Multitarget chimeric protein for immunotherapy of alzheimer's disease

Abstract

The present invention is related to the biomedical and biopharmaceutical sectors. Specifically, it relates to a chimeric antigen comprising the combination of the amino and carboxyl terminal regions of the amyloid beta peptide (Aβ), the amino and carboxyl terminal regions of the tau protein and a T cell epitope. The pharmaceutical composition comprising this chimeric antigen and at least one pharmaceutically acceptable vaccine adjuvant increases the efficacy of immunotherapy for the prevention and treatment of Alzheimer's Disease (AD). The chimeric antigen exerts its action by stimulating a multitarget humoral response with high titers of anti-Aβ and anti-tau antibodies simultaneously. This favors the combined elimination of toxic species of both Aβ and tau from the brain, which prevents or significantly improves the clinical symptoms and neuropathology of AD.

Description

[0001] Multi-target chimeric protein for immunotherapy of Alzheimer's disease

[0002] DESCRIPTION

[0003] The present invention relates to the biomedical and biopharmaceutical sectors. Specifically, it relates to a chimeric antigen comprising a combination of B-cell epitopes and the carboxyl-terminal region of the amyloid beta peptide (Ap), the amino- and carboxyl-terminal regions of the tau protein, and a T-cell epitope. The pharmaceutical composition comprising this chimeric antigen and at least one pharmaceutically acceptable vaccine adjuvant increases the efficacy of immunotherapy for the prevention and treatment of Alzheimer's disease (AD).

[0004] AD is the most common form of dementia, the leading cause of disability in adults over 65 years of age (Tahami Monfared et al., Neurology and therapy, 2022), and the sixth leading cause of death worldwide (Rayathala et al., Journal of Innovations in Applied Pharmaceutical Science, 2022:7: 28-31). It is a multifactorial neurodegenerative disease for which there is currently no effective treatment. The hallmarks of AD are the accumulation of amyloid beta (Ab) plaques and neurofibrillary tangles in the brain. Although the exact molecular mechanisms that trigger this pathology are not known, most experts agree that it begins with the accumulation of Ab peptides due to an imbalance between their production and clearance from the brain (Chen et al., Genes, Environment and Alzheimer's Disease, 2016).This process of Ap plaque formation is associated with multiple neuropathological mechanisms, including hyperphosphorylation of the tau protein and the subsequent formation and accumulation of paired helical filaments inside neurons. Protein aggregates of both Ap and tau synergistically trigger a cascade of events that lead to neurite dystrophy, asotrocytosis, altered ionic homeostasis, oxidative stress, and synapse loss, which together cause progressive neuronal death (Winblad et al., Alzheimer's Research and Therapy, 2014:6: 1-12, Davtyan et al., Alzheimer's Research & Therapy, 2019:11: 107)(Roda et al., Neural Regeneration Research, 2022:17: 1666-74).

[0005] The U.S. Food and Drug Administration (FDA) has approved very few medications for the treatment of AD. These include donepezil, rivastigmine, and galantamine, which are acetylcholinesterase inhibitors; memantine, which is a partial antagonist of the N-methyl-D-receptor.

[0006] SUBSTITUTE SHEET (RULE 26) aspartate, and the combination of donezepil with memantine. All of these drugs have limited effectiveness and do not reverse disease progression or improve cognitive dysfunction. They only delay the deterioration of symptoms for a limited period of time (Vassilakopoulou et al., Vaccines, 2021:9: 1 -27). Two disease-modifying drugs were recently approved by the FDA: Aducanumab and Lecanemab. Both are monoclonal antibodies that target aggregated Ap species. Both are controversial due to the high incidence of ARIAS in patients or insufficient evidence of efficacy.

[0007] In this context, the development of new disease-modifying therapies for AD remains an urgent need. Most of the disease-modifying drugs currently in development have as their main targets Ap peptides or the Tau protein (Cummings et al., Alzheimer's and Dementia: Translational Research and Clinical Interventions, 2021:7: 1-24). Immunotherapy is one of the most extensively studied strategies whose mechanism of action is to promote the clearance of malformed proteins from the brain, mediated by antibodies (Yiannopoulou & Papageorgiou, Journal of Central Nervous System Disease, 2020:12: 1-12).

[0008] Passive immunotherapy is one of the most active strategies (Cummings et al., Alzheimer's and Dementia: Translational Research and Clinical Interventions, 2021:7: 1- 24). However, it has several drawbacks, such as a high incidence of adverse reactions (amyloid-related imaging abnormalities, ARIAs), frequent administrations, high doses, and high production costs. Finally, there are still no conclusive reports on its clinical efficacy.

[0009] Active immunotherapy, on the other hand, is one of the most promising strategies and the best positioned for preventive intervention (Cacabelos, Expert Opinion on Drug Discovery, 2019:15: 1-6). It has advantages over passive immunotherapy because the production of specific antibodies is specific to the immunized patient and is therefore polyclonal, making it potentially multi-target. Furthermore, a sustained antibody response can be induced with a small number of immunizations. Recently, a meta-analysis on the efficacy and safety of immunotherapy in clinical trials showed that ARIAs are significantly less frequent in vaccinated patients than in patients treated with monoclonal antibodies (Foroutan et al., Clinical and Investigative Medicine, 2019:42: E53-65).However, because the progression of AD is closely linked to the aging process, active immunotherapy faces the challenge of developing strategies that stimulate the production of high antibody titers even in the context of the immunocenescence processes that occur in these elderly patients.

[0010] The efficacy of active immunization against the Ap peptide was first tested clinically with the AN1792 vaccine, which consists of a synthetic Ap1-42 peptide (Winblad et al., Alzheimer's Research and Therapy, 2014:6:1-12). However, phase II clinical trials showed that only 19.7% of patients with mild to moderate AD developed an anti-Ap antibody response (Vellas et al., Current Alzheimer Research, 2009:6:144-51). The emergence of ARIAs forced the discontinuation of this trial. These adverse events observed in patients treated with AN-1792 were attributed to the induction of a specific TH-1 (T-helper type 1) cell response generated by T cell epitopes contained in the synthetic Ap1-42 peptide that are generally located in its central region. However, follow-up of patients who responded to treatment revealed that approximately 4.Six years later, they maintained an anti-AN-1792 antibody response. This response, although low titer, was persistent despite receiving only 1-3 of the initially planned injections in the phase II study. Furthermore, the progression of cognitive decline was significantly reduced compared to the placebo-treated patient group, indicating that active immunotherapy may have long-term benefits (Vellas et al., Current Alzheimer Research, 2009 :6: 144-51 ).

[0011] In order to avoid a T-cell response, the second generation of vaccines was based on regions of the N-terminal end of Ab, which mainly contain B-cell epitopes. These strategies are based on administering short Ab peptides, fragmented peptides or mimetic peptides to activate the patient's immune system. They are usually conjugated to a carrier, such as virus-like particles or KLH (keyhole limpet hemocyanin), and are administered with an adjuvant to promote stimulation of the immune response (Winblad et al., Alzheimer's Research and Therapy, 2014:6: 1-12). With this new approach, several candidates have reached the clinical development stage, such as CAD106, ACC 001, AD01 -03, ACI-24, UB311, Lu AF20513 and ABvac40. In contrast, active immunotherapy targeting tau protein as a therapeutic strategy for AD is little explored.Epitope selection is more complex due to the flexibility of this protein and the abundance of post-translational modifications suffered by both normal and pathological forms (Novak et al., Frontiers in Neuroscience, 2018:12: 1-14). Furthermore, it is not yet known which of its conformations is the most toxic (Meeting & Bu, ALZFORUM, 2020 1-7). Approaches to such selection vary from domains of the tau protein to specific phospho-epitopes. Clinical studies carried out to date are limited; results have only been presented on the candidate AADvad , so there is little evidence of its efficacy in the treatment of AD.

