Vaccines for treating amyloidosis

Abstract

A peptide-based vaccine for use in the treatment or prevention of amyloid-to-thyroid amyloidosis (ATTR) is provided.

Description

Technical Field

[0001] This invention relates to vaccines comprising peptides derived from transthyretin (TTR) (i.e., TTR peptides), and the use of said vaccines in methods for treating or preventing transthyretin amyloidosis (ATTR). More specifically, said TTR peptides contain novel epitopes selectively presented and exposed in misfolded, oligomeric, and / or aggregated forms of TTR, respectively. The invention also relates to kits comprising said vaccines. Background Technology

[0002] Amyloid-to-thyroxine amyloidosis (ATTR) is a serious, age-related disease that leads to cardiomyopathy and / or sensorimotor polyneuropathy (Gertz syndrome). et al ., J. Am. Coll. Cardiol. 66 (2015), 2451-24661), and includes two subtypes—wild-type ATTR (ATTRwt) and variant ATTR (ATTRv)—with different pathogenesis. 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 its native form exists as a tetramer (Alshehri). et al. , J. Neuroendocrinol. 27 (2015), 303-3239). ATTRv, formerly known as hereditary / mutant ATTR, is an autosomal dominant genetic disorder. For both wild-type TTR (TTRwt) and mutant / variant TTR (TTRv) proteins, the pathogenic mechanism of ATTR is triggered by the partial unfolding of the TTR protein and its subsequent aggregation into β-sheets that form amyloid fibrils (Eisele). et al ., Nat. Rev. Drug Discov. 14 (2015), 759-780).

[0003] ATTR is characterized by two main clinical manifestations. The primary accumulation of amyloid fibrils in cardiac tissue leads to cardiomyopathy, while the deposition of fibrils in nerve fibers leads to polyneuropathy (Androgenetic adenomatous polyneuropathy). et al*Guideline of transthyretin-related hereditary amyloidosis for clinicians. Orphanet J. Rare Dis. 8 (2013), 31*. The factors that trigger organ-specific amyloid deposition remain unclear. Patients typically present with mixed symptoms, and only a small number of TTR mutations are known to cause isolated cardiac or neurological lesions (Maurer). et al ., J. Am. Coll. Cardiol. 68 (2016), 161-172).

[0004] There are currently three generally accepted approaches to ATTR treatment: (i) Following orthotopic liver transplantation, TTRwt synthesized by the donor liver almost completely replaces TTRv in the peripheral blood. In addition to the well-known organ shortage, patients receiving liver transplants with TTRv exhibit slow but persistent disease deterioration.

[0005] (ii) Low molecular weight compounds stabilize TTR tetramers, thereby minimizing the formation of amyloid precursors. Diflunisal, AG10, and tafamidis stabilize physiological TTR tetramers. Tafamidis has been approved for the treatment of stage 1 ATTR since 2011.

[0006] (iii) Gene silencing agents (mRNA repressive oligonucleotides) reduce hepatic secretion of TTRv and TTRwt. Inotersen is an antisense oligonucleotide administered subcutaneously (s.c.) once weekly. Patisiran is an siRNA oligonucleotide administered intravenously (i.v.) every three weeks in combination with preoperative medication. Both gene silencing agents were approved in 2018 for the treatment of stage 1 and 2 ATTR.

[0007] Meanwhile, a Phase I, open-label, multicenter trial of an in vivo gene-editing therapeutic agent called NTLA-2001 is exploring CRISPR / CAS9, a gene-editing technology capable of targeting in vivo genome editing, as an emerging therapy for ATTR amyloidosis. In addition, monoclonal antibodies (mAbs) are being investigated as another potential treatment for ATTR, such as PRX004 (NNC6019-0001), an investigational mAb designed to prevent fibrillation by specifically targeting and clearing misfolded TTR proteins found in ATTR-CM, and NI006, an investigational human mAb targeting TTR amyloid protein, which recently showed in a Phase I trial to be safe and able to eliminate cardiac ATTR (Garcia-Pavia) in a dose-dependent manner. et al ., N Engl J Med (2023), doi: 10.1056 / NEJMoa2303765). Tomasoni et al Different treatment strategies are outlined in Front Cardiovasc Med 10 (2023), 1154594.

[0008] Although every drug and concept currently available shows great potential to improve ATTR and its associated symptoms, alternative treatment strategies are still needed because each patient responds differently to a particular treatment, and therefore, it is always beneficial to choose different treatment options.

[0009] This technical problem is solved by the embodiments as defined in the claims, further described below, and illustrated in the examples and drawings. Summary of the Invention

[0010] This invention generally relates to vaccines that can be used to treat and prevent amyloid-to-thyroxine amyloidosis (ATTR) and diseases caused by or associated with TTR deposition. While TTR tetramer stabilizers, gene silencing agents, or antibodies are primarily used to treat acute and chronic diseases caused by ATTR, i.e., at the onset or presence of the disease, vaccines can be used to prevent the disease in high-risk subjects before its onset, for example, due to genetic predisposition or risk determined by changes in indicative biomarker levels. Furthermore, continuous administration of the therapeutic agent is generally not required, as vaccination provides durable protection due to the activation of the immune system. More specifically, this invention relates to a vaccine comprising an immunogen, in this invention, a peptide derived from TTR, i.e., a TTR peptide, which contains an epitope of TTR that is selectively presented or exposed only in misfolded, oligomeric, and / or aggregated forms of TTR, the same applies to novel epitopes, and / or that is at least not present in the physiologically active form of TTR, for example, exposed in wild-type or mutant TTR protein monomers but hidden in physiologically active tetramers and no longer exposed to antibody-binding epitopes. Most preferably, the epitope is not present on the natural TTR monomer to avoid any cross-reactivity. Typically, the peptide-based vaccines of the present invention comprise a TTR peptide and an adjuvant to stimulate an immune response. The vaccines of the present invention are preferably used for the treatment and prevention of ATTR.

[0011] About twenty years ago, Terazaki et al Lab Invest. 86 (2006) 23-31 describes immunization of transgenic mice carrying the most common FAP-associated TTR mutant, V30M (a transthyretin mutant with valine replaced by methionine at position 30), with the TTR mutant Y78F (a transthyretin mutant with tyrosine replaced by phenylalanine at position 78), designed to disrupt the stability of the native structure. This has been shown to expose cryptic epitopes recognized by monoclonal antibodies that react only with highly amyloidogenic mutants or amyloid fibrils exhibiting amyloid folding. Their results indicate that Y78F induces antibodies that specifically react with deposits, leading to an effective immune response that removes / prevents TTR deposition. Therefore, the authors conclude that TTR immunization with selected TTR mutants may have potential applications in the immunotherapy of FAP.

[0012] However, this approach has not been further used in vaccine development since then, likely because a significant concern associated with active immunotherapy is that the antigen (in this case, the mutant TTR protein) could still elicit an immune response against physiological TTR species. On the other hand, the Y78F mutant protein was used because it was believed that using this mutant TTR protein could reveal cryptic epitopes to trigger the desired immune response.

[0013] This invention is based on the following concept: identifying a segment in the TTR amino acid sequence that is exposed to the body's immune response and is unique to TTR protofibrils and amyloid protein; designing a corresponding peptide containing at least the immunogenic portion of this segment; and arranging the peptide and the immunogenic portion into 3D conformations that are similar to the conformations that the segment may have in the amyloid protein conformation. To this end, based on the possible mechanism of TTR protein misfolding, Cryo-EM studies were screened. These studies showed that after the native tetramer depolymerizes and unfolds, the polypeptide chain then assembles into an early protofibrillary state, forming a structurally disordered Ala36-His56 residue segment in a solvent-exposed conformation, which is not present in the native TTR tetramer; see Schmidt. et al ., Nat Commun 10 (2019), 5008, especially the Figure 3 Its illustrations and figures are incorporated herein by reference. In fact, recent publications have reported the 3D structure of ATTR fibrils extracted from patient tissue and determined using cryo-electron microscopy (cryo-EM); see Schmidt. et al (2019), ibid.; Iakovleva et al ., Nat Commun 12 (2021), 7141; Steinebrei et al ., Nat Commun 13 (2022), 6398.

[0014] In all three cases, the amyloid structure was nearly identical, characterized by a rigid core structure interrupted by unresolved segments from Lys35 to Gly57. This indicates the presence of loose segments with variable, non-rigid conformations. Nevertheless, according to the invention, the proximity of Lys35 and Gly57 within the amyloid core suggests that the unresolved segments form loops, which, as shown in the examples, were used to design the corresponding peptides.

[0015] Amino acid sequence analysis revealed that this loose region contains the epitope of NI006, namely WEPFA (SEQ ID NO:1), which is a selective anti-ATTR antibody known to remove cardiac amyloid; see Garcia-Pavia. et al .(2023), ibid., and Michalon et al., Nat Commun 12 (2021), 3142. Therefore, it is reasonable to hypothesize that NI006 binds to the loose segment in ATTR, which could be used to determine whether peptides derived from this segment can be engineered to mimic the 3D conformation in TTR amyloid, and that the antibody can bind selectively and with high affinity to TTR amyloid in a manner substantially similar to that of TTR amyloid.

[0016] In this context, it must be considered that peptides (i.e., immunogens) are typically formulated with adjuvants or immunogenic enhancers (such as protein carriers) to generate a sufficiently strong immune response to protect the subject from the disease prevented by the vaccine he or she received, and that the presence of, for example, protein carriers may negatively affect the structure of the peptide and lead to the loss of new epitope formation, as found in the 3D conformation of the loose segment of TTR amyloid.

[0017] Therefore, attempts have been made to stabilize the required 3D conformation of peptides by triggering cyclization of the corresponding peptide sequence.

[0018] Therefore, experiments conducted within the scope of this invention surprisingly revealed that TTR peptides conjugated with bovine serum albumin (BSA) as a carrier can serve as antigens for anti-TTR antibodies, and that cyclization of the peptides can increase antibody binding by more than 100-fold; see Examples 1 and 2. In particular, in ELISA assays, binding of anti-TTR antibodies (taking antibody NI-301.37F1 as an example) to linear TTR peptides TTR34-54 and cyclic peptides TTR34-54cyc, both containing the epitope WEPFA (SEQ ID NO: 1), was observed in nanomolar and picomolar ranges, respectively; see Example 1. Furthermore, conjugation of a carrier protein (here, BSA as an example) to TTR peptides, particularly cyclic peptides TTR34-54cyc and TTR39-50cyc, still resulted in antibody binding; see Example 2.

