NZ744411B2 - Amyloid conjugate and uses and methods thereof - Google Patents

Abstract

The present invention refers to a pharmaceutical composition which, in addition to allowing or producing an effective and specific immune response against Aβ40 (the antibodies produced are specific for Aβ40 without significantly binding to Aβ42), increases said response compared with the response produced by other conjugates also comprising CysAβ(33-40) peptide and KLH, and in which said elements have been bound or conjugated by means of another crosslinking agent that also allow the binding of a peptide to the transport protein. oduced by other conjugates also comprising CysAβ(33-40) peptide and KLH, and in which said elements have been bound or conjugated by means of another crosslinking agent that also allow the binding of a peptide to the transport protein.

Description

AMYLOID CONJUGATE AND USES AND METHODS THEREOF DESCRIPTION Field of the invention The present invention relates to the field of biochemistry, more specifically to the field of sector of protein conjugation. onally, the present invention has an application in the field of medicine and veterinary science in the treatment of d diseases.

Background of the invention Multiple conjugates useful for the active or passive immunization of patients with amyloid diseases, fundamentally for Alzheimer’s disease, have been described in the prior art. It should be pointed out that most of said prior art focuses on selecting peptides or transport proteins that allow generating a suitable immune response in patients, the crosslinking agent used not being given much ance or relevance. Said crosslinking agent is often sed in the form of a list of all those that are available or known up until now indicating that any of them can be used in an equivalent manner, or it is simply not even indicated.

For example, Spanish patent application with publication number ES2246105 ses the prevention or treatment of amyloid diseases, inter alia Alzheimer’s e, by means of the active immunization of patients with the conjugate formed by the Aβ33-40 peptide and the transport protein keyhole limpet hemocyanin nafter, KLH), with accession number 4BED in the Protein Data Bank. The use of said conjugate for generating antibodies (for example, by means of immunizing mammals or birds with said conjugate) which are uently used in a passive immunization method for the prevention or ent of amyloid diseases, inter alia Alzheimer’s disease, is also contemplated. Said patent document does not y the crosslinking agent used.

The only prior art document of which the inventors are aware and in which the crosslinking agent is considered important in the immune response ed in the conjugate is PCT patent application with ation number WO2005/072777. Said document discloses conjugates for the active or passive immunization of patients based on the crosslinking agent LPA with respect to which the capacity thereof for binding with or having two peptides at the same time is highlighted, making it appropriate for generating an immune se suitable in patients. Within the general explanation of an LPA-based conjugate it is plated that the e used may be an Aβ42 or Aβ40 C-terminal fragment, and fragments 33-42, 35-42, 36-42, 37-42, 38-42 and 39-42 are specifically mentioned and exemplified.

Additionally, said document mentions that the transport protein may be KLH. This same document describes generating other conjugates, using a different crosslinking agent (N-succinimidyl 3-(2- pyridyldithio) propionate, commonly known as SPDP).

Brief description of the invention After extensive and thorough experimentation, the inventors have surprisingly discovered that maleimidobutyric acid N-hydroxysuccinimide ester nafter, SM), a heterobifunctional crosslinking agent, in which each molecule thereof binds a peptide to the transport protein, used as a crosslinking agent for preparing CysAβ(33-40) peptide (SEQ ID NO: 1) and KLH conjugates, produces conjugates that allow generating an improved immune response in comparison to the immune response produced by conjugates generated with other homo- or heterobifunctional crosslinking agents of the prior art such as SPDP which also allow the binding of a peptide to the transport protein. Such improvement is clearly shown in examples 1 to 4 of the present specification. In addition, said examples show that said new conjugates or the pharmaceutical compositions comprising said conjugates produce: - an immune response that is as specific as possible, for the purpose of minimizing the side s associated with the (preventive or therapeutic) vaccine treatment; - the highest possible immune response for the purpose of assuring an effective immunization of patients and reducing the required doses of genic conjugate.

In a first aspect, the invention provides a composition comprising a conjugate of at least one CysAβ(33- 40) peptide (SEQ ID NO: 1) linked to the e limpet hemocyanin (KLH), n the crosslinking agent connecting each CysAβ(33-40) peptide to the keyhole limpet hemocyanin (KLH) of the conjugate is the maleimidobutyric acid N-hydroxysuccinimide ester (SM), wherein the composition further ses aluminum hydroxide gel.

In a second aspect the invention provides a pharmaceutical ition comprising i) a conjugate comprising at least one 33-40) peptide (SEQ ID NO: 1) linked to the keyhole limpet hemocyanin (KLH) and ii) aluminum hydroxide gel, wherein the crosslinking agent ting each CysAβ(33-40) e to the e limpet hemocyanin (KLH) of the conjugate is the maleimidobutyric acid N- hydroxysuccinimide ester (SM), and wherein the pH of the composition ranges from 5.8 to 7.0.

In a third aspect the invention provides a glass ampoule comprising the pharmaceutical composition of the first or second .