[0012] All of these anti-AP and anti-tau vaccine candidates have generally been successful in inducing specific antibody responses and have acceptable safety profiles, demonstrating the feasibility of active immunotherapy as a promising approach to treating AD. However, none of them has conclusive evidence of their efficacy in halting the progression of cognitive decline; and in some cases, such as AD02 and CAD106, worsening of symptoms has been observed compared to placebo (Francesco et al., Nature Reviews Neurology, 2019:2: 73-88). They all have in common that they are focused on a single therapeutic target, but experimental evidence indicates that AD symptoms appear in the presence of both characteristics: AP accumulation and loss of tau function (Zampar & Wirths, Peptides in Alzheimer's Disease, 2020, 23-50).This, coupled with the multifactorial and heterogeneous nature of this pathology, suggests that a multitarget strategy would have a greater impact on disease progression.

[0013] The solutions of combinations of Ap and tau epitopes that have been reported so far have not yet advanced to clinical stages, nor has their efficacy in humans been evidenced. These are described in patents US 201801539733 A1 , EP2883955 B1 , WO 2021236809 A2 and WO 2022032166. The first of these consists of a DNA vaccine composed of four Ap1-42 repeats fused to four repeats of the tau379-408 segment. Although no adverse effects were reported in mice, this candidate has the risk of inducing a pro-inflammatory cellular response because it contains the Th-1 cell epitopes of Ap in its structure (Orgogozo et al., Neurology, 2003:61:46-54).On the other hand, one of the main concerns regarding DNA vaccines is the possibility that the injected DNA could integrate into the cell's chromosomes and have no effect, or that cancer could develop in the long term due to the modifications it inflicts on the host cell's DNA. There is also a risk of developing antibodies against the DNA itself rather than the designed target (Hasson et al., Asian Pacific Journal of Tropical Biomedicine, 2015, p. 5: 344–53). The safety of this approach has not yet been demonstrated in humans.

[0014] The second proposal is a recombinant antigen consisting of three copies of the Ap1-11 fragment and three of tau2-18 based on the MultiTEP platform with the configuration 3Ap1-11-Mult¡TEP-3tau2-18 (EP2883955 B1 ). However, in animal models the anti-tau titers were significantly lower after treatment with 3Ap1-11-Mult¡TEP-3tau2-18 compared to treatment with 3tau2-18-Mult¡TEP or the mixture 3Ap1-11-MultiTEP and 3tau2-18-Mult¡TEP. These results, in combination with in silico studies of the three-dimensional structure of the antigens, suggested that tau epitopes were unfavorably presented on the surface of the construct proposed in this work (Davtyan et al., Scientific Reports, 2016:6:28912).Thus, the design of chimeric proteins that fuse ATP and tau epitopes into a single molecule requires strategies that allow their exposure on the surface of the three-dimensional structure so that they can be efficiently recognized by the immune system. Finally, this antigen contains a small segment of tau (tau2-18), which is not specific to the pathological forms of this protein, so the response it induces is unlikely to guarantee the effectiveness of the treatment.

[0015] The remaining two proposals are very similar in design (WO 2021236809 A2 and WO 2022032166). Both are based on the fusion of a copy of the Ap1-10 segment with a copy of the tau244-372 segment (a microtubule-binding motif). The difference between them lies in the addition of an additional peptide from α-synuclein to this antigen, a key protein in the pathogenesis of Parkinson's disease. Both are based on the classic B epitope region of the Ap peptide, which has been used in most immunotherapeutic strategies that have reached clinical stages, and which, on its own, has not been shown to be successful in halting the progression of the disease. Regarding the tau protein, both are based on a region that is characterized by being variable and poorly conserved in the six isoforms of the protein, so that both strategies limit their scope to only a portion of the species involved in the pathology.

[0016] The four multi-epitope proposals described have in common that none include specific fragments of the pathological forms of the Ap peptide, such as the C-terminal fragment of Ap42 and the phosphorylated forms of the tau protein, which are key in the development of the disease. Furthermore, considering that the phosphorylations of pathological tau have a heterogeneous profile that depends on the different stages of AD and the brain regions of the patients, it would be more effective if the vaccine candidates focused on the phosphorylated sites and included a greater number of key epitopes simultaneously or highly conserved immunogenic segments in the 6 isoforms. All of these elements could lead to treatment with these antigens not having sufficient effect on the elimination of the most toxic species and therefore having limited efficacy.

[0017] To date, there is no vaccine candidate whose antigen is composed of a single protein molecule that combines epitopes from both Ap and tau proteins, which is guaranteed to stimulate a multi-target response with high antibody titers, even in the context of immunosenescence in patients, and which can prevent or halt the progression of Alzheimer's disease.

[0018] The present invention solves the problem previously stated by providing an antigen composed of a chimeric protein comprising the combination of the amino and carboxyl terminal regions of the amyloid beta peptide (Ap1-42), the amino and carboxyl terminal regions of the tau protein and a T cell epitope. The pharmaceutical composition comprising this chimeric antigen and at least one pharmaceutically acceptable vaccine adjuvant increases the efficacy of AD immunotherapy.

[0019] The antigen of the present invention constitutes a unique construct, the design of which was based on the selection of segments with immunogenic potential from the Ap peptide and the tau protein. From the Ap peptide, the residues comprised in the Ap1-12 fragment of the N-terminal end were included because this is a B cell epitope in humans (Geylis et al., Neurobiol Aging. 2005: 26(5):597-606) and does not contain proinflammatory epitopes (Maier M et al., J Neurosci. 2006:3;26(18):4717-28). Also included were the residues comprised in the Ap28-42 fragment, of which there are no reports to date of its use as an antigen in vaccine candidates for AD. In addition, this segment confers specificity to the antigen against the more aggregative Ap1-42 isoform. The Ap13-27 segment was excluded from the construct because it contains T cell epitopes responsible for proinflammatory adverse effects.

[0020] The tau fragments selected to form the antigen comprise residues 1-48 and 320-441 because they are potentially successful in stimulating an effective immune response against AD and do not promote an exacerbated cellular inflammatory response (Selenica et al., Journal of Neuroinflammation 2014:11:152). The tau segment 1-48 is a conserved segment in the six isoforms of the tau protein (Wang and Mandelkow, Nature Reviews / Neurosciences, 2016:17:5-21). In addition, it contains the regions between amino acids 9-15 and 21-27 at the N-terminus that are highly immunogenic (Selenica et al., Journal of Neuroinflammation 2014:11:152). On the other hand, the fragment comprising tau residues 319–441 is conserved across all six isoforms and contains the highly immunogenic region 427–438 (Selenica et al., Journal of Neuroinflammation 2014:11 :152). It also includes serines 396, 404, and 422 (S396, S404, and S422), which are typically phosphorylated in AD.Phosphoserine 422 (pS422) is specific for pathological tau and is also associated with other neurodegenerative disorders (Troquier et al., Curr Alzheimer Res, 2012:9(4) :397-405). Regions 199-441 and 1 -368 were discarded because the segment between amino acids 274-315 may promote tau protein aggregation (Wang and Mandelkow, Nature Reviews / Neurosciences, 2016:17:5-21 ; Simic et al., Biomolecules, 2016:6(1 ):6), which is a drawback for the production of vaccine antigens. Furthermore, protein aggregates would cause the relevant epitopes to not be exposed to be recognized by the immune system. Tau fragments 1-198, 199-441 and 1-368 were also discarded because they have the disadvantage that they are not conserved for the six isoforms of this protein (Wang and Mandelkow, Nature Reviews / Neurosciences, 2016:17:5-21) and all of them are relevant in the development of AD (Buée et al., Brain Research Reviews, 2000:33:95-130).