[0019] The antibody NI-301.37F1, which binds to the TTR epitope WEPFA (SEQ ID NO: 1), is described in WO 2015 / 092077A1. Furthermore, the efficacy of the TTR peptide as an immunogen and therefore for vaccination methods has been demonstrated in in vivo mouse studies as described in Example 3. Specifically, mice were immunized with linear and cyclic BSA-conjugated TTR peptides of 12 and 21 amino acid lengths, and the immune response was characterized by ELISA against amyloid TTR (ATTR, mis.WT-TTR) and tetrameric TTR (TTR). Figure 3As shown, administration of the TTR peptides induced antibody formation against both TTR and ATTR after 38 days, with the tested sera exhibiting a stronger serum reactivity to ATTR than to TTR. Surprisingly, contrary to what might be expected from the initial ELISA assays described in Examples 1 and 2, both linear peptides TTR34-54 and TTR39-50 exhibited higher amyloid selectivity, EC50. 50(ATTR / TTR) The quotients were 15.8 and 14.7, respectively, in contrast to the amyloid-selective EC values ​​of the cyclic peptide TTR34-54cyc. 50(ATTR / TTR) With a quotient of 13.4, the cyclic peptide TTR39-50cyc exhibits selective EC of amyloid. 50(ATTR / TTR) The quotient is 4.3, see Table 1 and Figure 3 Furthermore, the highest ATTR-specific antibody titers were detected in the serum of mice immunized with the linear peptide TTR34-54, indicating that mice administered the TTR34-54 immunogen showed a higher immune response than mice administered other peptides; see [link to article]. Figure 4 .

[0020] Therefore, further evidence can be provided that, since the exemplary tested TTR peptide appears to mimic the 3D conformation of the loose segment in TTR amyloid and will elicit a novel epitope-specific immune response in mammals, it is reasonable to expect that the TTR peptide designed according to the present invention, when administered to subjects (e.g., in vaccine form), will trigger an immune response against the TTR peptide, including stimulation of T cells and other reactive immune cells, and resulting in the production of anti-TTR antibodies exhibiting high selectivity for amyloidogenic TTR. As mentioned above, antibodies binding to segments in the Lys35 to Gly57 range of TTR have the ability to clear misfolded TTR aggregates through phagocytosis; therefore, it is reasonable to expect that TTR peptides specifically inducing antibodies against this segment (preferably containing the amino acid sequence WEPFA (SEQ ID NO: 1)) can be used as immunogens in vaccines and induce their clearance before misfolded TTR aggregates further assemble into fibrils. This would avoid the manifestation of the disease phenotype, namely the accumulation of TTR fibrils. Therefore, it is reasonable to expect that the TTR peptide of the present invention can be used for immunization, i.e., to initiate an immune response against amyloidosis-inducing / misfolded TTRs to prevent the onset of ATTR, or to treat the disease when it has already manifested. The theory upon which the present invention is based is confirmed by Example 3, which demonstrates an effective immune response to the immunogen of the present invention, wherein serum antibodies preferentially bind to aggregated TTRs. Prevention of (further) formation of TTR aggregates is believed to prevent further disease exacerbation and / or complete regression of TTR aggregates, and to halt the disease cascade.

[0021] As described above, the disordered region of TTR residues Ala36-His56, which forms in a solvent-exposed conformation in TTR amyloid protein and is not present on the native TTR tetramer, constitutes a novel epitope and is a preferred basis for designing TTR peptides. The binding of antibodies to such novel epitopes is crucial to their safety and therapeutic efficacy; therefore, immunogen-induced antibody activation is key to the effectiveness of vaccines containing said immunogens. Accordingly, this invention relates to a vaccine comprising a peptide derived from TTR containing a TTR epitope that is selectively presented or exposed to 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), and preferably contains the amino acid sequences RKAADDTWEPFASGKTSESGE (SEQ ID NO: 2, TTR34-54) or DTWEPFASGKTS (SEQ ID NO: 3, TTR39-50) or composed thereof, with the most preferred amino acid sequence being RKAADDTWEPFASGKTSESGE (SEQ ID NO: 2, TTR34-54). In a preferred embodiment, the peptide is a linear peptide.

[0022] Subunit vaccines, primarily composed of peptides or proteins, may face limitations in immunogenicity, potentially requiring multiple immunizations to achieve a high level of immune response. This has spurred the development of various methods to enhance subunit vaccine responses, including the presentation of epitopes in polymeric forms such as virus-like particles, VLPs, or nanoparticles. This strategy can enhance the immune response by increasing the half-life of epitopes through reduced renal clearance and sensitivity to proteolytic degradation; see Malonis. et al. , Chem Rev. 120 (2020), 3210-3229. Not wishing to be bound by theory, the use of cyclic peptides as vaccines may have a similar effect, namely, enhanced vaccine response, due to the very stable conformation of cyclic peptides because their ends are linked together. Therefore, in one embodiment, the vaccine of the present invention comprises a TTR peptide capable of forming a cyclic compound, i.e., the vaccine of the present invention comprises a cyclized TTR compound in one embodiment. Cycling is preferably performed by linkers coupled to the N-terminal and C-terminal residues of the peptide. In a preferred embodiment, 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).

[0023] In Example 2, it was observed that BSA conjugation essentially did not interfere with antibody binding, which is crucial for the successful development of peptide-based vaccines. Particularly in typical peptide vaccination regimens, in addition to, or alternatively, methods of presenting epitopes in multimeric form, peptides containing epitopes of interest can be conjugated to carrier proteins. This can enhance the immune response by increasing the half-life of the epitope through reduced renal clearance and sensitivity to proteolytic degradation. The conjugation to carrier proteins is typically achieved through chemical conjugation. Carriers are generally known to possess immunogenic properties; therefore, simply covalently linking 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 vector. Therefore, TTR peptides appear to be suitable candidates for vaccines. Thus, in one embodiment, the vaccine of the present invention comprises a peptide, preferably a cyclic form of the peptide, coupled to a carrier protein (e.g., BSA).

[0024] As previously mentioned, presenting the correct 3D structure of the novel epitope is key to eliciting an immune response that preferentially recognizes ATTRs (i.e., aggregated TTRs, especially wild-type TTR species). Therefore, it was initially thought that, in addition to conferring serum stability, peptide cyclization might facilitate peptide folding to mimic the structures on misfolded and aggregated TTRs, and stabilize their conformation against potentially harmful interference with correct folding caused by immunogenic vectors, specifically BSA, a much larger polypeptide that is likely considered to negatively impact peptide folding or mask the epitope. However, experiments conducted according to the present invention surprisingly revealed that cyclization is neither necessary nor advantageous for the efficacy and specificity of TTR peptide immunogens; see Example 3, and... Figure 3 and Figure 4 .

[0025] Therefore, in a preferred embodiment of the invention, the TTR peptide-based immunogen is not cyclized and preferably not otherwise modified. Instead, the TTR peptide in the immunogen is preferably determined experimentally and / or by computer (…). in silico The minimal epitope / antigen sequence identified by analysis is used to represent a novel epitope specific to TTR pathological variants, oligomers, and / or aggregates, and optionally, about 1 to 10 amino acids are appended to its N-terminus and / or C-terminus, preferably wherein the appended amino acids are also present in the original TTR amino acid sequence; see also the examples.

[0026] As described above, the immunogen of the vaccine of the present invention can be produced by chemical synthesis or using recombinant DNA technology; see reviews, such as Wang et alSig Transduct. Target Ther. 7 (2022); https: / / doi.org / 10.1038 / s41392-022-00904-4. For any antigenic TTR peptide recombinantly expressed according to the invention, whether or not coupled to an immunogenic vector, the nucleic acid encoding said peptide or protein also constitutes an aspect of the invention, as do the expression vector containing said nucleic acid and the host cell containing said expression vector (autonomous or chromosomally inserted).

[0027] Therefore, in one or more embodiments, the present invention relates to a nucleic acid encoding the immunogen described herein; an expression vector comprising the nucleic acid; and / or a host cell comprising the nucleic acid or the expression vector. A method for producing an immunogen by recombinant expression and isolation of the immunogen from a host cell is another aspect of the invention.

[0028] The nucleic acids and expression vectors of the present invention may be part of a kit or composition, which optionally further comprises an immunogenic enhancer such as an immunogenic vector or an adjuvant.

[0029] The present invention also relates to a kit comprising the vaccine of the present invention.

[0030] Other embodiments of the present invention will be shown in the following description and examples. Attached Figure Description

[0031] Figure 1 ELISA results showed that the exemplary TTR peptide retained the 3D conformation of the loosely bound segment uniquely present in TTR amyloid protein by binding to the ATTR-specific antibody NI-301.37F1, and cyclization promoted antibody binding, i.e., TTR peptides, especially cyclic TTR peptides, are suitable as antigens and immunogens; (A) In ELISA-1, the binding of the antibody to peptide TTR34-54cyc and misfolded wild-type TTR (mis.WT-TTR) was analyzed, and the results showed that the binding of the antibody to the cyclic TTR34-54cyc peptide was much stronger than that to mis.WT-TTR, i.e., approximately (A) In ELISA-2, the binding of the antibody to peptides TTR34-54_bt and TTR34-54cyc_bt, as well as the BSA control, was analyzed. The results showed that the ATTR-specific antibody could bind to both linear and cyclic TTR peptides, but cyclization increased the binding of NI-301.37F1 by more than 100 times. (B) In ELISA-3, the binding of the antibody to peptides TTR34-54cyc and TTR40-49 was analyzed. No binding of the antibody to TTR40-49 was observed, indicating that a certain length of peptide and the antigenic / immunogenic part of the peptide are required for correct folding, respectively.

[0032] Figure 2 ELISA results showed that the BSA-conjugated TTR peptide exhibited a TTR amyloid-specific epitope, as demonstrated by its binding to antibody NI-301.37F1; (A) In ELISA-1, the binding of the antibody to peptides TTR34-54cyc_BSA, TTR34-54_BSA, TTR34-54SCRcyc_BSA, and TTR34-54SCR_BSA was analyzed, and the results showed that the antibody could bind both BSA-conjugated linear TTR peptides and BS-conjugated peptides. (A) A cyclic TTR peptide was conjugated, but cyclization increased the binding of NI-301.37F1 by more than 100-fold; (B) In ELISA-2, the binding of the antibody to peptides TTR39-50cyc_BSA, TTR39-50_BSA, TTR39-50SCRcyc_BSA, and TTR39-50SCR_BSA was analyzed. The results showed that the antibody bound to the cyclic BSA-conjugated TTR39-50cyc_BSA peptide, but almost no binding was detected with the corresponding linear peptide. No binding to the antigen control, i.e., the out-of-order (SCR) peptide, was observed in either ELISA assay.

