Vaccination methods against self-antigens in human patients - Patents.com

Abstract

To provide a method for vaccination against a self-antigen in a human patient.SOLUTION: The method of the present invention comprises administering a dose with an effective amount of a self-antigen to a patient to elicit a primary immune response, characterized in that the patient is subjected to a boost administration of the self-antigen, the amount of the self-antigen in the dose for the boost administration being higher than the amount of the self-antigen in the dose used in the administration for the primary immune response.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method of vaccination against self-antigens in human patients and to a set of vaccine formulations suitable for such vaccination. [Background technology]

[0002] Active immunization with self-protein antigens consists of generating an immune response involving both T and B cells, which leads to the induction of a highly efficient B cell differentiation pathway through specific structures (germinal centers, GCs) where antigen-specific B cells proliferate and differentiate into antibody-secreting plasma cells and memory B cells. In view of the fact that autoimmunity is the main side effect of vaccines against self-proteins (e.g., Non-Patent Document 1), a potentially successful vaccine must meet a series of criteria.

[0003] First, the antigenic component must not activate T cells but must act as a B cell antigen. Hence, Ags must be presented on their MHC molecules by antigen-presenting cells. Binding to them requires that the peptide Ag (i) has a certain length (8-9 aa for MHC class I) and (ii) presents certain amino acid residues at so-called anchor positions (Non-Patent Document 2). In general, antigen epitopes must be short enough to prevent T cell activation, but long enough to act as antibody epitopes. However, the generation of IgG-type Ab responses also depends on helper T (TH) cells. Thus, the vaccine must contain epitopes that can activate helper T (TH) cells. These must not be associated with the Ag of interest, as this could lead to cellular autoimmunity. Ideally, they must be strong TH epitopes and known not to be able to activate Abs that cross-react with molecules / structures found in humans. In this regard, various protein carriers have been shown to be potent activators of T cell help preclinically as well as clinically. Keyhole limpet hemocyanin (KLH) is a well-described "human immune system irrelevant" protein that generates strong Ab responses when physically bound to specific antigenic epitopes, e.g., antigenic peptide sequences.

[0004] Tailoring the Ab response of a vaccine targeting a self-protein is a real challenge. It is necessary to capture the desired structure without attacking related structures. This requires meeting two criteria simultaneously. First, the Abs raised must bind to the given target, which is essential for the intended activity of the vaccine. Second, however, they must not bind to related or homologous structures, which is an essential safety feature.

[0005] Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is a complex systemic disorder that causes a wide range of debilitating motor and non-motor symptoms. Typically, the presenting signs of PD are indefinite, and the non-motor symptoms include neuropsychiatric disorders (e.g., depression, REM sleep disorder), gastrointestinal disorders (e.g., constipation), and autonomic dysfunction. The cardinal motor symptoms of PD include resting tremor, bradykinesia, rigidity, and postural instability, and can develop up to 10 years after the onset of the non-motor symptoms. PD currently affects approximately 5 million people worldwide, and its prevalence is increasing with the aging of the global population.

[0006] Despite considerable investment, there are currently no drugs available with disease-modifying properties. Treatment of PD primarily addresses motor symptoms through the use of dopaminergic strategies (levodopa or dopamine agonists, COMT inhibitors and MAO-B inhibitors), anticholinergic drugs, or deep brain stimulation, which only have symptomatic effects and are associated with many side effects. More than 20 clinical trials with potential disease-modifying agents have been conducted or are currently ongoing. However, none have reached clinical endpoints (Non-Patent Document 3). As a result, there is an urgent need to develop novel disease-modifying treatments.

[0007] Increasing evidence indicates a causal role of alpha-synuclein (aSyn) oligomers in the process of causing neurodegeneration in PD. Lewy bodies or Lewy neurites are characteristic histopathological markers of PD. They are found mainly in neurons and are composed mainly of misfolded filamentous aSyn. Under physiological conditions, aSyn exists as a cytoplasmic intracellular unfolded protein that is abundant in the nucleus and presynaptic terminals, and it has been suggested that aSyn may be physiologically associated with synaptic plasticity. In PD, pathological aggregated forms of aSyn, possibly including oligomers of aSyn, appear to propagate not only the disease but also the Lewy body pathology by spreading prion-like from cell to cell. Furthermore, several lines of evidence in animal models support the theory that reducing the accumulation of oligomeric aSyn may have disease-modifying effects (Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, and Non-Patent Document 7). Thus, it has been recognized that treatments that reduce aSyn aggregates have the potential to prevent disease progression (Non-Patent Document 8, Non-Patent Document 9). In the last decade, significant progress has been made toward the development of immunotherapeutic approaches to remove aSyn aggregates. Immunization strategies against aSyn have already been shown to promote the degradation of aSyn aggregates (Non-Patent Document 10), prevent cell-to-cell spread (Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13), and reduce motor impairment (Non-Patent Document 14) in animal models. Taken together, these studies provide evidence that immunization against aSyn may have disease-modifying properties in humans.

[0008] AFFITOPE® PD01A has been developed for the treatment of synucleinopathies such as PD (see Non-Patent Document 15). PD01A is a peptide-KLH conjugate in which the peptide portion mimics the carboxyl-terminal region of human aSyn (Patent Document 1). It targets aSyn while avoiding closely related protein family members including β-synuclein (bSyn) and may have neuroprotective properties (Non-Patent Document 16). Increasing evidence indicates a causal role of alpha-synuclein (aSyn) oligomers in the process of causing neurodegeneration in PD. Lewy bodies or Lewy neurites are characteristic histopathological markers of PD. They are found mainly in neurons and are composed mainly of misfolded fibrillar aSyn. Furthermore, several lines of evidence in animal models support the theory that reducing the accumulation of oligomeric aSyn may have disease-modifying effects. Thus, treatments that reduce aSyn aggregates may have the potential to impede disease progression.