[0021] In one embodiment of this invention, the chimeric antigen comprises the arrangement of tau segments at the N- and C-termini of the chimera such that the antigen retains their positions in native tau protein. The two Aβ segments are located in the central region of the chimera. A T cell epitope is included between the two Aβ segments. In one embodiment of this invention, the N- and C-terminal regions of the Aβ peptide comprise 10-15 residues of the Aβ1-16 and Aβ28-42 fragments, respectively; and the N- and C-terminal regions of the tau protein comprise 40-121 residues of the tau1-48 and tau320-441 fragments; and the T cell epitope is a segment of Tetanus Toxoid (TT).In one embodiment of this invention, the serine residues comprised in the tau fragment 320-441 corresponding to the sites that are typically phosphorylated in AD are substituted by glutamic acid to simulate post-translational phosphorylation, which guarantees the incorporation of the negative charge that the phosphate group would supply to the molecule, in addition to a larger radical group to favor the adoption of the characteristic tertiary structure of the phosphonated site. All segments are separated by five amino acids that provide flexible regions and in an arrangement such that, according to in silico studies, all relevant epitopes are exposed on the surface of the tertiary structure of the polypeptide.

[0022] In one embodiment of this invention, the chimeric antigen is characterized by an amino acid sequence that is identified as SEQ ID NO: 1 in the Sequence Listing and is shown in Figure 1 , or an amino acid sequence that has at least 90% identity with SEQ ID NO: 1 , and is hereinafter referred to as ApT. This combination and arrangement of the immunogenic segments selected to form the chimeric antigen of the present invention has not been reported to date.

[0023] In one embodiment of the invention, the chimeric antigen also comprises an amino-terminal segment selected from the group of fragments of the LpdA protein of Neisseria meningitidis, glutathione-S-transferase, immunoglobulins, ovalbumin, diphtheria toxoid, menignococcal outer membrane protein complex, Haemophilus influenzae protein D, keyhole limpet hemocyanin. At the carboxyl terminus, said chimeric antigen also comprises a sequence selected from the group of poly-His, poly-Arg, FLAG.

[0024] In one embodiment of the invention, the antigen identified as SEQ ID NO: 1 is fused at the amino terminal end to the first 47 amino acids of the LpdA protein of Neisseria meningitidis, a 13 amino acid spacer region. In the carboxyl terminal region, it is fused to a poly-His. The chimeric antigen of interest for this invention was obtained from the insertion of the corresponding synthetic gene into the expression vector by means of a ligation reaction with T4 DNA ligase and subsequently confirmed by sequencing. The chimeric antigen obtained by the method described above can be cloned into expression vectors in viruses, bacteria, yeasts, phages, plants or mammalian cells, changing the insertion sites as appropriate. Once the genetic construct is obtained, its sequence must be verified using automatic sequencing methods.

[0025] In a particular embodiment, the pM238 vector for the Escherichia coli bacterium was used to obtain the chimeric antigen, which has, among other elements, a Trip promoter, the 47 amino acid sequence corresponding to the amino terminal region of the N. meningitidis LpdA protein (Patent EP0816506B1), a segment of six histidines and the T4 terminator. This vector also carries the ampicillin resistance gene as a selection gene. The chimeric fusion protein obtained was called PK-ApT. Once the polypeptide was purified by metal ion chromatography, it was verified by mass spectrometry analysis that it constituted more than 90% of the identified species with a molecular mass of 32.6 kDa, equal to the expected one.

[0026] In the present invention, the chimeric antigen fused to the Neisseria meningitidis PK-ApT LpdA protein fragment unexpectedly increased the efficacy of immunization without deteriorating the safety profile of the treatment in relation to the administration of the mixture of these individual fragments.This novel combination and arrangement of the different segments increased its immunogenic properties with respect to the mixture of individual fragments, which gives the chimeric antigen advantages over other immunotherapy varieties for AD, such as: 1) greater efficacy in raising high antibody titers, 2) it stimulates a multi-target response without compromising the safety profile of the treatment, 3) it overcomes the immunosenescence typical of elderly patients, 4) it requires fewer administrations than passive immunotherapy and 5) it facilitates vaccine formulation by requiring the production of a single protein molecule instead of the mixture of individual fragments. These elements provide the chimeric antigen with high potential for the treatment of neurodegenerative diseases characterized by the aggregation of the Ap peptide and the tau protein.The present invention describes specific active immunotherapy characterized by the administration of a recombinant chimeric antigen composed of segments of the Ap peptide, the modified tau protein and the tetanus toxoid, which is combined with a pharmaceutically acceptable vaccine adjuvant.

[0027] In one embodiment of the invention, the vaccine adjuvant in the pharmaceutical composition is selected from the group consisting of oily adjuvants, mineral salts, proteoliposomes, and ganglioside-conjugated proteoliposomes. In order to promote the development of the immune response, the chimeric antigen of the present invention may be combined with immunopotentiators and / or adjuvants.

[0028] The immunogen can be administered in pharmaceutically acceptable vehicles that are nontoxic and do not exhibit therapeutic effects, and which are known to those skilled in this art. The examples presented in this invention do not in any way restrict the use of a particular buffer or combination of buffers, or even the use of excipients that contribute to increasing their stability.

[0029] In another aspect, the invention provides the use of the chimeric antigen comprising the combination of segments of the Ap peptide, the modified tau protein and a T cell epitope, for the manufacture of a medicament for the prevention and treatment of Alzheimer's disease. This is because the combination and arrangement of these segments allows obtaining an antigen of stable conformation that presents all the epitopes of interest on its surface. This facilitates recognition by the immune system and stimulates a specific multi-target antibody response against the toxic species of the Ap peptide and the tau protein. The PK-ApT fusion protein, administered with an aluminum salt adjuvant, gives rise to a vaccine that is capable of breaking the tolerance of the immune system, and of inducing a specific humoral and cellular immune response to the Ap peptide and the tau protein simultaneously.This immune response is superior, both qualitatively and quantitatively, when compared to the immune response generated when a mixture of its peptides is used in the vaccine formulation. This increase in the immunogenicity of the PK-ApT chimeric antigen translates into an increase in the neuroprotective activity of the immune response effectors induced, greater than that generated by the other strategies evaluated.

