Human anti-TREM2 antibodies for treating neurodegenerative disorders

Abstract

The present invention provides a protein or antibody capable of binding to human TREM2, comprising an Ig light chain variable region and an Ig heavy chain variable region, wherein the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2.

Description

[Technical Field]

[0001] The present invention relates to proteins comprising or consisting of an immunoglobulin (Ig) heavy chain variable region or light chain variable region. The present invention also relates to proteins comprising an antibody light chain variable region and an antibody heavy chain variable region, and antibodies comprising an antibody light chain variable region and an antibody heavy chain variable region. The present invention also relates to pharmaceutical compositions comprising the proteins or antibodies. The proteins or antibodies are capable of binding to the stalk region of human TREM2, preferably hTREM2. The proteins or antibodies are generally agonists of hTREM2, preferably agonist antibodies against hTREM2. Thus, the proteins, antibodies, and pharmaceutical compositions can be used in therapy, particularly for the treatment or prevention of neurodegenerative disorders such as Alzheimer's disease. The present invention also relates to methods for treating or preventing neurodegenerative diseases such as Alzheimer's disease. [Background technology]

[0002] Neurodegenerative disorders, such as Alzheimer's disease (AD), result in the progressive deterioration of neuronal structure with age, ultimately leading to cognitive impairment and dementia. AD is the most common form of dementia, affecting millions of people worldwide. To date, only two antibody-based therapeutics have been approved for the treatment of AD. Aducanumab (marketed as Aduhelm) is an antibody against amyloid beta (Aβ), and its use and approval have been hotly debated. This antibody targets amyloid plaques, a key symptom of Alzheimer's disease, resulting in a reduction in plaque burden in the brain. Lecanemab (marketed as Leqembi) is another antibody recently approved by the FDA. Leqembi also targets Aβ, more specifically protofibrils. Both therapeutics have a high risk of infusion-related reactions, cerebral edema, and microhemorrhages (van Dyck et al., 2022). Among the numerous Aβ-based therapeutic approaches, Aduhelm and Leqembi are, to date, the only approved candidates for the treatment of AD. Therefore, new strategies to prevent plaque deposition are needed.

[0003] Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor expressed on myeloid cells and is essential for microglial cell activation. TREM2 mutations have been identified in neurodegenerative disorders such as AD, and the mutations result in loss of TREM2 function through a variety of different mechanisms (Gernot Kleinberger et al., 2017; Schlepckow et al., 2017; Song et al., 2017; Ulland et al., 2017). TREM2-mediated signaling in microglial cells induces the transition of homeostatic microglia into disease-associated microglia (DAM) (Keren-Shaul et al., 2017). This transition is phenotypically characterized by enhanced phagocytosis, migration, and cell survival. Activation of TREM2 signaling is mediated through the adaptor protein DAP12. Upon ligand binding to TREM2, the ITAM motif of DAP12 is phosphorylated, leading to the recruitment of phosphorylated spleen tyrosine kinase (pSYK) and activation of downstream signaling molecules. Signaling is terminated by α-secretase-mediated shedding of the extracellular domain of TREM2, resulting in the release of soluble TREM2 (sTREM2) (G. Kleinberger et al., 2014; Wunderlich et al., 2013). The function of sTREM2 is not entirely clear. Altered levels of sTREM2 have been reported in the CSF of AD patients, suggesting its use as a potential disease biomarker (Zhong & Chen, 2019). sTREM2 levels change dynamically during AD progression, with the highest levels occurring in the early symptomatic stages of the disease (Suarez-Calvet et al., 2016). Furthermore, a positive correlation between sTREM2 and phospho-tau and total tau can be observed (Suarez-Calvet et al., 2016).Furthermore, studies suggest a signaling function for sTREM2 (Zhong et al., 2017, 2019).

[0004] Departing from the prior art, it is an object of the present invention to provide therapeutic agents for the treatment or prevention of neurodegenerative diseases / disorders such as AD. Summary of the Invention

[0005] To solve this problem, the present invention provides: 1) A protein comprising or consisting of a heavy chain variable region, or comprising or consisting of a light chain variable region, the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2; A protein in which the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1, and preferably the protein is an agonist of human TREM2.

[0006] 2) The protein according to 1), which comprises an antibody heavy chain comprising the heavy chain variable region.

[0007] 3) The protein according to 1) or 2), comprising two identical antibody heavy chains, each of which comprises the heavy chain variable region and at least one, preferably at least two, Ig heavy chain constant regions.

[0008] 4) comprising a light chain variable region and a heavy chain variable region; the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1; The protein preferably according to any one of 1) to 3), wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2.

[0009] 5) comprising an antibody light chain and an antibody heavy chain; the light chain comprises a light chain variable region, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1; Preferably, the protein according to 4) above, wherein the heavy chain comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2.

[0010] 6) comprising a light chain variable region and a heavy chain variable region; the light chain (LC) variable region preferably comprises a segment in CDR-L3 having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; A protein preferably according to 4) or 5), wherein the heavy chain (HC) variable region comprises, preferably in CDR-H3, a segment having the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8.

[0011] 7) The light chain variable region preferably comprises, in CDRs L1 and L3, the following amino acid segments in the N-terminal to C-terminal direction: A segment (CDR-L1) having the amino acid sequence of SEQ ID NO: 3, and a segment (CDR-L3) having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; The heavy chain (HC) variable region preferably comprises, in CDRs H2 and H3, the following amino acid segments from N-terminus to C-terminus: A segment (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and The protein according to any one of 4) to 6), comprising a segment (CDR-H3) having the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8.

[0012] 8) The light chain (LC) variable region preferably comprises, in CDRs L1 to L3, the following amino acid segments from the N-terminus to the C-terminus: a segment having the amino acid sequence of SEQ ID NO: 3 (CDR-L1); A segment (CDR-L2) having the amino acid sequence of SEQ ID NO: 4, and comprising a segment (CDR-L3) having the amino acid sequence of SEQ ID NO: 5 or having an amino acid sequence with one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; The heavy chain (HC) variable region preferably comprises, in the CDRs H1 to H3, the following amino acid sequence segments in the N-terminal to C-terminal direction: a segment having the amino acid sequence of SEQ ID NO: 6 (CDR-H1); A segment (CDR-H2) having the amino acid sequence of SEQ ID NO: 7, and The protein according to 6) or 7), comprising a segment (CDR-H3) having the amino acid sequence of SEQ ID NO: 8 or having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8.

[0013] 9) the protein is a single-chain antibody (scFv), a Fab fragment, a F(ab)2 fragment, or an immunoglobulin Ig; and / or The protein according to any one of 4) to 8), wherein the protein is a fusion protein comprising a single-chain antibody (scFv), a Fab fragment, a F(ab)2 fragment, or an immunoglobulin (Ig) as the first segment of the fusion protein and the second fusion protein segment.

[0014] 10) antibody light chain (subunit) and antibody heavy chain (subunit), the light chain comprises a light chain variable region (VL) and a light chain constant region (CL); The heavy chain comprises a heavy chain variable region and at least one heavy chain constant region, preferably at least the constant region C H The protein according to any one of 4) to 9), including 1).

[0015] 11) The protein according to 10), wherein the heavy chain constant region comprises one, two, or three Ig heavy chain constant domains, preferably three heavy chain constant domains.

[0016] 12) The protein according to any one of 1) to 8), wherein the protein is an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM, or the protein is a fusion protein comprising the Ig and an additional fusion protein segment.

[0017] 13) The protein according to 12), wherein the Ig is an IgG or a fusion protein comprising an IgG and an additional fusion protein segment.

[0018] 14) The protein is a light chain, such as: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9, 10, or 11; or (b) The protein according to any one of 1) to 13), comprising a light chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10, or 11.

[0019] 15) The protein according to any one of 4) to 14), wherein the light chain is a κ light chain or a λ light chain.

[0020] 16) The protein is a heavy chain, such as: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15; or (d) The protein according to any one of 4) to 15), comprising a heavy chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

[0021] 17) The protein according to any one of 4) to 16), wherein the protein is a fully human Ig.

[0022] 18) A protein according to any one of 4) to 17), comprising an antibody heavy chain or Fc portion that cannot bind to an Fc receptor, or that has reduced binding to an Fc receptor, or that has a mutated constant region that reduces binding to an Fc receptor.

[0023] 19) The protein according to 18), wherein the amino acid sequence of the heavy chain has a P to G substitution at a position corresponding to position 334 of SEQ ID NO: 12 or 13 to prevent or reduce binding of the protein to an Fc receptor.

[0024] 20) A binding domain capable of binding to human transferrin receptor 1 (hTfR1) to enable the protein to cross the blood-brain barrier, preferably the protein is a modified C H a heavy chain having three domains or C capable of binding to hTfR1 H A protein according to any one of 1) to 3) and 4) to 19), comprising a C-terminal extension of three domains.

[0025] 21) A protein according to any one of 1) to 20), wherein the protein can bind to human TREM2 via its variable region, preferably to the stalk region of hTREM2, and generally, the protein or antibody is an hTREM2 agonist.

[0026] 22) An antibody comprising an Ig light chain variable region defined in 4) and an Ig heavy chain variable region defined in 4).

[0027] twenty three) A light chain that is: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9, 10, or 11; or (b) a light chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10, or 11; and A heavy chain comprising: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15; or (d) An antibody described in 22), comprising a heavy chain whose amino acid sequence is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

[0028] twenty four) Two light chains, such as: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 10 or 11; or (b) two light chains, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 10 or 11; and Two heavy chains, such as: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15; or (d) An antibody described in 22) or 23) comprising two heavy chains, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

[0029] 25) A pharmaceutical composition comprising the protein or antibody according to any one of 1) to 24) and a pharmaceutically acceptable carrier.

[0030] 26) The protein or antibody according to any one of 1) to 24) or the pharmaceutical composition according to 25) for use in treatment or prevention.

[0031] 27) A protein or antibody according to any one of 1) to 24) or a pharmaceutical composition according to 25) for use in a method for treating or preventing a neurodegenerative disease such as Alzheimer's disease, preferably in the early stages of the patient's disease or in the early stages of the patient's disease.

[0032] 28) A protein or antibody or pharmaceutical composition for use according to 26) or 27), wherein the use comprises parenteral administration, preferably intravenous, subcutaneous or intraperitoneal administration, of the protein or antibody to a mammal.

[0033] 29) A nucleic acid molecule encoding a protein, polypeptide, light chain, and / or heavy chain defined in any one of 1) to 24).

