METHOD FOR DETECTING THE PRESENCE AND QUANTITY OF THE LRP8 RECEPTOR AND OF ITS PROTEOLYTIC FRAGMENTS, DETECTION KIT AND RELATED ANTIBODIES

DESCRIPTION

FIELD OF THE INVENTION

[001] The present invention relates to a method for detecting the presence and quantity of Low-Density Lipoprotein Receptor- Related Protein 8 (LRP8) and of its carboxyl- terminal proteolytic fragments in biological samples isolated from the human body. The invention also relates to a kit for the detection of LRP8 and of its fragments, as well as the antibodies used in the kit and directed against LRP8 and its fragments, to be used in the method and for the diagnosis of neurodegeneration in diseases such as Alzheimer’s disease, Down syndrome and cancers.

BACKGROUND OF THE INVENTION

[002] AD, Alzheimer’s Disease, falls within the category of degenerative type dementia therefore it has a particularly insidious onset and does not have a specific elementary neurological framework. In general, however, AD is characterized by the progressive extension of neuronal degenerative lesions, the consequences of which are the progressive depletion of some neuronal populations and progressive presynaptic neurotransmitter insufficiency. Morphologically, cerebral atrophy can cause greater reductions of the weight of the brain than those due to normal ageing, saving the projection areas but compromising the association ones. Histologically, endoneuronal degenerations are found, among which the most common one is neurofibrillary, and extra neuronal degenerations, among which argyrophilic (or senile) plaques are the most common ones.

[003] The disorders presented by subjects affected by AD include both short term memory disorders (daily facts) and long term ones. They also have difficulty with the awareness and explanation of past events or they behave in the present as though they were in a past experience far from their current existence. Further typical disorders are connected with oral and written language that leads them to have difficulty planning speech, aphasia, inappropriate spontaneous speech, alexia (inability to read) and agraphia (inability to write); difficulty recognizing conventional graphic signs and common objects; alterations to mobility, sensitivity and coordination of movements and gestures that lead to significant daily problems such as, for example, wearing clothes correctly; the tendency to get lost in places; poor ability to concentrate; cognitive disorders.

[004] All of the aforesaid disorders obviously compromise social and working life both in subjects suffering from AD and the people who have to look after them such as relatives, caregivers and friends.

[005] It is to be noted that, according to statistical data, the incidence of cases of AD reaches 8-10% in subjects over 65 and even 30-50% in those over 80, considering that the average human life expectancy over recent decades has increased and is around 80- 85.

[006] Therefore, AD is a social problem of significant relevance and, therefore, it is the subject matter of numerous clinical studies that aim to identify early onset and to find new therapies that can effectively fight the causes of said disease.

[007] Unfortunately, the correct and early identification of AD, particularly in sporadic and late forms, cannot always be formulated, either due to the difficulty in distinguishing the symptoms thereof with respect to other forms of dementia, or due to the lack of indicators and precise and reliable markers. In other words, diagnostics are not currently able to offer valid and effective methods of identifying AD especially in the earlier stages so as to be able to intervene as soon as possible. Additionally, there are no effective drugs currently available for this disease.

[008] In fact, great efforts have been focused on the study of the proteolytic processing of amyloid precursor protein (AbRR), a Type I membrane protein, by b- enzymes, and y- secretase, as its alteration seems to be the triggering cause of AD. Therefore, the current aim is to find in particular g-secretase inhibitors as such enzymatic complex is considered to be a cornerstone in the development of the evolution of AD. In fact, it seems that an alteration of the proteolytic function of the latter leads to the production of neurotoxic b- amyloid peptides. Alternatively, other lines of research have focused on the production of murine and humanized monoclonal antibodies able to develop an immune response against Ab so as to block the neurotoxic potential thereof. However, such antibodies have been shown to be ineffective, not sufficiently specific (probably due to the cross-reactivity with other proteins and/or the target protein in a context of normal physiological conditions), and have also induced significant side effects. Therefore, to date, neither of the approaches are practical or therapeutically exploitable.

[009] For example, Larios J. et al. (Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signalling pathway, BMC NEUROSCIENCE, BIOMED CENTRAL, LONDON GB, vol. 15, no.1 , 19 September 2014) describe the use of a rabbit polyclonal antiserum against the cytoplasmic domain of ApoER2 recombinant human protein (A3481 by SIGMA Aldrich) which detects a band of 17 kDa. This commercial antibody recognizes a sequence corresponding to the C-terminal portion of LRP8 protein, i.e. the amino acids 928-945. However, this region is normally split into smaller fragments that are not identified by the antibody.

[0010] Pitts M. W. Et al. (Deletion of selenoprotein P results in impaired function of parvalbumin interneurons and alterations in fear learning and sensorimotor gating, NEUROSCIENCE, NEW YORK, NY, US, vol. 208, 10 February 2012) describe the interaction between selenoprotein P (Seppl ) and the ApoER2 receptor and the correlation with the release of selenium in the defence against oxidative stress. To evaluate the potential influence of Seppl on PV interneurons in mice, the expression profiles of ApoER2 were mapped. For this purpose a polyclonal goat antibody was used which binds close to the C-terminal portion of ApoER2. However, no fragment of the processing of ApoER2 is described.

[0011] Beffert U. et al. (ApoE Receptor 2 controls neuronal survival in the adult brain, CURRENT BIOLOGY, CURRENT SCIENCE, GB, vol. 16, no. 24, 19 December 2006) describe studies on mice brains selected for developing different forms of ApoER2. ApoER2 was therefore immunoprecipitated with a C-terminal antibody of ApoER2 (a-CT- ApoER2). Also in this case no specific fragment of ApoER2 is described, also considering that no Western Blotting experiment is described.

[0012] Hoe H. S. et al. (Effects of ApoE on neuronal signalling and APP processing in rodent brain, BRAIN RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 1 1 12, no. 1 , 27 September 2006) describe in vivo studies for confirming in vitro studies on the increased activation of ERK and decreased activation of JNK through ApoE receptors following the administration in primary neuronal cells of ApoE or an EP derived peptide thereof. For these studies a polyclonal rabbit antibody was used which recognizes the C-terminal portion of ApoER2. However, the target sequence of said portion is not specified.

[0013] Sogorb-Esteve Aitana et al. (Inhibition of g-secretase leads to an increase in presenilin-1 , MOLECULAR NEUROBIOLOGY HUMANA PRESS, US, vol. 5, no. 6, 16 August 2017) describe studies that are based on the amyloid hypothesis for the study of Alzheimer’s disease. In particular, the use of a polyclonal antiserum against the C- terminal portion of ApoER2 in brain extracts is described. However, the sequence of said portion is not specified. [0014] Furthermore, it is known that g-secretase is also involved in the onset of tumours. Therefore, g-secretase inhibitors are a very significant pharmaceutical target also for such diseases. In this case, however, there are potential clinical applications in the study phase.

[0015] Likewise, high levels of AbRR have been found in subjects affected by Down syndrome (DS). Therefore, an early analysis of such syndrome that is less invasive than chorionic villus sampling or amniocentesis and more precise than nuchal translucency is also of great interest.

SUMMARY OF THE INVENTION

[0016] The technical problem at the base of the present invention is therefore that of devising a method for the detection and quantification of specific and early markers in order to provide valid indications for the diagnosis of tumours and neurodegeneration in AD and in DS and, therefore, lead to the identification of a target for the development of effective therapies in the treatment thereof.