[0009] Recently, it was shown that vaccination with PD01A resulted in reduced accumulation of aSyn oligomers and improved memory and motor impairments in two mouse models of synucleinopathy covering PD and MSA (multiple system atrophy) (Non-Patent Document 17). Despite considerable clinical investment, there are currently no available therapeutic drugs with disease-modifying properties in humans. Treatment of PD mainly addresses motor symptoms by using dopaminergic strategies, anticholinergic drugs, or deep brain stimulation, which only have symptomatic effects and are associated with many side effects. More than 20 clinical trials with potential disease-modifying agents have been conducted or are currently ongoing. However, none have reached clinical endpoints. As a result, there is an urgent need for the development of novel disease-modifying treatments. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2009 / 103105A2 [Non-patent literature]

[0011] [Non-Patent Document 1] Gilman et al., Neurology 64(2005), 1553-1562 [Non-Patent Document 2] Rudolph, Ann.Rev.Immunol.24(2006),419-466 [Non-Patent Document 3] ALDakheel et al., Neurotherapeutics 11, (2014) 6-23 [Non-Patent Document 4] Games et al., Am. J. Pathol. 182 (2013), 940 [Non-Patent Document 5] Kim et al., J. Neurochem. 107 (2008), 303 [Non-Patent Document 6] Recasens et al., Ann. Neurol. 75 (2014), 351 [Non-Patent Document 7] Winner et al., J. Neurosci. 32 (2012), 16906 [Non-Patent Document 8] Lee et al., Neurosci. Res. 70 (2011), 339 [Non-Patent Document 9] Valera et al., Pharmacol.Ther.138(2013), 311 [Non-Patent Document 10] Masliah et al., Neuron. 46 (2005), 857 [Non-Patent Document 11] Bae et al., J. Neurosci. 32 (2012), 13454 [Non-Patent Document 12] Banner et al., PNAS 101 (2004), 9435 [Non-Patent Document 13] Games et al., J.Neurosci.34(2014), 9441 [Non-Patent Document 14] Masliah et al., PLoS One.6(2011), e19338 [Non-Patent Document 15] AFFiRiS press release reporting first clinical data on PD01A; http: / / www.affiris.com / news / first-clinical-data-of-therapeutic-parkinsons-disease-vaccine-encourage-s-continued-development / [Non-Patent Document 16] Vigneswara et al., PLos One8 (2013), e61442 [Non-Patent Document 17] Mandler et al., Molecular Neurodegeneration 10 (2015), 10 Summary of the Invention

[0012] It is an object of the present invention to provide more efficient disease-modifying treatments based on immunization with autoantigens.

[0013] Thus, the present invention provides a method of vaccination against an autoantigen in a human patient, in which an effective dose of an autoantigen is administered to the patient to induce a primary immune response, characterized in that the patient receives a boost dose of the autoantigen, wherein the amount of autoantigen in the boost dose is higher than the amount of autoantigen in the dose used in the administration for the primary immune response (i.e. higher than the amount of autoantigen in the dose used for priming the immune response).

[0014] Within the course of the present invention, the applicant has obtained clinical data showing that in the process of vaccination of patients against self-antigens, boost administration plays a central role in obtaining effective immunological and therapeutic effects. It has also become clear that the data obtained with conventional (non-self antigens, e.g. pathogen antigens) are not comparable or compatible with vaccination strategies against self-antigens. The results obtained by the present application show that it is advantageous if primary immunization (i.e. obtaining a primary immune response) is induced by a relatively small amount of antigen, and boost immunization is induced by a larger amount of self-antigen than in the administration for the primary immune response.

[0015] Despite the promising initial results in the AFF008 trial course (see Non-Patent Document 15 above), it was surprising that the specific selection of the booster vaccination scheme according to the invention significantly induced clinical outcomes of the overall performance of the different dose regimens applied. Especially over a long period of time, the dose of the vaccine in the boost phase (compared to the priming phase) was of paramount importance for the specific antibody response and the specific responsiveness of the vaccine. This was particularly surprising, because favorable results were obtained when the amount of autoantigen in the dose for boost administration was higher than the amount of autoantigen in the dose used in the administration for the primary immune response. This could not be derived from the prior art proposing vaccination against such antigens (e.g. WO2009 / 103105 A2, WO2011 / 009152 A1, WO2014 / 033158 A2, etc.), nor from scientific reports on model mice applying such vaccines (Mandler et al., Mol. Neurodeg. 10(2015):10; Mandler et al., Acta Neuropathol. 127(2014):861-879). This was even more unpredictable from the prior art. This is because the effects of priming and boosting administrations as previously reported are specific to a particular vaccine and a particular dose or even to a particular individual (e.g., Shete et al., J. Aids & Clin. Res. 5 (2014), DOI: 10.4172 / 2155-6113.1000293; Morera et al., Vaccine 30 (2012): 368-377; Gonzales et al., Hum. Vaccines 3 (2007): 8-13), but were considered not to be specific for the class of autoantigen vaccines according to the present invention, in particular for the autoantigens disclosed herein and shown in the Examples section of the present invention, the so-called amyloid beta, precursor protein convertase subtilisin / kexin type 9 (PSCK9), membrane-bound immunoglobulins of the IgE type, huntingtin protein, CD26 or alpha-synuclein (see below).It is essential for the present invention that the dose per administration for the primary immune response is low, i.e. lower than in the boost administration, so that the most specific B cells can be selected in this primary vaccination phase. This strategy prevents the induction of less specific B cells and antigen receptors. On the other hand, the boost administration should be performed with a high dose (i.e. higher than in the primary immune response) in terms of the amount of antigen in order to address as many B cells (produced in the primary immune response) as possible, regardless of where they are currently located (bone marrow, lymph nodes, spleen, etc.). The higher amount used according to the present invention therefore allows a boost that can establish (based on the most specific B cells primed together with the primary immune response) an efficient immunization against the autoantigen, and ultimately an efficient control of said autoantigen by the vaccination method of the present invention. In this regard, it is also important to note that the induction of the primary immune response can be achieved by administering one or more initial doses of the autoantigen to the individual to be vaccinated. Preferably, the primary immune response is induced by administering two, more preferably three, and especially preferably four, doses of the autoantigen. These administrations for the primary immune response are preferably spaced apart by at least 2 weeks, more preferably at least 3 weeks, and particularly preferably at least 4 weeks. Many vaccination schemes apply a vaccination interval of once every two weeks or once a month, which is therefore particularly preferred. The same applies to booster administrations. As already mentioned, the essential factor for achieving the object of the present invention, i.e. for generating an overall efficient immune response with respect to self-antigens, is the dosage per administration, not the (total) amount of all the administered dosages.