[0034] 30) A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 25 or 26.

[0035] 31) A eukaryotic cell comprising the protein according to any one of 1) to 24) or the nucleic acid molecule according to 29) or 30).

[0036] 32) A method for treating or preventing a neurodegenerative disease such as Alzheimer's disease, comprising administering a protein or antibody defined in any one of 1) to 24) or a pharmaceutical composition described in 25) to a mammal in need of such treatment or prevention.

[0037] We surprisingly identified antibodies that can potently induce human TREM2 / DAP-induced SYK phosphorylation, a crucial TREM2-dependent effector pathway in AD. SYK phosphorylation was increased up to 60-fold by antibody M07, whereas no or little activation was observed with other antibody clones. However, M07 binds to the same epitope on the extracellular domain of TREM2 as H08 and M03.

[0038] Using phage display technology, we obtained fully human anti-TREM2 antibodies that were initially screened for antigen binding. Selected fully human IgG1-LALA modified antibodies were used to determine their binding affinity to the extracellular domain of human TREM2, their activation of human TREM2 signaling (human TREM2 / DAP-dependent SYK phosphorylation), and most importantly, their efficacy in a complex and relevant AD model using human brain cells differentiated from human induced pluripotent stem cells (hiPSCs).

[0039] The LALA modification strongly reduces the effector function of Ig antibodies, particularly IgG, and especially IgG1 antibodies, which is important for research using human immune cells and neural cells. The fully human framework of the antibody we created is advantageous compared to existing humanized antibodies based on the identification of clones in non-human animal immune systems (e.g., U.S. Patent Application Publication No. 2017240631(A1) (Alector AL-002) and WO 2020172450(A1) (Denali)). This is because the use of fully human antibodies in repeated in vivo prophylactic or therapeutic applications in humans can be expected to result in fewer immunological complications.

[0040] First, we identified a number of antibodies that all possessed high affinity (10 -9 We identified a variety of structurally similar and related antibodies (heavy chain amino acid sequence homology of 90% or more) that bind to TREM2 at a heavy chain length less than M. To our great surprise, we then found that only one of these antibodies (antibody M07) was able to strongly induce human TREM2 / DAP-induced SYK phosphorylation, a crucial TREM2-dependent effector pathway in AD. While SYK phosphorylation was increased up to 60-fold by M07, no or little activation was observed with the other antibody clones, M07 binds to the same epitope on the extracellular domain of TREM2 as H08 and M03.

[0041] The increase in human TREM2 / DAP-dependent pSYK levels induced by M07 was much stronger than that observed with published and patented agonistic antibodies against human TREM2, particularly any human anti-human TREM2 antibody. The hT2AB antibody disclosed by AMGEN (International Publication No. 2022120373(A1)) was used in a pSYK assay equivalent to ours and caused a 12-fold increase over baseline (Ellwanger et al., 2021). Alector presented a number of antibodies against human anti-TREM2 in its patent application (US Patent Publication No. 2017240631(A1)). SYK phosphorylation was demonstrated at the protein level, with approximately a 3- to 4-fold increase reported for antibodies #22, #45, and #65 in human dendritic cells. A 6-fold increase in SYK phosphorylation was observed in human macrophages. Denali presented several anti-hTREM2 antibodies in its patent application (WO 2020172450(A1)), including CL0020188, which increased pSYK levels four-fold in TREM2-expressing HEK293 cells compared to a control antibody.

[0042] Furthermore, the inventors used an innovative, complex, and relevant AD model using human brain cells differentiated from human induced pluripotent stem cells (hiPSCs). Anti-TREM2 antibodies, known in the state of the art, have not been analyzed in relatively sophisticated AD models using hiPSC-derived neurons and microglia. For example, WO2020172450(A1) (Denali) discloses a phagocytosis assay using hiPSC-derived microglia and amyloid beta. However, analysis was not performed in coculture with neurons. Therefore, the benefit of amyloid beta phagocytosis to neurons cannot be determined. AD can also be studied in other disease models, all of which have inherent limitations. Most researchers still use mouse models, which often fail to predict the clinical efficacy of anti-AD drug candidates.

[0043] The present invention solves the above-identified problems and surprisingly provides fully human anti-human TREM2 antibodies that highly potently activate human TREM2-dependent human pSYK signaling, such that beneficial effects can be observed in relevant AD models in human brain cells. [Brief explanation of the drawings]

[0044] [Figure 1] Silver gel of human anti-TREM2 agonist antibody M07. The first (left) lane shows non-reducing, non-boiling (NRNB) conditions, while the second lane shows the sample after boiling in reducing buffer conditions. After reduction / boiling, the antibody separates into light and heavy chains, detected at 25 and 50 kDa.

[0045] [Figure 2] ELISA-based EC50 values ​​of antibodies binding to human ecTREM2. H01, H08, M03, and M07 bind to the human extracellular (ec) domain of TREM2 with similar EC50 values. M05 does not bind to ecTREM2. N=mean of 2 technical replicates

[0046] [Figure 3] Schematic diagram of human TREM2 with the epitope peptide within the stalk region highlighted. Modified from (Reifschneider et al., 2022). The amino acid sequence shown is that of SEQ ID NO: 27.

[0047] [Figure 4] ELISA-based EC50 values ​​for rat H01 and various human antibodies binding to epitope peptides derived from the human TREM2 stalk domain. H01 and M05 do not bind to peptide sequences present in the stalk region of human TREM2, while H08, M03, and M07 bind with measured EC50 values ​​ranging from 329 to 832 pM. N=2 mean, technical replicates.

[0048] [Figure 5]p-SYK signaling in HEK293-Flp-In hTREM2 / hDAP12 cells upon antibody treatment (40 μg / ml). AlphaLISA assays for pSYK show significant activation of the signaling pathway upon addition of human anti-TREM2 antibodies H08, M03, and M07, and rat anti-TREM2 antibody H01. Activation was much stronger with antibody M07 (average 36-fold increase over baseline) compared to any other antibody tested. H05 and isotype controls for rat or human antibodies did not result in activation of the signaling pathway. Means + / - SEM are shown. One-way ANOVA with Brown-Forsythe post-hoc test: n = 12 for H01 and H08, n = 16 for M03, n = 6 for M05, and n = 18 for M07. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0049] [Figure 6A] Titration of p-SYK signaling in HEK293-Flp-In hTREM2 / hDAP12 upon antibody treatment. Figure 6A. Titration curves of anti-TREM2 antibodies H01 and M07 show much stronger activation of pSYK signaling by M07 at different antibody concentrations. Figure 6B. Titration curve of anti-TREM2 antibody M07 shows stronger activation of pSYK signaling by M07 at different antibody concentrations. Mean + / - SEM is shown. n=9; EC50=4.387 nM; KD=2.19 nM. [Figure 6B] Titration of p-SYK signaling in HEK293-Flp-In hTREM2 / hDAP12 upon antibody treatment. A. Titration curves of anti-TREM2 antibodies H01 and M07 show much stronger activation of pSYK signaling by M07 at different antibody concentrations. B. Titration curve of anti-TREM2 antibody M07 shows stronger activation of pSYK signaling by M07 at different antibody concentrations. Mean + / - SEM is shown. n=9; EC50=4.387 nM; KD=2.19 nM.

[0050] [Figure 7A]An iPSC-derived microglia-neuron co-culture model of Alzheimer's disease detects neurite degeneration and dead nuclei with amyloid beta (Aβ). Figure 7A. Timeline of neuron and microglia co-culture. Addition of Aβ to the Alzheimer's disease model and parallel addition of TREM2 antibody to test for neuroprotective effects. Figure 7B. Representative images of co-cultures containing microglia (Iba1) and neurons (βIII-tubulin) in control conditions (medium) and 5 μM Aβ conditions. Figure 7C. Detection of neurite degeneration and increasing number of dead nuclei with increasing Aβ dose. Mean + / - SD is shown. n=6 technical replicates. Figure 7D. Representative images from ICC staining for MAP2 and DAPI, and Cellprofiler analysis of neurite and dead nuclei numbers. [Figure 7B] An iPSC-derived microglia-neuron co-culture model of Alzheimer's disease detects neurite degeneration and dead nuclei with amyloid beta (Aβ). Figure 7A. Timeline of neuron and microglia co-culture. Addition of Aβ to the Alzheimer's disease model and parallel addition of TREM2 antibody to test for neuroprotective effects. Figure 7B. Representative images of co-cultures containing microglia (Iba1) and neurons (βIII-tubulin) in control conditions (medium) and 5 μM Aβ conditions. Figure 7C. Detection of neurite degeneration and increasing number of dead nuclei with increasing Aβ dose. Mean + / - SD is shown. n=6 technical replicates. Figure 7D. Representative images from ICC staining for MAP2 and DAPI, and Cellprofiler analysis of neurite and dead nuclei numbers. [Figure 7C-1]An iPSC-derived microglia-neuron co-culture model of Alzheimer's disease detects neurite degeneration and dead nuclei with amyloid beta (Aβ). Figure 7A. Timeline of neuron and microglia co-culture. Addition of Aβ to the Alzheimer's disease model and parallel addition of TREM2 antibody to test for neuroprotective effects. Figure 7B. Representative images of co-cultures containing microglia (Iba1) and neurons (βIII-tubulin) in control conditions (medium) and 5 μM Aβ conditions. Figure 7C. Detection of neurite degeneration and increasing number of dead nuclei with increasing Aβ dose. Mean + / - SD is shown. n=6 technical replicates. Figure 7D. Representative images from ICC staining for MAP2 and DAPI, and Cellprofiler analysis of neurite and dead nuclei numbers. [Figure 7C-2] An iPSC-derived microglia-neuron co-culture model of Alzheimer's disease detects neurite degeneration and dead nuclei with amyloid beta (Aβ). Figure 7A. Timeline of neuron and microglia co-culture. Addition of Aβ to the Alzheimer's disease model and parallel addition of TREM2 antibody to test for neuroprotective effects. Figure 7B. Representative images of co-cultures containing microglia (Iba1) and neurons (βIII-tubulin) in control conditions (medium) and 5 μM Aβ conditions. Figure 7C. Detection of neurite degeneration and increasing number of dead nuclei with increasing Aβ dose. Mean + / - SD is shown. n=6 technical replicates. Figure 7D. Representative images from ICC staining for MAP2 and DAPI, and Cellprofiler analysis of neurite and dead nuclei numbers. [Figure 7D]An iPSC-derived microglia-neuron co-culture model of Alzheimer's disease detects neurite degeneration and dead nuclei with amyloid beta (Aβ). Figure 7A. Timeline of neuron and microglia co-culture. Addition of Aβ to the Alzheimer's disease model and parallel addition of TREM2 antibody to test for neuroprotective effects. Figure 7B. Representative images of co-cultures containing microglia (Iba1) and neurons (βIII-tubulin) in control conditions (medium) and 5 μM Aβ conditions. Figure 7C. Detection of neurite degeneration and increasing number of dead nuclei with increasing Aβ dose. Mean + / - SD is shown. n=6 technical replicates. Figure 7D. Representative images from ICC staining for MAP2 and DAPI, and Cellprofiler analysis of neurite and dead nuclei numbers.