[0017] Such problem is solved, according to the present invention, by a method for the detection and quantification of the full length LRP8 receptor protein, or LRP8-FL (LRP8 Full Length) and of its C-terminal fragments LICDs (LRP8 IntraCellular Domain) through selectively targeted antibodies against specific target sequences of the receptor itself and of said fragments thereof.

[0018] Further subject matter of the invention is therefore the selective antibodies to be used in such method, or fragments thereof that bind the aforesaid antigens.

[0019] Further subject matter is a kit for the implementation of said method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Further characteristics and advantages of the method for detecting and quantifying LRP8 and its fragments according to the invention will become clearer from the following description of an embodiment provided purely by way of non-limiting example with reference to the following figures, wherein:

- Figure 1 shows the amino acid sequence of the protein LRP8;

- Figure 2 shows the amino acid sequence of the alignment of the C-terminal region of the four different main splicing isoforms of the protein LRP8;

- Figure 3 shows the result of a Western blotting analysis with different monoclonal antibodies according to the present invention;

- Figure 4 shows the result of a Western blotting analysis performed after placing in contact a polyclonal antibody of the invention with samples of brain tissue taken both from patients affected by Alzheimer’s dementia and subjects not affected by Alzheimer’s;

- Figure 5 shows a graph of the quantitative data of the ratio between full length protein (LRP8-FL) and C-terminal fragments between sporadic AD subjects (SAD), relatives (FAD) and unaffected subjects (controls or CTL), following densitometric analysis;

- Figure 6 represents a graph of the comparison between the LRP8/LICDs ratio in control samples, SAD and FAD after ELISA testing;

- Figure 7 shows two photographs of the result of two immunofluorescence assays using antibodies according to the present invention on histological samples of subjects affected by AD and not affected by AD;

- Figures 8 and 9 show the results of two Western blotting analyses that highlight the presence of LICDs fragments of LRP8 both in liquor (Figure 8) and in plasma (Figure 9);

- Figure 10 represents the result of a Western blotting analysis of DDK antibody against tagged LRP8.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Current knowledge indicates that the processing of AbRR protein by b- and y- secretase is certainly a key event in the genesis of AD. It is likely that g-secretase could play quite a significant role in carcinogenesis, due to the processing of different substrates (including AbRR and LRP8) involved in the promotion or progression of tumours and in metastasization.

[0022] In particular, it is known that AbRR is a receptor of the cell surface that performs physiological functions on the surface of the significant neurons for the growth of neurites, neuronal adhesion and axonal genesis. It is also involved in cell motility and in the regulation of transcription through protein-protein interactions. It also functions as a kinesin-l membrane receptor, mediating the transport in the axon of presenilin 1 and b- secretase.

[0023] In relation to alterations connected with the processing of AbRR, as well as overexpression or specific punctiform mutations of the AbRR gene, familiar forms of AD are caused by mutations or deletions in presenilin 1 ( PSEN1 ) and 2 ( PSEN2 ) genes: the catalytic components of the proteolytic enzyme g-secretase. One of the most accredited hypotheses on the cause of said alterations, called“amyloid hypothesis”, is based on the assumption that the aberrant processing of AbRR through g-secretase induces the formation of specific b-amyloid (Ab) soluble neurotoxic peptides which, in turn, cause neurodegeneration (Selkoe and Hardy, 2016) which leads to the pathogenesis of AD.

[0024] However, this theory has recently highlighted important limits and, in particular, some points have emerged that lead to the debate:“function gain” vs.“function loss” of presenilins (as catalytic components of the g-secretase enzymatic complex) in the genesis of AD (Shen, 2014; Shen e Kelleher, 2007); the presence of numerous and different substrate proteins of g-secretase that interact with AbRR, potentially influencing the formation of Ab (Haapasalo e Kovacs, 201 1 ) and the pathogenesis of AD. This last consideration is interesting but, precisely because of the increasing number of y- secretase substrates and their mutual interaction with AbRR, it is significantly unexplored.

[0025] Therefore, to date, therapies based on g-secretase inhibitors (that aim to reduce the production of Ab), or on the immunological control of Ab peptides, have failed both at pre-clinical and clinical level, suggesting that other conditions contribute to the development of AD in parallel to the processing of AbRR through y-secretase.

[0026] Therefore, in the current state of knowledge, there is no clear information on which substrates of g-secretase are effectively involved in the development of AD. Therefore, such condition does not allow diagnostic methods and therapies to be developed that are actually effective in the early identification of AD and in the provision of targeted and effective pharmaceuticals.

[0027] As already mentioned, the involvement of g-secretase is also known in the development of tumours as some of its substrates (particularly Notch, DCC, cadherin, L1 - CAM, CD44, etc.) are directly connected with carcinogenic and/or metastatization processes. Therefore, particular scientific efforts have focused on therapeutic approaches that block the activity of g-secretase, using specific inhibitors or rather “modulators” also in tumours.

[0028] It is also known that, in general, scientific literature mentions more than ninety proteins that constitute putative substrates of g-secretase, suggesting involvement in a plethora of activities that are difficult to understand.

[0029] The idea at the basis of the present invention was therefore that of searching among these proteins for those which, as well as being substrates of g-secretase, were expressed at neuronal level, also interacted with AbRR, were a receptor for the apolipoprotein E (ApoE) and that had the transmembrane portion and the cleavage site of g-secretase most similar to AbRR. [0030] This idea was developed following a new hypothesis with respect to the so-called “amyloid hypothesis”, based on which there should be a parallel path to the formation of the protein b-amyloid which would involve different substrates of g-secretase. Therefore, among the approximately ninety different proteins known such as g-secretase substrates, the protein LRP8, also known as APOER2, was selected, the amino acid sequence of which is represented in figure 1 (Uniprot Q141 14). Efforts focused on the investigation of said protein in response to different conditions, i.e. the evaluation of the proteolytic pattern in subjects affected by AD with respect to subjects not affected by AD.

[0031] The investigation on the quantification of LRP8 was developed by means of a method according to the present invention, comprising a phase of placing in contact a biological sample isolated from the human body with a monoclonal or polyclonal antibody targeted against a specific amino acid sequence of the C-terminal portion of LRP8 identified by the amino acid residues 837-963 (according to the numbering of the isoforms Q141 14-1 and Q141 14-4 UniProtKB, figure 2). It was surprisingly observed that an antibody constructed in such a way was able to recognize both the full-length protein PM of about 100-105 kDa, and migrated C-terminal fragments between 18 and 75 kDa (y- secretase substrates and possibly of other proteases) and, even more surprisingly, smaller migrated fragments between 8 and 16 kDa in SDS, in particular between 8 and 12 kDa. Precisely the detection of these smaller fragments, i.e. smaller than 17kDa, was surprisingly important in providing an early diagnosis of AD more specifically with respect to the detection of larger fragments through the use of known antibodies. In fact, as reported below, thanks to the experiments at the basis of the present invention, it was found that in the aforesaid diseases the small fragments increase and the full-length protein decreases. Therefore, it is crucial to identify the quantity of these fragments less than 17 kDa together with the quantity of full-length LPR8.

[0032] In particular, the C-terminal amino acid sequence of the amino acid residues 837- 963 is constituted by the sequence:

IALLCMSGYLIWRNWKRKNTKSMNFDNPVYRKTTEEEDEDELHIGRTAQIGHVYPAAIS SFDRPLWAEPCLGETREPEDPAPALKELFVLPGEPRSQLHQLPKNPLSELPVVKSKRV ALSLEDDGLP (SEQ. I.D. 1 ).