[0030] The neuroprotective effects demonstrated in the invention may be based on different mechanisms, without ruling out their possible combinations. The polyclonal antibody response generated by vaccination with PK-ApT is capable of synergistically clearing toxic species from both the Aβ peptide and the tau protein from the brain. Clearance is favored because the antibodies can block and inhibit the aggregation process during the formation of Aβ plaques and neurofibrillary tangles by neutralizing the target. Furthermore, they can dissolve the aggregates and opsonize them by activating microglia and macrophages. Furthermore, the antibodies generated by vaccination can promote the transport of toxic species across the blood-brain barrier into the peripheral bloodstream. As a consequence of all this, the neurotoxicity induced by Aβ and tau aggregates is reduced.The polyclonal nature of the antibody response, which is induced as a result of immunization, is an important factor in the effectiveness of the biological events described above.

[0031] Immunization with PK-ApT induced a highly polarized response to the Th-2 type, through the majority production of IgG1 isotype antibodies. IgG1 production is an indirect measure of the relative contribution of Th-2 type cytokines, whereas IgG2a isotypes reflect the contribution to a Th-1 immune response. Furthermore, this Th-2 type cellular response was corroborated through the increase in the production of non-inflammatory cytokines such as IL-4 in splenocyte cultures from immunized mice.

[0032] All these properties of the immune response generated after immunization with the PK-ApT protein become more relevant when compared to the active immunotherapy strategy using Aβ and tau fragments separately. An important element is that a superior immune response is achieved with the same amount of antigen. This feature implies that the immunotherapy strategy involving PK-ApT has advantages from a production and safety perspective in humans.

[0033] The invention also provides a method for the prevention and treatment of AD. This comprises the administration of a therapeutically effective amount of a pharmaceutical composition comprising a) the chimeric antigen comprising segments of the Ap peptide, the modified tau protein and the tetanus toxoid, and b) at least one pharmaceutically acceptable vaccine adjuvant. This method, which involves immunization with said chimeric antigen, more effectively prevents or decreases aggregates of the Ap peptide and the tau protein, which offers advantages for halting the progression of cognitive decline in patients. In one embodiment of the invention, the method involving the administration of the composition comprising the chimeric antigen is combined with standard AD therapy, either simultaneously or sequentially.This treatment method can be used as first or second line therapy, in association or not with other chemical or biological agents.

[0034] In one embodiment of the invention, administration of the vaccine comprising the chimeric antigen is combined with a bolus injection or controlled release of monoclonal antibodies or fragments thereof specific for antigens relevant to the aggregation of the Ap peptide and the tau protein. Vaccine preparations comprising the chimeric antigen are administered to a human in a therapeutically effective amount. The mixture of antigen and adjuvant, also known as immunogen, can be administered by various routes, including subcutaneous, intramuscular, mucosal, peritoneal, intralymphatic, topical, or inhalation. In a preferred embodiment, the immunogen comprising the chimeric PK-ApT antigen is administered subcutaneously. The examples presented in this invention do not restrict the use of the antigen to a particular adjuvant or route of administration.

[0035] In one embodiment of the invention, said treatment or prevention method will be repeated at least once at intervals of between 15 and 28 days. The antigen doses to be used can be established according to different parameters. These doses depend on the route of administration, the stage of the disease, and the treatment period. A change in the dose administration interval, or a different administration route than those described in the examples below, does not deviate from the essence of the present invention, making it possible to optimize immunization schedules in order to obtain a better immune response specific to PK-ApT.

[0036] The following examples and figures, which should not be construed in any way as limiting the present invention, illustrate the multi-target action of immunotherapy with the proposed chimeric antigen on the main neuropathological mechanisms of AD.

[0037] Description of the content of the figures

[0038] Figure 1. Polypeptide that makes up the chimeric antigen ApT.

[0039] Figure 2. Anti-Ap and anti-tau antibody responses generated after immunization of mice with the PK-ApT polypeptide in combination with the aluminum hydroxide adjuvant, as measured by ELISA. Antibody levels are expressed as absorbance units at 450 nm for a 1:10,000 dilution of the tested sera. Different letters represent statistical significance according to Tukey's test (p<0.05).

[0040] Figure 3. Subclasses of anti-Ap and anti-tau antibodies generated by vaccination with PK-ApT in combination with the adjuvant aluminum hydroxide, determined by ELISA. Antibody levels are expressed as absorbance units at 450 nm for the 1:10000 dilution of the tested sera. Asterisks represent statistical significance according to the Tukey test (***p<0.001).

[0041] Figure 4. Cellular response induced by vaccination of mice with the chimeric antigen PK-ApT, its individual fragments or a mixture of them in combination with the adjuvant aluminum hydroxide. T cell proliferation (A) and levels of cytokines II-4 (B) and IFN-γ (C), measured in splenocyte cultures from individual mice after their in vitro restimulation with the peptides Ap42, tau320-441 or p30. Different letters represent statistical significance according to Tukey's test (p<0.05).

[0042] Figure 5. Dot blot showing the reactivity of sera from mice immunized with the PK-ApT polypeptide toward all monomeric and aggregated forms of the Ap peptide and human tau protein.

[0043] Figure 6: Inhibition of Ap or tau oligomer-induced cytotoxicity in SH-SY5Y human neuroblastoma cells mediated by serum antibodies from mice immunized with the PK-ApT polypeptide or a mixture of its fragments. Different letters represent statistical significance according to Tukey's test (p<0.05).

[0044] Figure 7: Effect of PK-ApT polypeptide vaccination on cognitive deficits in 3xTg Alzheimer’s disease transgenic mice. A: Performance on the Morris Water Maze in the two-day protocol of the different experimental groups. B: Long-term spatial memory, measured as the escape latency of the last trial with the platform visible (D1T4) minus the escape latency of the first trial with the platform visible (D2T1). Different letters represent statistical significance according to Tukey’s test; *** p < 0.001.

[0045] Figure 8: Effect of vaccination with PK-ApT polypeptide on Ap42 and tau deposition in the brains of 3xTg Alzheimer's disease transgenic mice, measured as the amounts of Ap42 and tau in the soluble (RIPA) and insoluble (GnHCI) protein fractions of the brain. Different letters represent statistical significance according to Tukey's test (p<0.05).

[0046] The following are examples of embodiments of the multi-target chimeric antigen, which should not be construed in any way as limiting the present invention.