In a fourth aspect, the invention relates to a method to manufacture a pharmaceutical composition according to the first or second , which comprises the following steps: a. Adding maleimidobutyric acid N-hydroxysuccinimide ester (SM) to a solution comprising keyhole limpet hemocyanin (KLH) in a buffer at a pH between 7.0 to 9.0; b. Eliminating the excess of maleimidobutyric acid N-hydroxysuccinimide ester from the 40 on of step a); c. Adding CysAβ(33-40) es (SEQ ID NO: 1) in DMSO to the solution of step b) at a pH of between 6.6 to 7.0 to produce the conjugates; d. Eliminating the free peptide from the solution from step c); e. Optionally filtrating the solution of step d); f. Adjusting the pH of the on of step d) or e) to a pH range of between 5.8 to 6.2; g. Adding aluminum hydroxide gel to the solution of step f) once the pH has been ed.

In a fifth aspect the invention relates to use of the pharmaceutical composition of the first or second aspect or the glass e of the third aspect in the manufacture of a medicament for the treatment of an amyloid disease.

In a sixth aspect, the invention relates to use of a conjugate of at least one CysAβ(33-40) peptide (SEQ ID NO: 1) linked to the keyhole limpet hemocyanin (KLH) in the manufacture of a medicament for the treatment or prevention of an amyloid disease, wherein the crosslinking agent connecting each CysAβ(33-40) peptide to the keyhole limpet hemocyanin (KLH) of the conjugate is the maleimidobutyric acid N-hydroxysuccinimide ester (SM), and wherein the medicament further comprises aluminum hydroxide gel.

Certain statements that appear below are broader than what appears in the statements of the invention above. These statements are provided in the interests of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which s the scope of the invention.

Described herein is a conjugate terized in that the crosslinking agent is SM.

Described herein is a composition comprising the conjugate described herein.

The use of said conjugate for ing a medicinal product, more specifically, a nal product intended for the treatment or prevention of amyloid es, is contemplated herein.

Described herein are itions comprising the ate bed herein, for use as a medicinal product, more specifically for use in the treatment or prevention of amyloid diseases.

Also described is a method of treatment or prevention of an amyloid disease by means of the delivery of a composition comprising the conjugate bed herein.

Described herein is a method of manufacturing antibodies based on the use of the conjugate described herein.

Detailed description of the invention Definitions As used herein, "amyloid disease" and the plural f refer to diseases associated with the β-amyloid accumulation. Said accumulation can fundamentally occur in the brain, producing diseases among which are found Alzheimer’s disease, Parkinson’s disease, cerebral amyloid athy, vascular dementia of an amyloid origin and dementia with Lewy bodies. β-amyloid accumulation can also fundamentally occur in skeletal muscle, producing inclusion-body myositis.

As used herein, "passive immunization" and the plural thereof refer to the ry of antibodies or fragments thereof to a patient with the intention of conferring immunity to said patient.

As used herein, e immunization" and the plural f refer to the delivery to a patient of peptides (in the form of ates) acting as immunogens, i.e., they allow antibody generation with the ion of conferring immunity to said patient.

As used herein, "adjuvant" and the plural thereof refer to immunomodulatory substances capable of being combined with the conjugate of the present invention for increasing, improving or otherwise modulating an immune response in a patient.

As used herein, "patient" and the plural thereof refer to any mammal, preferably human, in which the conjugate of the present invention or a composition comprising same can be administered for the purpose of treating or preventing an amyloid disease.

As used herein, "CysAβ(33-40)" refers to the sequence of positions 33 to 40 of Aβ40 (SEQ ID NO: 2) to which a cysteine has been added at the N-terminus. Said sequence is reflected in SEQ ID NO: 1 and is: CGLMVGGVV.

The term "comprising" as used in this ication and claims means sting at least in part of".

When interpreting statements in this ication and claims which include the term "comprising", other features besides the es prefaced by this term in each statement can also be present. Related terms such as "comprise" and ised" are to be reted in similar manner.

Description Described herein is a conjugate comprising at least one CysAβ(33-40) peptide (SEQ ID NO: 1) and keyhole limpet hemocyanin (KLH), terized in that the crosslinking agent connecting each of the components of the conjugate (each of said at least one peptide with KLH) is maleimidobutyric acid N- hydroxysuccinimide ester (SM).

Said conjugate, in on to allowing or producing an effective and specific immune response against Aβ40 (the antibodies produced are specific for Aβ40 without significantly binding to Aβ42), increases said response ed with the response produced by other conjugates also comprising CysAβ(33-40) 40 peptide (SEQ ID NO: 1) and KLH, and in which said elements have been bound or conjugated by means of another inking agent that also allow the binding of a peptide to the transport protein.

Also described is a composition comprising the conjugate as described.