[0033] Preferably, within the aforesaid sequence, particularly effective sub-sequences have been identified, selected from the group consisting of the amino acid residues: 837- 857 (IALLCMSGYLIWRNWKRKNTK, SEQ I.D. 2), 850-864 (NWKRKNTKSMNFDNP, SEQ I.D. 3), 863-878 (NPVYRKTTEEEDEDEL, SEQ I.D. 4), 874-887 (DEDELHIGRTAQIG, SEQ I.D. 5), 882-898 (RTAQIGHVYPAAISSFD, SEQ I.D. 6), 898- 917 (DRPLWAEPCLGETREPEDPA, SEQ I.D. 7), 915-940

(DPAPALKELFVLPGEPRSQLHQLPKN, SEQ I.D. 8), 940-963

(NPLSELPVVKSKRVALSLEDDGLP, SEQ I.D. 9).

[0034] More preferably, the sequences of the residues 874-887 (SEQ I.D. 5) and 940-963 (SEQ I.D. 9) are those that have provided the best results in terms of recognition of C- terminal fragments, LICDs as defined in the present description, and of the full-length protein LRP8.

[0035] As it is used here, the term "amino acid" is intended to indicate both natural amino acids and mimetic amino acids. Natural amino acids comprise 20 (L)-amino acids used during the biosynthesis of proteins, and others, for example 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine. Non natural amino acids include, for example, (D)-amino acids, norleucine, norvaline, p- fluorophenylalanine, ethionine and the like. Analogs of amino acids include modified forms of natural and non-natural amino acids. Such modifications can include, for example, the substitution or replacement of chemical groups and molecules on amino acids or through the derivatization of the amino acid (e.g. o-bromo-L-phenylalanine). Mimetic amino acids comprise, for example, organic structures that have functionally similar properties, such as the load and spacing of loads, to the characteristics of the reference amino acids. For example, an organic structure that imitates arginine (Arg or R) would have a positive load portion localized in the similar molecular space and having the same degree of mobility as the e-amino group of the lateral chain of the natural amino acid Arg.

[0036] The amino acid sequences according to the invention constitute polypeptides or fragments, variants or derivatives thereof isolated from the human body in the sense that they are not in their natural environment. They preferably derive from recombining technology and expression in host cells from which they are separated, fractioned, partially or substantially purified according to conventional methods.

[0037] With reference to the antibodies developed against the aforesaid sequences, fragments, variants, derivatives or analogs means any antibody or antibody peptide that maintains the property of antigen bond. Altered amino acid sequences are sequences in which there are substitutions, deletions or insertions of amino acids according to what is described above. [0038] Further subject matter is a nucleotide sequence that can be translated and transcribed in the aforesaid sequences that develop specific antibodies for use in the method of the present invention. These nucleotide sequences can be produced through conventional molecular biology methods that use for example reverse transcription in known viral systems (Molecular Cloning - A Laboratory Manual, Sambrook and Russel, Cold Spring Harbor).

[0039] It is to be noted that the terms antibody and immunoglobin in the present description can be used interchangeably and equally identify an antigen binding molecule that comprises at least the variable domain of a heavy chain and normally at least the variable domains of a heavy chain and of a light chain, or single chain antibodies, or minibodies (Harlow et al., Antibodies, “A laboratory Manual”, Cold Spring Harbor Laboratory Press, Second Edition, 2014).

[0040] The method of the invention can be selected from any one of the known analytical methods comprising the aforesaid step of placing the antibody in contact against any one or more of the sequences described above. The method is preferably selected from among ELISA (direct, sandwich, etc.), separation and detection by lateral flow, mono and bi-dimensional electrophoresis, Western Blotting, immunoprecipitation followed by any one of the preceding techniques, RIA, electrophoresis or HPLC or FPLC, or any chromatographic method followed by a detecting technique such as the preceding one, Surface Plasmon Resonance (SPR) or similar approaches based on interferometry, wherein the antibodies of the invention are used either as capturing or interacting molecules, mass spectroscopy after purification of the biological sample with HPLC, nano-HPLC chromatographic methodologies. As these methods are widely known to a person skilled in the art they will not be reported herein.

[0041] Regardless of the analytical method used, the biological samples separated from the human body can be pre-treated before being subjected to investigation techniques. In fact, biological samples can derive from body fluids or tissues. In particular, the fluids can be cerebrospinal fluid (CSF), plasma, complete blood, tear fluid, urine, body secretions, whereas the tissues may be brain tissue, nasal brush samples, epithelial tissue of the mucosa. In the event of using a biological sample of brain tissue, the detection method comprises a preparatory step of crushing the sample according to conventional methods (e.g. sonication, homogenization with solvents, buffers, detergents and other solutions) followed by precipitation/isolation of the protein fraction. The protein fraction is then incubated with the antibody of the present invention. Subsequently, the samples thus prepared are subjected to detection analysis according to any one of the aforesaid methods. If the biological sample is of the histological type and therefore intended for immunohistochemical analysis, it is subjected to preventive fixing, according to absolutely conventional methods.

[0042] The specific target sequences of recognition by the antibodies were identified after numerous experiments aiming to find the sequences that very selectively characterize both the protein LRP8-FL and its C-terminal fragments, defined as LICDs, and deriving from the processing of LRP8 by g-secretase or by other endoproteolytic enzymes such as metalloprotease, aspartyl-protease, serine protease and cysteine protease. It is to be noted that in physiological conditions LRP8 is processed so as to release a few C-terminal fragments within the cell.

[0043] In particular, it is known from scientific literature that C-terminal fragments of LRP8 with a length comprised between 20 and 24 kDa are produced following the block of y- secretase performed by the DAPT inhibitor (Hoe and Rebeck, 2005; Hoe et al., 2006). The present invention, instead, highlights how smaller fragments, i.e. fragments from 8 to 16 kDa, are more important in the diagnosis of many diseases as they have been found in large quantities in biological samples isolated from the human body in diseases subjects. Therefore, in the present description the term LICDs identifies the migrated C- terminal fragments of LRP8 in SDS-PAGE between 8 and 16 kDa, preferably between 8 kDa and 12 kDa. These fragments, as previously explained, comprise the aforesaid amino acid sequences and are advantageously recognized by the antibodies developed so as to recognize both LICDs, i.e. the smallest fragments between 8 and 16 kDa, and other fragments with a higher molecular weight (substrates of g-secretase and possibly also of other proteases) migrated between 18 and 75 kDa.

[0044] The analyses performed with the method according to the invention have led to the discovery of a particularly high quantity of LICDs and to the simultaneous reduction of the quantity of full-length LRP8 protein, in samples of AD patients with respect to non-AD controls.

[0045] LRP8 is a type I transmembrane receptor protein whose amino acid sequence is reported in figure 1 (SEQ. I.D. 14) and is available for example in the Uniprot database with identification number Q141 14. It is known that such protein has four different main splicing isoforms (SEQ I.D. 15, SEQ I.D. 16, SEQ I.D. 17, SEQ I.D. 18) whose alignment of the respective C-terminal regions is reported in figure 2. [0046] According to the present invention, the aforesaid sequences have been identified at the C-terminal end of LRP8 as target sites for recognition by specific selective antibodies.