[0051] [Figure 8A-1] Anti-hTREM2 M07 antibody reduces amyloid beta-dependent neurite degeneration and cell death in a microglia-neuron co-culture system. Figure 8A. Addition of the anti-hTREM2 antibody M07 in addition to amyloid beta (0.6 μM Aβ + M07 antibody) significantly reduces neurite degeneration and the number of dead nuclei compared to addition of Aβ alone (0.6 μM Aβ) or Aβ together with an isotype control (0.6 μM Aβ + isotype antibody). Figure 8B. Representative images of ICC staining for MAP2 and DAPI, and Cellprofiler analysis of the number of neurites and dead nuclei. Mean values ​​+ / - SD are shown. n=6 technical replicates. [Figure 8A-2] Anti-hTREM2 M07 antibody reduces amyloid beta-dependent neurite degeneration and cell death in a microglia-neuron co-culture system. Figure 8A. Addition of the anti-hTREM2 antibody M07 in addition to amyloid beta (0.6 μM Aβ + M07 antibody) significantly reduces neurite degeneration and the number of dead nuclei compared to addition of Aβ alone (0.6 μM Aβ) or Aβ together with an isotype control (0.6 μM Aβ + isotype antibody). Figure 8B. Representative images of ICC staining for MAP2 and DAPI, and Cellprofiler analysis of the number of neurites and dead nuclei. Mean values ​​+ / - SD are shown. n=6 technical replicates. [Figure 8B] Anti-hTREM2 M07 antibody reduces amyloid beta-dependent neurite degeneration and cell death in a microglia-neuron co-culture system. Figure 8A. Addition of the anti-hTREM2 antibody M07 in addition to amyloid beta (0.6 μM Aβ + M07 antibody) significantly reduces neurite degeneration and the number of dead nuclei compared to addition of Aβ alone (0.6 μM Aβ) or Aβ together with an isotype control (0.6 μM Aβ + isotype antibody). Figure 8B. Representative images of ICC staining for MAP2 and DAPI, and Cellprofiler analysis of the number of neurites and dead nuclei. Mean values ​​+ / - SD are shown. n=6 technical replicates.

[0052] [Figure 9] Titration of p-SYK signaling in iPSC-derived microglia upon antibody treatment. The titration curve of the anti-TREM2 antibody M07 shows activation of pSYK signaling by M07 at different antibody concentrations. n=1. DETAILED DESCRIPTION OF THE INVENTION

[0053] The proteins and antibodies of the present invention can bind to the extracellular (ec) domain of human TREM2, particularly its stalk region. Furthermore, the proteins and antibodies of the present invention have excellent abilities to activate human TREM2, particularly to activate p-SYK signaling. Therefore, the proteins and antibodies are TREM2 agonists, preferably agonists of p-SYK signaling of hTREM2. Therefore, the proteins and antibodies of the present invention are highly promising active agents for treating neurodegenerative disorders such as AD.

[0054] definition As used herein, a protein can be a monomeric protein, i.e., a protein containing one subunit or polypeptide molecule, or a polymeric protein, i.e., a protein containing two or more subunits or polypeptide molecules. Examples of monomeric proteins are single-chain antibodies (scFv) or single-domain antibodies. Examples of multimeric proteins are Fab fragments of Ig, F of Ig, and the like. ab The protein may have modifications in the side chains of the amino acid residues, such as those described below for polypeptides.

[0055] A polypeptide is a polypeptide molecule, as opposed to a sequence stretch or portion of a molecule. The amino acid residues of a polypeptide may have chemical modifications in the side chains of the residues, such as a disulfide bond between two cysteine ​​residues of the same polypeptide or between two cysteine ​​residues of different polypeptides. Other examples of chemical modifications of amino acid residue side chains are glycosylation, such as modification of asparagine residues in heavy chains, markers, tags, labels, or oxidized side chains, addition (linkage) of other proteins or polypeptides. The modifications of amino acid residue side chains are not limited to moieties of small molecules, but may also be other polypeptides or protein domains.

[0056] The term "region" of a protein or polypeptide refers to a domain of said protein or polypeptide, i.e., a stretch (or segment) of the amino acid sequence of the polypeptide or protein. A polypeptide or protein comprises said region as a stretch or segment of the amino acid sequence of said polypeptide or protein. A protein or polypeptide comprises at least one more amino acid residue than that region, stretch or segment. The terms "region" and "domain" are used interchangeably herein.

[0057] The term "amino acid sequence" refers to the primary structure of a polypeptide, region, domain, segment, or stretch. Amino acid sequences are often defined by reference to a reference sequence, which is identified by a SEQ ID NO. Unless a subrange of the reference sequence is specified, reference to a reference sequence is to the entire amino acid sequence of the reference sequence.

[0058] A stretch or segment (of a polypeptide) refers to a number of (contiguous) amino acid residues within a polypeptide molecule, where the polypeptide molecule contains more amino acid residues than the stretch or segment.

[0059] The term "antibody" refers to a protein having an immunoglobulin fold that specifically binds to an antigen via its variable region, herein the extracellular (ec) domain of human TREM2, particularly the epitope of SEQ ID NO: 16. This term encompasses polyclonal and monoclonal antibodies, single-domain antibodies, heavy-chain antibodies, and single-chain antibodies. As used herein, the term "antibody" also includes Ig fragments that retain binding specificity via their variable region, including, but not limited to, Fab, F(ab')2, scFv, and bivalent scFv. Antibodies may contain light chains classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Preferred antibodies herein are immunoglobulins (Igs). Antibodies and Igs used in the present invention are preferably monoclonal and fully human.

[0060] Immunoglobulins (Ig) are proteins of the globulin type (naturally found in serum or other body fluids) that have antibody activity, i.e., they specifically bind to antigens via their variable regions. An Ig molecule contains two light (L) chains and two heavy (H) chains (or polypeptide chains or subunits) linked together by disulfide bonds. Ig can form oligomeric structures, such as IgM, which is a pentameric Ig. Ig is classified into five classes, IgG, IgM, IgA, IgD, and IgE, based on differences in antigen binding and structure in the H chain. Ig regions are the domains of Ig. The domains of Ig are the variable domain and the constant domain. An Ig heavy chain contains one variable domain (or region) and two C H 1. C H 2, and C H Ig light chains have two domains, a variable domain and a constant domain.

[0061] The term "light chain" or "antibody light chain" refers to a polypeptide comprising an immunoglobulin (Ig) light chain (LC) variable region (or domain) and an Ig light chain constant region (or domain).

[0062] The term "heavy chain" or "antibody heavy chain" refers to an Ig heavy chain (HC) variable region and at least one Ig constant region, generally at least C H Preferably, a heavy chain or antibody heavy chain is a polypeptide comprising an Ig heavy chain variable region (or domain) and three Ig constant regions (or domains), C H 1. C H 2. C H 3 means a (complete) Ig heavy chain.

[0063] Proteins and antibodies of the present invention The proteins of the invention comprise or consist of an (Ig) heavy chain variable region, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence which has one or two amino acid residue substitutions compared to SEQ ID NO: 2. Alternatively, the proteins of the invention comprise or consist of an (Ig) light chain variable region, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence which has one or two amino acid residue substitutions compared to SEQ ID NO: 1. Examples of such proteins are single domain antibodies or nanobodies. Single domain antibodies consist of the variable domain of either the heavy or light chain, preferably the heavy chain.

[0064] In another embodiment, the protein of the present invention may be a heavy chain antibody. In such an embodiment, the protein generally comprises or consists of (one or preferably two) heavy chains, each comprising a heavy chain variable region, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2. The heavy chain preferably further comprises, apart from the variable region, at least one constant region (domain), e.g., two constant regions or five constant regions. The protein may comprise or consist of two (preferably identical) subunits or polypeptides, each polypeptide comprising the variable region and at least one, preferably at least two heavy chain constant regions. Examples of such heavy chain antibodies are V H Alternatively, the protein may comprise or consist of two (preferably identical) subunits or polypeptides, each polypeptide comprising the variable region and five heavy chain constant regions. An example is the V NAR In all of these embodiments, the CDRs of the heavy chain variable domain are as set forth below.

[0065] In a preferred embodiment described below, the protein of the invention comprises a light chain variable region and a heavy chain variable region.

[0066] Thus, the protein of the present invention may comprise a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1; The amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO:2.

[0067] Preferably, the protein comprises an antibody light chain and an antibody heavy chain. The light and heavy chains are polypeptides comprising a light chain variable region as defined above and a heavy chain variable region as defined above, respectively. The variable regions (both light and heavy chains) comprise three hypervariable regions, generally referred to as complementarity determining regions (CDRs) and numbered CDR1 to CDR3 from the N-terminus to the C-terminus. The CDRs are preceded and separated by less variable regions, generally referred to as framework regions (FRs), and numbered FR1 to FR4 from the N-terminus to the C-terminus of the chain.

[0068] The light chain (LC) variable region may preferably comprise, in CDR-L3, a segment of the amino acid sequence of SEQ ID NO: 5 or a segment of an amino acid sequence which has one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; the heavy chain (HC) variable region may preferably comprise, in CDR-H3, a segment of the amino acid sequence of SEQ ID NO: 8 or a segment of an amino acid sequence which has one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 8. The absence of such amino acid substitutions is preferred for both these CDRs and chains in this and the following embodiments.

[0069] Preferably, the light chain variable region comprises, preferably in CDRs L1 and L3, the following amino acid segments in the N-terminal to C-terminal direction: A segment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), and a segment (CDR-L3) of the amino acid sequence of SEQ ID NO: 5 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; and The heavy chain (HC) variable region preferably comprises, in CDRs H2 and H3, the following amino acid segments from N-terminus to C-terminus: A segment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and It comprises a segment (CDR-H3) of the amino acid sequence of SEQ ID NO:8 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO:8.