In a preferred embodiment, the composition additionally comprises one or more adjuvants, which are ably selected from mineral salts (such as aluminum hydroxide, aluminum phosphate or calcium phosphate), microparticles and active surface agents [such as nonionic block polymer tants, virosomes, saponins, meningococcal outer membrane proteins (proteosomes), immune stimulating complexes, cochleates, dimethyl dioctadecyl ammonium bromide, avridine, vitamin A or vitamin E], bacterial products [such as the cell wall skeleton of Mycobacterium phlei, muramyl dipeptides and tripeptides (threonyl-MDP, MDP-butyl ester, dipalmitoyl phosphatidylethalonamine MTP), osphoryl lipid A, Klebsiella pneumoniae glycoprotein, Bordetella pertussis, Bacillus Calmette- Guérin, V. ae and E. coli heat-labile enterotoxin, trehalose dimycolate, CpG oligodeoxynucleotides], hormones and cytokines (for example, eukin-2, interferon α, interferon-β, granulocyte-macrophage colony-stimulating factor, dehydroepiandrosterone, Flt3 ligand, 1,25 dihydroxyvitamin D3, interleukin-1, interleukin-6, interleukin-12, human growth hormone, β-microglobulin and lymphotactin), single antigen constructs (such as multiple peptide antigens bound to a lysine nucleus or cytotoxic T-cell epitopes bound to helper T-cell epitopes and palmitoylated at the inus), polyanions (such as dextrans or double-stranded polynucleotides), rylics (such as polymethylmethacrylate or acrylic acid cross linked with allyl sucrose), carriers [such as tetanus toxoid, diphtheria toxoid, group B ococcal outer membrane proteins (proteosomes), Pseudomonas ins A, a cholera toxin B subunit, a abile ng enterotoxigenic E. coli enterotoxin, the hepatitis B virus nucleus, cholera toxin A fusion proteins, CpG dinucleotides, thermal shock proteins or fatty acids], live vectors (such as vaccinia virus, canarypox virus, irus, attenuated ella typhi, Bacillus Calmette-Guérin, Streptococcus gordonni, herpes simplex virus, vaccine-derived poliovirus, rhinovirus, Venezuelan equine encephalitis virus, Yersinia colitica, Listeria monocytogenes, Shigella, Bordetella pertussis or Saccharomyces siae), vehicles [such as water-in-oil emulsions (for e mineral oils such as complete Freund’s adjuvant or incomplete Freund’s adjuvant; vegetable oils; squalene or ne); oilin-water emulsions, such as a e of squalene, Tween-80 and Span 85; liposomes; or biodegradable polymer microspheres of, for example, es and glycolides, polyphosphazones, betal-glucans or proteinoids], others (such as N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine, gamma insulin and aluminum ide, transgenic plants, human dendritic cells, lysophosphatidylglycerol, stearyl- ne or tripalmitoyl pentapeptide), or combinations thereof.

In a preferred embodiment, said adjuvant is aluminum hydroxide, and more preferably aluminum hydroxide gel. Therefore, a preferred embodiment refers to a composition, preferably a pharmaceutical composition, comprising i) a conjugate comprising at least one CysAβ(33-40) peptide (SEQ ID NO: 1) linked to the keyhole limpet hemocyanin (KLH) and ii) aluminum hydroxide gel, wherein the crosslinking agent connecting each CysAβ(33-40) e to the keyhole limpet hemocyanin (KLH) of the conjugate is the idobutyric acid N-hydroxysuccinimide ester (SM).

It is noted that, as shown in example 5, the adsorption rates of the conjugate onto the aluminum 40 hydroxide gel of said preferred composition depends on the pH. In fact, the adsorption rate increases at lower pH-values. A reduction from pH 7.4 to pH to 7.2 or even 7.0 does not result in significant higher adsorption rates. An adsorption at pH 6.8 shows an adsorption rate around 90%. At pH 6.0 the adsorption ratio is the highest. At a conjugate concentration based on 200 μg/mL net peptide and pH 6.7% of conjugate is still free.

Based on these analyses, in order to increase the immunogenic capacity of the ceutical composition, a pH value of between 5.8 to 7.0 should be used in order to increase the adsorption rate of the conjugate onto the adjuvant and significantly increase its immunogenic capacity.

Therefore, another preferred embodiment refers to a ition, preferably a pharmaceutical composition, sing i) a conjugate comprising at least one CysAβ(33-40) peptide (SEQ ID NO: 1) linked to the keyhole limpet hemocyanin (KLH) and ii) aluminum hydroxide gel, wherein the crosslinking agent connecting each CysAβ(33-40) peptide to the e limpet anin (KLH) of the conjugate is the maleimidobutyric acid N-hydroxysuccinimide ester (SM), and wherein the pH of the composition ranges from 5.8 to 7.0, ably from 6.2 to 7.0, more preferably from 5.8 to 6.2.

In further preferred embodiments, the concentration of CysAβ(33-40) peptides (SEQ ID NO: 1) in the pharmaceutical composition is of at least 100μg, preferably at least 150μg, more preferably from 150μg to 400μg, still more preferably from 160μg to 240μg, still more preferably about 200μg.

In yet r preferred embodiments, the KLH-SM-CysAβ(33-40) conjugates present in the pharmaceutical composition se a ratio of at least 45 33-40) peptides (SEQ ID NO: 1) per keyhole limpet hemocyanin (KLH) protein.

In still r preferred embodiment, the pharmaceutical composition in contained in glass ampoules, preferably of 1 or 1.2 ml.

It is r noted that, as reflected above, the adsorption rates of the conjugate onto the aluminum hydroxide gel of in the composition as described (when aluminum hydroxide gel is used) s on the pH. Moreover, surprisingly the pH increases during the storage of the pharmaceutical composition once it has been manufactured thus reducing the adsorption rates if such increases reached pH values above 7.0, or preferably above 6.8. In order to diminish such drawback that clearly affects the immunogenic capacity of the pharmaceutical composition, it is important to adjust the pH of the pharmaceutical composition to a range of n 5.5 to 6.5, preferably 5.8 to 6.2, more preferably 5.9 to 6.1, at the time of manufacturing the pharmaceutical composition so that storage does not or affects minimally the genic capacity of the composition.