[0033] Figure 3 ELISA results showed that the immune response, as measured by serum antibody titers, increased over time (day 0 (d0) compared to day 38 (d38)), and serum obtained after immunizing BalbC mice with TTR peptides was more responsive to ATTR than to TTR; (A) ELISA against TTR and ATTR showed that serum obtained after immunizing mice with peptide / immunogen TTR39-50 was more responsive to TTR (ECG). 50 The EC50 value was 1038, and the ATTR value was 15271, therefore the amyloid selectivity (EC50) was high. 50(ATTR / TTR) (A) The quotient value was 14.7; (B) ELISA for TTR and ATTR showed that the serum responsiveness to TTR obtained after immunizing mice with peptide / immunogen TTR39-50cyc was (EC50) 50 The EC50 value is 1813, and the ATTR value is 24343, therefore the amyloid selectivity (EC50) is high. 50(ATTR / TTR) The quotient was 15.4; (C) ELISA for TTR and ATTR showed that the serum responsiveness to TTR obtained after immunizing mice with peptide / immunogen TTR34-54 was (EC50) 50 The EC50 value is 3400, and the ATTR value is 53809, therefore the amyloid selectivity (EC50) is high. 50(ATTR / TTR) The quotient was 15.8; (D) ELISA for TTR and ATTR showed that the serum responsiveness to TTR obtained after immunizing mice with peptide / immunogen TTR34-54cyc was (EC50) 50The EC value is 7785, and the ATTR value is 33587, therefore the amyloid selectivity (EC) is high. 50(ATTR / TTR) The quotient was 4.3; (D) Control ELISA against TTR and ATTR showed that the reactivity (ECG) of serum obtained after immunizing mice with the control peptide / immunogen PR906 was 4.3. 50 ). Measure serum OD 450 The values ​​were plotted on a logarithmic scale relative to serum dilutions (dilution range from 1:100 to 1:5,904,900).

[0034] Figure 4 ELISA results showed that sera obtained after immunizing BalbC mice with TTR peptides TTR39-50, TTR39-50cyc, TTR34-54, and TTR34-54cyc were more reactive to ATTR than to TTR. Furthermore, the highest antibody titers, particularly ATTR-specific antibody titers, were detected in sera obtained from mice immunized with the linear peptide TTR34-54. The serum dilution used in this assay was 1:24,300. Detailed Implementation

[0035] This invention relates to a peptide-based vaccine comprising a peptide derived from transthyretin (TTR), namely a TTR peptide, which contains an epitope of TTR that is selectively presented or exposed to antibody binding only in misfolded, oligomeric, and / or aggregated forms of TTR.

[0036] Unless otherwise defined in this application, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials described herein may be used to practice or test the invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Materials, methods, and examples are illustrative only and are not intended to be limiting.

[0037] Other embodiments of the invention will become apparent from the following description, examples, and claims. Those skilled in the art will understand that the following description of the general features of the general embodiments is intended to be combined with the description of one or more other features of the general embodiments. For the avoidance of any doubt, it is hereby emphasized that the use and meaning of expressions such as “in some embodiments,” “in certain embodiments,” “in some cases,” “in some situations,” “in another embodiment,” “in one embodiment,” etc., are that any embodiments described therein should be considered in combination with each feature of those embodiments, and this disclosure must be treated in the same manner as if the features of those embodiments were combined in the same embodiment. The same applies to any combination of embodiments and features illustrated in the appended claims and examples, which are also intended to be combined with the features of the corresponding embodiments described in the specification, wherein the embodiments are characterized by subordination only for the sake of consistency and brevity, and in practice, each combination of embodiments and features (which may be interpreted due to (multiple) subordinations) must be considered as a literal disclosure, not as a choice among different options.

[0038] Unless otherwise stated, the terms used in this article have the definitions provided in the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford University Press, 1997, revised in 2000, reprinted in 2003, ISBN 0 19 850673 2; second edition published in 2006, ISBN 0-19-852917-1 978-0-19852917-0).

[0039] The term "cyclic peptide" as used herein can refer to a compound composed entirely of protein, for example, in which the linker is 2, 3, 4, 5, 6, 7, or 8 amino acids, or in which the linker is absent. For example, it is possible that the native protein sequence (i.e., the amino acid fragment containing the antibody epitope) allows cyclization, for example due to the presence of two appropriately spaced cysteine ​​residues, without the addition of additional amino acids. It should be understood that the properties described for the cyclic peptides identified in the examples can be incorporated into other compounds, such as cyclic compounds containing non-amino acid linker molecules. "Cyclic peptide" and "cyclic compound" are used interchangeably when the cyclic compound is composed of amino acids.

[0040] The term "immunogenic" refers to a substance that induces antibody production and activates lymphocytes or other reactive immune cells that target the antigenic portion of an immunogen (in this invention, the TTR peptide).

[0041] As used herein, “immunogen” or “immunogenic compound” refers to a substance that triggers an immune response and leads to antibody production, and may include, for example, the TTR peptide described herein, particularly the cyclic peptide described herein, as a multi-antigenic peptide conjugate and / or fused with an immunogenic enhancer (such as a carrier protein (e.g., BSA)). In addition to the conjugates described herein, immunogenic peptide mimics that trigger cross-reactions with the epitopes described herein also constitute immunogens.

[0042] The term “corresponding linear compound” in relation to cyclic compounds refers to a compound (optionally a peptide) that contains the same sequence or chemical motif as a cyclic compound or is composed of it but in a linear (non-cyclic) form.

[0043] As used herein, the term "linker" refers to a chemical moiety, preferably weakly immunogenic or non-immunogenic, that can be directly or indirectly covalently linked to a protein fragment or peptide as defined herein. Linker ends can, for example, be linked to create a cyclic compound. Linkers can be located at N-terminal and C-terminal positions. Alternatively, linkers can be located internally at a "distance" from the ends. Linker ends can, for example, be linked to create a cyclic compound. Linkers can contain one or more functionalizable moieties, such as one or more cysteine ​​(C) residues. Linkers can also be linked to other proteins or components via functionalizable moieties. Linkers can be linked to carrier proteins or immunogenic enhancers, such as keyhole hemocyanin (KLH) or bovine serum albumin (BSA), via functionalizable moieties. Cyclic compounds containing linkers are longer than the peptide or protein fragment itself. Linkers can include, but are not limited to, non-immunogenic moieties, such as the amino acids glycine (G) and alanine (A), or polyethylene glycol (PEG) repeating units.

[0044] As used herein, the term "functionalizable moiety" refers to a chemical entity having a "functional group," which is defined as a group of atoms or a single atom that will react with another group of atoms or a single atom (the so-called "complementary functional group") to form a chemical interaction between the two groups of atoms or between atoms. In the case of cysteine ​​(C), the functional group can be -SH, which can react to form a disulfide bond. The reaction with the other group of atoms can be a covalent or a strong non-covalent bond, such as the biotin-streptavidin bond, whose dissociation constant (Kd) can be approximately 1e-14. A strong non-covalent bond, as used herein, refers to 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.

[0045] Proteins and / or other reagents can be coupled to cyclic compounds, for example, to aid immunogenicity. For this purpose, any functionalizable moiety capable of reacting (e.g., forming covalent or strong non-covalent bonds) can be used. In one specific embodiment, the functionalizable moiety is a cysteine ​​residue that reacts with an unpaired cysteine ​​residue on a protein of interest to form a disulfide bond, which can be, for example, an immunogenic enhancer (such as a carrier protein like bovine serum albumin (BSA)) or a T helper cell epitope. The term "reacts with" as used herein generally implies the presence of electron flow or electrostatic charge transfer, resulting in the formation of a chemical interaction.

[0046] As used herein, the phrase "effective amount" refers to the amount of an antigenic / immunogenic composition that elicits an immune response when administered to humans or animals. The effective amount can be readily determined by those skilled in the art following standard procedures.

[0047] As used herein, the term "carrier protein" refers to an immunogenic protein used to enhance the immune system's response to compounds that are otherwise insufficiently immunogenic (such as another protein / peptide). Carrier proteins are particularly suitable for small compounds with a very limited number of possible epitopes, for which the immune response can only target, or for compounds with very low immunogenicity. Preferred carrier proteins have a high compound-to-carrier ratio, allowing a large number of compounds to be coupled to a single carrier protein to increase the immunogenicity of the carrier protein antigen complex.

[0048] The term "adjuvant" refers to a compound that, when administered with an antigen (in this case, the TTR peptide), enhances, stimulates, activates, synergizes, or modulates the immune response to that antigen, but does not elicit an immune response 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 can be natural compounds, modified or derivative forms 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, as commonly known as Alhydrogel®), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1B, IL-2, IL-7, IL-12, IL-18, GM-CSF, and IFN-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virions, bacterial adjuvants (e.g., monophosphoryl lipid A (MPL) or muramyl peptides), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or polynucleotide adjuvants (e.g., CpG oligodeoxynucleotides). QS-21 is a product derived from soapberry ( Quillaja saponariaSaponins extracted from [the following components]. 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 an 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 ODNI. Hiltonol (Poly-ICLC) is a synthetic complex of carboxymethyl cellulose, polyinosinic acid-polycytidylic acid, and poly-L-lysine double-stranded RNA.

[0049] The term "immunity" refers to the process of activating, strengthening, or enhancing an individual's immune system against agents ("vaccines") that typically cause or induce disease or condition. Thus, by immunizing a healthy individual against these agents, the onset of a disease or condition can be prevented. Immunizing a patient with a disease or condition can treat the disease or condition or prevent its further development. Immunization can be achieved through various techniques, the most common being vaccination of a healthy individual or a patient with a disease or condition. The basic principle of active immunization via vaccines (containing TTR peptides) is the creation of immune "memory." In the context of persistent ATTRs (i.e., cellular antigens, i.e., amyloid-inducing TTRs), the aim of active immunization is to maintain persistently high antibody levels through lifelong, periodic immunization. Challenging an individual's immune system (e.g., with a vaccine containing a disease-specific immunogen) induces the formation and / or proliferation of immune cells that specifically recognize the immunogens contained in the vaccine. At least a portion of these immune cells remain viable for a period of time after vaccination (which can extend to 10, 20, or 30 years). Compared to the immune response of an individual who has not been challenged with a vaccine and is encountering the immunogen for the first time, if an individual's immune system encounters the immunogen again within the aforementioned time period, the immune cells generated by vaccination are reactivated, and the immune response against the immunogen is enhanced. In many cases, a single dose of vaccine is insufficient to produce the number of long-lived immune cells required for effective protection against the disease or condition. Therefore, repeated challenges with a disease-specific biologic are necessary to establish durable protective immunity against the disease or to cure a given disease.