[0047] Example 1. Cloning and expression of the chimeric fusion protein

[0048] The gene corresponding to the chimeric antigen ApT was synthesized through the services of the company Genewiz (USA). In order to have representative polypeptides of the Ap peptide and the tau protein separately, two additional genes called Ap and tau respectively were synthesized. The synthesis of the three genes was carried out taking into account: 1) codon optimization for protein expression in E. coli systems, and 2) the inclusion of the cleavage sites of the enzymes Nhe I at the 5' position and BamH I at the 3' position of the sequence for assembly into the pM238 vector. The sequence of the three synthetic genes ApT, Ap and tau was 100% verified by complete sequencing. The nucleotide sequences of the synthetic genes ApT, Ap and tau are identified in the Sequence Listing as SEQ ID NO: 3-5, respectively.The assembly of the synthetic genes encoding the chimeric antigen ApT and the Ap and tau fragments separately, in the pM238 vector was carried out by double digestion with the enzymes Nhe I and Bam H I of both the vector and each gene, the purification of the restriction fragments from agarose gels with the Monarch DNA Gel Extraction Kit (New England Biolabs) and the subsequent ligation reaction with the T4 DNA ligase enzyme. With the inclusion of the synthetic gene in the pM238 vector, each of the ApT, Ap and tau antigens was fused to 47 amino acids from the amino terminus of the N. meningitidis LpdA protein (Patent EP0816506), connected by 13 amino acids that function as a bridge. A region of six histidines was located towards the carboxyl terminus of each fusion protein to facilitate its purification. The plasmids resulting from the cloning of the three synthetic genes were named pPK-ApT, pPK-Ap and pPK-tau, respectively.The nucleotide sequence of these plasmids was 100% verified. The nucleotide sequence of the coding region for each of the three chimeric fusion proteins is identified in the Sequence Listing as SEQ ID NO: 6-8. The fusion polypeptides were named PK-ApT, PK-Ap, and PK-tau, and their amino acid sequences are identified in the Sequence Listing as SEQ ID NO: 2, 9, and 10, respectively. For the expression of the three chimeric proteins PK-ApT, PK-Ap, and PK-tau, E. coli bacteria of the BL21 strain transformed with the plasmids pPK-ApT, pPK-Ap, or pPK-tau were grown for 3 h 37. e C in a 5 L thermoregulator. Luna Bertani medium supplemented with 100 pg / mL ampicillin (pH 7.0-7.5) was used. To induce expression of the three chimeric proteins, 10 to 100 pg of 3p-indoleacrylic acid per mL of medium was added, and each culture was grown at 37 °C. e C for 16 hours.

[0049] Example 2. Obtaining, purifying and characterizing the chimeric fusion protein

[0050] The bacterial biomass obtained by fermentation (as described in Example 1 ) was subjected to chemical cleavage in phosphate buffer (100 mM NaFLPC, 10 mM Tris-Cl, 8M urea, pH 8). Each of the PK-ApT, PK-Ap or PK-tau proteins contained in the soluble fraction was purified by metal chelate affinity chromatography under denaturing conditions, using a Ni-NTA matrix (Qiagen), and then subjected to gel filtration.

[0051] To characterize the chimeric fusion proteins PK-ApT, PK-Ap, and PK-tau, western blot techniques were used for immunodetection of Ap, tau, and the N. meningitidis LpdA protein fragments; and ESI-MS mass spectrometry using a QTOF-Ultima orthogonal hybrid configuration mass spectrometer was used to verify their molecular mass. The presence of fragments of Ap, tau and the N. meningitidis LpdA protein in the three chimeric fusion proteins was verified because they were recognized by a 1:2500 dilution of the anti-Ap antibody A3981 (Sigma), a 1:5000 dilution of the anti-tau antibody MAB 2239 (Sigma) or a 1:4000 dilution of the anti-P64K antibody MAB-Ntp64k-HRP (CBSS), respectively. ESI-MS / MS mass spectra confirmed that the proteins were intact, as designed, and their molecular masses matched those expected.

[0052] Example 3. Immunogenicity of the chimeric fusion antigen in mice with Alzheimer's disease

[0053] To evaluate the immunogenicity of the chimeric fusion protein PK-ApT, three-month-old male 3xTg-AD transgenic mice were immunized subcutaneously. In addition, the chimeric auxiliary proteins PK-Ap and PK-tau injected separately, as well as their mixture (PK-Ap + PK-tau), were used as controls in this experiment. In all cases, each mouse received 50 pg of chimeric antigen per dose in aluminum hydroxide as adjuvant. As a negative control, mice were immunized with the mixture of vehicle (40 mM Tris pH 7.4) and adjuvant. All mice received one injection every two weeks for eight weeks. In total, each mouse received five injections, and blood was drawn one week after the last immunization.

[0054] To detect the response of specific anti-Ap and anti-tau antibodies, indirect ELISA assays were performed. Soluble proteins 2.5 pM Ap1-42 (Sigma) or 10 pg / ml human tau (Abeam) were immobilized in a 96-well plate at pH 9.7. e C overnight. Mouse sera were added to the plate at a 1:10,000 dilution. Detection was performed with a conjugated mouse IgG-specific sheep antibody (Sigma). Conjugated anti-IgG1, IgG2ab, IgG2b, and IgM antibodies (Abeam) were used to characterize the subclass profile of the anti-Ap and anti-tau antibodies generated by vaccination in the mice.

[0055] The means and variances of antibody titers were compared between experimental groups using ANOVA (p<0.05) with the tools of the Statistica 8.0 program (Stat Soft.).

[0056] Figure 2 shows the results of the evaluation of the levels of specific IgG antibodies to the Ap peptide and the human tau protein, expressed in absorbance units. The administration of the PK-ApT chimeric antigen in aluminum hydroxide as an adjuvant was able to generate a significantly greater antibody response than that generated by the Ap and tau fragments separately or by mixing them with this same adjuvant (pcO.001). This result indicates that the PK-ApT chimeric antigen has greater immunogenicity, which can be explained by the fact that the chimera presents a primary structure with marked differences in relation to the native structures of the Ap peptide and the tau protein, which facilitates its recognition as foreign by the immune system and manages to break the immunological tolerance. Furthermore, PK-ApT generated a multi-target response against the two main structures that are poorly formed in AD.

[0057] The majority of antibodies generated by vaccination with the chimeric antigen PK-ApT was IgG1, and the ratio of IgG1 to IgG2a subclasses was always greater than 1 (Figure 3, ANOVA, p<0.001). The production of IgG1 antibodies is an indirect measure of the relative contribution of Th-2 cytokines, whereas IgG2a reflects the contribution of the Th-1 response. This result indicates that the chimeric antigen PK-ApT induces a highly polarized Th-2 response. Thus, immunization generated a potent antibody response without contributing to the neuroinflammation characteristic of AD.

[0058] Example 4. Cellular response generated after immunization with the chimeric fusion antigen

[0059] To determine whether vaccination with the chimeric antigen PK-ApT induces T cell activation and proliferation, which are key steps in the development of neuroinflammation, spleens were removed from mice immunized with either the chimeric antigen PK-ApT, its individual fragments PK-Ap or PK-tau, or a mixture of them (PK-Ap + PK-tau) two weeks after the last immunization. Splenocytes were resuspended in RPMI 1640 at 2x10 6 cells / mL and 100 pL were added to each well of a 96-well flat-bottom plate. Splenocytes were re-stimulated in vitro with the addition of 100 pL of the Ap42 or tau320-441 peptide at 20 pg / mL dissolved in RPMI 1640 and mixed. After incubation at 37 eC for 72 h, 5% CO2, the supernatants were collected and the proliferative response was measured in the plates with the CelITiter 96 Aqueous One Solution Cell Proliferation Assay reagent set (Promega) according to the manufacturer's instructions. The stimulation index was calculated by the ratio of the optical density values ​​(OD490nm) of the wells containing the cells stimulated by any of the peptides and those of the cells that only contained medium. The levels of IL-4 and IFN-γ were determined in the culture supernatants by ELISA using the Mouse IL-4 Quantikine ELISA Kit and Mouse IFN-γ Quantikine ELISA Kit reagent sets (R&D Systems) according to the manufacturer's instructions. The values ​​obtained for each of the experimental groups were compared by ANOVA.

[0060] T cell proliferation in splenocyte cultures from mice treated with the chimeric antigen PK-ApT, its individual fragments PK-Ap or PK-tau, or a mixture of these (PK-Ap + PK-tau) was moderate in all treatments tested (Figure 4). However, surprisingly, treatment with the PK-ApT polypeptide showed significantly lower T cell proliferation than the other treatments (Figure 4A). The T cell epitopes in the formulations containing the PK-Ap and PK-tau antigens separately are likely to be more exposed than those in the more complex tertiary structure of the PK-ApT antigen, thereby protecting the chimera from generating a proinflammatory response.