Therefore, yet another preferred embodiment refers to a method to manufacture a pharmaceutical composition, which comprises the following steps: a. Adding maleimidobutyric acid oxysuccinimide ester (SM) to a composition comprising keyhole limpet hemocyanin (KLH) in a buffer at a pH between 7.0 to 9; 40 b. Eliminating the excess of maleimidobutyric acid N-hydroxysuccinimide ester from the solution of step a), preferably by using 0,02 M Na-Phosphate-buffer at a pH of about 6.6 to 7.0; c. Adding CysAβ(33-40) peptides (SEQ ID NO: 1) in DMSO to the solution of step b) at a pH of between 6.6 to 7.0 to produce the conjugates; d. Eliminating the free peptide from step c), preferably by using 0,01 M PBS-buffer at a pH of 6,6 to 7.0; e. Optionally filtrating the solution of step d), preferably by using a filter of about 0.2 μm.; f. ing the pH of the solution of step d) or e) to a pH range between 5.5 to 6.5, preferably 5.8 to 6.2, more preferably 5.9 to 6.1, still more preferably about 6.0; and g. Adding aluminum hydroxide gel to the solution of step f) once the pH has been adjusted.

Described herein is the use of a ition comprising the ate described herein for preparing a medicinal product. ably said composition is the composition bed herein or in any of its preferred ments. Also in a preferred embodiment, said medicinal product is for use in the treatment or prevention of an amyloid disease, more preferably of a amyloid e selected from Alzheimer’s disease, Parkinson’s disease, cerebral amyloid angiopathy, vascular dementia of an amyloid origin, inclusion-body myositis and dementia with Lewy bodies. In the most preferred embodiment, said medicinal product is used for the prevention or treatment of Alzheimer’s disease.

Described herein is a method of treatment or of prevention of an amyloid disease in a patient in need of same comprising the delivery of a eutically ive amount of a composition comprising the conjugate described herein. Preferably said composition is the composition bed herein or in any of its preferred embodiments. Also in a preferred embodiment, said amyloid disease is a amyloid disease selected from Alzheimer’s disease, Parkinson’s disease, cerebral amyloid angiopathy, vascular dementia of an amyloid origin, ion-body myositis and dementia with Lewy bodies, even more preferably Alzheimer’s disease. bed herein is a method of manufacturing dies characterized in that it comprises an immunization step for immunizing mammals or birds with a composition comprising the conjugate described herein. Preferably said composition is the composition described herein, or in any of its preferred embodiments. It is contemplated that the mammals used in such method can be ruminants, equidae, lagomorphs, primates (preferably ) or any other mammal that allows obtaining the suitable amounts of serum for ting or obtaining ient amounts of antibodies. It is contemplated that the birds used in the method described herein are any fowl-like birds, waterfowl, pigeons and doves or any other bird that allows obtaining suitable amounts of serum for extracting or obtaining sufficient amounts of antibodies. It is further contemplated the protection of the antibodies obtained or obtainable by the method described herein, as well as their use to manufacture a pharmaceutical composition for use in the treatment of an amyloid e selected from Alzheimer’s disease, Parkinson’s disease, cerebral amyloid angiopathy, vascular dementia of an amyloid origin, inclusion-body myositis and dementia with Lewy bodies, even more preferably mer’s e. bed herein is a conjugate generated using the crosslinking agent SM, and compositions comprising same that allow generating a greater immune response compared with conjugates generated with other crosslinking agents of the prior art.

Additionally, the immune response generated by said conjugates described herein or the compositions comprising said conjugates is ic for Aβ40, i.e., it allows generating anti-Aβ40 specific dies without generating anti-Aβ42 antibodies.

For better understanding, the present ion is described in further detail below in reference to the attached drawings presented by way of example and in reference to illustrative non-limiting examples.

In this specification where reference has been made to patent specifications, other al documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the ion. Unless specifically stated ise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common l knowledge in the art.

Example 1. Preparation of KLH-SM-CysAβ(33-40) conjugates.

For preparing these conjugates, KLH was used as a transport protein, SM as a crosslinking agent and CysAβ(33-40) (SEQ ID NO: 1) as an immunogenic peptide (peptide with residues 33-40 of the amyloid peptide to which a cysteine has been added at the N-terminus).

Binding took place between the available lysine residues of the KLH and the cysteine added at the N- terminal end of the peptide. In this case, the binding of the crosslinking agent to KLH was done first (KLH activation step), and, in a second step, the immunogenic peptide was added to the ted KLH so that conjugation could take place.

The protocol ed for the foregoing is as follows: • A 250 mM stock solution of SM was ed by dissolving 100 mg of SM in 680 μL of dry DMSO.

Aliquots of said stock solution were made and stored at -20°C.

• KLH was dissolved at a concentration of 5 mg/mL in PBS/5mM EDTA pH 7.4. • 16 μL of stock solution of SM were added for each milliliter of KLH that was conjugated.

• Said solution was left to react for 2 hours and 30 minutes at room temperature under gentle stirring.