[0047] In particular, polyclonal and monoclonal antibodies have been developed. Both polyclonal and monoclonal antibodies have been created through conventional methods that shall therefore not be described in detail herein. In relation to monoclonal antibodies, it was chosen to use as an immunogen preferably the sequence SEQ. I.D. 5 with the addition of a cysteine Cys at the C end for coupling with KLH or an analogous protein or peptide that has the capacity to bond in turn to target proteins of interest, such as the sequences according to the present invention or more generally haptenes that however have poor immunogenic capacity, so as to reinforce such capacity thereof. A reference to the methods for the production of such antibodies can be found for example in“Critical Steps in the Production of Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations”, Marlies Leenaars, Coenraad F. M. Hendriksen (ILAR Journal, Volume 46, Issue 3, 1 January 2005, Pages 269-279) in which specific bibliographical references can also be found on the conditions of the individual operating steps.

[0048] In any case, when polyclonal antibodies are used, the serum taken from the immunized mammal (e.g. mouse, rabbit, goat, horse) with the aforesaid antigens is treated conventionally so as to purify the antibodies developed through, for example, immunoaffinity chromatography. Furthermore, the antibodies can be humanized, xenogeneic or human-murine chimeric.

[0049] As for the production of monoclonal antibodies, the hybridoma technique is widely known and generally envisages the use of immortal cell lines created by cell fusion or other techniques such as the direct transformation of B lymphocytes with oncogenic DNA or transfection with virus (e.g. M. Schreier et al. “Flybridoma Techniques”, 1980; Hammerling et al.,“Monoclonal Antibodies and T-cell Flybridomas”,1981 ; Kennet et al., Monoclonal Antibodies, 1980; Harlow et al., Antibodies, “A laboratory Manual”, Cold Spring Harbor Laboratory Press, Second Edition, 2014). In particular, clones of hybridomas deposited at the Advanced Biotechnologies Centre (CBA)/lnterlab Cell Line Collection of San Martino University Hospital 1ST, National Institute for Cancer Research, of Genoa (Italy) with numbers 17E1 1 C2 and 25F7F1 were produced.

[0050] The sequences of antibodies of the cell lines 17E1 1 C2 and 25F7F1 were identified by GenScript and are reported respectively in the following tables. For identification, the materials used were the reactant TRIzol® (Ambion, Cat. No.: 15596-026), the synthesis kit PrimeScript™ 1 st Strand cDNA (Takara, Cat. No.: 61 10A). The method envisages the isolation of the total RNA from the cells of the hybridomas following the TRIzol®Reagent manual. The total RNA was then reverse transcribed in cDNA using both the anti-sense isotype-specific primer and universal primers following the PrimeScript™ 1 st Strand cDNA Synthesis Kit manual. Fragments of antibody of the heavy chain and of the light chain were amplified according to the standard operating procedure (SOP) of rapid amplification of the ends of cDNA (RACE) of GenScript. The fragments of amplified antibodies were cloned separately in a standard cloning vector. PCR on colonies was performed in order to select clones with inserts of the correct size. The consensus sequence was provided. For the analysis of the sequences any suitable free online program can be used, preferably NCBI Nucleotide BLAST, IMGT/V Quest program and NCBI IgBLAST.

HYBRIDOMA 17E1 1 C2

Table 1

Heavy chain: DNA sequence (414 bp)

HYBRIDOMA 17E11C2

Table 2

Heavy chain: amino acid sequence (138 aa)

HYBRIDOMA 17E11C2

Table 3

Light chain: DNA sequence (393 bp) (SEQ I.D. No.21)

HYBRIDOMA 17E1 1 C2

Table 4

Light chain: amino acid sequence (131 aa) (SEQ I.D. No. 22)

HYBRIDOMA 25F7F1

Table 5

Heavy chain: DNA sequence (414 bp) (SEQ I.D. No. 23)

HYBRIDOMA 25F7F1

Table 6

Heavy chain: amino acid sequence (138 aa)

HYBRIDOMA 25F7F1

Table 7

Light chain: DNA sequence (393 bp) (SEQ I.D. No. 25)

HYBRIDOMA 25F7F1

Table 8

Light chain: amino acid sequence (131 aa)

[0051] Further subject matter of the present invention is therefore a monoclonal or polyclonal antibody able to bind to any one of the aforesaid sequences for use in an identification and quantification method of LRP8 and its LICDs fragments. These antibodies can be produced through recombinant host cell culture modified after the injection of an expression vector such as a plasmid constructed so as to transfer into the genetic heritage of the host the nucleotide sequences coding the amino acid sequences of interest. The nucleotide sequences that code for said antibodies can comprise any polyribonucleotide or polydeoxyribonucleotide that may be a non-modified or modified RNA or DNA. These expression systems can be designed so as to include signal molecules in order to effectively identify and separate the molecules of interest from the culture. Among the expression vectors that can be used gB324 is the preferred one, but also pcDNA3, pFICMV/Zeo, pCR3.1 , pEF1 /His, pIND/GS, pRc/FICMV2 and others known in the sector can be used.

[0052] Likewise, preferably, the antibodies produced according to the present invention comprise a signal molecule, i.e. a molecule able to be detected through the aforesaid analytical methods such as, for example, a fluorophore, a radioisotope, an enzyme, a heavy metal. Furthermore, antibodies or immunoglobulins can be whole molecules or antibody fragments. Immunoglobulins are notoriously IgG, IgM, IgA, IgE, IgD and related sub-classes, composed of two light chains (L) and two heavy chains (H), both chains being constituted by constant (CL, CH) and variable regions (VL, VH). The fragments can for example be single chain fragments F(ab’), F(ab), F(ab’)2 fragments.

[0053] Different subject matter of the present invention is an amino acid sequence selected from the sequences SEQ I.D. 1 -9, as described previously, for use in a detection and quantification method of LRP8 and its LICDs fragments.

[0054] Further subject matter of the present invention is a diagnostic kit for the determination of diseases associated with the aberrant processing of the protein LRP8. Such kit preferably comprises a micro-plate whose wells are coated with an antibody produced according to the above, a container for a control sample comprising the protein LRP8 and/or its C-terminal fragments of 8-16 kDa, a container for a diluent substance of the control sample and one for the testing sample, a container for a buffer, a container for a substrate substance, a container for a stopping-reaction solution, a container for a washing buffer. Additionally, the kit comprises an illustrative sheet with the explanations of how to prepare the samples, incubation and washing times, and photometric reading conditions of the detected signal.

[0055] According to a preferred form of the invention, the kit can comprise a reference sample or a table of pre-established data for the comparison between values and/or types of molecules detected and corresponding quantities in reference to conditions not considered pathological and pathological conditions.

[0056] As well as the techniques through ELISA as summarized above (paragraph 0048) and subsequently described in detail (see paragraph 0059), the kit can comprise the use of applications such as“SPR” (Surface Plasmon Resonance) using an optical method to measure the refraction index close to the surface of a sensor. This surface is a side of a flow cell, through which an aqueous solution (running buffer) is passed through at a continuous speed. To allow the detection of an interaction, a molecule (ligand) is immobilized on the surface of the sensor. The molecule with which an interaction is to be evaluated (analyte) is injected in aqueous solution, which has the same composition as the running buffer, through the flow cell, through a continuous flow. When the analyte is bonded to the ligand, the accumulation of proteins on the surface of the sensor causes an increase of the refraction index. This change of the refraction index is measured in real time and the result is shown as a response unit (UR) as a function of time (sensorgram).