[0050] As detailed above, this invention is based on the concept of identifying a segment in the amino acid sequence of TTR that is exposed to the body's immune response but is unique to TTR fibrils and amyloid proteins. Examination of the amino acid sequence reveals that this segment contains the epitope of NI006, namely WEPFA (SEQ ID NO: 1). As shown in the examples, different TTR peptides containing the epitope WEPFA (SEQ ID NO: 1) (which is contained in a loose segment and is a novel epitope in this sense, hidden in the natural folded conformation of the TTR protein but exposed to antibody binding after unfolding and aggregation) maintain an amyloid-specific conformation and are therefore suitable as immunogens. As explained in more detail above, it is reasonable to expect that once such peptides are administered to a subject (e.g., in the form of a vaccine), they will trigger a specific immune response against TTR aggregates, leading to the clearance of TTR amyloid proteins, as observed in passive immunotherapy with NI006, and thus also preventing the manifestation of ATTR. Therefore, the TTR peptides described herein can be used as immunogens in vaccines.

[0051] Therefore, this invention relates to a vaccine comprising a peptide derived from TTR, i.e., a TTR peptide, which contains an epitope of TTR that is selectively presented or exposed only in misfolded, oligomeric, and / or aggregated forms of TTR, and the use of said TTR peptide as an immunogen, i.e., an immunogenic compound. In other words, this invention relates to a peptide derived from TTR, i.e., a TTR peptide, which contains an epitope of TTR that is selectively presented or exposed only in misfolded, oligomeric, and / or aggregated forms of TTR, for use as a vaccine.

[0052] 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 other TTR peptides, comprising epitopes present in or overlapping with said loose segment and exposed to TTR misfolded variants, as well as aggregates, protofibrils, and / or oligomers, are also suitable for the vaccines of the present invention. 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 present in or overlapping with the loose TTR fragment and exposed to TTR misfolded variants and aggregates, fibrils and / or oligomers, such as 54-ELXGLTXE-61 (SEQ ID NO: 13), a peptide recognized by antibody NI-301.35G11 disclosed in WO 2015 / 092077 A1, where X can be any amino acid; WEPFASG (SEQ ID NO: 14), a peptide recognized by antibody NI-301.12D3 disclosed in WO 2015 / 092077 A1; and 30-VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE-66 (SEQ ID NO: 20), a peptide recognized by antibody described in WO 2014 / 124334 A2. Furthermore, the amino acid sequence used in TTR peptides can be altered, for example by amino acid substitution / deletion / addition compared to the original amino acid sequence in a loose segment, as long as the 3D conformation is not substantially affected. For example, a TTR peptide may include an epitope containing the amino acid sequence WXPFA (SEQ ID NO: 11), such as the peptide recognized by antibody NI-301.28B3 disclosed in WO 2015 / 092077 A1, where X can be any naturally occurring amino acid.

[0053] Furthermore, it is reasonable to expect that other TTR peptides, which contain epitopes exposed to TTR misfolded variants and in aggregates, protofibrils and / or oligomers, may also promote the induction of antibodies capable of clearing pathological TTR aggregates when administered to subjects, and thus could be used as immunogens in 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 exposed to the amino acid sequence in TTR misfolded variants and aggregates, protofibrils and / or oligomers, such as EEFFXEGIY (SEQ ID NO: 12), for example, the peptide recognized by antibody NI-301.59F1 disclosed in WO2015 / 092077 A1, where X can be any amino acid; TTAVVTNPKE (SEQ ID NO: 15), for example, the peptide recognized by antibody NI-301.18C4 disclosed in WO 2015 / 092077 A1; KCPLMVK and VFRK (SEQ ID NOs: 16 and 17), representing peptides containing conformational epitopes, requiring at least C of the first sequence and V and F of the second sequence, and being epitopes recognized by antibody NI-301.44E4 in WO 2015 / 092077 A1; EHAEVVFTA (SEQ ID NO: 18), for example, especially Higaki et al The peptides recognized by antibodies 14G8 / PRX004 / NN-6019 disclosed in Amyloid 23 (2016), 86-97; GPRRYTIAA (SEQ ID NO: 19), such as the peptide recognized by antibody 18C5 described in WO 2019 / 071205 A1; ALLSPYSYSTTAV (SEQ ID NO: 21), such as the peptide recognized by antibody binding to TTR109-121 described in WO 2014 / 124334 A2; WKALGISPFHE (SEQ ID NO: 22), such as the peptide recognized by antibody 371M described in WO 2015 / 115332 A1; SYSTTAVVTN (SEQ ID NO: 23), such as the peptide recognized by antibody 313M (RT24) described in WO 2015 / 115331 A1; or LLSPYSYSTTAVVTNPKE (SEQ ID NO: 23). 24), such as the peptide recognized by the antibody that binds to TTR100-127 as described in WO 2014 / 124334 A2.

[0054] 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 TTR (TTRv). Therefore, in one embodiment, the TTR peptide contained in the vaccine of the present invention contains an epitope composed of the wild-type TTR amino acid sequence, and thus, the vaccine of the present invention can be used to prevent the occurrence of sporadic wild-type ATTR triggered by misfolded wild-type TTR.

[0055] As described above, the exemplary TTR epitope WEPFA (SEQ ID NO: 1) is a novel epitope that is selectively presented and exposed not only in the misfolded, oligomeric, and / or aggregated forms of TTR, but also not in the physiologically active tetrameric form of TTR, nor on the natural TTR monomer. Therefore, according to the present invention, the TTR peptide contains an epitope selectively presented or exposed in the misfolded, oligomeric, and / or aggregated forms of TTR. Alternatively, the TTR peptide may contain an epitope not present in the physiologically active tetrameric form of TTR (but possibly present on natural and / or mutant TTR monomers), but preferably, this epitope is also not present on the natural TTR monomer. Further exemplary epitopes not present on natural TTR monomers may be obtained from the applicant’s prior work disclosed in WO 2015 / 092077 A1, and include, for example, the amino acid sequence EEFXEGIY (SEQ ID NO: 12), an epitope recognized by antibody NI-301.59F1, where X can be any amino acid, or ELXGLTXE (SEQ ID NO: 13), a peptide recognized by antibody NI-301.35G11, where X can be any amino acid. Therefore, in one embodiment, the TTR peptide contained in the vaccine of the present invention comprises an epitope comprising the amino acid sequence WEPFA (SEQ ID NO: 1), EEFXEGIY (SEQ ID NO: 12), or ELXGLTXE (SEQ ID NO: 13), or an epitope composed thereof. Most preferably, the TTR peptide comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

[0056] As further shown in the examples, particularly in Example 2, the tested anti-TTR antibody showed that it could bind to both TTR34-54 and TTR39-50.

[0057] Therefore, in one embodiment of the invention, the TTR peptide comprises 5 to 40 amino acids, 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, or constitutes thereof, of the TTR protein. In one embodiment, the TTR peptide comprises at least 5 amino acids, preferably at least 10, more preferably at least 12, more preferably at least 15, and most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues, or constitutes thereof, of the TTR protein. As described above, these amino acid residues comprise epitopes presented or exposed in misfolded, oligomeric, and / or aggregated forms of TTR. More specifically, at least the epitope should be present, and those skilled in the art know that the epitope may be as few as four amino acids and may be supplemented with an appropriate number of amino acids and / or other portions sufficient to provide a stable peptide, such as a linker portion required for cyclization.

[0058] However, in principle, there is no limitation on the length of the peptide, provided that it is stable and immunogenic (i.e., can induce antibodies upon administration to a subject) and optionally can be cyclized. Therefore, the TTR peptide contained in the vaccine of the present invention comprises 4 to all amino acids of the TTR protein. Preferably, the TTR peptide comprises 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.

[0059] These amino acids represent only epitopes, or represent epitopes and adjacent amino acids in the TTR protein. In a preferred embodiment, the TTR peptide comprises amino acid residues of the TTR protein, wherein these amino acid residues comprise the epitope and adjacent amino acids.

[0060] Most preferably, the peptide contained in the vaccine of the present invention comprises the amino acid sequence shown in SEQ ID NO: 2 (TTR34-54) or SEQ ID NO: 3 (TTR39-50).

[0061] Relatively short immunogenic peptides (i.e., TTR peptides, less than about 50 amino acids) are typically synthesized using standard chemical peptide synthesis techniques, such as solid-phase synthesis, in which the C-terminal amino acid of the sequence is linked to an insoluble carrier, followed by the sequential addition of the remaining amino acids in the sequence. Solid-phase synthesis techniques are well known to those skilled in the art. Alternatively, TTR peptides can be synthesized using recombinant nucleic acid methods. Typically, 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, if necessary, refolding the peptide. Techniques sufficient to guide those skilled in the art to perform such procedures can be found in the literature. Once expressed, the recombinant peptide can be purified according to standard procedures, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, gel electrophoresis, etc.

[0062] In Examples 1 and 2, the superior performance of cyclic peptides relative to their corresponding linear peptides in ELISA detection was demonstrated. Specifically, the binding affinity of TTR antibodies to cyclic TTR peptides was approximately 10 to 100 times higher than that to their corresponding linear peptides. Therefore, in one embodiment, the peptides contained in the vaccine of the present invention form cyclic compounds. In other words, in one embodiment, the present invention relates to a vaccine comprising cyclic compounds, said cyclic compounds comprising peptides containing TTR protein epitopes, said epitopes preferably being exposed to antibody binding only in misfolded and / or aggregated forms of said protein (as in the case of novel epitopes), and / or said epitopes being at least not present in the physiologically active form of said protein, for example, in the case of antibody-binding epitopes exposed in TTR protein monomers but hidden in physiologically active tetramers and no longer exposed. In particular, in one embodiment, the vaccine of the present invention comprises a TTR peptide in a cyclizable form as defined above. In a preferred embodiment, the TTR peptide comprises a linker covalently linked to N-terminal and C-terminal residues of said peptide to form a cyclic compound.

[0063] As in Example 3 and Figure 3 and Figure 4 As shown, immunization with the cyclic peptides TTR34-54cyc and TTR39-50cyc, and surprisingly, even more significantly, with the corresponding linear peptides TTR34-54 and TTR39-50, both resulted in selective immune responses, as antibodies specific to ATTR were generated after immunization with the peptides, as measured by serum reactivity and amyloid selectivity.

[0064] Therefore, the present invention also relates to the use of TTR peptides as immunogens, i.e., immunogenic compounds. The vaccines of the present invention may contain one immunogenic compound, i.e., one TTR peptide, or more than one TTR peptide, such as two, three, or four different TTR peptides.

[0065] The linear TTR peptides used in the examples consist of the amino acid sequence RKAADDTWEPFASGKTSESGE (TTR34-54; SEQ ID NO: 2), containing a total of 21 amino acids of amyloidogenic TTR, including the five-amino acid epitope WEPFA; and the amino acid sequence DTWEPFASGKTS (TTR39-50; SEQ ID NO: 3), containing a total of 12 amino acids of amyloidogenic TTR, including the five-amino acid epitope WEPFA. Therefore, in a preferred embodiment, the linear peptide consists of a total of 5 to 30, preferably 10 to 30, more preferably 10 to 25, and 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.