• Then, the reaction buffer was changed to remove on byproducts and excess unreacted SM. For this purpose, a PD10 column (GE Healthcare; reference 1701) was used as follows: o Step a): The column was equilibrated with 25 mL (5 mL, 5 times) PBS (80 mM sodium hydrogenophosphate dihydrate, 20mM sodium ogenophosphate drate, 100mM sodium chloride) /5mM EDTA pH 7.4. o Step b): 2.5 mL of the already reacted solution were added in the column and said solution was left to penetrate, discarding the eluate. o Step c): 3.5 mL of PBS were added in the column and the eluate was collected. o Step d): The column was re-equilibrated with 25 mL (5 mL, 5 times) of 5mM EDTA pH 7.4, as explained above in step a). 40 o Steps b) to d) were repeated all the times needed ing on the volume of solution that reacted. After that, the column was ed equilibrated. o Step e): The column was stored at 4°C in order to use it on other occasions with the same peptide conjugate in the same manner. Preceding protocol.

• Then the peptide that was to be mixed with activated KLH was calculated. To that end, was taken into account both the molecular weight of the peptide and the molecular weight of KLH, in addition to the active sites therein (measured according to the methods known in the prior art based on measuring absorbance at 343 nm before and after treating the activated KLH solution with dithiothreitol and performing the necessary conversion). For example, for 10 mg of KLH with 1724 active sites: /6725000 (mean molecular weight of KLH)= 1.48x10-6 mmol of KLH 1.48x10-6 mmol of KLH x 1724 active sites= 2.55x10-3 mmol of peptide required for covering all the active sites.

A 3-fold peptide excess was placed to favor the conjugation reaction: 2.55x10-3 x 3= 7.65x10-3 mmol of peptide ed. 7.65x10-3 x weight molecular of the e (834.4 Da)= mg of e required (6.38 mg of peptide) (applying the ed conversion factors).

• Once the peptide/activated KLH ratio (i.e., KLH with bound SM) was determined, the following reaction was prepared: mixture of the peptide with the activated KLH with SM in a suitable proportion. To that end, the peptide was prepared at 6 mg/mL in DMSO and was slowly added to activated KLH. The proportion of DMSO in the final reaction must not exceed 30%. If it were higher than that in any case, PBS/5mM EDTA pH 7.4 is added until the amount thereof was reduced to values of less than 30%.

• The solution was left to react at room temperature and under stirring between 18 and 24 hours.

• Finally, the obtained solutions were stored at 4°C. The peptide that has not conjugated with KLH and is therefore free may or may not be removed. If said free peptide is not removed, the e concentration in the final product is determined by the total peptide used in the reaction, not only that which is bound to native KLH, and the final reaction volume.

Example 2. Comparison of the immune response of KLH-crosslinking agent-CisAβ(33-40) conjugates generated using different crosslinking agents.

In this case, the immune se strength generated in mice (4 per group) with the following conjugates was compared: • KLH-SM-CysAβ(33-40) (produced according to Example 1).

• KLH-SPDP-CysAβ(33-40): this ate uses a inking agent commonly used in the prior art, imidyl 3-(2-pyridyldithio)propionate (SPDP) and is produced by means of a protocol very similar to the one described in Example 1 (introducing the necessary adaptations) and known in the prior art.

The immune response strength test was conducted in BALB/c strain mice. The protocol that was followed was: 1. A week before the first inoculation, blood was drawn from all the mice participating in the study to 40 obtain pre-immune serum. 2. Depending on the group to which each mouse was assigned (see Table 1 for the ent analyzed ), each mouse was inoculated the corresponding vaccine once a week for three weeks ht. 3. A week after the third shot, blood was drawn again from each of the mice participating in the study to measure the response ed in the serum.

Table 1. Groups used in the study and description of the conjugate administered to each of them.

Group Transport protein Crosslinking agent Adjuvant Alhydrogel® (aluminum 1 KLH, Manufacturer 1 SM hydroxide gel) Alhydrogel® (aluminum 2 KLH, cturer 1 SPDP hydroxide gel) Alhydrogel® (aluminum 3 KLH, Manufacturer 2 SM hydroxide gel) 4 KLH, Manufacturer 2 SM None Alhydrogel® (aluminum KLH, Manufacturer 2 SPDP hydroxide gel) Alhydrogel® (aluminum 6 KLH, Manufacturer 3 (GMP) SM hydroxide gel) The peptide dose administered to the mice in each of the groups shown in Table 1 is ted in Table 2 included below.

Table 2. Peptide dose, including both total peptide and peptide conjugated to KLH administered to each of the mice in each of the groups of the study.

Total peptide dose (sum of free peptide and KLH- KLH-bound e dose (in Group bound peptide) (in μg) μg) 1 120 40 2 120 40 3 120 40 4 120 40 120 40 6 60 60 Table 3 shows a tabulated summary of the immune response strength results obtained for the different groups (analyzing serum from the mice obtained the week after finishing the therapeutic or vaccination regimen explained in the t example), together with the increase observed in the immune response (fold number the immune response sed a week after ing the therapeutic regimen with respect to the pre-immune response). The measurement of the immune response was taken on the serum obtained from each mouse by means of ct ELISA, according to the protocol known in the prior art, with respect to which it should be pointed out that the ELISA plates were sieved with Aβ40 peptide. Once the corresponding steps of washing, blocking, subsequent washing, incubation with the samples to be analyzed of plasma/serum (1:3 serial dilutions starting with a 1:30 dilution) and additional washing were performed, each well was incubated with the Anti-mouse IgG (H+L) antibody HRP (the secondary antibody dilution was 1:2000 in e on at pH 8). After incubating with said antibody and washing the wells, the plate was developed by adding 100 μL per well of an ABTS solution (diammonium 2,2'-azinobis-[3- ethylbenzothiazolinesulfonate]; Roche; reference: 102 946 001) with 0.375 mg/mL in ABTS buffer (Roche; reference: 11 112 597 001). This substrate turned green upon ng with the peroxidase bound to the secondary antibody. The color intensity depended on the amount of antibodies bound to the plate. The reaction was incubated for 55 minutes at room temperature and in the dark, and then the absorbance was read in an ELISA plates reader at 405 nm. The obtained absorbance results were analyzed with the GraphPad Prism 3.02 program. The "One Site Competition" equation was used for the analysis: (Maximum Minimum ) Y = Minimum + 1+10x LogEC50 The EC50 data, which is the inflection point of the curve, i.e., the point at which 50% of the maximum effect observed was produced, was obtained from the entioned analysis. In the present case, it was interpreted as the serum dilution at which 50% of the peptide present in the well bound to the antibody t in the serum.