[0061] On the other hand, the higher levels of IL-4 than INF-γ in the supernatants of splenocyte cultures from mice treated with PK-ApT (Figure 4B and C) correspond to a stronger antibody response polarized to the Th-2 type generated by the chimeric antigen compared to that stimulated by its individual fragments or the mixture of them.

[0062] Example 5. Evaluation of the ability of antibodies resulting from vaccination with the chimeric fusion antigen to recognize the toxic species of Ap and tau

[0063] The ability of sera from mice immunized with the PK-ApT antigen to recognize different Ap and tau structures was determined by dot blot assay. First, polymerization of both the synthetic peptide Ap1-42 (Sigma) and recombinant human tau protein (441 aa soform, Abeam) was induced in vitro according to procedures previously described by (Stine et al., Methods in Molecular Biology, 2011 :670: 13-32) and (Combs et al., Neurobiology of Disease, 2016 :94: 18-31), respectively. Briefly, Ap1-42 monomers were obtained after dissolving the peptide in cold hexafluoro-2-propanol (HFIP). Then, HIFP was removed by evaporation and Ap1-42 was dissolved in anhydrous dimethyl sulfoxide (DMSO). A 5mM aliquot of Ap was added with cold water to a final concentration of 100pM immediately before use.Ap oligomers were prepared from a fresh solution of 5mM Ap42 in DMSO to which cold F12 culture medium was added to a final concentration of 100pM and incubated for 24h at 4. e C. Finally, the preparation of the Ap fibrils was also started from a fresh solution of 5mM Ap42 in DMSO to which 10mM HCl was added to a final concentration of 100pM and incubated at 37°C. eC for 24 h. On the other hand, aggregated tau species were obtained after adding arachidonic acid in ethanol to recombinant tau protein in polymerization buffer (5 mM DTT, 100 mM NaCl, 100 pM EDTA, and 10 mM HEPES at pH 7.64), up to a final concentration of 75 pM, and left to polymerize overnight. To perform Dot Blot, drops of solutions containing monomers, oligomers or fibrils of the synthetic peptide Ap1-42, or monomers or fibrils of recombinant human tau protein (2 pg / drop), were placed directly onto a nitrocellulose membrane. The membranes were incubated in 3% skim milk for 1 h at room temperature, followed by washing with PBST. The membranes were then incubated separately for 2 h with the pool of sera from mice immunized with the PK-ApT chimeric antigen. Anti-Ap 6E10 (BioLegend) or anti-tau (DAKO) antibodies were used as positive controls.Membranes were washed with PBST and incubated for 1 h at room temperature with peroxidase-conjugated anti-mouse IgG (Abeam) or anti-rabbit IgG (Abeam) secondary antibodies at a 1:1000 dilution. Reactivity was visualized with the substrate 3,3'-diaminobenzidine (SIGMA).

[0064] The immune serum of mice vaccinated with the PK-ApT chimeric antigen effectively recognized both monomeric and aggregated species of both Aβ and tau (Figure 5). This demonstrated the first function of antibodies generated as a result of treatment with the PK-ApT chimeric antigen: their simultaneous binding to a broad range of Aβ and tau therapeutic targets.

[0065] Unlike familial AD, where the cause is genetic, sporadic AD is triggered by a deficiency in the clearance of ATP from the brain. This peptide accumulates through an aggregation pathway involving different species: ATP monomers, oligomers, fibrils, and plaques. Of these, the most toxic species are the oligomers, which cause calcium dysfunction, mitochondrial failure, inflammatory response, oxidative stress, transcriptional impairment, synapse loss, and ultimately neuronal death. Furthermore, ATP oligomers induce tau hyperphosphorylation, causing it to aggregate, forming oligomers that evolve from paired helical filaments to neurofibrillary tangles. Tau oligomers are equally toxic, with strong effects on synapses, and in turn induce the formation of ATP oligomers.In addition to this positive feedback between Ap and tau aggregation, both proteins act cooperatively to lead to neuronal death (Roda et al., Neural Regeneration Research, 2022:17: 1666-74). Therefore, antibodies generated by vaccination with the PK-ApT chimeric antigen have the potential to act both systemically and centrally to promote the clearance mechanism of monomeric and oligomeric Ap and tau species from the brain and thus prevent their accumulation and subsequent aggregation. Since the different aggregation states of Ap peptides and tau protein coexist in Alzheimer's disease brains (Roda et al., Neural Regeneration Research, 2022:17: 1666-74), the elimination of all these structures simultaneously would have greater neuroprotective effects in vivo.

[0066] Example 6. Evaluation of the ability of antibodies resulting from vaccination with the chimeric fusion antigen to inhibit Ap and tau-induced cytotoxicity

[0067] The function of antibodies generated by vaccination with the chimeric antigen PK-ApT in inhibiting ApT- and tau-induced toxicity in cell cultures was evaluated in vitro using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) cell viability assay. The ApT oligomer (Davtyan et al., Journal of Translational Medicine, 2011:9:127) and tau oligomer (Sun et al., Journal of Neuroinflammation, 2021:18:131) induced toxicity models were used in the human neuroblastoma cell line SH-SY5Y (ATCC, VA). Cells were inoculated into each well of a 96-well plate at 1x10 4 cells / 100pL of medium (45% DMEM, 45% Ham's Modified F12 Medium, 10% FBS and 2mM L-glutamine). The plates were incubated at 37 eC for 24h in 5% CO2. Antibodies from the immune serum of mice vaccinated with the chimeric antigen PK-ApT, PK-Ap, PK-tau, the mixture of PK-Ap + PK-tau were purified by affinity chromatography according to the protocol previously described (Mamikonyan et al., Journal of Biological Chemistry, 2007:282: 22376-86). For the induced toxicity assay, Ap or tau oligomers, prepared as described in example 5, were incubated for 1 h at room temperature with or without the purified antibodies, with occasional shaking. After incubation, the purified oligomer / antibody mixtures were diluted in culture medium until the oligomers and antibodies remained at a final concentration of 2pM and 0.2pM, respectively. 100pL of these dilutions were added to SH-SY5Y cells and incubated for 72h. Untreated cells were incubated in parallel as a control.Viability was then measured in both toxicity models using the MTT Assay Kit (Abeam) according to the manufacturer's instructions. Cell viability was calculated by dividing the absorbance of each well containing treated cells by the absorbance of the wells containing untreated cells and expressed as a percentage.

[0068] Figure 6 shows that incubation of serum antibodies from vaccinated mice with the PK-ApT chimeric antigen or a mixture of its peptides (PK-Ap + PK-tau) significantly protected cells against toxic stimuli in both the in vitro model of Ap and tau oligomer-induced toxicity. These results demonstrated that the best cell viability was obtained with antibodies resulting from vaccination with the PK-ApT chimeric antigen. In this way, the therapeutic potency of vaccination with the PK-ApT chimeric antigen was confirmed by the greater protective effects of the antibodies stimulated by the treatment.