[0066] The cyclic TTR peptides used in the examples consist of the amino acid sequence GCGGGRKAADDTWEPFASGKTSESGEGGGCG (TTR34-54cyc; SEQ ID NO: 4), totaling 31 amino acids, including the 21 amino acids of the amyloidogenic protein TTR (including the five-amino acid epitope WEPFA) and a 10-amino acid linker sequence (five amino acids each at the N-terminus and C-terminus of the 21 amino acid fragment of TTR); and the amino acid sequence GCGGGDTWEPFASGKTSGGGCG (TTR39-50cyc; SEQ ID NO: 5), totaling 22 amino acids, including the 12 amino acids of the amyloidogenic protein TTR (including the five-amino acid epitope WEPFA) and a 10-amino acid linker sequence (five amino acids each at the N-terminus and C-terminus of the 12 amino acid fragment of TTR). Therefore, in a preferred embodiment, the cyclic compound consists of a total of 15 to 40, preferably 20 to 40, more preferably 20 to 35, or even more preferably 30 ± 1, 2, 3 or 4 amino acids, or consists of 20 ± 1, 2, 3 or 4 amino acids, but most preferably consists of 30 ± 1, 2, 3 or 4 amino acids, or, in the case of incorporation of non-amino acid residues (e.g., as linkers), its structure is configured to resemble 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, preferably 10 to 25, more preferably 20 ± 1, 2, 3, or 4 amino acids, but most preferably 20 ± 1, 2, 3, or 4 amino acids, and optionally a linker may be added. The linker is preferably 5 to 20 amino acids long, more preferably 5 to 15, and most preferably 10 ± 1, 2, 3, or 4 amino acids, and may be distributed at both ends (N-terminus and C-terminus) or only at one end. It is also conceivable that the linker sequence or "filler" sequence is located inside the amino acid sequence derived from the amyloidogenic protein, for example, if the epitope of the target-binding molecule is a conformational epitope or a discontinuous epitope.

[0067] Therefore, the cyclic compounds present in the vaccines of the present invention may comprise or be composed of TTR peptides, said TTR peptides being composed of or containing TTR novel epitopes, which means that additional amino acids or other chemical entities may be present in the protein fragments or peptides that form the cyclic compounds, for example for peptide or protein fragment cyclization as described further below.

[0068] The additional amino acids can be naturally located adjacent to the epitope sequence (i.e., flanking amino acids of the epitope sequence) and are present in the TTR protein sequence from which the peptide originates. That is, the TTR peptide forming the cyclic compound contains a TTR epitope and other amino acids flanking the epitope, respectively. The number of these adjacent / flanking amino acids can vary, for example, from 1, 2, or 3 amino acids to 50 amino acids, preferably from 1, 2, or 3 amino acids to 40 amino acids, more preferably from 1, 2, or 3 amino acids to 30 amino acids, more preferably from 1, 2, or 3 amino acids to 20 amino acids, and even more preferably from 10 to 20 amino acids. These amino acids can be evenly distributed at the N-terminus and C-terminus of the epitope sequence, or unevenly distributed, for example, with 7 additional amino acids at the N-terminus and 9 amino acids at the C-terminus.

[0069] Alternatively, in one embodiment, the TTR peptide may include a linker, i.e., the protein fragment or peptide may contain an epitope without any adjacent amino acids, and a linker; or it may contain an 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 comprises a novel epitope and an amino acid adjacent to the epitope, and a linker. Preferably, the linker is covalently linked directly or indirectly to the N-terminal residue and the C-terminal residue of the TTR peptide.

[0070] In one embodiment, the linker amino acid is selected from non-immunogenic or weakly immunogenic amino acid residues, such as G or A. For example, the linker can be GG, GGG, GAG, G(PEG)G, PEG-PEG (also known as PEG2)-GG, etc. It may include one or more functionalizable moieties, such as amino acids having functional groups, for example, for coupling the compound with an immunogenic enhancer (such as a carrier such as BSA).

[0071] Methods for cyclizing peptides are generally known in the art. For example, cyclization can be achieved by chemical crosslinking (using methods particularly with chemical scaffolds). Crosslinking requires functional groups, and only a few protein chemical targets account for the vast majority of crosslinking techniques, such as primary amines (-NH2), where this group is present at the N-terminus of each polypeptide chain and in the side chain 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 thiol groups (-SH), where this group is present in the side chain of cysteine.

[0072] Scaffold-based cyclization is one of the most commonly used methods because it can be applied to chemically or biosynthesized peptides. Typically, scaffold compounds such as organohalides (most commonly organobromines) selectively react with the thiol group of cysteine. Non-thiol groups, such as the primary amine of lysine or the N-terminal amino group in the peptide, can also be used for cyclization, for example, using chemicals containing N-hydroxysuccinimide (NHS). Specially designed non-natural amino acids can also be used for peptide cyclization via bioorthogonal reactions. For example, if the peptide contains an azide-containing amino acid such as azidohomalanine or azidophenylalanine, a copper-mediated click reaction with an alkyne-containing scaffold can lead to cyclization.

[0073] In addition, cysteine ​​can be linked together between its side chains via disulfide bonds (-SS-), or it can undergo amide cyclization without any scaffold (head-to-tail cyclization or main chain cyclization).

[0074] For example, peptides having "C" residues at their N-terminus and C-terminus, such as the cyclic TTR compounds used in Examples 1 and 2, GCGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 4) and GCGGGDTWEPFASGKTSGGGCG (SEQ ID NO: 5), can be converted into cyclic peptides via an SS cyclization reaction. Prior to cyclization, the cyclic compound can be synthesized as a linear molecule with a linker covalently attached to the N-terminus or C-terminus of a peptide containing the TTR peptide or the relevant epitope mentioned herein, or in its vicinity. Alternatively, prior to cyclization, a portion of the linker is covalently attached to the N-terminus or its vicinity, and a portion is attached to the C-terminus or its vicinity. In either case, the linear compound is cyclized, for example, via SS bond cyclization. Therefore, the compound can be cyclized in the following ways: 1) by covalently bonding a peptide bond to the C-terminus or its vicinity at or near the N-terminus of the peptide linker (e.g., cyclizing the backbone); 2) by covalently bonding a side chain within the peptide linker to the N-terminus or C-terminus or its vicinity; or 3) by covalently bonding two side chains within the peptide linker. In this context, "vicinity" is defined as within 1, 2, or 3 amino acid residues from the N-terminus or C-terminus. Preferably, the linker is coupled to the N-terminus or C-terminus.

[0075] As described above, peptides can be cyclized by oxidizing thiol- or sulfhydryl residues (including, for example, cysteine ​​and homocysteine) at their N-terminus or C-terminus or internally. For example, two cysteine ​​residues flanking a peptide can be oxidized to form a disulfide bond. Oxidizing agents that can be used 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 that may be used in such methods. Crosslinking agents are also known in the art and can be selected, for example, based on the functional groups used for crosslinking, see, for example, the crosslinking agent selection tool provided by Thermo Fisher Scientific.

[0076] Therefore, in one embodiment, the linker comprises a functionalizable portion, such as an amino acid having one of the aforementioned functional groups, such as lysine, aspartic acid, glutamic acid, or cysteine, a non-natural amino acid such as azido-homalanine or azido-phenylalanine, or a functionally equivalent molecule such as polyethylene glycol (PEG).

[0077] If the functionalizable moiety is a naturally occurring amino acid, such as lysine, aspartic acid, glutamic acid, serine, threonine, or cysteine, then the functionalizable moiety does not necessarily have to be in the linker; it can also be present in the epitope or in adjacent amino acids within the protein fragment forming the cyclic peptide or within the peptide. Therefore, the cyclization of the TTR peptide can also occur without a linker. Thus, in one embodiment, the cyclic compound present in the vaccine of the present invention is formed from the TTR peptide without a linker. The linking can occur via the side chains of one or more amino acids, such as the thiol moiety of a cysteine ​​residue, the carboxylic acid moiety of an aspartic or glutamic acid residue, the hydroxyl group of a serine or threonine residue, or the amino group of a lysine or arginine residue.

[0078] 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 comprise or consist of any amino acid, including or composed of non-natural amino acids, but preferably comprises at least any of the aforementioned functionalizable moieties, i.e., lysine, aspartic acid, glutamic acid, or cysteine, non-natural amino acids such as azido-homalanine or azido-phenylalanine, or functionally equivalent molecules 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.

[0079] 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), or glycine (G), or serine (S), or selected from alanine (A) and glycine (G), or selected from glycine (G) and serine (S), but preferably glycine (G).

[0080] Even more preferably, the linker amino acid is selected from alanine (A), or glycine (G), or serine (S), or alanine (A) and glycine (G), or glycine (G) and serine (S), preferably glycine (G), and the functionalizable moiety is cysteine ​​(C). Therefore, preferably, cyclization is carried out using a scaffold compound such as an organohalide (preferably an organobromide) that selectively reacts with the thiol group of cysteine, or via a disulfide bridge. Most preferably, cyclization is carried out via a disulfide bridge.

[0081] 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 functionally equivalent molecules, and / or combinations thereof, wherein, when the linker comprises only amino acids, at least one of these amino acids preferably has any of the functional groups described above, preferably cysteine. 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), with glycine (G) being preferred.

[0082] As described above, the length of the linker can vary, for example, it can be 9 amino acids, such as GGGGCGGGG (SEQ ID NO: 27); or 8 amino acids, such as GGGCGGGG (SEQ ID NO: 28), GGCGGGGG (SEQ ID NO: 29) or GCGGGGGG (SEQ ID NO: 30); or 7 amino acids, such as GGGGCGG (SEQ ID NO: 31), GGGCGGG (SEQ ID NO: 32), GGCGGGG (SEQ ID NO: 33) or GCGGGGG (SEQ ID NO: 34); 6 amino acids, such as GGGCGG (SEQ ID NO: 35), GGCGGG (SEQ ID NO: 36) or GCGGGG (SEQ ID NO: 37); 5 amino acids, such as GCGGG (SEQ ID NO: 25) or GGGCG (SEQ ID NO: 26); 4 amino acids, such as GCGG (SEQ ID NO: 27). NO: 38) or GGCG (SEQ ID NO: 39); or 3 amino acids, such as GCG. Most preferably, the linker in the cyclic compound comprises or consists of GCGGG (SEQ ID NO: 25) or GGGCG (SEQ ID NO: 26).