Table 3. Mean immune response results ed for each of the groups of the study. It includes results for the serum after the vaccine treatment (1 week after the three injections according to the protocol described in the t example) and the increase observed between said point and the pre-immune se (before starting the eutic regimen). The mean pre-immune EC50 was 18.11.

Group Post-3 inoculation EC50 Increase in EC50 (fold number) 1 12974.95 716.33 2 580.35 32.04 3 6325.50 349.22 4 924.97 51.07 1.87 0.10 6 5056.00 279.14 In view of the results shown in Table 3 and the conjugate used in each of the study groups, the following can be concluded: • The different KLHs used as transport proteins (from ent manufacturers), despite having a certain effect on the immune response, have not been shown to be relevant in determining the magnitude of the immune response (high immune response in groups 1, 3, 4 and 6, i.e., groups with an increase in EC50 of 50 or more, versus a weak or inexistent immune response in groups 2 and 5, i.e., groups with an increase in EC50 of less than 50).

• The differences between obtaining a high immune se and a weak or tent immune response lie in the crosslinking agent used. Based on the experimental results obtained, it is deduced that the conjugates in which SM were used produce an immune response that is significantly greater that the one observed for the conjugates in which SPDP was used. In fact, the groups treated with ates with the crosslinking agent SM allow generating a high immune response whereas the groups treated with conjugates in which SPDP was used showed an immune response that was much weaker or inexistent.

• In addition to the foregoing, the results obtained for group 4 must be highlighted, as they clearly show that the response of the conjugates generated using SM is still greater than the one observed for the conjugates generated using SPDP, even without the use of an adjuvant.

The foregoing shows that the use of SM as a crosslinking agent for preparing the KLH-crosslinking agent-CysAβ(33-40) conjugates provides vaccines with an immune response that is surprisingly greater whether said conjugate is used with an adjuvant or without an adjuvant. e 3. Comparison of the degree of conjugation de binding to KLH) of KLH-crosslinking agent-CysAβ(33-40) conjugates generated using different crosslinking agents.

As in the case of Example 2, the conjugates for which the degree of ation or number of peptides bound per molecule of transport protein (KLH) was compared are: • KLH-SM-CysAβ(33-40) (produced ing to Example 1).

• KLH-SPDP-CysAβ(33-40) (produced as ted in Example 2).

Table 4 shows a tabulated summary of the experimental s obtained for the experiment for binding the e to the transport protein that was carried out.

Table 4. Description of the analyzed ates and of the number of peptide molecules bound to each KLH molecule observed by mass spectrometry. With respect to said bonds, the table indicates one or two values depending on if one or two s of the ponding conjugate were analyzed.

Number of peptide molecules bound Transport protein Crosslinking agent to each KLH molecule KLH, cturer 1 SM 81 KLH, Manufacturer 1 SPDP 35/33 KLH, Manufacturer 2 SM 70/68 KLH, Manufacturer 2 SPDP 44/31 As seen in Table 4, those KLHs from different manufacturers had no effect on in the bonds result obtained. In contrast, the crosslinking agent did indeed have an enormous effect on the results obtained given that SM allowed obtaining twice the number of bonds or more, i.e., by using SM as a crosslinking agent twice the number of peptide molecules binds for each KLH molecule. This result is surprising and unexpected given that the peptide used incorporates a cysteine at the N-terminus for reacting with the crosslinking agents. According to the prior art, the incorporation of said cysteine should allow the peptide conjugation to be ent and equivalent to any of the crosslinking agents known in the prior art. However, it was observed in this case that SM allows a more efficient conjugation reaction than SPDP does.

These binding results allowed explaining part of the results shown in Example 2 (i.e., part of the improvement observed in immune response induction and subsequent antibody tion). heless, said immune response results are not completely assimilable to the obtained binding results, which is also sing in view of said obtained results and suggesting that the crosslinking agent butes to increasing the immune response not only by mediating greater binding of the peptide to the transport protein.

Example 4. Immune response assays in rabbits and icity of the antibodies generated.

A potency assay of vaccines comprising the KLH-SM-CysAβ(33-40) conjugates was conducted in rabbits. In this case, the s were vaccinated with 200 μg of total bound peptide, said 200 μg being bound to the transport protein (chosen dose depending on preliminary studies). Each rabbit was inoculated with 1 mL of the vaccine with the previously specified dose, using 2% Alhydrogel® (aluminum hydroxide gel) as an adjuvant.