[0070] More preferably, the light chain (LC) variable region preferably comprises, from the N-terminus to the C-terminus, the following amino acid segments in CDRs L1 to L3: a segment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), A segment of the amino acid sequence of SEQ ID NO: 4 (CDR-L2), and a segment (CDR-L3) of the amino acid sequence of SEQ ID NO: 5 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; and The heavy chain (HC) variable region preferably comprises the following amino acid sequence segments in the N-terminal to C-terminal direction in CDRs H1 to H3: a segment of the amino acid sequence of SEQ ID NO: 6 (CDR-H1), A segment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and It comprises a segment (CDR-H3) of the amino acid sequence of SEQ ID NO:8 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO:8.

[0071] CDRs that do not have amino acid residue substitutions compared to a given reference sequence are preferred. As explained above, these segments (CDRs) are generally not contiguous in the chain or region, but are separated by framework regions, as can be seen from SEQ ID NOs: 1 and 2.

[0072] The protein of the present invention may be a single-chain antibody (scFv). In this case, the protein is a polypeptide comprising or consisting of a light chain variable region and a heavy chain variable region in this order or in the reverse order from N-terminus to C-terminus. The variable region is as defined above, and the CDRs are preferably as defined above. scFvs typically do not comprise a constant region.

[0073] However, apart from the variable regions, the light and heavy chains of the proteins of the invention generally further comprise one or more Ig constant domains or all domains of the constant regions of the (complete) Ig light and heavy chains, respectively. Thus, the proteins may comprise an antibody light chain and an antibody heavy chain, the light chain comprises a light chain variable region, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1; The heavy chain comprises a heavy chain variable region, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2. Embodiments having none or no (i.e., none) amino acid residue substitutions are preferred. CDRs are preferred as described above.

[0074] Since the light chain of a naturally occurring immunoglobulin contains one Ig constant domain or region, the light chain of the protein of the invention preferably contains (especially one) constant region. The light chain may be a kappa or lambda light chain, the former being preferred. Since the heavy chain of a naturally occurring immunoglobulin contains three constant domains (generally C H 1. C H 2 and C H 3), the heavy chain of the protein of the invention therefore generally comprises at least one constant domain, preferably C H In one embodiment, the heavy chain comprises two (Ig) constant domains, preferably C H 1 and C H Even more preferably, the heavy chain of the protein of the invention comprises three (Ig) constant domains, e.g., CH 1. C H 2 and C H It contains three domains (from N- to C-terminus).

[0075] Thus, the protein of the present invention may be an Ig Fab fragment, i.e., comprise a light chain and a heavy chain. The former comprises or consists of a polypeptide comprising a light chain (kappa or lambda), and the latter comprises a heavy chain variable region and one or more (Ig) heavy chain constant regions, generally C. H 1. The light and heavy chains are generally covalently linked by disulfide bridges.

[0076] The protein of the invention may alternatively be a F(ab)2 fragment of Ig, which comprises two Fab fragments linked by one or more disulfide bridges.

[0077] The proteins of the present invention may comprise an Ig light chain and (i.e., a complete or complete) Ig heavy chain. A complete heavy chain means that the heavy chain comprises, apart from the variable domain, the three Ig constant domains. Thus, the heavy chain preferably comprises, apart from the variable domain, the three constant domains C H 1. C H 2 and C H 3. In a more preferred embodiment, the protein of the present invention comprises two light chains and two (complete) heavy chains. Thus, the protein is preferably an immunoglobulin of any isotype, such as IgG, IgM, IgA, IgD, and IgE. Preferably, it is an IgG. Ig comprises two (generally identical) Ig light chains and two Ig heavy chains, which are generally (but not necessarily) identical. Within IgG, the protein may be IgG1, IgG2, IgG3, or IgG4, depending on the heavy chain. Preferably, it is IgG1, IgG2, or IgG3, more preferably an IgG1, such as clone M07, described and used in the examples.

[0078] A preferred protein or antibody of the invention is a light chain, (a) the amino acid sequence of the light chain is or comprises SEQ ID NO: 9, 10 or 11, preferably SEQ ID NO: 10 or 11; or (b) the amino acid sequence of the light chain is or comprises an amino acid sequence based on a kappa 1 light chain framework having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9 or 10, preferably SEQ ID NO: 10; or (c) A light chain, the amino acid sequence of which may also be or include an amino acid sequence based on a lambda light chain framework having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 11.

[0079] In the above embodiments, particularly embodiments (a), (b) and (c), the protein or antibody preferably comprises a heavy chain comprising: (d) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15; or (e) The heavy chain further includes a heavy chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15.

[0080] In the above embodiment, it is further preferred that the protein or antibody is an immunoglobulin comprising two identical (Ig) light chains and two (Ig) heavy chains (which may also be identical), more preferably an IgG1 antibody. Thus, the protein or antibody comprises two light chains, (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9, 10 or 11, preferably SEQ ID NO: 10; or (b) may comprise or consist of two light chains, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10 or 11, preferably SEQ ID NO: 10; Also, two heavy chains, (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15; or (d) It may comprise or consist of two heavy chains, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15.

[0081] The two light chains and the two heavy chains are preferably identical in amino acid sequence. Preferred are embodiments with one or no amino acid residue substitutions. The CDRs are as defined above.

[0082] Further embodiments of the protein or antibody of the invention are as follows: the protein or antibody comprises two subunits (chains) of the kappa 1 light chain of SEQ ID NO: 9 or SEQ ID NO: 10 (the latter is preferred) or a subunit (chain) of the lambda light chain of SEQ ID NO: 11 and two subunits of the heavy chain of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, preferably SEQ ID NO: 13 or 15.

[0083] In another embodiment, the protein comprises two light chains of SEQ ID NO: 10, one heavy chain of SEQ ID NO: 13, and one heavy chain of SEQ ID NO: 15. In a further embodiment, the protein comprises two light chains of SEQ ID NO: 10 and two heavy chains of SEQ ID NO: 13. In a further embodiment, the protein comprises two light chains of SEQ ID NO: 10 and two heavy chains of SEQ ID NO: 15. In these embodiments, the heavy chains may have one or two amino acid residue substitutions in the sequence portion outside the heavy chain CDRs defined above.

[0084] In yet another embodiment, the protein comprises two light chains of SEQ ID NO: 11, one heavy chain of SEQ ID NO: 13, and one heavy chain of SEQ ID NO: 15. In a further embodiment, the protein comprises two light chains of SEQ ID NO: 11 and two heavy chains of SEQ ID NO: 13. In a further embodiment, the protein comprises two light chains of SEQ ID NO: 11 and two heavy chains of SEQ ID NO: 15. In these embodiments, the heavy chains may have one or two amino acid residue substitutions in the sequence portion outside the heavy chain CDRs defined above.

[0085] As stated above, the protein of the invention is preferably an antibody, more preferably an Ig, comprising an (Ig) light chain variable region as defined above and an (Ig) heavy chain variable region as defined above. The antibody preferably has a light chain comprising: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9 or 10; or (b) a light chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9 or 10, preferably SEQ ID NO: 10; and A heavy chain comprising: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15; or (d) A heavy chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15.

[0086] Preferred embodiments are similar to those set out above for the proteins of the invention.

[0087] Alternatively, the protein of the invention may be an antibody, preferably an Ig, comprising an (Ig) light chain variable region as defined above and an (Ig) heavy chain variable region as defined above. The antibody preferably has a light chain comprising: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 11; or (b) a light chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 11; and A heavy chain comprising: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15; or (d) A heavy chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15, preferably SEQ ID NO: 13 or 15.

[0088] Preferred embodiments are similar to those set out above for the proteins of the invention.

[0089] The protein or polypeptide of the protein of the present invention may be a fusion protein. The fusion protein comprises a polypeptide comprising any of the chains, regions, or domains described above as a first segment and a second segment (preferably the C-terminus of the first segment). The second fusion protein segment may provide the protein or antibody with additional functionality, such as binding to a receptor (other than hTREM2). The fusion protein may be a fusion protein comprising an Ig selected from IgG, IgA, IgD, IgE, and IgM, and a second fusion protein segment, with IgG being preferred. Preferably, the fusion protein is a fusion protein of any such Ig, comprising two Ig light chains and two heavy chains, with at least one, preferably both, heavy chain polypeptides comprising the second fusion protein segment.

[0090] The proteins or antibodies of the present invention may have further modifications to facilitate transport of the protein or antibody to the brain of a subject. In the case of parenteral administration, e.g., intravenous administration, the protein or antibody may have modifications that facilitate crossing the blood-brain barrier (BBB). BBB crossing and brain delivery of proteins or antibodies is known in the art; for a review, see Pardridge (2015), Expert Opinion on Drug Delivery, 12:2, 207-222 (DOI: 10.1517 / 17425247.2014.952627). An established solution utilizes binding of the protein to be delivered to the human transferrin receptor (hTfR). Denali Therapeutics' WO 2018152285 and WO 2018152326 and Kariolis et al. (Science Translational Medicine, Vol. 12, No. 545; DOI: 10.1126 / scitranslmed.aay1359) describe amino acid residue substitutions in the CH3 region of an antibody heavy chain to achieve specific binding to TfR. WO 2014033074 and WO 2015101588 (Roche) describe BBB shuttle modules comprising a brain effector entity, a linker, and a monovalent binding entity that binds to a BBB receptor such as TfR.

[0091] Thus, the protein or antibody of the present invention may comprise a binding domain capable of binding to hTfR1 to enable the protein to cross the BBB, and preferably, the protein or antibody comprises a heavy chain having a modified CH3 domain that binds to hTfR1. The heavy chain CH3 domain may be modified as described in WO2018152285, WO2018152326, or Kariolis et al. Alternatively, for a similar purpose, the protein of the present invention may comprise the above-mentioned fusion protein.