[0016] This has notably been demonstrated for the present invention in clinical trials of a vaccination strategy for PD targeting alpha-synuclein ("aSyn") as an autoantigen, utilizing aSyn mimotopes to modulate brain levels of aSyn aggregates, which in the case of PD are pathological protein aggregates.

[0017] The applicants of the present invention recently completed a Phase I first-in-human clinical trial evaluating the safety and tolerability of two doses (15 μg and 75 μg) of the therapeutic vaccine AFFITOPE® PD01A administered to early stage PD patients. All adverse events were mild or moderate in severity and the treatment was deemed well tolerated. Antigen (PD01A) administration (applied once a month for four times) was demonstrated to induce an extrafollicular response resulting in the appearance of (low) IgG Abs reactive with both (i) the peptide component of the vaccine, PD01A, and (ii) the target aSyn peptide sequence (Figure 1). As B cells proliferated in the germinal center and differentiated into plasma cells, IgG Ab titers increased to peak values, which were reached 4 weeks after the third immunization (V8).

[0018] To better understand the induction of functional Abs, e.g., aSyn-specific Abs, by immunization, the relationship between the dose at the time of priming immunization and the resulting Ab response, and how the primed immune response (PD01A-induced response) against self-antigens (e.g., aSyn) can be "reactivated" in humans, patients who received PD01A treatment in the AFF08A study were analyzed in a second clinical trial. For this purpose, PD01A-immunized PD patients were boosted with a single dose of either 15 μg or 75 μg PD01A after 115 weeks (on average). In the AFF08A trial, "booster" exposure to the antigen PD01A reactivated immune memory, resulting in a rapid rise in Ab titers against PD01A and the targeted aSyn peptide sequence (see Figure 1), demonstrating a true secondary immune response. In general, short-lived plasma cells maintain peak Ab levels for 2-3 weeks. Thereafter, serum Ab titers initially decline at a rapid rate similar to that following primary immunization. Long-lived plasma cells that reach a survival niche in the bone marrow continue to produce antigen-specific Abs and then decline at a slower rate.

[0019] Many determinants regulate the strength of vaccine-induced GC and therefore the peak Ab response. The major determinants are the nature of the vaccine antigen and its inherent immunogenicity.

[0020] Another important factor of primary vaccine Ab response is the use of optimal dose of vaccine antigen, which can be experimentally determined. Generally, up to a certain threshold, higher dose of antigen induces higher primary Ab response. Above the threshold, a bell-shaped or plateau-shaped response can be seen. This can be particularly useful when immunocompetence is limited.

[0021] A limited dose of vaccine may limit the primary immune response, but increase the competition of B cells for follicular dendritic cell-associated antigens, thus leading to a more stringent selection of higher affinity GC-B cells and a stronger secondary "boost" response. Thus, in humans, only a specific combination of antigenic doses, e.g., optimal antigenic dosing-window, in the "priming" phase and again in the "boosting" phase, will provide a sufficient Ab response capable of modulating the expression of pathological protein aggregates, e.g., aSyn aggregates in the case of PD.

[0022] As already mentioned above, the vaccine according to the invention meets the relevant criteria as a suitable vaccine dealing with self-antigens ("self-proteins"), directed against antigens that are "self" to the individual to be vaccinated, but must not cause autoimmunity as a side effect when administered. Thus, the antigenic components of the self-antigen must not activate T cells, but act as B cell antigens. Thus, the antigen must be presented by antigen-presenting cells on their MHC molecules. The requirements reported by Rudolph, Ann. Rev. Immunol. 24 (2006), 419-466 also apply. In general, antigenic epitopes must be short enough to prevent T cell activation and long enough to act as antibody epitopes. Furthermore, the generation of IgG-type antibody responses is also dependent on helper T (TH) cells, therefore the vaccine must contain epitopes capable of activating helper T (TH) cells (not related to the antigen of interest (to prevent cellular autoimmunity)). Ideally, a vaccine according to the invention is a strong Th epitope, known to be unable to activate antibodies that cross-react with other molecules / structures found in humans. Thus, (protein) carriers such as keyhole limpet hemocyanin (KLH) have been shown to be potent activators of T cell help, not only clinically but also preclinically. For example, KLH is a well-described protein that is "foreign to the human immune system" and generates a strong Ab response when physically bound to a specific antigenic epitope, e.g., an antigenic peptide sequence. The immune response of the vaccine of the invention needs to be directed to the desired structure without attacking related structures. Thus, the antibodies raised in the vaccinated individual need to bind to the predetermined target (self-antigen), which is essential for the intended activity of the vaccine. Furthermore, these vaccine-induced antibodies should not bind to related or homologous structures (as an essential safety feature).

[0023] Preferably, the autoantigen according to the present invention is an antigen that has been reported or suggested in the art as a scientifically plausible vaccine candidate or vaccination target.

[0024] The method according to the invention is therefore in principle applicable to any kind of autoantigen vaccination strategy. However, it is preferred to use the method according to the invention for autoantigens for which it is known that it is problematic to obtain an efficient immune response. Thus, preferred autoantigens according to the invention are selected from the group consisting of amyloid beta, proprotein convertase subtilisin / kexin type 9 (PSCK9), membrane-bound immunoglobulins of the IgE type, huntingtin protein, CD26 and alpha-synuclein antigens, preferably alpha-synuclein antigens, in particular alpha-synuclein antigens containing the DMPVDPDN epitope and / or the KNEEGAP epitope.

[0025] It has been shown that vaccination immunity can be further improved when a variation of the natural sequence of the autoantigen is applied, which has at least the same (preferably improved) immunity-conferring properties compared to the natural sequence.Therefore, in the context of the present invention, it is clear that such a variation of the natural autoantigen amino acid sequence should be included within the scope of the term "autoantigen" according to the present invention, as long as the immunity-conferring properties are (at least) preserved (or preferably improved) in terms of inducing specific (natural) autoantigen-binding antibodies in vaccinated individuals.Such a variation can be provided by AEEITOMES technology (for example, as disclosed in Schneeberger et al., Human Vacc. 6 (2010), 1-5). These variations provided by the AEEITOME® technology are called AFFITOPE® and are disclosed, inter alia, in WO2004 / 062556A and WO2006 / 005707A for amyloid beta, in WO2009 / 103105A and WO2011 / 020133A for aSyn, in WO2011 / 009152A for AngII, in WO2013 / 174920A for C5a, and in WO2014 / 033158A and WO2015 / 128287A for PCSK9. The AFFITOPE® disclosed in these documents is also a preferred autoantigen according to the present invention.