[0069] Example 7. Efficacy of PK-ApT polypeptide administration in the treatment of Alzheimer's disease in mice

[0070] To evaluate the effect of vaccination with the PK-ApT polypeptide on the cognitive functions of immunized mice, the Morris water maze test was performed (Morris, Journal of Neuroscience Methods, 1984:11 (1 ):47-6). This behavioral model has demonstrated its validity as a measure of hippocampus-dependent spatial navigation and reference memory. A two-day protocol is reported for it, based on an initial series of training trials with the visible platform, followed by a memory test 24 h later, but with the hidden platform (Gulinello et al., Behav. Brain Res, 2009:196:220-227). Thus, animals that develop a correct spatial strategy move quickly toward the hidden platform area, indicating that they maintain intact long-term memory capacity.In contrast, animals that take a long time to locate the hidden platform have defects in long-term spatial memory, with longer escape latencies.

[0071] All mice were transported to the behavioral testing room in their home cages at least 1 day before testing, and experiments were conducted between 8:00 a.m. and 12:30 p.m. A 120 cm diameter and 51 cm deep pool with a water temperature of 24 ± 2 °C was used. eC, in which high-contrast visual cues were placed on the wall in each quadrant and external cues were placed in the room. The training phase consisted of four trials with the platform visible (V1–V4), and the last trial was considered the mouse's baseline post-habituation escape latency. This was designated D1 VT4 (day 1, visible platform, test 4). Training was staggered so that each mouse had the same inter-trial interval, i.e., 30 ± 10 minutes between trials, and was initiated on each occasion from different randomly selected positions in the maze. To make the platform visible, it was marked with a high-contrast object relative to the tank background. Animals that failed to escape within 1 minute were manually guided to the platform and allowed to remain on it for 5–10 seconds before being removed.They were then dried and placed in their respective cages, lit by incandescent light to prevent hypothermia. Twenty-four hours after the last visible platform test (D2), the animals were evaluated in a series of three trials with the hidden platform (T1-T3). Trials were conducted every 30 minutes, and each trial lasted a maximum of 1 minute. For all trials, the platform remained in the same location. The difference in escape latencies was calculated: that of the last training trial with the visible platform and that of the first trial with the hidden platform (24 hours later).

[0072] Escape latencies during training with the visible or hidden platform were analyzed using ANOVA. For the long-term memory study, the last escape latency value from the training phase with the visible platform (D1 VT4) and the first value from the test with the hidden platform performed 24 h later (D2T1) were used. Mice with intact long-term memory capacity should quickly reach the hidden platform on the first test. A scoring system was generated with the difference D2T1 -D1 VT4, which in a successful subject should be close to 0. Higher values ​​indicate a longer escape latency after 24 h, implying a deficit in the animals' spatial performance. This comparison was performed using a variance analysis, followed by Tukey's post-hoc test with a significance level of α=0.05.

[0073] After behavioral testing, animals were sacrificed and the brain was immediately removed to analyze Ap and tau deposition. Extraction of brain proteins was performed by serial fractionation of brain homogenate in (1) TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl), (2) RIPA (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50 mM Ths pH 7.4) and (3) GndHCI (5 M guanidium hydrochloride, 50 mM HEPES (pH 7.3), 5 mM EDTA) as previously described (Wu et al., Neuropharmacology, 2018 :131 : 351-63). Briefly, frozen tissue was homogenized in nine volumes of TBS (m / v) containing protease inhibitors (Roche). Samples were centrifuged for 2 h at 16,000 g at 4 e C and the soluble fraction of TBS was stored in aliquots at -70 e C. The pellet was washed in TBS and then resuspended in nine volumes of RIPA (m / v) containing protease and phosphatase inhibitors (Roche). It was mixed gently at 4 eC for 30 min and then centrifuged for 2 h at 16,000 g at 4 e C. The RIPA-soluble fraction was stored in aliquots at -70°C. The pellet was washed with RIPA and resuspended in GndHCl at a rate of 150 mg of pellet / mL. The pellet was gently mixed at room temperature for 2 h, vortexing occasionally. The samples were centrifuged for 2 h at 16,000 g at 4 °C. e C and the soluble GndHCI fraction was neutralized with 20 volumes of 1 M Tris-HCI. The deposition of Ap42 and tau in the soluble RIPA and GndHCI fractions was measured by ELISA with the Amyloid beta 42 ELISA Kit (Invitrogen) and Tau ELISA Kit (Invitrogen), respectively, according to the manufacturer's recommendations. The concentration of Ap42 and tau in the brain fractions resulting from the different treatments was compared by ANOVA with a significance level of 0.05.

[0074] Figure 7 (A and B) shows the results of behavioral assessment after vaccination of 3xTg-AD transgenic mice with PK-ApT polypeptide. The results indicate that mice receiving placebo have long-term spatial memory deficits, and that treatment with PK-ApT, PK-Ap, PK-tau, or PK-Ap+PK-tau polypeptides significantly improved cognitive loss. The arithmetic difference in the latency of the last trial of training with the visible platform and that of the first trial with the hidden platform demonstrates that the group vaccinated with PK-ApT polypeptide was significantly lower than the rest of the treatments (Figure 7B). These results indicated that this treatment allows the recovery of spatial learning and memory functions in Alzheimer's-type transgenic mice and is more effective than individual peptides or a mixture of them.

[0075] Analysis of Ap42 and tau levels in the brain of 3xTg-AD mice after treatment is shown in Figure 8. These results show that treatment with PK-ApT polypeptide significantly decreases the Ap42 load (the aggregative form of Ap) in the brain because its levels are decreased in both the RIPA fractions (soluble proteins) and the GndHCI fractions (insoluble proteins) when compared to the rest of the treatments tested or placebo. Surprisingly, soluble tau levels were not affected by treatment, but insoluble tau levels were not (Figure 8). Immunotherapy significantly reduced the deposition of insoluble tau in the brain of transgenic mice, and the outcome with PK-ApT polypeptide was better than with the rest of the treatments (Figure 8).This result showed that treatment with the polypeptide preferentially eliminated toxic tau species, maintaining normal levels of functional tau protein.

[0076] Example 8. Evaluation of the safety profile after administration of the PK-A0T polypeptide and the adjuvant aluminum hydroxide

[0077] Female Balb / c mice (n=5) and female New Zealand rabbits (n=4) were immunized subcutaneously with 200 μg of the chimeric PK-ApT polypeptide adjuvanted with 0.7 mg of aluminum phosphate. Doses were administered every 14 days for a total of 4 immunizations. A placebo group was included in each experiment and was immunized with the excipient of the vaccine antigen formulation and the adjuvant.

[0078] In the experimental block corresponding to mice, three additional passive immunotherapy groups were included. One group was administered a mouse monoclonal antibody specific to the Ap peptide. Another group was administered a mouse monoclonal antibody specific to the tau protein. The third group was administered an unrelated antibody control of the same isotype. These antibodies were previously obtained using traditional hybridoma technology. In all cases, administration was performed at a dose of 12.5 mg / kg intraperitoneally, with a weekly frequency, for a total of 4 doses.

[0079] In both studies, weekly body weight assessments were performed at different times than immunizations. Once immunizations were completed, biological samples were collected, which was possible in 100% of the animals included in the experiment, across both animal species.

[0080] In the case of mice, the heart, lung, spleen, and ten other organs were removed at the end of the immunization regimen. The organs underwent qualitative evaluation, both macroscopically and microscopically. For microscopic evaluation and histological diagnosis, the samples were processed using well-known methods. Any abnormalities found in the actively or passively immunized groups that were not found in the corresponding placebo group were considered treatment-related adverse events.