[0092] For the treatment or prevention of neurological disorders or diseases, the proteins or antibodies of the present invention may have reduced Fc effector function to prevent undesirable or unwanted effects on the immune system. To this end, the proteins of the present invention may lack the CH2 and CH3 domains of the antibody heavy chain. However, preferably, the proteins comprise a heavy chain comprising the CH2 and CH3 domains, with the CH2 domain having one or more amino acid residue substitutions that reduce Fc effector function. An example of such a mutation is, inter alia, the well-known L234A and L235A double mutation reviewed by Wang et al. (Protein Cell 2018, 9(1), 63-73; doi.org / 10.1007 / s13238-017-0473-8), which reduces binding of the protein to Fc receptors and thereby reduces effector function. Corresponding mutations are generally preferred for all embodiments of the present invention in which the protein comprises a heavy chain having a CH2 domain. Each of SEQ ID NOs: 12 to 15 contains this double mutation at its corresponding position, positions 239 and 240 of SEQ ID NO: 13. Furthermore, to further reduce the effector function of the protein or antibody, the heavy chain may further have a P to G substitution (resulting in a LALA-PG triple mutation) at position corresponding to position 334 of SEQ ID NO: 13 or 15 to prevent or reduce binding of the protein to Fc receptors. This mutation is described in detail in WO2012130831A1.

[0093] nucleic acid molecule The present invention provides nucleic acid molecules encoding the proteins, polypeptides, light chains, and / or heavy chains defined above. The nucleic acid molecules may be plasmids or vectors containing one or more constructs or cistrons encoding the proteins, polypeptides, light chains, and / or heavy chains, as well as regulatory genetic elements for their expression in a suitable cell. In embodiments in which the protein comprises two or more different polypeptide molecules, the plasmid or vector may contain two or more constructs or cistrons, one for each polypeptide to be expressed. Alternatively, the present invention provides kits for two nucleic acid molecules, one encoding a first polypeptide of the protein of the invention and one encoding a second polypeptide of the protein of the invention. The nucleic acid molecules may comprise the nucleotide sequence of SEQ ID NO: 25 or 26.

[0094] cell The present invention provides a cell, preferably a eukaryotic cell, comprising a protein according to the invention or a nucleic acid molecule according to the invention, such as those described above. The cell is preferably used to produce and express the protein of the invention. For use of the protein in humans, the cell is preferably a human cell to impart human-like glycosylation to the protein or antibody. However, the cell may have engineered glycosylation machinery to impart the desired glycosylation to the protein.

[0095] Protein or antibody production The proteins or antibodies of the present invention can be expressed from nucleic acid molecules encoding them in a suitable expression system, as generally known in the art. In the case of hetero-oligomeric proteins such as immunoglobulins, the light and heavy chains can be expressed from a bicistronic plasmid in the same cell, preferably a eukaryotic cell, as described in the Examples. The light and heavy chains can be expressed in a form containing an N-terminal leader sequence that directs secretion of the leader. The leader should be such that after secretion or in the secretory pathway of the cell, the light and heavy chains can then assemble to form the oligomeric protein, preferably without the leader sequence. For use in humans, the cell line used for expression is preferably human, to confer human-like glycosylation to the protein or antibody, if desired.

[0096] Pharmaceutical Compositions and Formulations The present invention also provides pharmaceutical compositions comprising the proteins or antibodies of the present invention. The compositions generally further comprise one or more pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers include any solvent or dispersion medium that is physiologically compatible and does not interfere with or otherwise inhibit the activity of the active agent. A preferred solvent is water, which may further contain excipients.

[0097] Examples of excipients are carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, stabilizers, and / or buffers. Pharmaceutical compositions can be prepared in a manner known to those skilled in the art by mixing the protein or antibody, for example, by conventional mixing, dissolving, or lyophilizing processes.

[0098] For parenteral administration, pharmaceutical compositions can generally be administered as a solution by injection or infusion. For injection, proteins or antibodies can be formulated into preparations by dissolving, suspending, or emulsifying them in an aqueous solvent, which may contain conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives. In some embodiments, the compounds can be formulated in aqueous solutions, for example, in physiologically compatible buffers such as saline buffer. Injectable preparations can be provided in unit dosage form, for example, in ampoules or in multi-dose containers, with or without added preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles.

[0099] However, the pharmaceutical composition may alternatively be a solid composition, for example in lyophilized form, which can be reconstituted before use with a suitable solution or vehicle, as described above.

[0100] Typically, pharmaceutical compositions for use in in vivo administration are sterile, which can be accomplished according to methods known in the art, such as sterile filtration of a solution or irradiation.

[0101] Treatment or prevention The protein or antibody of the present invention is used for treatment or prevention. The disease disorder to be prevented or treated is a neurodegenerative disease such as Alzheimer's disease. To treat or prevent the disorder or disease in the patient, the protein or antibody is administered to a subject in need of treatment / prevention. The subject or patient is a mammal, preferably a human. The present invention also provides a method for treating or preventing a neurodegenerative disease such as Alzheimer's disease in a mammal, preferably a human, comprising administering the protein or antibody or pharmaceutical composition of the present invention to a mammal or human in need of such treatment or prevention.

[0102] In treating or preventing neurodegenerative diseases, the patients to whom the protein or antibody is administered are preferably those in the early stages of neurodegenerative diseases such as Alzheimer's disease, since it is expected that the efficacy will be higher in patients in the early stages of neurodegenerative diseases such as Alzheimer's disease than in those in the advanced stages. Thus, treatment at an early stage prevents or inhibits the progression of the disease to a more advanced or severe stage.

[0103] The stage of neurodegenerative diseases such as Alzheimer's disease can be determined by established methods. One such method is the Mini-Mental State Examination (MMSE) or Folstein test, which is based on a 30-point questionnaire widely used in clinical and research settings to measure cognitive impairment. The MMSE may be used in the version described by Tombaugh, Tom N.; McIntyre, Nancy J. (1992) "The Mini Mental Status Examination: A Comprehensive Review." Journal of the American Geriatrics Society. 40(9):922-935. doi:10.1111 / j.1532-5415.1992.tb01992. The MMSE is commonly used in medical practice to screen for dementia. It is also used to estimate the severity and progression of cognitive impairment and to track the course of cognitive changes in individuals over time. Therefore, the MMSE is an effective method for recording an individual's response to treatment. Any score of 24 or higher (out of 30) indicates normal cognition. Below this, scores may indicate severe (≦9 points), moderate (10-18 points), or mild (19-23 points) cognitive impairment. Raw scores must also be corrected for educational level and age. Low to very low scores correlate closely with the presence of dementia, although other psychiatric disorders can also result in abnormal findings on the MMSE test. The presence of purely physical problems can hinder interpretation if not properly noted. For example, a patient may be physically unable to properly hear or read instructions, or may have a motor disorder that affects their writing and drawing skills.

[0104] Another method for determining the stage of neurodegenerative diseases such as Alzheimer's disease is the CDR global score (Clinical Dementia Rating Scale). The CDR is a global rating scale for staging patients diagnosed with dementia. The CDR assesses cognitive, behavioral, and functional aspects of Alzheimer's disease and other dementias. Rather than mental status tests or surveys, the assessor makes a judgment on six categories based on all available information. Although the CDR rating system relies heavily on memory scores, the CDR has good inter-rater reliability for dementia staging. The CDR is a widely used scale in both Alzheimer's disease facilities and dementia research. The CDR is calculated based on semi-structured interviews with the subject and caregiver (informant) and the clinician's clinical judgment. The CDR is calculated based on testing six different cognitive and behavioral domains, including memory, orientation, judgment and problem-solving, community tasks, household and hobby performance, and personal care. The CDR is based on a scale of 0 to 3: no dementia (CDR = 0), suspected dementia (CDR = 0.5), mild cognitive impairment (CDR = 1), moderate cognitive impairment (CDR = 2), and severe cognitive impairment (CDR = 3). Two sets of questions are asked, one to the informant and one to the subject. The set for the informant includes questions about the subject's memory problems, the subject's judgment and problem-solving abilities, the subject's community activities, the subject's home life and hobbies, and personal questions related to the subject. The set for the subject includes memory-related questions, orientation-related questions, and questions about judgment and problem-solving abilities. This method is described in Handbook of Clinical Neurology, Volume 167, 2019, Pages 89-104, Chapter 6 - Cognitive and neuropsychological examination of the elderly.

[0105] Thus, the present invention provides a protein or antibody for use in the treatment or prevention of a neurodegenerative disease, such as Alzheimer's disease, preferably in a patient having an early stage of a neurodegenerative disease, such as Alzheimer's disease, as follows: - cognitive impairment in the patient with a score of 23 or less on the Mini-Mental State Examination (MMSE) test, preferably a score of 10 to 23, more preferably a score of 19 to 23, or - The patient's cognitive impairment is measured by a score of 0.5 or more and 2 or less on the Clinical Dementia Rating Scale (CDR Global Score).

[0106] The protein or antibody is preferably administered parenterally. Examples of preferred routes of administration are intravenous, subcutaneous and intraperitoneal.

[0107] The protein or antibody can be administered to a subject in a therapeutically effective amount or doses. Dosage ranges per administration of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg can be used. However, dosages can vary according to several factors, including frequency of administration, the selected route of administration, the formulation of the composition, the patient's response, the severity of the condition, and the judgment of the prescribing physician. Dosages can be increased or decreased over time, depending on the needs of an individual patient. Patients can be given a low dose initially, then increased to an effective dose tolerated by the patient. Determining such effective amounts is well within the capabilities of those skilled in the art.

[0108] The protein or antibody can be administered once every 1 to 6 weeks, preferably once every 2 to 4 weeks. [Example]

[0109] The present invention is not limited to the examples described below.

[0110] I. Materials and Methods Identification of antibody clones A portion of the human TREM-2 extracellular domain was cloned into a complete repertoire of human antibody sequences (a diverse set of at least 5×10 10 The IgG1 heavy chain was used as a coating antigen in a phage display screening of a phage library containing the IgG1 heavy chain variable domains (clones) at Proteogenix, Strasbourg, France. Several phage binders that bound to the antigen with high affinity were identified. Using standard techniques, the human DNA and protein sequences of the variable regions of each of the light and heavy chains were identified from monoclonal phage preparations. Clones were specifically engineered for optimized codons with input from other partners. Each of these heavy chain variable domain sequences was used to synthesize a fully human IgG1 heavy chain by synthetic gene assembly at GeneArt, Regensburg, using further codon optimization for Cricetus griseus by combining it with a constant domain sequence containing L-to-A mutations at the appropriate positions 239 / 240 (corresponding to consensus sequence positions 234 and 235) (gene accession number UniProtKB-P0DOX5). Each light chain variable domain was then combined with the constant region sequence of the kappa 1 light chain (accession number UniProtKB-P0DOX7) and similarly assembled. An AvrII restriction site was added to the beginning of the heavy chain gene and a BstZ171 restriction site to the end. Similarly, an EcoRV restriction site was added to the beginning of the light chain gene and a PacI restriction site to the end. These DNA fragments were then used to construct the Freedom TSThe heavy and light chain genes were inserted into the respective multiple cloning sites (AvrII-BstZ171 and EcoRV-PacI) of the bicistronic vector pCHOv1, part of the CHO-S kit (Thermo Fisher cat# A13696-01). The resulting vector was prepared at GeneArt, Regensburg, subjected to complete quality control, and purified using a column. This plasmid allows the expression of two different proteins in one cell under two hybrid modifications of the cytomegalovirus (CMV) promoter, specifically the expression of antibody heavy and light chains in one cell, such as Chinese hamster ovary cells (CHO).