[0026] Therefore, in the present invention, it is preferred to use as autoantigens variations of the natural sequence of autoantigens, i.e. AFFITOPEs (registered trademarks) (which may also be called "mimotopes", which are peptides that contain sequence variations compared to the original natural sequence of an autoantigen, but which exhibit similar (the same or improved) immunoconferring properties, i.e. are able to induce an immune response similar or higher than that obtained with the natural sequence; mimotopes are therefore recognized by the same mAbs or pAbs as the natural autoantigen, but induce a stronger immune response or an immune response that is not associated with severe side effects such as neuroinflammation). Thus, in the vaccination according to the invention, as autoantigens, aSyn mimotopes, angiotensin II mimotopes or PCSK9 mimotopes are used, more preferably DQPVLPD, DMPVLPD, DSPVLPD, DSPVWAE, DTPVLAE, DQPVLPDN, DMPVLPDN, DSPVLPDN, DQPVTAEN, DSPVWAEN, DTPVLAEN, HDRPVTPD, DRPVTPD, DVPVLPD, DTPVYPD, DTPVIPD, HDRPVTPDN, DRPVTPDN, DNPVHPEN, DVPVLPDN, DTPVYPDN, DTPVIPDN, DQPVLPDG, DMPVLPDG, DSPVLPDG, DSPVWAEG, DRPVAPEG, DHPVHPDS, DMPVSPDR, DSPVPPDD, DQPVYPDI, DRPVYPDI , DHPVTPDR, EYPVYPES, DTPVLPDS, DMPVTPDT, DAPVTPDT, DSPVVPDN, DLPVTPDR, DSPVHPDT, DAPVRPDS, DMPVWPDG, DAPVYPDG, DRPVQPDR, YDRPVQPDR, DMPVDPEN, DMPVDADN, EMPVDPDN, DNPVHPE, KNDEGAP, ANEEGAP, KAEEGAP , KNAEGAP, RNEEGAP, HNEEGAP, KNEDGAP, KQEEGAP, KSEEGAP, KNDDGAP, RNDEGAP, RNEDGAP, RQEEGAP, RSEEGAP, ANDEGAP, ANEDGAP, HSEEGAP, ASEEGAP, HNEDGAP, HNDEGAP, RNAEGAP, HNAEGAP, KSAEGAP, KSDEGAP, KSEDGAP,aSyn mimotopes selected from the group consisting of RQDEGAP, RQEDGAP, HSAEGAP, RSAEGAP, RSDEGAP, RSEDGAP, HSDEGAP, HSEDGAP, and RQDDGAP, in particular selected from the group consisting of DQPVLPD, DSPVLPD, DVPVLPD, DSPVLPDG, YDRPVQPDR, DHPVHPDS, DAPVRPDS, KNDEGAP, KQEEGAP or KSEEGAP, DPVYIHPF, DAVYIHPF, DRHYIHPF, DAAYIHPF, DRAYAHPF, DPGYIHPF, DRAYDHPF, AAYIHPF, RAYAHPF, and PGYIHPF. It is preferred to use an angiotensin II mimotope selected from the group consisting of SIPWSLERIT, SIPWSLERITPPR, SIPWSLERTTPPR, VIPWNLERILPPR, SVPWNLERIQPPR, SIPWSLERTT, SIPWSLERLT, SIPWSLERLTPPR, SIPWSLERIQ, SIPWSLERIQPPR, VIPWNLERIL and SVPWNLERIQ, in particular SIPWSLERIT, VIPWNLERIL or SVPWNLERIQ.

[0027] It is preferred to use a significantly higher amount of autoantigen in the boost dose than in the dose for primary immune induction. Thus, the amount of autoantigen in the boost dose is at least 20%, preferably at least 50%, more preferably at least 100%, and especially preferably at least 200% higher than the amount used in the dose for primary immune response.

[0028] In absolute terms, it is preferred that the amount of autoantigen in the boost dose is at least 20 μg, more preferably 50 μg. In this context, it is important to note that the "μg autoantigen" referred to in the present invention means the amount of antigen peptide in the dose and does not include the carrier or linker portion (if any) of the vaccine complex.

[0029] In the context of the present invention, it is important to perform boost vaccination when the primary immune response has already ended, i.e. when the antibody titer induced by this primary vaccination (induced by one, two, three, four or more vaccine administrations in the course of primary immune response induction) has fallen beyond a significant level (e.g., beyond a given threshold level or even beyond the detection limit of a (preferably rather insensitive) assay suitable for testing a large number of samples), or has fallen to at least 30%, preferably 20%, in particular 10% or less of the maximum antibody level present in the course of said primary vaccination. Such a level may differ between different autoantigen vaccines, but is usually present at least 6 months after primary vaccination. Thus, in a preferred embodiment, said boost administration can be administered at least 6 months, preferably at least 12 months after the first administration of autoantigen to induce primary immune response. There are also strategies in which boost administration is preferably performed at a later time, for example 18 months, 2 years, 3 years or 5 years after primary immune response vaccination.

[0030] The route of administration according to the present invention is generally the same as the current vaccination route. Thus, preferred administration of autoantigens is subcutaneous, intradermal or intramuscular.