The animals were treated with the previously indicated dose by means of subcutaneous injection of the vaccine once a week for 3 straight weeks drawing blood a week before cing the vaccination protocol and a week after finishing it.

The titration of the generated antibodies and the analysis of their specificity was done by means of ELISA, according to the protocol known in the prior art and briefly indicated in Example 2, with the following differences: • The ELISA plates were sieved with Aβ40 or Aβ42 peptide (included as SEQ ID NO: 3), depending on if specific antibodies are to be detected or those that bind to Aβ40 or to Aβ42, respectively.

• The antibody used to detect the presence of antibodies in the analyzed sera was Anti-Rabbit IgG (H+L) HRP (Invitrogen; reference: 65-6120) (the secondary antibody dilution was 1:2000 in vehicle solution at pH 8).

The development reagents were those indicated in Example 2, and therefore the plates were also read at 405 nm and the same equation was applied to the s. The EC50 data, which is the inflection point of the curve, i.e., the point at which 50% of the maximum effect ed was produced, was obtained from the analysis. As indicated in Example 2, said result was interpreted as the serum dilution at which 50% of the peptide present in the well bound to the antibody present in the serum.

According to the aforementioned titration protocol, all the samples obtained were titrated to detect Aβ40 and Aβ42 peptide antibodies. The obtained s are shown in tabulated form in Tables 5 and 6.

Table 5. Amount of anti-Aβ40 specific antibodies in rabbits before and after the vaccination protocol. A column ng to the increase observed as a consequence of treatment is also ed.

Increase in EC50 (fold fication of Rabbit mune EC50 Post-3 inoculation EC50 number) 71 36.17 47406 4 72 7.483 4021 537.35 73 14.07 73421 5218.27 74 4.482 12467 2781.57 75 45.48 5173 113.74 Increase in EC50 (fold Identification of Rabbit Pre-immune EC50 Post-3 inoculation EC50 76 20.5 98381 4799.07 Table 6. Amount of Aβ42 specific antibodies in rabbits before and after the vaccination protocol. A column ng to the increase observed as a uence of ent is also included.

Increase in EC50 Identification of Rabbit Pre-immune EC50 Post-3 inoculation EC50 (fold number) 71 53.88 0.049 0.00 72 29.24 0.069 0.00 73 22.49 2.761 0.12 74 24.05 15.49 0.64 75 73.18 0.063 0.00 76 23.37 34.04 1.46 Based on what is shown in Tables 5 and 6, it is deduced that the conjugate of the present invention (KLH-SM-CysAβ(33-40)) allows not only obtaining a high immune response in s but also said response is specific for Aβ40 (without a significant humoral response to Aβ42), i.e., anti-Aβ40 specific antibodies are generated in the immune response that do not bind to Aβ42.

The results included in Examples 1 to 4 prove and confirm the technical advantages and effects explained above in the description, g that the use of SM as a crosslinking agent allows generating KLH-crosslinking agent-CysAβ(33-40) conjugates that generate a greater immune response with respect to when another crosslinking agent of the prior art is used. onally, -CisAβ(33-40) conjugates allow generating high immune responses in mice and rabbits, specific for Aβ40 (without a significant humoral response to Aβ42), i.e., anti-Aβ40 specific antibodies that do not bind to Aβ42 are generated in said immune ses. Said examples validate the usefulness of the conjugate of the present invention in the treatment of amyloid diseases, preferably Alzheimer’s disease, in mammals, preferably in humans.

Example 5. Adsorption studies for KLH-SM-CysAβ(33-40) conjugates onto the aluminum hydroxide gel. 1.35 mg/ml, peptide is lent to 14.4 mg/ml conjugate KLH-SM-CysAβ(33-40).

Tables 7.

In these studies the influence of the tration of conjugate on the adsorption rate as well as the influence of the pH on the adsorption rate were ined. In the first experiment the tion rate of ent concentrations of conjugate in a defined amount of aluminium hydroxide (0.35 % corresponds to the permitted dose of 1.25 mg Al per single dose) and the defined pH 7.4 (physiological pH) was tested. At the physiological g conditions (pH 7.4) 12 to 18 % free conjugate was detected in the supernatant.

A reduction of the amount of peptide to 100 μg peptide does not yield higher adsorption rates.

In a second study the influence of the pH on the adsorption rate was determined. The pH was altered (pH 6.0 to pH 7.4) at defined conjugate concentrations (based on 100 μg, 150 μg and 220 μg peptide net/mL). As illustrated in the results shown above, the adsorption rate depends on the pH. In fact, the adsorption rate increases at lower ues (see tables): A reduction from pH 7.4 to pH to 7.2 or even 7.0 does not result in significant higher tion rates. An adsorption at pH 6.8 shows an adsorption rate around 90 %. At pH 6.0 the adsorption ratio is the highest. At a conjugate concentration based on 200 μg/mL net peptide and pH 6, 7% of conjugate is still free.

Based on these analyses, in order to increase the immunogenic capacity of the pharmaceutical composition of the invention, a pH value of between 5.8 to 7.0 should preferably be used in order to increase the adsorption rate of the conjugate onto the adjuvant.

Although the invention has been described with respect to preferred embodiments, the latter must not be considered to be limiting of the ion, which will be defined by the broadest interpretation of the following claims.