[0111] The IgG1-LALA isotype control was generated using the heavy and light chain sequences of an anti-green fluorescent protein (GFP) antibody.

[0112] E. coli DH5α was transformed with 1 ng of plasmid DNA. Transformed bacteria were plated onto agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. The next day, colonies were picked, grown in LB medium containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Maxi Prep was performed using NucleoBond Xtra Maxi EF, a Maxi kit for endotoxin-free plasmid DNA (Machery Nagel cat# 740424.50).

[0113] The coding sequences for the light and heavy chains of clone M07 are shown in SEQ ID NOs: 25 and 26, respectively.

[0114] The term "clone M07" refers to an IgG1 antibody of the invention expressed from a coding sequence encoding a kappa-1 light chain of SEQ ID NO: 9 and a heavy chain of SEQ ID NO: 12. Upon secretion, the N-terminal leader is cleaved. Thus, the mature light chain has the amino acid sequence of SEQ ID NO: 10, and the mature heavy chain has the amino acid sequence of SEQ ID NO: 13. The experiments described below are performed with antibody M07, which comprises two of the mature light chains and two of the heavy chains. The heavy chain polypeptides of SEQ ID NOs: 12 and 13 are C H The three domains contain a binding segment (TFN) for binding to the human transferrin receptor (hTfR). SEQ ID NOs: 14 and 15 are heavy chains corresponding to SEQ ID NOs: 12 and 13, but with C H The heavy chains lack the binding segment (TFN) in the 3 domain and have the corresponding wild-type sequence in place of TFN. All heavy chains contain the LALA double mutation.

[0115] HEK293-Flp-In cell culture HEK293-Flp-In cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing GlutaMAX I, supplemented with 10% (v / v) fetal calf serum (FCS), 1% (v / v) penicillin / streptomycin, and 0.4% (v / v) hygromycin B.

[0116] CHO-S kit and gene transfer, selection and purification Freedom TSCHO-S cells, commercially available as part of a CHO-S kit (Thermo Fisher cat# A13696-01), were thawed and grown in cell culture using the recommended medium as recommended in the manufacturer's instructions. Transfection of the pCHOv1 plasmid, containing the heavy and light chain sequences under the control of two different hybrid CMV promoters, was performed according to the manufacturer's instructions. After positive selection of successfully transfected CHO-S cells, the cells were maintained in culture under selective pressure as recommended in the manufacturer's instructions. At intervals, the supernatant was harvested and prepared for antibody purification. Briefly, the supernatant (approximately 250 ml) was diluted 1:4 with binding buffer (20 mM sodium phosphate; 50 mM sodium phosphate, pH 7.0) and loaded onto a 1 ml Protein A column (Cytiva cat# 17040201) using a peristaltic pump. The column was then washed with 10 column volumes of binding buffer, and the antibody was eluted with elution buffer (0.1 M glycine-HCl, pH = 2.7) into neutralization buffer (1 M TRIS-HCl, pH = 9). Fractions containing protein (checked by Nanodrop) were pooled and dialyzed overnight against 1x PBS. The dialyzed sample was concentrated, and the concentration was determined by Nanodrop.

[0117] Differentiation of hiPSC-derived microglia (hiMGL) We differentiated hiMGLs from iPSCs as described (Abud et al., 2017), with modifications to improve efficiency and yield. When iPSCs reached 70–90% confluence, they were split 1:100–200 onto GelTrex-coated 6-well plates for HPC differentiation using EDTA, yielding approximately 30 small colonies per well. Cells were fed with 2 ml of HemA medium (HPC Differentiation Kit, StemCell Technologies) on day 0, half-fed with 1 ml on day 2, and half-fed with 2 ml of HemB on day 3, half-fed on days 5 and 7, and topped with 1 ml on day 10. On day 12, HPCs were harvested as nonadherent cells and either frozen or continued with microglial differentiation. HPCs were frozen at 1 million cells per ml in BamBanker (Wako). They were then thawed directly onto a GelTrex-coated 6-well plate, and 1 million cells were evenly distributed among 6 wells in 2 ml of iMGL medium containing freshly added 25 ng / ml M-CSF, 100 ng / ml IL-34, and 50 ng / ml TGF-β. 1 ml of β medium was added to the top every other day. During microglial differentiation, cells were split 1:2 every 6–8 days depending on confluency. A very similar differentiation protocol was recently published (McQuade et al., 2018). We did not use CD200 or CX3CL1 because these genes did not appear to affect hiMGL gene expression, as determined by Nanostring analysis (data not shown). hiMGL cells were used for experiments on day 16 of differentiation.

[0118] Differentiation of hiPSC-derived cortical neurons For cortical neuron differentiation, initial neural progenitor cells (NPCs) were differentiated from hiPSCs and NPCs and further differentiated into cortical neurons as described (Gregg et al., 2016) with modifications. Specifically, hiPSCs were grown to 100% confluence on GelTrex-coated plates. Neural induction (NI) medium containing 10 μM SB431542 and 250 nM LDN193189 was added to the cells (in vitro day 0 (DIV0)) and maintained for 12 days. Cells were split as single cells using Accutase with ROCK inhibitor at approximately DIV2 and DIV8. At DIV2, 0.26 mio cells per 12-well plate were split onto GelTrex-coated 12-well plates. At DIV8, 200 μl of a 30 mio cell / ml cell suspension was added to 1.1 cm wells. 2 NPCs were seeded onto 6-well plates coated with poly-L-ornithine and laminin (PLO / lam). Rosettes were visualized during neural induction. From DIV12, NI medium was replaced with neural maintenance (NM) medium, and 20 ng / ml bFGF was added within the first 4 days. At DIV22, rosettes were isolated with STEMdiff Neural Rosette Selection Reagent (STEMCELL Technologies) and plated onto PLO / lam-coated 6-well plates in NM medium containing bFGF. At DIV29, rosettes were split using accutase. At DIV39, NPCs were frozen in neural progenitor cell freezing medium (STEMCELL Technologies).

[0119] For cortical neuron differentiation, NPCs were thawed in NM medium containing bFGF on PLO / lam-coated 6-well plates. 1.5 ml of NPCs were differentiated on PLO / lam-coated 6-well plates in maturation medium consisting of NB / B27 medium (Neurobasal medium, penicillin-streptomycin, 1x B27 supplement) with additional factors (4 μM PD033291, 20 ng / ml BDNF, 20 ng / ml GDNF, 100 μM ascorbic acid, 0.5 mM cAMP, 1 μg / ml laminin). After 1 week, 50,000 cells were split into 96-well plates using Accutase and ROCK inhibitor. Half of the medium was replaced every 2–3 days. Neurons were allowed to differentiate for 2 weeks before co-culture with microglia. An Integra ASSIST PLUS pipetting robot was used for coating, cell plating, and medium changes.

[0120] Neuron-microglia co-culture PD0332991 was removed from the maturation medium two days before adding microglia to neurons. hiMGLs at day 14 of differentiation were used for co-culture. 8,500 cells per 96-well plate were added to neurons in NB / B27 medium supplemented with 25 ng / ml M-CSF, 100 ng / ml IL-34, and 50 ng / ml TGF-β1. Neurons and microglia were co-cultured for 2 weeks before addition of amyloid-β.

[0121] Amyloid-β treatment Human amyloid-β (1-42) (rPeptide, A-1170-02) or scrambled human amyloid-β (1-42) (rPeptide, A-1004-1) was incubated overnight at 37°C for aggregation. Amyloid-β was added to the co-culture system at the indicated concentrations every 3–4 days by replacing half of the medium. Anti-TREM2 antibody was added in parallel with amyloid-β for 1–2 weeks.

[0122] p-SykAlphaLISA Phosphorylated SYK (p-Syk) was measured using the AlphaLISA SureFire Ultra p-Syk Assay Kit (PerkinElmer, ALSU-PSYK-A-HV) according to the manufacturer's instructions. Briefly, HEK293 cells overexpressing human TREM2 and human DAP12 were seeded in 50 μl of medium in a 96-well plate at a density of 50,000 cells / well and incubated overnight at 37°C in a cell culture incubator. The next day, the medium was removed, and 50 μl of antibody diluted in medium was added to the cells. After 5 minutes of incubation at 37°C, the treatment solution was removed, and the cells were lysed with 50 μl of lysis buffer supplemented with phosphatase inhibitor (VWR) on a plate shaker (approximately 350 rpm) for 10 minutes. Then, 30 μl of the lysate was transferred to a CLARIOstar Plus An additional incubation step (1 h each) with acceptor and donor beads was used for subsequent analysis using a plate reader (BMG Labtech).

[0123] For microglial cells, 96-well plates were pre-coated with antibodies overnight at 4°C, and the following day, microglial cells were added at a density of 60,000 cells / well. After 10 min of incubation at 37°C, the treatment solution was removed, and cells were lysed with 50 μl of lysis buffer supplemented with phosphatase inhibitor (VWR) for 10 min on a plate shaker (approximately 350 rpm). 30 μl of the lysate was then transferred to a CLARIOstar Plus An additional incubation step (1 h each) with acceptor and donor beads was used for subsequent analysis using a plate reader (BMG Labtech).