[0083] As described above, the cyclic compound comprises a peptide containing a TTR epitope, most preferably containing the epitope of the amino acid sequence WEPFA (SEQ ID NO: 1) and adjacent amino acids and linkers located at the N-terminus and C-terminus of the peptide, wherein the linkers may in principle contain any of the above-described linker sequences, and preferably contain the amino acid sequences GCGGG (SEQ ID NO: 25) or GGGCG (SEQ ID NO: 26). Therefore, in a preferred embodiment, the cyclic compound comprises or consists of the amino acid sequences H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 4) or H-GCGGGDTWEPFASGKTSGGGCG-OH (TTR39-50cyc; SEQ ID NO: 5), which have been shown as suitable target antigens in Examples 1 and 2.

[0084] In some cases, the immunogenicity of peptides is limited. Techniques for conferring immunogenicity to peptides are well known in the art, including, for example, conjugation with immunogenicity enhancers such as carriers or T helper cell epitopes, or administration in the presence of adjuvants.

[0085] In one embodiment, the TTR peptide contained in the vaccine of the present invention is formulated together with an immunogenicity enhancer, i.e., with a reagent that helps to elicit an immune response against the peptide.

[0086] 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. Proteins such as BSA and / or other reagents may be conjugated to the TTR peptide, for example, to help enhance immunogenicity. Carriers suitable for this purpose are known in the art and include, for example, but not limited to: maltose-binding protein “MBP”, bovine serum albumin (BSA), KLH (keyhole hemocyanin), ovalbumin, flagellin, serum albumin, immunoglobulin molecules, thyroglobulin, ovalbumin, polymers of D- and / or L-amino acids, tetanus toxoid (TT), diphtheria toxoid (DT), genetically modified cross-reactive substance (CRM) of diphtheria toxoid, CRM197, meningococcal outer membrane protein complex (OMPC) and Haemophilus influenzae protein D (HiD), and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Exotoxin A. Various methods for chemically crosslinking peptides with carrier proteins are known in the art and typically involve reactive thiol and / or amino groups. Many of these systems are commercially available (e.g., ThermoFisher Scientific's Imject). TM The most common chemical cross-linking is carried out using thiol groups in the antigen, which can be introduced by adding cysteine ​​residues to the antigenic peptide (e.g., C-(GA)10), or via primary amino groups (e.g., ThermoFisher Scientific's Imject). TM Maleimide and Imject TM EDC products). Furthermore, methods for conjugating peptides with immunogenic enhancers (such as KLH) or carriers (such as BSA) are described in Lateef. et al ., Journal of Biomolecular Techniques 18 (2007), 173-176, the contents of which are incorporated herein by reference.

[0087] In one embodiment, the carrier coupled to the TTR peptide is BSA or KLH, meaning the TTR peptide comprises a carrier and an immunogenic enhancer, such as BSA or KHL. In a preferred embodiment, the carrier coupled to the TTR peptide is BSA, meaning the TTR peptide comprises a carrier and an immunogenic enhancer, such as BSA. The immunogenic enhancer / carrier can be coupled to the peptide directly (e.g., via an amide bond or disulfide bond) or indirectly via a chemical linker. In particular, BSA is covalently coupled using a free amino reaction, which links the free amino group on the peptide to the free amino group on the BSA protein.

[0088] It is known that the use of KLH may lead to the production of large amounts of antibodies against KLH, which is an unwanted side effect and should therefore be avoided. Therefore, in one implementation, the vaccine does not contain KLH as an immunogenic enhancer.

[0089] In one embodiment, the immunogen comprising the TTR peptide and the TTR peptide each comprise a T helper cell epitope. More specifically, the TTR peptide may be linked to a heterologous T helper cell epitope peptide to form a peptide immunogen construct. Optionally, the linking may be performed using a heterologous spacer. The term "heterologous" as used herein refers to an amino acid sequence derived from an amino acid sequence that is not of the wild-type TTR sequence or homologous to it. A heterologous spacer may be any molecule or chemical structure capable of linking two amino acids and / or peptides together, and may include chemical compounds, natural amino acids, non-natural amino acids, or any combination thereof. The heterologous T helper cell epitope may be any T helper cell epitope capable of enhancing an immune response to the TTR epitope. The T helper cell epitope may also have a hybrid binding motif that binds to MHC class II molecules of multiple species, or may contain multiple hybrid MHC class II binding motifs to allow maximal activation of T helper cells, thereby initiating and modulating an immune response. T helper cell epitopes are preferably immune-silencing, meaning that antibodies generated from the TTR peptide immunogen construct are almost (if any) targeting the T helper cell epitope, thus allowing a highly focused immune response directed towards the targeted TTR epitope peptide. T helper cell epitopes may include amino acid sequences derived from foreign pathogens. Several universal T helper cell epitopes are known in the art for this purpose, such as universal T helper cell epitopes derived from tetanus and diphtheria toxins, such as P30 (Deithelm-Okita). et al ., The Journal of Infectious Diseases 181 (2000),1001-1009; Swartz et al ., npj Vaccines 6 (2021), 12.

[0090] Therefore, in one embodiment, the peptide is formulated together with an immunogenic enhancer, preferably linked to a carrier molecule (such as a carrier protein, e.g., BSA) or to a T helper cell epitope, forming a conjugate that facilitates the induction of an immune response against the peptide, preferably such that the induced antibody specifically binds to and clears amyloid-inducing TTRs, i.e., TTR aggregates in the subject's body, thereby inhibiting the formation of TTR deposits, thereby achieving treatment or prevention of the disease.

[0091] Alternatively, the immunogen can be a multiantigen peptide (MAP) containing a TTR peptide. MAP vaccine systems have been developed to avoid the adverse reactions associated with conventional vaccines (i.e., live attenuated pathogens, inactivated pathogens), carrier proteins, and cytotoxic adjuvants. Two main approaches to developing MAP vaccine systems are: (1) adding functional components such as T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic methods using well-defined nanomaterials as antigen display platforms, such as self-assembled peptides, non-peptide dendritic polymers, and gold nanoparticles. The use of MAP systems can improve the sometimes poor immunogenicity of subunit peptide vaccines. In MAP systems, multiple copies of the antigenic peptide simultaneously bind to the α- and ε-amino groups of a lysine-based, non-immunogenic dendritic scaffold, helping to confer anti-degradation stability, thereby enhancing molecular recognition by immune cells and inducing a stronger immune response than a single small antigenic peptide. In some compositions, MAP comprises one or more of the following: lysine-based dendritic scaffolds, helper T cell epitopes, immunostimulatory lipophilic moieties, cell-penetrating peptides, free radical-induced polymerization, self-assembled nanoparticles as antigen presentation platforms, and gold nanoparticles. Therefore, in one embodiment, the TTR peptide contained in the vaccine of the present invention is prepared as a MAP.

[0092] This invention does not include a MAP in which the sequence GGEHAEVVFTAGGGK is synthesized in eight copies on a MAP dendritic core ([fluorenemethyloxycarbonyl(Fmoc)Fmoc-Lys(Fmoc)]4-Lys2-Lys-bAla) and ligated to Wang resin. This MAP is described in Higaki. et al ., Amyloid, 23:2 (2016), 86-97, and described in Bugyei-Twum 2012 (Inhibition of transthyretin fibrillation by using conformation-specific antibodies, a compliant Master of Science thesis submitted to Antoinette Bugyei-Twum, Graduate School of Biochemistry, University of Toronto).

[0093] In one embodiment, the vaccine of the present invention, in addition to the TTR peptide, also comprises an adjuvant. Most vaccines are injected together with an adjuvant to stimulate an immune response. Incorporating an adjuvant into a vaccine formulation aims to enhance, accelerate, and prolong the specific immune response required against the vaccine antigen. Advantages of adjuvants include enhancing the immunogenicity of the antigen, altering 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 implement the present invention generally include any adjuvant known in the art that enhances the immune response to the immunogenic composition, i.e., the vaccine, and / or results in more durable immunity without itself inducing an immune response. Preferred adjuvants allow for a reduction in the amount of immunogenic composition administered to obtain a sufficient immune response. However, the nature of adjuvants can vary considerably. 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 one that does not affect the structure of the peptide, such as its cyclization.

[0094] Suitable adjuvants can be selected and evaluated by those skilled in the art, for example, based on the European Medicines Agency's "Guideline on adjuvantsin vaccine for human use" (EMEA / CHMP / VEG / 134716 / 2004), published on 20 January 2005 and last updated on 16 February 2023. Adjuvants can be administered as a single composition with the immunogen. Alternatively, adjuvants can be administered before, simultaneously with, and / or after the immunogen. Exemplary adjuvants are listed above and below: different groups of adjuvants may be used, namely (i) delivery systems, such as mineral salts, such as aluminum salts (aluminum hydroxide, aluminum sulfate, and aluminum phosphate), emulsions, such as Freund's adjuvant, MF59, or AS03, and microparticles, such as virus-like particles, virions, PLA / PLGA; (ii) immunostimulants, such as TLR1 / 2 agonists, such as L-pampo, MALP-2, Pam2CSK4, and Pam3CSK4; TLR3 agonists, such as Poly(I:C) (polyinosinic acid: polycytidylic acid) Poly-ICLC; TLR4 agonists, such as monophospholipid A (MPL); and TLR5 agonists. Examples include flagellins, TLR7 / 8 agonists such as imiquimod (R837; 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine) and requimod (R848, 4-amino-2-(ethoxymethyl(etoximetil))-a,a-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol), and TLR9 agonists such as CpGODNs, (iii) combination adjuvants such as AS01 and AS02, AS04, and (iv) mucosal adjuvants such as cholera toxin (CT), heat-sensitive enterotoxins (LTK3 and LTR72), chitosan; see review Facciolà et al Vaccines 10 (2022), 819. Some preferred adjuvants include incomplete Freund's adjuvant (IFA) (Montanide ISA-51) or CpG oligodeoxynucleotides (ODNs).

[0095] According to the present invention, the immunogen of the vaccine can be produced by chemical synthesis or using recombinant DNA technology, wherein the TTR peptide disclosed herein can be coupled or not coupled to an immunogenic vector. Corresponding means and methods are well 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, edited by Glick and Patten; ISBN: 978-1-683-67366-8, February 2022; Textbook on Cloning, Expression and Purification of Recombinant Proteins, 2022 Edition, Kakoli Bose; ISBN: 978-981-16-4986-8.

[0096] Therefore, the present invention also relates to nucleic acids encoding the immunogens described herein, preferably in combination with immunogenic enhancers such as immunogenic carriers or adjuvants (e.g., one of those described above).

[0097] Typically, the nucleic acid of this invention is in an expressible form, meaning that when placed in a cell, the immunogen or a portion thereof is expressed by that nucleic acid. In one embodiment, this can be achieved by using a translatable nucleic acid (such as mRNA, for example, for in vitro translation) or by incorporating the nucleic acid into an expression vector capable of expressing that nucleic acid, which is also part of this invention.