[0081] In the case of rabbits, two blood draws were performed at different times, and each blood draw was used to obtain plasma (for hematology determinations) and serum (for clinical chemistry determinations). The draws were performed before starting immunization and one week after the last immunization. Hematology determinations included the count and / or percentage of various cell types, among other parameters. Blood chemistry determinations included hemoglobin concentration, alanine aminotransferase enzyme concentration, aspartate aminotransferase enzyme concentration, creatinine concentration, among others. For each parameter, comparisons were made between the values ​​obtained before and after immunizations, between the treated group and the corresponding placebo group, and whether these values ​​were within the physiological range reported for the species.An adverse event was considered treatment-related when alterations were found in the groups immunized with the PK-ApT polypeptide that were not found in the corresponding placebo group.

[0082] No macroscopic or microscopic alterations were observed in the organs of mice belonging to the placebo groups or those immunized with the PK-ApT polypeptide. The hematology and clinical biochemistry results obtained in rabbits indicated the absence of significant changes when the determinations made before and after immunization with the PK-ApT polypeptide were compared. No significant differences were also found between the immunized group and its corresponding placebo group. In general, all hematology and biochemistry parameters for the immunized and placebo groups were within the physiological range. In both mice and rabbits, there were no significant effects on body weight in the immunized groups with respect to that found in the corresponding placebo groups, at the different times evaluated.

[0083] The overall analysis of these results demonstrates that immunization with the chimeric polypeptide PK-ApT, adjuvanted with aluminum phosphate, generates a safe immune response. This active immunotherapy strategy offers advantages over passive immunotherapy with specific monoclonal antibodies, due to the absence of adverse effects.

Claims

MODIFIED CLAIMS received by the International Bureau on 3 February 2025 (03.02.2025) 1 . A chimeric protein for immunotherapy of Alzheimer's disease comprising epitopes from the N-terminal region of Tau protein comprising the first 48 amino acids from residues 1 to 48 of SEQ ID NO: 1 , N-terminal region of amyloid beta peptide (Ap) comprising the first 12 amino acids from residues 54 to 65 of SEQ ID NO: 1 , p30 T cell epitope tetanus toxoid peptide comprising 21 amino acids from residues 71 to 91 of SEQ ID NO: 1 , the C-terminal region of Ap comprising the last 15 amino acids from residues 97 to 111 of SEQ ID NO: 1 , and the C-terminal region of Tau protein comprising 122 amino acids from residues 117 to 238 of SEQ ID NO: 1 , with substitutions of Serine to Glutamic acid at residues 193, 201 , and 219.

2. The chimeric protein of claim 1, further comprising a linker to a segment selected from group (i) and / or a linker to a segment selected from group (i) at a C-terminal portion of the protein or an N-terminal portion of the protein: i) Fragments of Neisseria meningitidis LpdA protein, glutathione-S-transferase, immunoglobulins, ovalbumin, diphtheria toxoid, meningococcal outer membrane protein complex, Haemophilus influenzae protein D, keyhole limpet hemocyanin, virus-like particles. i) Poly-His, poly-Arg, FLAG.

3. The chimeric protein of claim 2, wherein the amino-terminal segment comprises the first 47 amino acids of the Neisseria meningitidis LpdA protein from residue 1 to 47 of SEQ ID NO: 2, and the carboxyl-terminal segment is a poly-His.

4. A nucleic acid comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1 to 3. 28 MODIFIED SHEET (ARTICLE 19) 5. A pharmaceutical composition for use in the treatment of Alzheimer's disease, comprising a) the antigen of claims 1 to 4, and b) at least one pharmaceutically acceptable vaccine adjuvant.

6. The pharmaceutical composition of claim 5, wherein the vaccine adjuvant is selected from the group consisting of oily adjuvants, mineral salts, nanoparticles and nanoparticles conjugated with gangliosides.

7. The pharmaceutical composition of claim 6, wherein the vaccine adjuvant is an aluminum salt selected from the group of aluminum hydroxide, aluminum phosphate, aluminum sulfate.

8. The pharmaceutical composition of claim 7, wherein the vaccine adjuvant is aluminum hydroxide.

9. The pharmaceutical composition of any one of claims 5 to 8, wherein the composition comprises at least one pharmaceutically acceptable diluent.

10. The pharmaceutical composition of claims 5 to 9 for use in the manufacture of a medicament for the prevention and treatment of Alzheimer's disease.

11. Pharmaceutical composition for use according to claim 10, wherein the medicament for the prevention and treatment of Alzheimer's disease is a vaccine for active immunotherapy.

12. Pharmaceutical composition for use according to claim 10, wherein the vaccine is administered at least once at intervals of between 15 and 28 days.

13. Pharmaceutical composition for use according to claim 11, wherein the vaccine is administered parenterally.

14. Pharmaceutical composition for use according to claim 11, wherein the vaccine is administered by a parenteral route selected from the group comprising the intradermal route, the subcutaneous route, the intramuscular route and the intravenous route. 29 MODIFIED SHEET (ARTICLE 19) Declaration according to Article 19.1 Patent No. PCT / CU 2024 / 050004 of International Application of the Center for Neurosciences of Cuba. Claim 1 is modified by specifying the regions and sequences of the respective elements comprised in the chimeric protein, following the defined epitope distribution, according to the design strategy. Claims 2, 3 and 4 are deleted and the remaining claims are renumbered. Claim 5 (new claim 2) is modified by updating the dependency and adding the words "furthermore" and "the", and the phrases "a linker to a" and "at a C-terminal portion of the protein or an N-terminal portion of the protein" to improve the sufficiency of the disclosure and the clarity of this claim. Claim 6 (new claim 3) is modified by updating the dependency, replacing the phrase "characterized by" with the word "comprises" to improve the clarity of this claim, and specifying the regions of the Neisseria meningitidis LpdA protein in the chimeric protein. Claim 7 (new claim 4) is modified by updating the dependency, and replacing the phrase "characterized by a nucleotide sequence encoding the chimeric proteins" with the phrase "comprising a nucleic acid sequence encoding any polypeptide" to improve the clarity of this claim. Claim 8 (new claim 5) is modified by updating the dependency, reformulating the claim to a pharmaceutical composition for use in a medical treatment. Claims 9, 10 and 11 (new claims 6, 7 and 8) are modified by updating the dependency and adding the word “pharmaceutical” before the word “composition”. New claim 6 is further amended by adding the word “consistent” after the word “group”, and by replacing the word “proteoliposomes” with the word “nanoparticles”. 30 DECLARATION ACCORDING TO ARTICLE 19 (1) New claim 9 is included to specify that the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Claims 12-14 (new claims 10-12) are amended by updating the dependency, reformulating these claims to a pharmaceutical composition for use in the manufacture of a medicament that is a vaccine, and specifying the frequency and timing of dosing. New claim 10 is further amended by replacing the word "medicine" with the word "medicament" to improve clarity and establish the connection between this claim and amended claim 13 (new claim 11), and by deleting the word "the" before the word "prevention". Claim 15 is deleted. New claims 13 and 14 are included to specify the route of administration of the vaccine. 31 DECLARATION ACCORDING TO ARTICLE 19 (1)