WE

Claims (26)

CLAIM :

1. A composition comprising a conjugate of at least one CysAβ(33-40) peptide (SEQ ID NO: 1) linked to the keyhole limpet hemocyanin (KLH), wherein the crosslinking agent connecting each CysAβ(33-40) 5 peptide to the keyhole limpet hemocyanin (KLH) of the conjugate is the maleimidobutyric acid N- hydroxysuccinimide ester (SM), wherein the composition further comprises aluminum ide gel.

2. A pharmaceutical composition sing i) a conjugate comprising at least one CysAβ(33-40) 10 e (SEQ ID NO: 1) linked to the e limpet hemocyanin (KLH) and ii) aluminum hydroxide gel, wherein the crosslinking agent connecting each CysAβ(33-40) peptide to the keyhole limpet hemocyanin (KLH) of the conjugate is the maleimidobutyric acid oxysuccinimide ester (SM), and wherein the pH of the composition ranges from 5.8 to 7.0. 15

3. The pharmaceutical composition according to claim 2, wherein the pH of the composition ranges from 6.2 to 7.0.

4. The ceutical composition according to claim 2, wherein the pH of the composition ranges from 5.8 to 6.2.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein the amount of CysAβ(33-40) peptides (SEQ ID NO: 1) in the pharmaceutical ition is at least 100μg.

6. The pharmaceutical composition according to any one of claims 2 to 4, n the amount of 25 CysAβ(33-40) peptides (SEQ ID NO: 1) in the pharmaceutical composition is at least 150μg.

7. The pharmaceutical composition according to any one of claims 2 to 4, wherein the amount of CysAβ(33-40) es (SEQ ID NO: 1) in the ceutical composition is from 150μg to 400μg. 30

8. The pharmaceutical composition according to any one of claims 2 to 4, wherein the amount of CysAβ(33-40) peptides (SEQ ID NO: 1) in the pharmaceutical composition is from 160μg to 240μg.

9. The pharmaceutical composition according to any one of claims 2 to 8, wherein the conjugates in turn comprise a ratio of at least 45 CysAβ(33-40) peptides (SEQ ID NO: 1) linked to each keyhole limpet 35 hemocyanin (KLH).

10. A glass ampoule comprising the pharmaceutical composition of any one of claims 2 to 9.

11. A method to manufacture a pharmaceutical composition according to any one of claims 2 to 9, which 40 comprises the following steps: a. Adding maleimidobutyric acid N-hydroxysuccinimide ester (SM) to a solution comprising keyhole limpet hemocyanin (KLH) in a buffer at a pH between 7.0 to 9.0; b. ating the excess of maleimidobutyric acid N-hydroxysuccinimide ester from the solution of step a); c. Adding CysAβ(33-40) peptides (SEQ ID NO: 1) in DMSO to the solution of step b) at a pH of between 6.6 to 7.0 to produce the conjugates; 5 d. Eliminating the free peptide from the solution from step c); e. Optionally filtrating the solution of step d); f. Adjusting the pH of the solution of step d) or e) to a pH range of between 5.8 to 6.2; and g. Adding aluminum ide gel to the solution of step f) once the pH has been ed. 10

12. The method of claim 11, wherein the excess of step b) is eliminated by using 0.02 M Na-Phosphatebuffer at a pH of about 6.6 to 7.0.

13. The method of claim 11 or claim 12, wherein the excess of step d) is eliminated by using 0.01 M PBS-buffer at a pH of 6.6 to 7.0.

14. The method of any one of claims 11 to 13, n the on is filtrated in step e) by using a filter of about 0.2 μm.

15. The method of any one of claims 11 to 14, wherein the pH of step f) is adjusted to a pH range of 20 about 6.0.

16. Use of the pharmaceutical composition of any one of claims 2 to 9 or the glass ampoule of claim 10, in the manufacture of a ment for the ent of an amyloid disease. 25

17. The use according to claim 16, wherein the amyloid disease is selected from the list ting of Alzheimer’s disease, Parkinson’s disease, cerebral amyloid angiopathy, vascular ia of an amyloid origin, inclusion-body myositis and dementia with Lewy bodies.

18. The use according to claim 17, wherein the amyloid disease is Alzheimer’s disease.

19. Use of a ate of at least one CysAβ(33-40) peptide (SEQ ID NO: 1) linked to the keyhole limpet hemocyanin (KLH) in the manufacture of a medicament for the treatment or prevention of an d disease, wherein the crosslinking agent connecting each CysAβ(33-40) peptide to the e limpet hemocyanin (KLH) of the conjugate is the maleimidobutyric acid N-hydroxysuccinimide ester (SM), and 35 wherein the medicament further comprises um hydroxide gel.

20. The use of claim 19, wherein the amyloid disease is selected from the list consisting of Alzheimer’s disease, Parkinson’s disease, cerebral amyloid angiopathy, vascular dementia of an amyloid origin, inclusion-body myositis and dementia with Lewy bodies.

21. The use of claim 20, wherein the amyloid disease is Alzheimer’s disease.

22. A composition according to claim 1, substantially as herein described with reference to any example thereof.

23. A pharmaceutical composition according to any one of claims 2 to 9, substantially as herein described with reference to any example thereof.

24. A glass ampoule according to claim 10, substantially as herein described with nce to any example thereof. 5

25. A method according to any one of claims 11 to 15, substantially as herein described with reference to any example thereof.

26. A use according to any one of claims 16 to 21, substantially as herein described with reference to any example thereof. 10985845_1