[0098] In another embodiment, the present invention relates to a host cell comprising the nucleic acid or expression vector of the present invention, and the use of said nucleic acid and expression vector for producing an immunogen in the cell. The cell may be, for example, a bacterial, yeast, or mammalian cell.

[0099] In one embodiment, the immunogen can be purified from a cell culture and formulated into a vaccine, optionally including the step of conjugating the immunogen (e.g., a TTR peptide) with an immunogenic enhancer (such as an immunogenic carrier and / or adjuvant). Therefore, the method of recombinantly producing an immunogen by expressing and isolating it from a host cell is another aspect of the invention. Any suitable host cell can be used, such as prokaryotic or eukaryotic cells; in specific embodiments, the host cell is selected from the group consisting of bacteria, fungi (including yeast), and mammalian cells.

[0100] 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 immunogenic enhancer such as an immunogenic vector), an expression vector containing said nucleic acid, a host cell containing said nucleic acid or expression vector, and / or the immunogen of the present invention, and optionally at least one immunogenic enhancer (such as an immunogenic vector, if applicable) and / or adjuvant.

[0101] The present invention also relates to a kit containing the vaccine of the present invention, and optionally includes means for administering the vaccine and / or instructions, such as dosage recommendations and / or a syringe for administering the vaccine. In a preferred embodiment, the kit contains the vaccine of the present invention in a pharmaceutical container, such as a pre-filled vial or syringe.

[0102] Furthermore, a method is disclosed for preparing a vaccine composition for inducing an immune response in an organism (particularly an animal or human affected by ATTR or a healthy organism at risk of developing ATTR, thus requiring such treatment), for the prevention, treatment, or mitigation of the effects of ATTR, said method comprising formulating the TTR peptide according to the invention into a pharmaceutically acceptable form. Pharmaceutically acceptable carriers for vaccine formulation are known in the art.

[0103] As described above, the vaccine of the present invention can be used to prevent ATTR in subjects by triggering an immune response, e.g., by inducing anti-TTR antibodies capable of binding to and clearing amyloid-inducing TTRs (i.e., TTR aggregates). Anti-TTR antibodies have been shown to be used to treat ATTR, such as ATTR-CM; see Garcia-Pavia. et al ., 2023, same as above. Garcia-Pavia et al The described results further highlight the usefulness of anti-TTR antibodies in the treatment of musculoskeletal disorders, because, as Figure 2 As shown, TTR deposits in the shoulder joint were cleared during treatment with antibody NI006. Therefore, the vaccine of the present invention may also be useful in treating ATTR, particularly in early ATTR in subjects. Therefore, the present invention also relates to the vaccine and kit of the present invention for the prevention or treatment of ATTR (e.g., ATTR cardiomyopathy (ATTR-CM), ATTR polyneuropathy (ATTR-PN), particularly ATTR-CM) and musculoskeletal diseases or conditions in subjects, wherein the latter are preferably associated with TTR deposits in the joint, most preferably selected from the group consisting of osteoarthritis, carpal tunnel syndrome, joint pain, shoulder pain, amyloid arthritis, lumbar spinal stenosis, biceps tendon rupture, trigger finger, and rotator cuff disease, preferably wherein the musculoskeletal disease or condition is osteoarthritis or amyloid arthritis, preferably amyloid arthritis.

[0104] The immunization process using the vaccine of this invention can be monitored by detecting antibody titers in plasma or serum. The immunogen can be used as the antigen, and antibody levels can be assessed using standard ELISA or other immunoassay procedures. Following immunization, antiserum can be obtained, and polyclonal antibodies can be isolated from the serum if necessary; see also Example 3.

[0105] ATTR includes two subtypes—wild-type ATTR (ATTRwt) and variant ATTR (ATTRv). Cardiac TTR deposits caused by ATTRwt are found in 10%-15% of individuals aged 65 and older. An epitope containing the amino acid sequence WEPFA (SEQ ID NO:1) can be found in TTRwt; 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 test negative for the TTR mutation gene.

[0106] The present invention also 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 wild-type TTR accumulation, the method comprising administering an effective amount of the vaccine of the present invention to a subject. Administering may be performed using known techniques, such as via intravenous, intramuscular, intradermal, or subcutaneous routes.

[0107] The vaccine of this invention can be administered to patient populations at risk of developing ATTR, such as the elderly, or individuals known to carry known ATTR-promoting mutations and thus considered "high-risk." More specifically, subjects eligible for treatment include individuals at risk but asymptomatic, as well as patients currently exhibiting symptoms, including treatment-naïve subjects who have not previously received treatment for the disease. Subjects at risk include the elderly and asymptomatic subjects with a known genetic risk of the disease. Such individuals include those with a family history of the disease and those whose risk has been determined through genetic or biochemical marker analysis.

[0108] In preventative applications, the vaccine of the present invention can be administered to subjects susceptible to ATTR or at other risk for other diseases. The administration regimen (dosage, frequency, and route of administration) effectively reduces the risk of at least one disease sign or symptom, lessens its severity, or delays its onset. In particular, the regimen effectively inhibits or delays the formation of amyloid-inducing TTR.

[0109] In therapeutic applications, the vaccine of the present invention can be administered to subjects suspected of having ATTR or patients who already have ATTR, and the administration regimen (dosage, frequency, and route of administration) effectively improves or at least inhibits the further deterioration of at least one disease sign or symptom. In particular, the regimen preferably effectively reduces or at least inhibits the further increase in ATTR levels.

[0110] The regimen is considered to have a therapeutic or preventative effect if the treated individual achieves a more favorable outcome compared to the average outcome of a comparable control group of subjects not treated using the method of the present invention, or if, in a controlled clinical trial, the treated subject shows a more favorable outcome compared to the control subjects. The effective dose varies depending on many different factors, such as the method of administration, target site, patient's physiological state, whether other drugs are administered, and whether the treatment is preventative or therapeutic.

[0111] This specification incorporates numerous references. All cited references (including bibliographic references, granted patents, published patent applications, background sections cited in full in this application, manufacturer's instructions, user manuals, etc.) are hereby expressly incorporated by reference; however, it is not acknowledged that any cited reference is prior art to this invention.

[0112] A more complete understanding can be obtained by referring to the following specific embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0113] Example Example 1: TTR peptide displays a novel epitope of TTR amyloid protein To test the immunogenic potential of the TTR peptide, namely its ability to elicit protective antibodies (which requires the peptide to bind to the antibody), corresponding binding studies were initially conducted.

[0114] In particular, the ability of anti-TTR antibodies to bind to TTR peptides has been exemplarily evaluated using an ELISA assay. A cyclic peptide containing amino acid residues 34 to 54 of wild-type TTR (TTR34-54cyc, biotinylated and non-biotinylated forms) and a linear peptide in its corresponding biotinylated form (TTR34-54) were used as target antigens, and antibody NI-301.37F1 was used as the anti-TTR antibody. Furthermore, TTR peptides TTR40-49 and misfolded wild-type TTR (mis.WT-TTR) were used as antigen controls.

[0115] The cyclic peptide TTR34-54cyc (1.36 mg / mL) was manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. Specifically, a peptide containing the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 4) was synthesized via solid-phase peptide synthesis and cyclized via a disulfide bridge between two cysteine ​​residues within a polyglycine fragment. 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 aminocaproic acid (Ahx) spacer between their N-terminus and biotin residues, 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)).

[0116] Wild-type TTR protein purified from human plasma was obtained from Bio-Rad Laboratories, Inc. (California, USA; 7600-0604) and custom-purified by protein A / G chromatography followed by lectin column purification to remove residual immunoglobulins. Plasma-purified wild-type TTR was provided in 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 aggregation buffer (50 mM acetate-HCl, 100 mM KCl, 1 mM EDTA, pH 3.0) and then incubating with shaking at 1000 rpm for 4 hours at 37°C. mis.WT-TTR was aliquoted and stored at -20°C until use. The quality of mis.WT-TTR was confirmed by ELISA and biolayer interference (BLI) technique.

[0117] Three ELISA tests were performed. In the first ELISA test (ELISA-1), the binding of the antibody to peptides TTR34-54cyc and mis-WT-TTR was analyzed. In the second ELISA test (ELISA-2), the binding of the antibody to peptides TTR34-54_bt and TTR34-54cyc_bt, as well as the BSA control, was analyzed. In the third ELISA test (ELISA-3), the binding of the antibody to peptides TTR34-54cyc and TTR40-49 was analyzed.

[0118] 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, with each target antigen diluted to a concentration of 10 µg / ml in PBS buffer (pH 7.4). Non-specific binding sites were blocked for 1 hour at room temperature (RT) with blocking buffer containing 2% (w / v) bovine serum albumin (BSA) and 0.1% polysorbate 20 (pH 7.4) in 1×PBS buffer. The NI-301.37F1 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted twice in blocking buffer at the indicated concentration (dilution series from 400 nM to 4 pM and 0) and incubated overnight at 4°C. Binding was determined using an anti-human IgG antibody conjugated to horseradish peroxidase (HRP), followed by HRP activity measurement using a standard colorimetric assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA). Data were analyzed using Prism software in GraphPad. EC was estimated for each data point using a nonlinear regression fitted with a log(agonist) response model with a variable slope. 50 Values. The least squares regression method was used for data fitting.

[0119] ELISA results showed that antibody NI-301.37F1 could bind to the TTR peptide. ELISA-1 assays showed that the binding of NI-301.37F1 to the cyclic TTR34-54cyc peptide was much stronger than its binding to mis.WT-TTR, approximately 10 times stronger. Specifically, in ELISA-1, the ECGs of NI-301.37F1 binding to the cyclic TTR34-54cyc peptide... 50 The EC value is 0.022 nM, and the combination of NI-301.37F1 and mis.WT-TTR is used for EC. 50 It is 0.19 nM; see Figure 1 A. ELISA-2 assays further revealed that NI-301.37F1 can bind to both linear and cyclic TTR peptides, but cyclization increased NI-301.37F1 binding by more than 100-fold. Specifically, in ELISA-2 assays, NI-301.37F1 bound to the cyclic TTR34-54cyc_bt peptide at EC50... 50 The EC50 of NI-301.37F1 combined with linear TTR34-54_bt is 0.11 nM. 50 It is 19.5 nM; see Figure 1 B. No binding of NI-301.37F1 to TTR40-49 was observed in the control ELISA assay, i.e., ELISA-3; see [link to ELISA test]. Figure 1 C.