[0124] ELISA-based binding assay for anti-TREM2 antibodies Antibody binding to the extracellular domain of TREM2 (ecTREM2) or human or mouse peptide fragments of the stalk region of TREM2 was quantified in an ELISA assay. All procedures were performed at room temperature, and incubations were performed on a microtiter plate shaker. ELISA plates were coated with the corresponding ligand (ecTREM2 (Holzel Diagnostika, 11084-H08H) or human or mouse TREM2 peptide fragments) in coating buffer (NaHCO3) at 60 μl / well (final concentration 0.5 μg / ml) for 1 hour. The coated plates were washed three times with PBS-T (PBS, 0.1% Tween-20), blocked with 100 μl / well of blocking solution (PBS-T, 3% milk powder) for 1 hour, and washed again. Antibodies were prediluted 1:10 in PBS to a concentration of 1 μg / ml. A dilution series was performed with dilution steps of 1:3 and 1:10. 50 μl / well of diluted AB was transferred to the blocked ELISA plate and incubated for 1 hour. The plate was washed three times with PBS-T and incubated for 1 hour with anti-human-Strep-POD (Jackson Immunoresearch, #109-035-098) diluted 1:10,000 in PBS-T. After three washes, bound POD was detected by incubating with 100 μl / well of TMB substrate (Thermo Scientific, #34029) until a maximum optical density (OD) of approximately 1–2 was reached. Finally, the colorimetric reaction was stopped with 100 μl / well of stop solution (1 M H2SO4), and the OD was determined at a wavelength of 450 nm with a reference wavelength of 595 nm in a plate reader (SpectraMax i3xl).

[0125] Immunocytochemistry (ICC) and imaging Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and washed three times with PBS. For permeabilization and blocking, cells were incubated in 3% normal goat serum (Abcam) and 0.3% Triton X-100 in PBS for 1 hour at room temperature, followed by three washes. The following primary antibodies were used for overnight incubation at 4°C: rabbit anti-synapsin 1 (1:500, Synptic Systems, #106 103) and mouse anti-MAP 2 (1:1500, Sigma-Aldrich, #M9942). After three washes, cells were incubated with the following secondary antibodies for 1 hour at room temperature: donkey anti-rabbit 488 (Thermo Fisher, #A32790) and donkey anti-mouse 647 (Thermo Fisher, #A32787). After three washes with PBS, cells were stained with 10 μM DAPI. After washing three times with PBS, cells were imaged using an EVOS M7000 imaging system with a 20x objective, with 13 pictures taken per well, six wells per condition.

[0126] CellProfiler analysis CellProfiler software (Carpenter et al., 2006) was applied for image analysis. A customized CellProfiler pipeline was created for automated analysis of synapse counts, total and dead nuclei, and neurite area. Binary image analysis was applied to control correct image segmentation and object identification. In rare cases, cell culture issues, such as pipetting errors, can cause outliers for specific wells. Each well was visually inspected for visible differences from the mean, and outliers were statistically identified using the Grubbs method (α = 0.05). Identified outliers were then removed.

[0127] Neurite analysis: The MAP2 channel was used for neurite detection. To enhance the neurite structure, a nodule enhancement method was applied. Neurites were detected using the minimum cross-entropy threshold method. To account for cell density, the neurite area was normalized to the total number of nuclei per photograph.

[0128] Nucleus analysis: The DAPI channel was used for nuclei detection. DAPI images were illumination-corrected, and nuclei were detected using the Otsu threshold method. Dead nuclei were defined as the sum of apoptotic nuclei and small bright nuclei. Apoptotic nuclei were defined as a minimum of two small apoptotic bodies located close to each other. Speckle enhancement was applied to enhance the apoptotic body structure. Apoptotic bodies were detected using the Otsu threshold method. Small bright nuclei were filtered from nuclei based on intensity and area.

[0129] statistical analysis For comparisons of more than two groups, one-way ANOVA was used. Statistical significance was * ,<0.05; ** , p<0.01; and *** , p<0.001; and **** , p<0.0001.

[0130] II. Results Example 1: Cloning and analysis of antibodies against the hTREM2 stalk region As described in the Methods, we identified several antibody clones that bound with high affinity to the extracellular domain of TREM2 and to epitope peptides derived from the human TREM2 stalk region. These clones were structurally highly related to each other, but not identical.

[0131] Based on amino acid sequence comparisons by BLAST, the heavy chain gene, including the 19 amino acid (aa) leader peptide, of clone H08 is 95% homologous to that of clone M07 (22 of 471 amino acids differ, 87% homology when considering only the variable regions of both antibody clones). The light chain gene, including the 22 amino acid leader peptide, of clone H08 is 88% homologous to M07 (27 of 243 amino acids differ).

[0132] The heavy chain gene of clone M07, including its 19 amino acid leader peptide, is 96% homologous to M05 (18 of 471 amino acids differ, and 90% homology is achieved when only the variable regions of both antibody clones are considered), and the light chain gene of clone M07, including its 22 amino acid leader peptide, is 85% homologous to M05 (40 of 239 amino acids differ).

[0133] Similarly, the heavy chain gene of clone M07, including its 19 amino acid leader peptide, is 96% homologous to M03 (18 of 471 amino acids differ, with 90% homology when considering only the variable regions of both antibody clones), and the light chain gene of clone M07, including its 22 amino acid leader peptide, is 82% homologous to M03 (41 of 239 amino acids differ).

[0134] Example 2: Analysis of antibody M07 against the hTREM2 stalk region All antibody clones, including M07, were compared with each other, a previously established agonist rat antibody (H01) against hTREM2, and a fully human control antibody (IgG1-LALA isotype). The rat anti-human TREM2 antibody H01 was identified and purified after immunization in rats. This antibody binds to the extracellular domain of TREM2. No specific DNA sequence is available for this antibody.

[0135] SDS gels (silver stained) of M07 under reducing and non-reducing conditions are shown in Figure 1. The antibody can be detected at approximately 150 kDa under non-reducing, non-boiling (NRNB) conditions. Under reducing / boiling (RB) conditions, we detect the antibody light chain at 25 kDa and the heavy chain at 50 kDa (Figure 1).

[0136] We determined the binding affinity of all antibody clones, including M07, as well as the control antibody H01 and a negative control (not shown), to ecTREM2 by an ELISA-based binding assay (Figure 2). H08, M03, and M07, as well as the rat control antibody H01, showed affinities of less than 350 pM to human ecTREM2 (190 pM, 350 pM, 204 pM, and 274 pM, respectively), whereas M05 did not bind to the ecTREM2 domain (Figure 2).

[0137] In a next step, we sought to identify the precise antibody-binding epitope of TREM2. We investigated a peptide fragment (DAGDLWFPG, SEQ ID NO: 16) representing a specific epitope from the human TREM2 stalk region (Figure 3: oval) and again performed an ELISA binding assay.

[0138] Antibodies H08, M03 and M07 showed affinities of less than 850 pM for the specific epitope peptide (832 pM, 329 pM and 335 pM, respectively), whereas M05 and the rat control antibody H01 did not bind to this epitope (Figure 4).

[0139] Example 3: Activation of TREM2 signaling using p-SYK assay To test the activation of TREM2 signaling via anti-TREM2 antibodies, we used a p-SYK AlphaLISA assay, which allows for the detection of human pSYK phosphorylation that occurs upon ligand binding to human TREM2. To analyze activation of the TREM2 pathway, HEK293 cells stably expressing human TREM2 and human DAP12 were incubated with medium or with medium containing various antibodies at a concentration of 40 μg / ml for 5 minutes at 37°C, after which SYK phosphorylation was determined. Appropriate controls (human IgG1-LALA and rat IgG isotype antibodies) were also used in the assay. Treatment of HEK293-Flp-In hTREM2 / hDAP12 cells with M07 resulted in a strong increase in p-SYK levels compared to baseline controls (isotype or medium alone). M07 induced an average of 36-fold activation over baseline, thus activating pSYK much more potently than H01 or M03 (Figure 5), while H08 induced very little and M05 did not induce any activation at all. The increase over baseline is shown in the table below.

[0140] [Table 1]

[0141] We also performed a titration series of the best activating antibody clones (H01 and M07) (Figure 6).

[0142] To test activation of TREM2 signaling in cell types that endogenously express the TREM2 receptor, we differentiated microglial cells from hiPSCs. Using the previously described pSYK AlphaLISA assay, we assessed pSYK phosphorylation in hiPSC-derived microglial cells using increasing concentrations of M07 antibody. Treatment of microglial cells with M07 resulted in a strong, concentration-dependent increase in p-SYK levels (Figure 9).

[0143] Example 4: Induction of neurodegeneration in an Alzheimer's disease model Amyloid beta was added to a microglia-neuron coculture system to induce neurodegeneration as a model of Alzheimer's disease. Microglia exhibited the expected branched morphology in control conditions containing medium alone, whereas they exhibited an amoeboid morphology upon the addition of amyloid beta (Figure 7B). A dose-dependent effect of amyloid beta on neurodegeneration was observed. Neuritic degeneration, characteristic of neurodegeneration, was measured by MAP2+ neurite area / viable nuclei. Neuronal death, another hallmark of neurodegeneration, was quantified by detecting the number of dead nuclei / total nuclei (Figure 7C, D). This neurodegeneration model was used to determine the neuroprotective effect of the anti-hTREM2 antibody M07 on neurons.

[0144] Figure 8 shows that amyloid-β-dependent neurodegeneration was significantly reduced by the addition of the anti-hTREM2 antibody M07 compared with the isotype antibody. Specifically, the M07 antibody reduced neurite degeneration at 1 and 2 weeks after antibody addition. Regarding the number of dead cells per nucleus, the M07 antibody reduced the number of dead nuclei at 2 weeks after antibody addition.

[0145] Example 5: hTREM2 antibodies reduce neurodegeneration in a hiPSC-derived microglia-neuron co-culture model of Alzheimer's disease Amyloid-β was added to a microglia-neuron coculture to induce neurodegeneration as a model of Alzheimer's disease (Figure 7A). Microglia exhibited the expected branched morphology in control conditions containing medium alone, whereas they exhibited an amoeboid morphology upon the addition of amyloid-β (Figure 7B). The dose-dependent effects of amyloid-β on neurodegeneration could be measured. Neuritic degeneration, characteristic of neurodegeneration, was measured by MAP2+ neurite area / viable nuclei. Neuronal death, another hallmark of neurodegeneration, was quantified by detecting the number of dead nuclei / total nuclei (Figure 7C+D). This neurodegeneration model was used to determine the neuroprotective effect of the anti-hTREM2 antibody M07 on neurons.

[0146] Figure 8 shows that amyloid-β-dependent neurodegeneration was significantly reduced by the addition of the anti-hTREM2 antibody M07 compared with the isotype antibody. Specifically, the M07 antibody reduced neurite degeneration at 1 and 2 weeks after antibody addition. Regarding the number of dead cells per nucleus, the M07 antibody reduced the number of dead nuclei at 2 weeks after antibody addition.