[0057] Such technology could be used to detect the presence and affinity of the LRP8 receptor and/or its fragments, present in a biological sample, towards the monoclonal and/or polyclonal antibodies described in the present invention. In particular, such approach envisages that the kit provides the antibodies (e.g. 17E1 1 C2 or 25F7F1 ) as ligands and that they are immobilized on the surface of the sensor, with an appropriate buffer, in order to search for the LRP8 receptor or fragments thereof, in a biological sample of interest. Such sample should be presented in liquid form (plasma, liquor, saliva, urine...) and be appropriately treated before being injected into the flow cell to measure the binding thereof and therefore the SPR with respect to a negative reference control sample (a sample from a healthy subject or a sample without LRP8 or LICDs) and a positive one (e.g. a calibration curve with known quantities of LRP8 or LICDs). Therefore, the kit will comprise at least a bio-sensor or chip provided with a transparent support carrying on one of its surfaces a metal layer (generally gold or silver) having a free surface on which the antibodies according to the present invention have been fixed, at least a support microfluidic cartridge in whose interior a buffer containing an analyte to be detected is made to flow, a container for the analyte buffer.

[0058] Another possibility for the development of diagnostic tests that aim to detect the presence of LRP8, its fragments or LICDs in biological samples of interest, such as those mentioned above, is based on a stick of plastic material which has a buffer inside it. Such kit exploits a liquid sample to be migrated along this buffer by capillarity (lateral flow). In the buffer exposed to the biological liquid to be analysed there is a specific antibody for the antigen searched for (in a typical example the monoclonal 17E1 1 C2 could be used conjugated to coloured indicator molecules such as colloidal gold for example). The antigen-antibody complex moves by capillarity following the biological sample as far as the two strips that are initially invisible: the first is the one where the other antibody that recognizes the antigen is immobilized (in our example it could be the antibody 25F7F1 fixed onto the support), while the second strip is the control one and is where an antibody is immobilized that can recognize the crystallizable fragment (Fc) of the antibodies that can be found on the buffer. This strip represents a positive control for the successful migration of the sample to the buffer by capillarity. The successful bond of LRP8 and/or its fragments and/or of the LICDs to the first strip and of the control antibodies to the second strip is highlighted by the colouring of such bands.

If the control strip is coloured it means that the specific antibodies for LRP8, its fragments and the LICDs transported by the capillary flow of the sample have necessarily crossed the first strip; if the first strip is coloured it means that LRP8 and/or its fragments and/or the LICDs are present in the biological sample. It is also possible to use optical readers to evaluate the different colour intensities developed by the reaction between the sample and the first strip. Therefore, the kit will comprise a support having the shape of a long strip whose surface is coated with a plurality of capillary elements in succession, a first element (the sample pad) which absorbs a sample fluid, a second element (conjugated bearing) storing an antibody-signal molecule conjugate (dye), a third element carrying one or more areas (often called strips) wherein a capturing molecule has been fixed.

[0059] Below are some embodiments of the invention provided by way of non-limiting example of the invention.

PREPARATION OF SPECIFIC ANTIBODIES FOR LRP8

[0060] According to the present invention, the isoform 1 of LRP8, which is sequentially the longest, was used for in vitro studies through cloning techniques that use cDNA for the expression both of its wild-type form and of a marked isoform at the C-terminal with the tag sequence myc-DDK. The corresponding clone was purchased from OriGene, Cat. No. RC220963. Subsequently, the transmembrane region was identified on said clones (described in the Uniprot website: http:/7www.uniprot.org/uniprot/Q141 14, and can be identified using the public software AGADIR or TMPred available on the website https://expasy.orQ/). It has been seen that this region was conserved in all four isoforms and is represented by the sequence VIGIIVPIVVIALLCMSGYLI (SEQ. I.D. 10). Furthermore, the sequence VV-IA (SEQ I.D. 1 1 ) was identified as the putative cleavage site by g-secretasi, such site being identical to the AbRR site that generates the fragment Ab-40. The sequence DNPVY (SEQ I.D. 12) was also identified as the necessary site for the internalization and interaction with adaptor proteins such as JNK and Dab1 . It is also known that the sequence

AISSFDRPLWAEPCLGETREPEDPAPALKELFVLPGEPRSQLHQLPKNPLSELPVVK SK

(SEQ I.D. 13), present only in isoforms 1 and 4, is typical of mammals, is determined by the exon 18 in human beings and is apparently connected to the memory processes and activities of Relina, of which LRP8 is a receptor (Telese et al., 2015).

[0061] In particular, the aforesaid sequence DEDELFIIGRTAQIG (SEQ. I.D. 5) is present in all four isoforms and consists of a short sequence with loaded amino acids and is localized between the internalization sequence DNPVY and the sequence of the exon 18. The aforesaid sequence NPLSELPVVKSKRVALSLEDDGLP (SEQ I.D. 9) is instead localized at the end of the sequence 18 of the exon.

[0062] These sequences SEQ I.D. 5 and SEQ I.D. 9 were selected as the best antigens for developing monoclonal antibodies. A total of two polyclonal antibodies and two monoclonal cell lines of hybridomas against said sequences were obtained. In summary, the technique for the production of monoclonal antibodies from hybridomas envisages the immunization (with an antigen peptide sequence of the protein of interest) of an animal (generally mouse, rat or rabbit) from which spleen plasma cells are taken. In particular, BALB/c mice were immunized in the present invention. The plasma cells were immortalized through fusion with SP2/0 murine myeloma cells and then those secreting direct antibodies against the antigen of interest were selected, used in the immunization of the animal. In more detail, the individual plasma cells secrete direct antibodies against a specific epitope of the antigen protein and the monoclonal antibodies can therefore be obtained from a cell line derived from a single plasma cell. Once they are stable, the clonal lines of hybrodomas provide an almost infinite quantity of antibodies. The antibodies secreted by the cells can be used as such (supernatants) or further purified and/or concentrated by means of chromatography techniques on a column e.g. by affinity with peptides (appropriately fixed onto a support) with the sequences previously identified. In the present case the antibodies obtained from hybridomas coming from mice immunized with peptides SEQ I.D. 5 and SEQ I.D. 9 were initially used as such as cell supernatants obtained from the derived hybridomas and, subsequently, they were purified by means of chromatography by affinity using peptides with the same sequences SEQ I.D. 5 and SEQ I.D. 9 as capture peptides in the binding resin.