[0120] Example 2: BSA-conjugated TTR peptide retains novel epitopes of TTR amyloid-specific antibodies. In typical peptide vaccination regimens, the peptide containing the epitope of interest is conjugated to a carrier protein. This conjugation enhances the immune response by increasing the epitope's half-life, for example, by reducing renal clearance and sensitivity to proteolytic degradation. Therefore, further ELISA assays were performed to evaluate the ability of an anti-TTR antibody (NI-301.37F1, used here as an example) to bind to a BSA-conjugated TTR peptide.

[0121] In particular, the ability of anti-TTR antibodies to bind BSA-conjugated TTR peptides was exemplarily evaluated by ELISA assay, using BSA-conjugated cyclic peptides containing amino acid residues 34 to 54 of wild-type TTR (TTR34-54cyc_BSA), BSA-conjugated cyclic peptides containing amino acid residues 39 to 50 of wild-type TTR (TTR39-50cyc_BSA), and corresponding BSA-conjugated linear peptides (TTR34-54_BSA and TTR39-50_BSA) as target antigens, and antibody NI-301.37F1 as the anti-TTR antibody. Furthermore, as antigen controls, BSA-conjugated disordered cyclic and linear peptides (TTR34-54SCRcyc_BSA, TTR39-50SCRcyc_BSA, TTR34-54SCR_BSA, and TTR39-50SCR_BSA) were used.

[0122] The cyclic peptides coupled with 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. Specifically, 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 via solid-phase peptide synthesis and cyclized through a disulfide bridge between two cysteine ​​residues within a polyglycine fragment. Furthermore, BSA was coupled to the cyclic peptides using a divalent free amino coupling agent. 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.

[0123] Misfolded wild-type TTR aggregates were prepared as described in Example 1.

[0124] Two ELISA assays were performed. In the first ELISA assay (ELISA-1), the binding of the antibody to peptides TTR34-54cyc_BSA, TTR34-54_BSA, TTR34-54SCRcyc_BSA, and TTR34-54SCR_BSA was analyzed. In the second ELISA assay (ELISA-2), the binding of the antibody to peptides 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.

[0125] ELISA results showed that antibody NI-301.37F1 could bind to the BSA-conjugated TTR peptide. ELISA-1 assay showed that antibody NI-301.37F1 had a high binding affinity (ECG) to the cyclic BSA-conjugated TTR34-54cyc_BSA peptide. 50 Value: 3.8 nM), and it also shows that the antibody can bind to the corresponding BSA-conjugated linear peptide (TTR34-54_BSA) (EC). 50 Value: >400 nM). Cyclation increased NI-301.37F1 binding by more than 100-fold. No binding was observed with the antigen control (i.e., with the disordered peptide); see [link to relevant documentation]. Figure 2 A. ELISA-2 assay showed binding to the TTR39-50cyc_BSA peptide conjugated to cyclic BSA (EC). 50 Value: >400 nM), but almost no binding was detected with the corresponding linear peptide. No binding was observed with the antigen control (i.e., with the out-of-order peptide). Therefore, TTR39-50 is approximately the minimum peptide length required for NI-301.37F1 binding; see [link to relevant documentation]. Figure 2 B.

[0126] Example 3: Immunogenic potential of TTR peptides in mammals The immunogenic potential of TTR peptides has been validated in in vivo mouse studies, demonstrating that immunization with TTR peptides triggers a highly selective immune response against amyloid-inducing TTRs. Specifically, laboratory animals were immunized with a target peptide conjugated to the carrier protein BSA (using an amine-reactive cross-linking agent), the peptide conformation of which resembled that present in TTR amyloid fibrils. Following repeated injections of the immunogenic peptide, the immune response was monitored by serum titers to characterize its ability to specifically bind to the target protein of the amyloid conformation.

[0127] immunity Five groups of BalbC mice, six in each group (30 mice in total), were injected with BSA-conjugated TTR peptides (linear or cyclic conformation). Peptides of 12 and 21 amino acid lengths in cyclic and linear conformations were used to trigger a specific immune response against ATTR (groups 1–4). An unrelated peptide (referred to herein as peptide PR906) was used as a control to characterize response selectivity.

[0128] Mice were subcutaneously injected with the peptide in three consecutive doses, approximately two weeks apart. The antigen was mixed with RIBI adjuvant for the first and second injections. Pre-injection blood was injected without adjuvant. Blood samples were collected before the first injection (day 0) and 38 days later (after the third injection). Antigen-specific titers against the peptide antigen and ATTR were determined by ELISA.

[0129] List of antigens and immune groups Group 1 TTR34-54_BSA Group 2 TTR34-54cyc_BSA Group 3 TTR39-50_BSA Group 4 TTR39-50cyc_BSA Group 5 PR906 Immune response characterization Immunoreactivity monitoring (serum titer monitoring) and immunoreactivity characterization were performed using ELISA targeting WT-TTR and mis.WT-TTR (also known as 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 with BSA-free blocking buffer at room temperature for 1 hour. Serum samples were serially diluted in PBS from 1:100 to 1:5,904,900, transferred to the corresponding ELISA plates, and incubated at room temperature for 1 hour. After washing, antibodies binding to the immunogen were detected using HRP-conjugated anti-mouse IgG secondary antibody at a dilution of 1:20,000.

[0130] Immune responses were monitored on days 0 and 38, after which mice were sacrificed and blood samples were collected. Each serum sample was tested to determine the immune response rate for each immunogen.

[0131] like Figure 3 As shown, all administered TTR peptides induced antibody formation against both ATTR and TTR, with the highest OD detected in mouse serum samples collected after a 38-day incubation period. 450Furthermore, it was evident that the levels of ATTR-specific antibodies in the serum of all mice were significantly higher than those of TTR-specific antibodies. Therefore, after immunization of BalbC mice with the corresponding TTR peptide, serum antibody titers increased over time (day 0 vs. day 38), and serum reactivity to mis.WT-TTR / ATTR was higher than that to WT-TTR. Specifically, the serum reactivity to TTR obtained after immunization of mice with the peptide / immunogen TTR39-50 was [evaluated / statistically]. 50 The EC50 value was 1038, and the ATTR value was 15271, therefore the amyloid selectivity (EC50) was high. 50(ATTR / TTR) The quotient is 14.7; see Figure 3 A and Table 1. Serum reactivity (EC50) of mice immunized with peptide / immunogen TTR39-50cyc to TTR. 50 The EC50 value is 1813, and the ATTR value is 24343, therefore the amyloid selectivity (EC50) is high. 50(ATTR / TTR) The quotient is 15.4; see Figure 3 B and Table 1. Serum reactivity (EC50) of mice immunized with peptide / immunogen TTR34-54 to TTR. 50 The EC50 value is 3400, and the ATTR value is 53809, therefore the amyloid selectivity (EC50) is high. 50(ATTR / TTR) The quotient is 15.8; see Figure 3 C and Table 1. Serum reactivity (EC50) of mice immunized with peptide / immunogen TTR34-54cyc to TTR. 50 The EC value is 7785, and the ATTR value is 33587, therefore the amyloid selectivity (EC) is high. 50(ATTR / TTR) The quotient is 4.3; see Figure 3 D and Table 1.

[0132] Table 1: Serum reactivity and amyloid selectivity after immunogen administration

[0133] Figure 4 This was also confirmed by ELISA results obtained using a serum dilution of 1:23,300. Specifically, the study showed that serum obtained after immunizing BalbC mice with TTR peptides TTR39-50, TTR39-50cyc, TTR34-54, and TTR34-54cyc exhibited significantly higher responsiveness to ATTR than to TTR (by the corresponding OD). 450 (Values ​​are expressed as values). Furthermore, the highest antibody titers were detected in the serum of mice immunized with the linear peptide TTR34-54.

[0134] In summary, immunization of mice with the above-mentioned TTR peptides (including the carrier protein, BSA in this case) triggered an immune response, whether linear or cyclic, and resulted in the production of anti-TTR antibodies that exhibit high selectivity for amyloid-inducing TTRs.

Claims

1. A vaccine comprising an immunogen, said immunogen comprising a TTR peptide derived from transthyretin (TTR), said TTR peptide comprising a neoepitope selectively presented or exposed in a misfolded, oligomeric and / or aggregated form of TTR.

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

3. The vaccine according to claim 1 or 2, wherein said TTR peptide and 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 said TTR peptide comprises at least 4 amino acid residues from the TTR amino acid sequence Lys35 to Gly57.

5. The vaccine according to any one of claims 1 to 4, wherein said 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 said 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 said TTR protein.

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

3.

8. The vaccine according to any one of claims 1 to 7, wherein said TTR peptide comprises a linker covalently coupled to the N-terminal residue and the C-terminal residue of said peptide to form a cyclic compound.

9. The vaccine according to claim 8, wherein said linker comprises or consists of 1-8 amino acids and / or one or more functionalizable moieties, preferably wherein said linker amino acids are selected from glycine (G) or alanine (A), and / or wherein said functionalizable moiety is cysteine (C), most preferably wherein said 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 said TTR peptide has the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO:

5.

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

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

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

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

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

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

17. Use of the nucleic acid of claim 14 or the expression vector of claim 15 in the production of the immunogen in a cell.

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

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

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

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

22. Use of the vaccine of any one of claims 1 to 13, the kit of claim 20 or 21, or the composition of claim 18 or 19 in a method of treating or preventing transthyretin amyloidosis (ATTR) in a subject.

23. Use of the vaccine of any one of claims 1 to 13 or 22, the kit of any one of claims 20 to 21, or the composition of claim 18 or 19 in a method of treating or preventing a musculoskeletal disease or disorder in a subject.

24. Use of the vaccine, kit or composition of claim 22 or 23, wherein the subject has sporadic wild-type transthyretin-mediated amyloidosis cardiomyopathy (ATTRwt-CM), and / or wherein the subject tests negative for a TTR mutation gene.

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

26. A method of treating or preventing a musculoskeletal disease or disorder, the method comprising administering to a subject in need thereof the vaccine of any one of claims 1 to 13, or the composition of claim 18 or 19.

27. The method of claim 25 or 26, wherein the subject has sporadic wild-type transthyretin-mediated amyloidosis cardiomyopathy (ATTRwt-CM), and / or wherein the subject tests negative for a TTR mutation gene.

28. Use of the vaccine of any one of claims 1 to 13, or the composition of claim 18 or 19, in the manufacture of a medicament for treating or preventing transthyretin amyloidosis (ATTR) in a subject.

29. Use of the vaccine of any one of claims 1 to 13, or the composition of claim 18 or 19, in the manufacture of a medicament for treating or preventing a musculoskeletal disease or disorder in a subject.

30. The use of claim 28 or 29, wherein the subject has sporadic wild-type transthyretin- mediated amyloidosis cardiomyopathy (ATTRwt-CM), and / or wherein the subject has a negative TTR mutation gene test.

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