[0031] According to a preferred embodiment of the invention, the autoantigen is administered together with an adjuvant, preferably aluminum hydroxide. According to its most preferred embodiment, the invention relates to the use of a European Pharmacopoeia grade (Aluminium-oxyhydroxide, monograph 1664), in particular the product manufactured by Buttentag Biosector (2% Alhydrogel) tested for European Pharmacopoeia compliance. Alhydrogel is available in three varieties: Alhydrogel 1.3%, Alhydrogel 2% and Alhydrogel "85". Alhydrogel 2% was selected as the international standard for aluminum hydroxide gel. The pharmaceutical preparations according to the invention are formulated aseptically in a suitable buffer, preferably an isotonic phosphate buffer (1 mM to 100 mM), preferably at an alhydrogel concentration of 1.0 mg / mL or more (given as an equivalent value of Al2O3. This metric (Al as "equivalent value of Al2O3") is generally used for the present invention. Accordingly, all dosages and amounts mentioned in this application, insofar as they relate to aluminum hydroxide, refer to equivalent values ​​of Al2O3 (of aluminum hydroxide (alhydrogel)), even more preferably at an alhydrogel concentration of 1.5 mg / mL or more, most preferably at an alhydrogel concentration of 2.0 mg / mL or more (given as an equivalent value of Al2O3). The amount of aluminum salt for the alhydrogel is given as equivalent value of Al2O3 according to the strength as stated by the manufacturer (i.e. 2% alhydrogel is equivalent to 2% Al2O3, i.e. 20 mg / mL of Al2O3). The concentrations can be directly converted to respective aluminum concentrations by using the respective molecular masses (20 mg / mL Al2O3 (molecular weight 101,96) is equivalent to 10.6 mg / mL aluminum (molecular mass 26,98)).

[0032] Preferred autoantigens according to the invention are polypeptides comprising 7 to 30, preferably 7 to 20, more preferably 7 to 16, most preferably 8 amino acid residues. It is also preferred (and this is often essential to obtain an immune response) to conjugate these peptide antigens to a pharma- ceutically acceptable carrier, preferably a protein carrier, especially keyhole limpet hemocyanin (KLH), Crm-197, tetanus toxoid (TT) or diphtheria toxin (DT).

[0033] According to a further preferred embodiment, the boost administration is repeated after some time, for example after 1, 2, 3, 5 or 10 years. Preferably, the second or subsequent boost administration is performed in the same or similar manner as the first boost administration, i.e. with an increased amount of autoantigen compared to the dose of the primary vaccination.

[0034] According to a further aspect, the present invention provides a kit for use in vaccination against an autoantigen in a human patient, comprising: a first vaccine formulation containing an effective amount of an autoantigen to induce a primary immune response against the autoantigen; and a second vaccine formulation containing an effective amount of the autoantigen to induce a boost immune response against the autoantigen. wherein the amount of autoantigen in the second vaccine formulation is higher than in the first vaccine formulation.

[0035] In the kit, a vaccine formulation according to the invention (ie as disclosed herein) is provided.

[0036] The present invention also relates to the use of a kit according to the invention for the manufacture of a vaccine for inducing an immune response against a self-antigen in a human patient.

[0037] The present invention also provides a vaccine for use in vaccinating a human patient against an autoantigen, in which a dose comprising an effective amount of an autoantigen, particularly a mimotope of an autoantigen, is administered to the patient to induce a primary immune response, wherein the patient receives a boost dose of the autoantigen, the amount of the autoantigen in the boost dose being higher than the amount of the autoantigen in the dose used in administration for the primary immune response.

[0038] The invention is further disclosed in the following non-limiting examples and figures. [Brief description of the drawings]

[0039] [Figure 1] Figure 1A-D shows that injection of the aSyn mimotope PD01A 20 months after priming induces an immunological boosting effect. (C) The most significant immunological effect is seen when priming with four monthly injections of 15 μg PD01A and boosting with 75 μg PD01A. (D) Boosting with 75 μg still enhanced the antibody response above the level reached during priming. Figure 1E-H shows aSyn-specific antibodies in human serum. Figure 1I-L shows KLH-specific antibodies in human serum. [Diagram 2] 2A-D show titers against AngII (3×2-weekly vaccinations with 5 μg, with boosts of 5 μg at week 22 and 50 μg at week 33). [Diagram 3] FIG. 13: Titers against huPCSK9 (Long Term (3×2 week vaccinations (2 doses) and 52 week follow up). Re-vaccination with 30 μg at week 52). [Figure 4] FIG. 1 shows the vaccine formulation for the AFF008A clinical trial. [Diagram 5] FIG. 1 shows baseline characteristics of patients in the AFF008A clinical trial. [Figure 6] FIG. 1 shows the immunization schedule and follow-up of the AFF008A clinical trial. [Figure 7] FIG. 1 shows adverse events by study group in the AFF008A clinical trial. [Figure 8] FIG. 1 illustrates SOC terms and adverse events by study group for the AFF008A clinical trial. [Figure 9] FIG. 1 shows PD01-specific antibody titers in the AFF008A clinical trial. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0040] PD vaccination boost AFFITOPE® PD01A was developed for the treatment of synucleinopathies such as PD (WO2009 / 103105A2). PD01A is a peptide-KLH conjugate, where the peptide portion mimics the carboxyl-terminal region of human aSyn (with the natural epitope DMPVDPDN). It targets aSyn while avoiding closely related protein family members including β-synuclein (bSyn) and may have neuroprotective properties (Vigneswara et al., PLoS One8 (2013), e61442). It has recently been shown that vaccination with PD01A resulted in reduced accumulation of oligomers of aSyn and improved memory and motor impairment in two mouse models of PD (Mandler et al., Acta Neuripathol. 127 (2014), 861; Mandler et al., Molecular Neurodegeneration 10:10 (2015)).

[0041] Participants: Study participants were recruited from Vienna, Austria, and the surrounding area. For entry into the clinical trial, patients were required to have a confirmed diagnosis of PD. Individuals with idiopathic Parkinson's disease were known to have had the disease for less than 4 years, exhibited Hoehn-Jahr severity stage I / II, and fulfilled the UK Parkinson's Disease Society Brain Bank Criteria (Hughes et al., J. Neurol. Neurosurg. Psychiatry 55 (1992), 181).

[0042] Patients' dopamine transporter-single photon emission computed tomography (DAT-SPECT) brain scans and magnetic resonance imaging (MRI) scans had to be consistent with a diagnosis of PD. All potential participants receiving conventional PD treatment had to have had at least 3 months of stable treatment prior to the study period and be willing to continue for the entire study period.

[0043] All study subjects provided free informed consent. Study protocols, patient information, informed consent and all other required study documentation were submitted to an independent ethical committee.