The two lines of hybridomas described above develop antibodies that recognize both the entire protein LRP8 and its different C-terminal fragments. In particular, it was found that hybridomas developed by immunizing animals against the sequences SEQ. I.D. 5 and 9 with a cysteine at the C-terminus are able to produce antibodies that recognize both the protein LRP8 as such, and the fragments of 18-25 kDa, which are substrates of y- secretase and different shorter fragments of 8-16 kDa, collectively defined as LICDs- derived intracellular domain LRP8, similar to the AICDs fragments derived from the cleavage of AbRR by g-secretase or by other proteases such as those previously specified. The polyclonal antibodies (rabbit) GS1 and GS2 were made using SEQ I.D. 5 and SEQ I.D. 9, respectively. By way of example, Figure 3 shows the result of a Western blotting analysis is represented in which the cell lysate is obtained from N2A cells transfected with a cDNA coding for the protein LRP8 (Origene, Cat. No. RC220963) and subjected to SDS-PAGE analysis and Western blotting with the polyclonal antibodies GS1 and GS2 (lanes 1 , 5), with the monoclonals 25F7F1 and 17E1 1 C2 (lanes 2 and 3) and with other polyclonals according to SEQ I.D. 3, 4 and 8 (lanes 4, 6 and 7, respectively). In lane 8 there is a negative control whereas in lane 9 the hybridization was performed using a commercial antibody vs LRP8 (Sigma Aldrich Cat No. SAB1306331 ) as a positive control. All the antibodies were used at the final concentration of 1 pg/ml. It is therefore clear that both the polyclonal antibodies and the antibodies of clones of hybridomas according to the present invention are able to identify not only the full-length protein LRP8 and the fragments from 18-25 kDa, but also advantageously the fragments 8-16 kDa, as indicated by the boxes in Figure 3. In other words, the result of the analysis highlighted how the antigen sequences of LRP8 previously described are a selective and specific target for the identification of LRP8 and its fragments in cell cultures. Similar results were also obtained with sequences SEQ I.D. 1 , 2, 6-8.

ANALYSIS ON HUMAN BRAIN SAMPLES

[0063] In principle the protein levels of LRP8 and its C-terminal fragments were analysed using SDS-PAGE in Tris-Tricine buffer followed by Western Blotting. The samples derive from the temporal and frontal cortex of the human brain of 10 patients suffering from AD compared with 9 non-AD patients, both groups being subjected to the antibodies known as GS1 and GS2 developed respectively against sequences SEQ. I.D. 5 and SEQ. I.D. 9. Samples of different brain areas (frontal and temporal cerebellar cortex) of a group of subjects (5) with familial AD (FAD) having mutations on the PSEN1 gene, coding for presenilin 1 , were analysed.

[0064] In particular, for each patient, about 100 mg of brain tissue were homogenated in RIPA buffer for 30” in a rotor-stator homogenizer and subsequently centrifuged at 10OOOxg at 4°C for about 20 min. The supernatant was subjected to analysis of the protein fraction extracted by means of the electrophoretic technique (SDS-PAGE) in 12.5% acrylamide gel (separating) in Tris-Tricine buffer. The gel with the proteins separated was then transferred onto a 0.22 pm PVDF membrane in transfer buffer, and the same membrane marked with GS1 specific primary antibody for LRP8 and secondary anti rabbit, such as to recognize the primary antibody and marked with HRP (GE Healthcare, Cat. No. RPN4201 ). Subsequently, through chemiluminescence, the detection of protein bands on an X-ray plate was performed.

[0065] Figure 4 shows an X-ray plate obtained with the aforesaid process, in which the lanes represent the signals obtained from the same number of brain samples of non-AD subjects (control) and AD previously prepared and incubated with GS1 . As can be noted, the lanes indicated with SAD and FAD show a signal that identifies a significant presence of C-terminal fragments of LRP8 comprised between 8 and 16 kDa and a parallel reduction of the full-length protein of 105 kDa with respect to the controls (Control lanes), as indicated by the exemplifying arrows of figure 4. On the right hand side of the WB image, the molecular weights of the marker are shown.

[0066] The photographic plates obtained from the Western Blot experiments were digitalized by acquisition with an imaging tool (Gel Doc EZ System, Biorad). A densiometric analysis of such digital images was performed which consists of the evaluation of the density of the protein signal with the IT software ImageLab (Biorad): such software allows the protein bands to be selected and a numerical value to be assigned to them according to the intensity of the bands. The densiometric analysis was performed as follows: for each patient the value of the full-length protein LRP8 and the normalized value of the LICDs fragments was expressed as a ratio. The mean values of the ratio between full-length protein LRP8/LICDs fragments for each group analysed (controls, SAD and FAD) were obtained with the related mean standard error. The analysis indicates that in the SAD samples a mean of 4.2 times more fragments of LICDs were found with respect to the controls. Such value comes from the ratio between the mean value of full-length LRP8 protein/LICDs fragments of the control samples (20.51 ) vs SAD (4.82). In fact, Figure 5 shows the ratio between full-length LRP8 protein and LICDs respectively in control samples (CTL), samples of patients with SAD and samples of patients with FAD. If the analysis is extended to the comparison of the ratio between the full-length LRP8 protein and its LICDs fragments in the samples analysed, it is possible to note the significant reduction of the full-length protein and the parallel increase of LICDs in the FAD and SAD cases (Figure 4) with respect to the samples of control subjects without dementia, with a consequent reduction of the ratio between the full- length protein and the fragments comprised between 8 and 16 kDa.

[0067] Superposable results were also obtained using the ELISA Sandwich technique. In order to be able to specifically evaluate the presence of LICDs, the samples were prepared using centrifugal concentrators (Centricon 30, Millipore, Cat. No. MRCF0R030) that have a molecular filter able to separate the proteins below 30 kDa, from those that have a higher weight, so as to be able to isolate the fraction that contains the LICDs (below 30 kDa) from that with the full-length receptor (fraction above 30 kDa). The multi well plates with 96 wells were prepared with a substance that acts as a coating (0.1 M Na carbonate/bicarbonate - ratio 70:30) for 16 h in order to be able to immobilize the antibody (GS1 or GS2) on the wall of the multi-well plates with 96 wells. The following day the wells were washed with a solution containing PBS+0.05% Tween 20 and then a solution of PBS+10% of goat serum (NGS) was added for 3 h at ambient temperature to block the non-specific sites. The samples previously prepared (fractions above 30 kDa and fractions below 30 kDa) were added to the well, after the removal of the blocking solution, and incubated for 16 h at + 4°C. Subsequently, 5 washes were performed with solution containing PBS+0.05% Tween 20 and the second anti-LRP8 antibody (antibody produced by 25F7F1 mouse hybridomas) was added which can bond to the antigens immobilized by the first antibody. The wells were washed again 5 times with the solution containing PBS+0.05% Tween 20 to remove the excess antibody and a secondary anti-mouse antibody conjugated with FIRP was added (GE Healthcare, Cat. No. RPN4201 or analog). After incubation at 37°C for 1 hour, the wells were washed five times as previously described and a solution of tetramethylbenzidine (TMB, Sigma), peroxidase substrate, was added. After leaving it to react for 10 minutes at ambient temperature, the reaction was stopped with a 1 M sulphuric acid solution and the absorbance readings were performed at 450 nm using a spectrophotometer with housing for multi-well plates (VICTOR 3, Perkin Elmer). In Figure 6 it is possible to observe the results obtained from the ELISA experiments: as also observed in the WB experiments, with this other technique it is also possible to observe a reduction of the ratio between the full-length receptor (fraction above 30 kDa) and LICD (fraction below 30 kDa) in patients with sporadic and familial AD with respect to the control group. In fact, Figure 6 shows a graph related to the ratio between the full-length receptor and LICDs (fractions reported above) of the values obtained through ELISA technique, for each of the samples belonging to the three analysed groups: non-AD controls (CTL), SAD and FAD. Also from this analysis it is possible to see how the trend of the values of the ratio between the full-length receptor and LICDs is significantly higher in the controls than in subjects with AD (SAD and FAD). Alternatively, with this technique it is however possible, for diagnostic purposes, to compare only the data related to LICDs or to the full-length protein (LRP8-FL) that can be obtained from the samples with the diagnostic bias, with respect to the levels thereof obtained from reference samples not affected by dementia or with respect to a calibration curve.

