at least 15, most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues of the TTR protein.

7. The vaccine according to any one of claims 1 to 6, wherein the TTR peptide has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

8. The vaccine according to any one of claims 1 to 7, wherein the TTR peptide includes a linker that is covalently bonded to the N-terminal and C-terminal residues of the peptide to form a cyclic compound.

9. The vaccine according to claim 8, wherein the linker comprises or consists of one to eight amino acids and / or one or more functionalizable moieties, preferably the amino acids of the linker are selected from glycine (G) or alanine (A), and / or the functionalizable moiety is cysteine ​​(C), most preferably the linker comprises or consists of GCGGG (SEQ ID NO: 25) or GGGCG (SEQ ID NO: 26).

10. The vaccine according to any one of claims 1 to 9, wherein the TTR peptide has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:

5.

11. The vaccine according to any one of claims 1 to 10, wherein the immunogenicity enhancer is a carrier protein, and the TTR peptide is coupled to the carrier protein.

12. The vaccine according to claim 11, wherein the carrier protein is bovine serum albumin (BSA).

13. The vaccine according to any one of claims 1 to 11, wherein the immunogenicity enhancer is a heterologous T helper cell epitope, and the epitope is preferably linked to the peptide via a heterologous spacer.

14. A nucleic acid encoding the immunogen of a vaccine according to any one of claims 1 to 13.

15. An expression vector capable of expressing the nucleic acid described in claim 14.

16. A host cell comprising the nucleic acid described in claim 14 or the expression vector described in claim 15.

17. A nucleic acid according to claim 14 or an expression vector according to claim 15 for producing the immunogen within a cell.

18. A composition comprising the vaccine and adjuvant according to any one of claims 1 to 13.

19. A kit or composition comprising the nucleic acid described in claim 14 or the expression vector described in claim 15, and optionally an immunogenicity enhancer and / or at least one adjuvant.

20. A kit comprising a vaccine according to any one of claims 1 to 13, or a composition according to claim 18 or 19, and optionally means and / or instructions for administering the vaccine.

21. The kit according to claim 19 or 20, wherein the vaccine or composition is present in a pre-filled vial or syringe.

22. A vaccine according to any one of claims 1 to 13, a kit according to claim 20 or 21, or a composition according to claim 18 or 19, for use in a method for treating or preventing TTR amyloidosis (ATTR) in a subject.

23. A vaccine according to any one of claims 1 to 13 or 22, a kit according to claim 20 or 21, or a composition according to claim 18 or 19, for use in a method for treating or preventing musculoskeletal disorders or conditions in a subject.

24. The vaccine, kit, or composition for use according to claim 22 or 23, wherein the subject has sporadic wild-type transthyretin-mediated amyloidosis associated with cardiomyopathy (ATTRwt-CM), and / or the subject is negative in a genetic test for TTR mutations.

25. A method for treating or preventing TTR amyloidosis (ATTR), the method comprising administering a vaccine according to any one of claims 1 to 13, or a composition according to claim 18 or 19, to a subject in need of treatment or prevention of TTR amyloidosis (ATTR).

26. A method for treating or preventing a musculoskeletal disorder or condition, comprising administering a vaccine according to any one of claims 1 to 13, or a composition according to claim 18 or 19, to a subject in need of treatment or prevention of a musculoskeletal disorder or condition.

27. The method according to claim 25 or 26, wherein the subject has sporadic wild-type transthyretin-mediated amyloidosis associated with cardiomyopathy (ATTRwt-CM), and / or the subject is negative in a genetic test for TTR mutations.

28. Use of the vaccine according to any one of claims 1 to 13, or the composition according to claim 18 or 19, for the manufacture of a drug for the treatment or prevention of TTR amyloidosis (ATTR) in a subject.

29. Use of the vaccine according to any one of claims 1 to 13, or the composition according to claim 18 or 19, for the manufacture of a drug for the treatment or prevention of musculoskeletal disorders or conditions in a subject.

30. The use according to claim 28 or 29, wherein the subject has sporadic wild-type transthyretin-mediated amyloidosis associated with cardiomyopathy (ATTRwt-CM), and / or the subject is negative in a genetic test for TTR mutations.

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