[0044] material and method: AFFITOPE® PD01A was initially administered to 24 individuals with early stage PD in AFF008, a Phase I clinical trial designed primarily to evaluate the safety and tolerability of the vaccine and then to evaluate its immunological and clinical activity (preliminary analysis). To this end, two doses (15 μg or 75 μg of AFFITOPE® PD01A (i.e. 15 μg or 75 μg of peptide CDMPVDPDN bound to the corresponding amount of KLH) adsorbed to 1 mg of Alhydrogel (as equivalent Al2O3) were applied four times, four weeks apart. Each dose was applied to 12 patients. In addition, up to eight patients were invited to participate in a non-treated control group. The obtained results (i) confirmed the safety and tolerability of the vaccine, (ii) demonstrated the induction of a vaccine-specific IgG response, and (iii) provided evidence in support of the intended clinical activity. All 32 patients were enrolled in a follow-up study (AFF00 AFF008E) was offered to participants, 31 of whom (24 vaccine patients and 7 controls) agreed, with 30 completing the study. One patient in the low-dose group was lost to follow-up. AFF008E participants were offered one "boost injection" of either the low-dose (15 μg; derived from the high-dose by clinical mixing) or high-dose (75 μg) formulation and were divided into four different treatment groups: low-dose (AFF008)-low-dose (AFF008), low-dose-high, high-dose-low, and high-dose-high. Patients in the control group were left untreated. A total of 28 (22 / 6) agreed, with all 28 completing the AFF008E study.

[0045] Injections were applied by the investigator into the subcutaneous tissue. All administration took place at the investigational site. Patients in the treatment groups were randomized to receive either 15 μg or 75 μg of AFFITOPE® PD01A. Both formulations contain the equivalent of 0.5 mg aluminum.

[0046] The study was conducted in compliance with Good Clinical Practice (GCP), the revised Declaration of Helsinki (2013), and local statutory and regulatory requirements (Austrian Pharmaceutical Affairs Act) and applicable international regulations. The study is registered in a recognized clinical trial repository (www.clinicaltrials.gov, identifiers: NCT01568099, NCT02216188).

[0047] The results are shown in Figure 1A-L. 1A-D show that injection of the aSyn mimotope PD01A 20 months after priming induces an immunological boosting effect. (C) The most striking immunological effect is seen when priming with four monthly injections of 15 μg PD01A and boosting with 75 μg PD01A. (D) Boosting with 75 μg still enhanced the antibody response above the level reached during priming.

[0048] Figure 1E-H shows aSyn epitope responses. Figure 1L shows KLH responses. All Figures 1A-L show antibody titers from human sera.

[0049] Figures 1E-H and 1I-L are organized similarly to Figures 1A-D. The red dotted lines represent group means. The figure headings represent the vaccine doses: priming dose (AFF008) / boosting dose (AFF008A). Group size: n=4 (15μg / 15μg), all other groups n=6. Time period for all figures: 3.5 years. Early stage PD patients (defined by Hoehn-Yarr severity stage I / II) received four priming immunizations with doses of 15 or 75μg, 28 days apart. Antibodies against the immunizing peptide (PD01), against the target aSyn epitope (aa110-130; formulated as a BSA conjugate), and against KLH were quantified by ELISA. The priming dose was mainly beneficial with regard to the duration of the antibody response. The low dose induces longer-lasting antibody response than the high dose, and the strength of both responses is comparable.Similarly, the boost result is determined by the dose.The low dose achieves reactivation of the antibody response to the level of priming response.In contrast, the high dose enhances the antibody response by up to 10 times compared to the response obtained during the priming phase.The best response that forms the basis of the present invention was found with the combination of low and high doses for priming and boosting. EXAMPLES

[0050] AngII vaccination boost vaccine Peptides DRAYAHPF, RAYAHPF, DPGYIHPF and PGYIHPF were conjugated to keyhole limpet hemocyanin (KLH) or CRM197 (Cross Reactive Material197) via the heterobifunctional linker GMBS (4-maleimidobutyric acid N-hydroxysuccinimide ester). The indicated amounts of the conjugated peptides (1, 5, 30, 150 μg) were suspended in aluminum hydroxide (final concentration of aluminum hydroxide was 0.2%). As buffer, either PBS or mannitol / phosphate was used.

[0051] Animal testing Female BALB / c mice were kept under a 12-h light / dark cycle with free access to food and water and were approximately 8–10 weeks old at the start of the experiment.

[0052] Ten Balb / c mice per group were immunized subcutaneously. Mice were initially injected three times with a total volume of 1 mL (2 x 500 μL in the left and right shoulder regions) spaced two weeks apart. Blood was collected at intervals several times after the third injection. Data shown here are from plasma collected approximately 8 months after the third injection (designated as P3i). Approximately 8.5 months after the third injection, half of the remaining mice in each group were boosted with the same amount of antigen as in the first three immunizations or immunized with higher / lower amounts (see scheme below). The final plasma (designated as P4b) was collected 6 weeks after the fourth vaccination.

[0053] [Table 1]

[0054] [Table 2]

[0055] Peptide ELISA To determine the immunogenicity of the vaccine, 96-well Nunc-Maxisorb plates were coated with 1 μM of each injected peptide conjugated to bovine serum albumin (BSA) in 0.1 M NAHC03, pH 9.2-9.4. Non-specific binding was blocked by incubation with blocking buffer (5% BSA in PBS). Appropriate serum dilutions were added to the wells, serially diluted 1:2, and incubated for approximately 1 hour at 37°C. Bound antibodies were detected using biotinylated goat anti-mouse IgG, followed by incubation with horseradish peroxidase conjugated to streptavidin. The substrate ABTS was added and the optical density (OD) at 405 nm was measured in a microwell plate reader. Titers were determined by OD in the assay. max The serum dilution rate was defined as the dilution rate at which the serum concentration reached 50%.

[0056] To calculate the fold induction of antibodies after reboost, the anti-injected peptide titers of individual mice determined after the fourth immunization were divided by the titers determined up to 8 months after the third immunization. The average factors obtained for each group are shown on the graph.