[0068] Immunoistochemical analyses have also confirmed the aforesaid results. In fact, it was observed that on the same samples as above there is an intense and widespread colouring (image on the left in Figure 7) in the parenchymal portion of the AD samples due to the presence of antibodies against SEQ. I.D. 5 marked with the specific fluorescent secondary antibody which, after its bond with the primary antibody, is excited at the wavelength of 578 nm and emits at 603 nm in the red range (Alexa Fluor 568 by the company Thermo Fisher or analogs). On the contrary, in the control samples neuronal and intracellular colouring is observed (image on the right in Figure 7).

ANALYSIS IN PLASMA AND IN LIQUOR (CSF)

[0069] In parallel to the aforesaid investigations, samples of plasma and liquor of AD and non-AD subjects were processed in order to identify and quantify the presence of LRP8 and LICDs as a peripheral marker. Samples of MCI (Mild Cognitive Impairment) subjects were also evaluated in which according to some scholars AD could be found in its earliest stage. This detection was also performed through SDS-PAGE technique followed by Western Blotting (Figure 8) after placing the antibody GS1 in contact in samples of CSF coming from the cerebral ventricles (taken post-mortem) of control subjects (lines 1 and 4 of Figure 8), of a subject with SAD (Line 2) and a familial case (Line 3, FAD) with a mutation in the PSEN1 gene. The procedure is identical to the one described in paragraph 0056. As can be noted from the result of the analysis through Western Blotting of Figure 8, the LICDs fragments are only present in AD cases, with respect to non-AD cases, while the full-length protein is present in all subjects, as indicated by the boxes. Likewise, in human plasma samples, a significant increase in LICDs has been detected in AD cases (Lines 7-10 of the Western Blotting of Figure 9) with respect to the controls (Lines 1 -6 of Figure 9).

[0070] The data obtained from the aforesaid analyses performed both on human brain tissues and CSF and plasma, suggest that an increase in LICDs represents a valid indicator or diagnostic marker for the identification of AD also in the early stages.

[0071] Additionally, alternatively or in parallel to what is described above, some techniques can also make use of the assessment of the reduction of LRP8 FL (see 0059); in fact also the simultaneous reduction of the quantity of the full-length protein LRP8 would confirm the presence of AD and early onset AD.

COMPARATIVE EXAMPLE

[0072] For comparative purposes only, a murine cell line (N2A) stably transfected with a plasmid expressing isoform 1 of the human protein LRP8 (Uniprot Q141 14-1 ) was prepared, comprising a label or tag of the DDK-myc type bonded to its C-terminal end. The murine monoclonal antibody anti-DDK (Origene, clone OTI4C5) against such tag showed the result of Figure 10 which represents the SDS-PAGE technique followed by Western Blotting performed under the same conditions previously described. In particular, it is clear that also this type of antibody is able to bond only to sequences of no less than 25 kDa (lines 3-4). Lines 1 -2 indicate two negative controls, i.e. two samples of N2A cells not expressing LRP8-DDK-myc.

LISTING OF SEQUENCES

<110> RUSSO, Claudio

<120> Method for detecting the presence and quantity of the LRP8 receptor and of its proteolytic fragments, detection kit and related antibodies

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20 25 30

Ala Asn Glu Asp Ser Lys Met Gly Ser Thr Val Thr Ala Ala Val He

35 40 45

Gly lie lie Val Pro He Val Val lie Ala Leu Leu Cys Met Ser Gly 50 55 60

Tyr Leu He Trp Arg Asn Trp Lys Arg Lys Asn Thr Lys Ser Met Asn

65 70 75 80

Phe Asp Asn Pro Val Tyr Arg Lys Thr Thr Glu Glu Glu Asp Glu Asp

85 90 95

Glu Leu His He Gly Arg Thr Ala Gin He Gly His Val Tyr Pro Ala

100 105 110

Ala He Ser Ser Phe Asp Arg Pro Leu Trp Ala Glu Pro Cys Leu Gly 115 120 125

Glu Thr Arg Glu Pro Glu Asp Pro Ala Pro Ala Leu Lys Glu Leu Phe

130 135 140

Val Leu Pro Gly Glu Pro Arg Ser Gin Leu His Gin Leu Pro Lys Asn 145 150 155 160

Pro Leu Ser Glu Leu Pro Val Val Lys Ser Lys Arg Val Ala Leu Ser 165 170 175

Leu Glu Asp Asp Gly Leu Pro

180

<210> 19

<211> 57

<212> DNA

<213> Homo sapiens

<400> 19

atgggatgga gctggatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactct 57

<210> 20

<211> 90

<212> DNA

<213> Homo sapiens

<400> 20

gaggtccagc tgcaacagtc tggacctgtg ctggtgaagc ctggggcttc agtgaagatg 60 tcctgtaagg cttctggata caaattcact 90

<210> 21

<211> 15

<212> DNA

<213> Homo sapiens

<400> 21

gactacttta tgaac 15 <210> 22

<211> 42

<212> DNA

<213> Homo sapiens

<400> 22

tgggtgaaga agagccatgg aaagagcctt gagtggattg ga 42

<210> 23

<211> 51

<212> DNA

<213> Homo sapiens

<400> 23

gttattaatc cttacaacgg tggtactaga tacaaccaga agttcaaggg c 51

<210> 24

<211> 96

<212> DNA

<213> Homo sapiens

<400> 24

aaggccacat tgactgttga caagtcctcc agcacagcct acatggagct caacagcctg 60 acatttgagg actctgcagt ctattactgt gcaagg 96

<210> 25

<211> 30

<212> DNA

<213> Homo sapiens <400> 25

gagacggtag accttgcctg gtttgttcac 30

<210> 26

<211> 33

<212> DNA

<213> Homo sapiens

<400> 26

tggggccaag ggactctggt cactgtctct gca 33

<210> 27

<211> 19

<212> PRT

<213> Homo sapiens

<400> 27

Met Gly Trp Ser Trp lie Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15

Val His Ser

<210> 28

<211> 30

<212> PRT

<213> Homo sapiens

<400> 28

Glu Val Gin Leu Gin Gin Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr 20 25 30

<210> 29

<211> 5

<212> PRT

<213> Homo sapiens

<400> 29

Asp Tyr Phe Met Asn

1 5

<210> 30

<211> 14

<212> PRT

<213> Homo sapiens

<400> 30

Trp Val Lys Lys Ser His Gly Lys Ser Leu Glu Trp lie Gly

1 5 10

<210> 31

<211> 17

<212> PRT

<213> Homo sapiens

<400> 31

Val He Asn Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gin Lys Phe Lys 1 5 10 15

Gly <210> 32

<211> 32

<212> PRT

<213> Homo sapiens

<400> 32

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu 1 5 10 15

Leu Asn Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 33

<211> 10

<212> PRT

<213> Homo sapiens

<400> 33

Glu Thr Val Asp Leu Ala Trp Phe Val His

1 5 10

<210> 34

<211> 11

<212> PRT

<213> Homo sapiens

<400> 34 Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210> 35

<211> 57

<212> DNA

<213> Homo sapiens

<400> 35

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagt 57

<210> 36

<211> 69

<212> DNA

<213> Homo sapiens

<400> 36

gatgttttga tgacccaaac tccaccctcc ctgcctgtca ctcttggaga tcaggcctcc 60 atctcttgc 69

<210> 37

<211> 48

<212> DNA

<213> Homo sapiens

<400> 37

agatctagtc agagtattgt atatagtaat ggaaacacct acttagaa 48

<210> 38 <211> 45

<212> DNA

<213> Homo sapiens

<400> 38

tggtacctgc agaagccagg ccagtctcca aagctcctga tctac 45

<210> 39

<211> 21

<212> DNA

<213> Homo sapiens

<400> 39

aaagtttcca accgattttc t 21

<210> 40

<211> 96

<212> DNA

<213> Homo sapiens

<400> 40

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 60 agagtggagg ctgaggatct gggaatttat tactgc 96

<210> 41

<211> 27

<212> DNA

<213> Homo sapiens

<400> 41 tttcaaggtt cacatgttcc gctcacg 27

<210> 42

<211> 30

<212> DNA

<213> Homo sapiens

<400> 42

ttcggtgctg ggaccaagct ggagctgaaa 30

<210> 43

<211> 19

<212> PRT

<213> Homo sapiens

<400> 43

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp lie Pro Ala 1 5 10 15

Ser Ser Ser

<210> 44

<211> 23

<212> PRT

<213> Homo sapiens

<400> 44

Asp Val Leu Met Thr Gin Thr Pro Pro Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Asp Gin Ala Ser He Ser Cys

20

<210> 45

<211> 16

<212> PRT

<213> Homo sapiens

<400> 45

Arg Ser Ser Gin Ser lie Val Tyr Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15

<210> 46

<211> 15

<212> PRT

<213> Homo sapiens

<400> 46

Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr 1 5 10 15

<210> 47

<211> 7

<212> PRT

<213> Homo sapiens

<400> 47

Lys Val Ser Asn Arg Phe Ser 1 5

<210> 48

<211> 32

<212> PRT

<213> Homo sapiens

<400> 48

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15

Leu Lys lie Ser Arg Val Glu Ala Glu Asp Leu Gly lie Tyr Tyr Cys

20 25 30

<210> 49

<211> 9

<212> PRT

<213> Homo sapiens

<400> 49

Phe Gin Gly Ser His Val Pro Leu Thr

1 5

<210> 50

<211> 10

<212> PRT

<213> Homo sapiens <400> 50

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

<210> 51

<211> 57

<212> DNA

<213> Homo sapiens

<400> 51

atgggatgga gctggatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactct 57

<210> 52

<211> 90

<212> DNA

<213> Homo sapiens

<400> 52

gaggtccagc tgcaacagtc tggacctgtg ctggtgaagc ctggggcttc agtgaagatg 60 tcctgtaagg cttctggata caaattcact 90

<210> 53

<211> 15

<212> DNA

<213> Homo sapiens

<400> 53

gaccacttta tgaac 15 <210> 54

<211> 42

<212> DNA

<213> Homo sapiens

<400> 54

tgggtgaaga agaaccatgg aaagagcctt gagtggattg gg 42

<210> 55

<211> 51

<212> DNA

<213> Homo sapiens

<400> 55

gttattaatc cttacaacgg tggtactagg ctcaacccga agtttaaggg c 51

<210> 56

<211> 96

<212> DNA

<213> Homo sapiens

<400> 56

aaggccacat tgactgttga caagtcctcc agcacagtct acatggagct caacagcctg 60 acatttgagg actctgcagt ctattactgt gcaagg 96

<210> 57

<211> 30

<212> DNA

<213> Homo sapiens <400> 57

gagactatag accttgcctg gtttgcttat 30

<210> 58

<211> 33

<212> DNA

<213> Homo sapiens

<400> 58

tggggccagg ggactctggt cactgtctct gca 33

<210> 59

<211> 19

<212> PRT

<213> Homo sapiens

<400> 59

Met Gly Trp Ser Trp lie Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15

Val His Ser

<210> 60

<211> 30

<212> PRT

<213> Homo sapiens

<400> 60 Glu Val Gin Leu Gin Gin Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr

20 25 30

<210> 61

<211> 5

<212> PRT

<213> Homo sapiens

<400> 61

Asp His Phe Met Asn

1 5

<210> 62

<211> 14

<212> PRT

<213> Homo sapiens

<400> 62

Trp Val Lys Lys Asn His Gly Lys Ser Leu Glu Trp lie Gly

1 5 10

<210> 63

<211> 17

<212> PRT

<213> Homo sapiens

<400> 63 Val lie Asn Pro Tyr Asn Gly Gly Thr Arg Leu Asn Pro Lys Phe Lys 1 5 10 15

Gly

<210> 64

<211> 32

<212> PRT

<213> Homo sapiens

<400> 64

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Tyr Met Glu 1 5 10 15

Leu Asn Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 65

<211> 10

<212> PRT

<213> Homo sapiens

<400> 65

Glu Thr lie Asp Leu Ala Trp Phe Ala Tyr

1 5 10

<210> 66

<211> 11

<212> PRT <213> Homo sapiens

<400> 66

Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210> 67

<211> 57

<212> DNA

<213> Homo sapiens

<400> 67

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgctac cagcagt 57

<210> 68

<211> 69

<212> DNA

<213> Homo sapiens

<400> 68

gatgttttga tgactcaaac tccaccctcc ctgcctgtca ctcttggaga tcaggcctcc 60 atctcttgc 69

<210> 69

<211> 48

<212> DNA

<213> Homo sapiens

<400> 69 agatctagtc agagtattgt atatagtaat ggaaaaacct atttagaa 48

<210> 70

<211> 45

<212> DNA

<213> Homo sapiens

<400> 70

tggtacctgc agaagccagg ccagtctcca aagctcctga tgtac 45

<210> 71

<211> 21

<212> DNA

<213> Homo sapiens

<400> 71

aaagtttcca accgattttc t 21

<210> 72

<211> 96

<212> DNA

<213> Homo sapiens

<400> 72

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 60 agcgtggagg ctgaggatct gggaatttat tactgc 96

<210> 73

<211> 27 <212> DNA

<213> Homo sapiens

<400> 73

tttcaaggtt cacatgttcc gctcacg 27

<210> 74

<211> 30

<212> DNA

<213> Homo sapiens

<400> 74

ttcggtgctg ggaccaagct ggagctgaaa 30

<210> 75

<211> 19

<212> PRT

<213> Homo sapiens

<400> 75

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp lie Pro Ala 1 5 10 15

Thr Ser Ser

<210> 76

<211> 23

<212> PRT

<213> Homo sapiens <400> 76

Asp Val Leu Met Thr Gin Thr Pro Pro Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Asp Gin Ala Ser lie Ser Cys

20

<210> 77

<211> 16

<212> PRT

<213> Homo sapiens

<400> 77

Arg Ser Ser Gin Ser lie Val Tyr Ser Asn Gly Lys Thr Tyr Leu Glu 1 5 10 15

<210> 78

<211> 15

<212> PRT

<213> Homo sapiens

<400> 78

Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Met Tyr 1 5 10 15

<210> 79

<211> 7

<212> PRT

<213> Homo sapiens <400> 79

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 80

<211> 32

<212> PRT

<213> Homo sapiens

<400> 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15

Leu Lys lie Ser Ser Val Glu Ala Glu Asp Leu Gly lie Tyr Tyr Cys

20 25 30

<210> 81

<211> 9

<212> PRT

<213> Homo sapiens

<400> 81

Phe Gin Gly Ser His Val Pro Leu Thr

1 5

<210> 82

<211> 10

<212> PRT <213> Homo sapiens

<400> 82

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

1 5 10

* * * * *