[0057] The results are shown in Figure 2A-D. Groups of BALB / c mice (n=10) received priming immunizations at weeks 0, 2 and 4 by injection with 5 μg of angiotensin II pseudopeptide conjugated to KLH (A and B), 5 μg of angiotensin II pseudopeptide conjugated to CRM (C and D). Immunization with either vaccine also led to the induction of AngII-specific IgG Ab responses. All vaccines showed similar responses in terms of response kinetics, but to some extent differed in terms of intensity. A boost with a low dose, i.e., 5 μg, of each vaccine applied at week 22 did not significantly enhance the AngII-specific Ab responses, regardless of vaccine type / formulation. In contrast, application of each vaccine at a high formulation, i.e., 50 μg, at week 33 boosted the AngII-specific Ab responses clearly beyond the levels reached during the priming phase. EXAMPLES

[0058] PCSK9 Immunity Boost material and method vaccine: The peptides SIPWSLERIT, VIPWNLERIL and SVPWNLERIQ were conjugated to keyhole limpet hemocyanin (KLH) via the heterobifunctional linker GMBS (4-maleimidobutyric acid N-hydroxysuccinimide ester).

[0059] Animal testing: Five BALB / c mice were immunized subcutaneously. Mice were allowed free access to food and water and kept under a 12-h light / dark cycle. Mice were 8-10 weeks old at the start of the 21 experiments. Mice were injected three times, at 2-week intervals, with either 5 μg or 25 μg net peptide conjugated to KLH and adsorbed to Alhydrogel as an adjuvant, in a total volume of 1 mL. Blood was collected approximately 2 weeks after each injection and at 1-month intervals (up to 1 year) after the final third immunization. 52 weeks after the prime immunization, mice were revaccinated (fourth immunization) with 25 μg net peptide conjugated to KLH and adsorbed to Alhydrogel as an adjuvant, in a total volume of 1 mL. Blood was collected 2 and 4 weeks after the fourth immunization (plasma 4a and 4b, respectively).

[0060] Protein ELISA: To determine the immunogenicity of the vaccine, and therefore to identify the amount of PCSK9-specific antibodies in the plasma of the immunized animals, an ELISA immunoassay was performed. The ELISA immunoassay produces a signal that can be easily quantified and represents a quantitative measure of the amount of vaccine-induced PCSK9-specific antibodies. Thus, the titer as measured by the ELISA method directly correlates with the amount of target-specific antibodies (μg / mL) in the plasma samples of treated animals. All plasma samples were taken two weeks after the last immunization and treated equally. To make a direct comparison, quantitative evaluation of the vaccine-induced PCSK9-specific antibodies by PCSK9 protein ELISA immunoassay and comparison with their associated controls (original sequence and negative control) were performed simultaneously for all samples. For this purpose, ELISA plates were coated with recombinantly expressed human PCSK9 protein. Non-specific binding was blocked by incubation with blocking buffer (1% BSA in PBS). Appropriate serum dilutions (pool with starting dilution of 1:100) were added to wells, serially diluted 1:2 (12 dilution steps) and incubated for approximately 1 hour. Bound antibodies were detected by incubation with anti-mouse IgG antibody, ABTS was added as substrate and OD at 405 nm was measured. As a negative control, serum from a control group injected with an irrelevant peptide was analyzed. The titer was determined in the assay by measuring the OD max The serum dilution factor at which the antibody titer reached 50% was defined as the serum dilution factor at which the antibody titer reached 50%.

[0061] The results are shown in Figure 3. Groups of mice received a priming immunization at 0, 2 or 4 weeks by injection with either 5 μg or 25 μg of AFFITOPE vaccine targeting PCSK9. Negative control mice received an unrelated peptide conjugated to KLH and adjuvanted with Alum (aluminum hydroxide). Abs against human PCSK9 were still detectable 48 and 52 weeks after priming for both treatment groups. However, the response was more pronounced in the group of animals receiving the low dose. On the other hand, negative control animals did not show an Ab response against human PCSK9. Application of a single boost with 25 μg of AFFITOPE vaccine at week 52 resulted in a more pronounced increase in huPCSK9Ab, with the levels reached in the low dose (5 μg) / high dose (25 μg) group being approximately twice as high as those reached in the high dose (25) / high dose (25) group. A single injection of 25 μg of AFFITOPE vaccine did not elicit an Ab response in control mice (not previously exposed to AFFITOPE vaccine). EXAMPLES

[0062] AFF008A: Successful boosting of pre-existing immune responses to alpha-synuclein ("aSyn") In a course of clinical trials ("AFF008A"), the safety and immunological analysis of a vaccine approach based on AFFITOPE® using an aSyn targeting sequence (peptide DQPVLPD; "PD01A") was analyzed in patients with early stage Parkinson's disease (PD).

[0063] PD is a common progressive neurodegenerative disease, the prevalence of which correlates with age and reaches up to 4% of people over 85 years of age. The first clinical sign of the disease is movement disorder. However, non-motor symptoms such as anosmia, constipation, depression and orthostatic hypotension are also characteristic of the disease onset, and cognitive impairment or dementia may occur during disease progression. Despite intensive scientific efforts in understanding the pathogenesis of the disease, currently available treatments are limited to symptomatic but lack disease-modifying properties. Alpha-synuclein (aSyn), especially its oligomeric structure, is considered to be the main pathological feature of the disease pathogenesis and is therefore an attractive target in the development of therapeutics for PD.

[0064] The aim of this study was to develop an AFFITOPE®-based vaccine (called PD01A; Figure 4 ) targeting aSyn for the long-term treatment or prevention of PD.

[0065] Research Design and Methods AFF008A (NCT02216188-additive boost), a phase I study evaluating peptide-based immunization with AFFITOPE® PD01A, was a blinded, single-center, randomized, controlled, parallel group evaluating two "boost" doses (15 μg and 75 μg). The study was conducted in early-stage PD patients (FIG. 5). The primary endpoint was the tolerability and safety of a single subcutaneous boost injection (FIG. 6). Each dose was tested in patients who had previously received four priming immunizations with either 15 μg or 75 μg AFFITOPE® PD01A (NCT01568099, "AFF008"; see Example 1). The secondary endpoint was the post-boost immune response induced by the two AFFITOPE® PD01A dosing regimens. Furthermore, the antibody titers of IgG Ab specific to the immunization peptide, KLH (carrier protein) and aSyn target sequence were monitored by ELISA. In addition, the response to aSyn recombinant protein was also monitored by ELISA.

[0066] result The boost using two different doses of PD01A AFFITOPE®-based vaccine was well tolerated (Figures 7, 8). Investigational efficacy variables showed no clinical deterioration in the treated groups compared to the untreated control-arm of the study. The boost vaccination with PD01A resulted in a dose-dependent reactivation of specific immune responses approximately 2 years after priming immunization. That is, PD patients immunized four times with a low dose (AFF008) and boosted with a high dose (AFF008A) showed a clear immunological boost. The immune response was maintained throughout the entire 24-week observation period (Figure 9).

[0067] Immune responses against AFFITOPE® PD01A were observed in 19 of 22 (86%) vaccinated PD patients. Of these 19 PD patients, 14 (73%) produced antibodies specific for the original epitope of aSyn.

[0068] conclusion The AFFITOPE® PD01A vaccine approach in early stage PD patients is well tolerated, elicits long-lasting immune responses, and can be boosted.

Claims

1. 1. A kit for use in vaccination against an autoantigen in a human patient, comprising: a first vaccine formulation containing an effective amount of an autoantigen to induce a primary immune response against the autoantigen; and A second vaccine formulation containing an effective amount of an autoantigen to induce a boost immune response against the autoantigen. wherein the amount of autoantigen in the second vaccine formulation is at least 200% higher than in the first vaccine formulation and the dosage in the second vaccine formulation is at least 50 μg of autoantigen; Here, the autoantigen is an alpha-synuclein antigen, and the alpha-synuclein antigen is DQPVLPD, DMPVLPD, DSPVLPD, DQPVLPDN, DMPVLPDN, DSPVLPDN, HDRPVTPD, DRPVTPD, DVPVLPD, DTPVYPD, DTPVIPD, HDRPVTPN, DRPVTPDN, DVPVLPDN, DTPVYPDN, DQPVLPDG, DMPVLP alpha-synuclein mimotopes selected from the group consisting of: DG, DSPVLPDG, DHPVHPDS, DMPVSPDR, DRPVYPDI, DHPVTPDR, DTPVLPDS, DMPVTPDT, DAPVTPDT, DSPVVPDN, DLPVTPDR, DSPVHPDT, DAPVRPDS, DMPVWPDG, DRPVQPDR, YDRPVQPDR, and DMPVDADN; kit.

2. 2. The kit of claim 1, wherein the booster administration is administered at least 6 months after the first administration of the autoantigen to induce the primary immune response.

3. The kit according to claim 1 or 2, wherein the alpha-synuclein antigen is an alpha-synuclein antigen containing the DMPVDPDN epitope.

4. The kit of any one of claims 1 to 3, wherein the booster administration is administered at least 12 months after the first administration of the autoantigen to induce a primary immune response.

5. The kit according to any one of claims 1 to 4, wherein the administration of the autoantigen is subcutaneous, intradermal or intramuscular.

6. The kit of claim 1 , wherein the autoantigen is administered together with an adjuvant.

7. The kit of claim 6, wherein the adjuvant is aluminum hydroxide.

8. The kit of any one of claims 1 to 7, wherein the autoantigen is bound to a pharma- ceutically acceptable carrier.

9. The kit of claim 8 , wherein the pharma- ceutically acceptable carrier is a protein carrier.

10. The kit according to claim 9, wherein the protein carrier is KLH (keyhole limpet hemocyanin), Crm-197, tetanus toxoid (TT) or diphtheria toxin (DT).

11. The kit of claim 1 , wherein the booster administration is repeated.

12. 12. The kit of claim 11, wherein the boost administration is repeated at least three years after the first boost administration, using the same amount as in the first boost administration.

13. 13. The kit of any one of claims 1 to 12, wherein the alpha-synuclein antigen is an alpha-synuclein mimotope selected from the group consisting of DQPVLPD, YDRPVQPDR, DSPVLPD, DTPVLPDS, DQPVLPDN, DVPVLPDN, DMPVLPDN, DSPVLPDN, DQPVLPDG, DMPVLPDG, DLPVTPDR, DSPVLPDG, DSPVHPDT, DVPVLPD, DHPVHPDS and DAPVRPDS.

14. 14. Use of a kit according to any one of claims 1 to 13 for the manufacture of a vaccine for inducing an immune response against a self-antigen in a human patient.

15. A vaccine for use in vaccinating a human patient against an autoantigen, comprising administering to the patient an effective amount of a dose of an autoantigen to induce a primary immune response, wherein the patient receives a boost dose of the autoantigen, the amount of the autoantigen in the boost dose being at least 200% higher than the amount of the autoantigen in the dose used for administration for the primary immune response, and wherein the dose in the second vaccine formulation is at least 50 μg of autoantigen; Here, the autoantigen is an alpha-synuclein antigen, and the alpha-synuclein antigen is DQPVLPD, DMPVLPD, DSPVLPD, DQPVLPDN, DMPVLPDN, DSPVLPDN, HDRPVTPD, DRPVTPD, DVPVLPD, DTPVYPD, DTPVIPD, HDRPVTPDN, DRPVTPDN, DVPVLPDN, DTPVYPDN, DQPVLPDG, DMPVLP alpha synuclein mimotopes selected from the group consisting of: DG, DSPVLPDG, DHPVHPDS, DMPVSPDR, DRPVYPDI, DHPVTPDR, DTPVLPDS, DMPVTPDT, DAPVTPDT, DSPVVPDN, DLPVTPDR, DSPVHPDT, DAPVRPDS, DMPVWPDG, DRPVQPDR, YDRPVQPDR and DMPVDADN; vaccine.

16. 16. The vaccine for use in vaccination according to claim 15, wherein the alpha synuclein antigen is an alpha synuclein mimotope selected from the group consisting of DQPVLPD, YDRPVQPDR, DSPVLPD, DTPVLPDS, DQPVLPDN, DVPVLPDN, DMPVLPDN, DSPVLPDN, DQPVLPDG, DMPVLPDG, DLPVTPDR, DSPVLPDG, DSPVHPDT, DVPVLPD, DHPVHPDS and DAPVRPDS.

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