[0147] III. Discussion and Conclusions Using phage display technology, we obtained fully human anti-TREM2 antibodies that were initially screened for antigen binding. Selected fully human IgG1-LALA modified antibodies were used to determine their binding affinity to the extracellular domain of human TREM2, their activation of human TREM2 signaling (human TREM2 / DAP-dependent SYK phosphorylation), and most importantly, their efficacy in a complex and relevant AD model using human brain cells differentiated from human induced pluripotent stem cells (hiPSCs).

[0148] The LALA modification strongly reduces the effector function of IgG1 antibodies, which is important for studies using human immune and neural cells. The fully human backbone of the antibody we created is advantageous compared to existing humanized antibodies based on the identification of clones in non-human animal immune systems (e.g., U.S. Patent Application Publication No. 2017240631(A1), Alector AL-002 and WO 2020172450(A1), Denali). This is because the use of fully human antibodies in repeated in vivo prophylactic or therapeutic applications in humans can be expected to result in fewer immunological complications.

[0149] First, we identified a number of antibodies that all bind to the extracellular domain of human TREM2 with high affinity (10 -9We identified a variety of structurally similar and related antibodies (heavy chain amino acid sequence homology of 90% or more) that bind to TREM2 at a heavy chain length less than 100 kJ / s. To our great surprise, we then found that only one of these antibodies could potently induce human TREM2 / DAP-induced SYK phosphorylation, a crucial TREM2-dependent effector pathway in AD. SYK phosphorylation was increased up to 60-fold by M07, whereas no or little activation was observed with the other antibody clones. However, M07 binds to the same epitope on the extracellular domain of TREM2 as H08 and M03.

[0150] The increase in human TREM2 / DAP-dependent pSYK levels induced by M07 was much stronger than that observed with published and patented agonistic antibodies against human TREM2, particularly any human anti-human TREM2 antibody. The hT2AB antibody disclosed by AMGEN (International Publication No. 2022120373(A1)) was used in a pSYK assay equivalent to ours and caused a 12-fold increase over baseline (Ellwanger et al., 2021). Alector presented a number of antibodies against human anti-TREM2 in its patent application (US Patent Publication No. 2017240631(A1)). SYK phosphorylation was demonstrated at the protein level, with approximately a 3- to 4-fold increase reported for antibodies #22, #45, and #65 in human dendritic cells. A 6-fold increase in SYK phosphorylation was observed in human macrophages. Denali presented several anti-hTREM2 antibodies in its patent application (WO 2020172450(A1)). One of them is CL0020188. This antibody increased pSYK levels four-fold in TREM2-expressing HEK293 cells compared to a control antibody. Data from Fassler et al. showed pSYK activation at the protein level mediated by anti-hTREM2 antibodies, but did not quantify the increase (Fassler et al., 2021).

[0151] Furthermore, the present inventors used an innovative, complex, and relevant AD model using human brain cells differentiated from human induced pluripotent stem cells (hiPSCs). Anti-TREM2 antibodies, known in the state of the art, have not been analyzed in a relatively sophisticated AD model using hiPSC-derived neurons and microglia. For example, WO2020172450(A1) (Denali) disclosed a phagocytosis assay using hiPSC-derived microglia and amyloid beta. However, no analysis was performed in co-culture with neurons. Therefore, the benefit of amyloid beta phagocytosis on neurons cannot be determined.

[0152] AD can also be studied in other disease models, all of which have inherent limitations. Most researchers still work with mouse models, which often fail to predict the clinical efficacy of anti-AD drug candidates.

[0153] The present invention solves the above-identified problems and surprisingly provides fully human anti-human TREM2 antibodies that strongly activate human TREM2-dependent human pSYK signaling, such that beneficial effects can be observed in relevant AD models in human brain cells. Previously, potent activators were only described for mouse TREM2 in mouse cells.

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[0155] Nucleotide and amino acid sequences SEQ ID NO: 1 Variable region or domain of the light chain of clone M07 (107 amino acids): DIQLTQSPLSLSASAGDRVTITCRASQSIRDYLGWYQQKPGKAPKLLIYAASKLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYHTPPFTFGQGTKVEI

[0156] SEQ ID NO: 2 Variable region or domain of the heavy chain of clone M07 (122 amino acids): EVQLLESGGGLVQPGGSLRLTCAASGFTFSSYAMSWVRQAPGKGLEWVSVINGRGSNTYYADSVKGRFTITRDNSKNTLYLEMNSLRAEDTAVYYCARVRAYSGPSYGFDYWGQGTLVTVSS

[0157] CDR-L1 of SEQ ID NO: 3 M07: QSIRDY

[0158] CDR-L2 of SEQ ID NO: 4 M07:AAS

[0159] CDR-L3 of SEQ ID NO: 5 M07: QQSYHTPPFT

[0160] CDR-H1 of SEQ ID NO: 6 M07:GFTFSSYA

[0161] CDR-H2 of SEQ ID NO: 7 M07: NGRGSNT

[0162] CDR-H3 of SEQ ID NO: 8 M07: ARVRAYSGPSYGFDY

[0163] SEQ ID NO: 9 Kappa 1 light chain of M07 (237 amino acids) with an N-terminal leader of 22 amino acid residues, the sequence of predicted CDRs 1-3 is underlined: [Table 2]

[0164] Kappa 1 light chain (215 amino acids) of SEQ ID NO: 10 M07, sequence of predicted CDRs 1-3 is underlined: [Table 3]

[0165] Lambda light chain (213 amino acids) of SEQ ID NO: 11 M07, sequence of predicted CDRs 1-3 is underlined: [Table 4]

[0166] SEQ ID NO: 12 Heavy chain of clone M07 complete IgG1-LALA-TFN (C H The sequence of putative CDRs 1 to 3 is underlined: [Table 5]

[0167] SEQ ID NO: 13 Heavy chain of clone M07 complete IgG1-LALA-TFN (452 ​​amino acids), sequence of predicted CDRs 1 to 3 is underlined: [Table 6]

[0168] SEQ ID NO: 14. Heavy chain complete IgG1-LALA (471 amino acids) with N-terminal leader, sequence of predicted CDRs 1-3 is underlined: [Table 7]

[0169] SEQ ID NO: 15 Heavy chain complete IgG1-LALA (452 ​​amino acids), sequence of predicted CDRs 1 to 3 is underlined: [Table 8]

[0170] SEQ ID NO: 16 Peptide fragment representing an epitope derived from the hTREM2 stalk region: DAGDLWFPG

[0171] Kappa 1 light chain of SEQ ID NO: 17 H08, sequence of predicted CDRs 1-3 is underlined: [Table 9]

[0172] SEQ ID NO: 18. Heavy chain complete IgG1-LALA of clone H08, sequence of predicted CDRs 1 to 3 is underlined: [Table 10]

[0173] SEQ ID NO: 19 M03 complete IgG1 kappa 1 light chain, predicted CDRs 1-3 sequence is underlined: [Table 11]

[0174] SEQ ID NO: 20 M03 complete IgG1 lambda light chain, sequence of predicted CDRs 1-3 is underlined: [Table 12]

[0175] SEQ ID NO: 21 Heavy chain complete IgG1-LALA of clone M03; sequence of predicted CDRs 1 to 3 is underlined: [Table 13]

[0176] SEQ ID NO: 22 M05 complete IgG1 kappa 1 light chain, predicted CDRs 1-3 sequence is underlined: [Table 14]

[0177] SEQ ID NO: 23 Complete IgG1 lambda light chain of M05, sequence of predicted CDRs 1-3 is underlined: [Table 15]

[0178] SEQ ID NO: 24. Heavy chain complete IgG1-LALA of clone M05, sequence of predicted CDRs 1 to 3 is underlined: [Table 16]

[0179] SEQ ID NO: 25 Coding sequence of the kappa 1 light chain of antibody M07 with N-terminal leader (723 bp): CATCATGGACATGAGAGTGCCCGCTCAGCTGCTGGGACTGCTGTTGTTGTGGCTGTCTGGCGCTAGATGCGACATCCAGCTGACCCAGTCTCCACTGTCTCTGTCTGCCTCTGCTGGCGACAGAGTGACCATCACCTGTCGGGCCTCTCAGTCTATCAGAGACTACCTCGGCTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACGCTGCCTCTAAACTGCAGTCCGGCGTGCCCTCTAGATTCTCTGGCTCTGGATCTGGCACCGACTTCACCCTGACCATCAGTTCTCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTCCTATCACACCCCTCCATTCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCACCATCTGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCTGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCCACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCTTTCAACAGAGGCGAGTGCTGATTAAT

[0180] Nucleotide sequence (1424bp) of the heavy chain of antibody M07 with the sequence number 26 N-terminal leader:

[0181] SEQ ID NO: 27 Amino acid sequence of hTREM2 as shown schematically in FIG.

Claims

1. A protein capable of binding to human TREM2, comprising or consisting of an immunoglobulin (Ig) heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2, and wherein the protein is capable of activating human TREM2-dependent pSYK signaling.

2. comprising an Ig light chain variable region and an Ig heavy chain variable region; the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1; 2. The protein according to claim 1, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO:

2.

3. A protein capable of binding to human TREM2, comprising or consisting of a heavy chain variable region and / or comprising or consisting of a light chain variable region; the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 2; the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one or two amino acid residue substitutions compared to SEQ ID NO: 1; A protein, wherein said protein is capable of activating human TREM2-dependent pSYK signaling.

4. comprising a light chain variable region and a heavy chain variable region, the light chain (LC) variable region comprises, preferably in CDR-L3, a segment of the amino acid sequence of SEQ ID NO: 5 or a segment of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; The protein according to claim 1, 2 or 3, wherein the heavy chain (HC) variable region comprises, preferably in CDR-H3, a segment of the amino acid sequence of SEQ ID NO: 8 or a segment of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO:

8.

5. The light chain variable region preferably comprises, in CDRs L1 and L3, the following amino acid segments in the N-terminal to C-terminal direction: a segment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), and comprising a segment (CDR-L3) of the amino acid sequence of SEQ ID NO: 5 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5, The heavy chain (HC) variable region preferably comprises, in CDRs H2 and H3, the following amino acid segments from N-terminus to C-terminus: A segment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and 5. The protein according to any one of claims 2 to 4, comprising a segment (CDR-H3) of the amino acid sequence of SEQ ID NO: 8 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO:

8.

6. The light chain (LC) variable region preferably comprises, in CDRs L1 to L3, the following amino acid segments in the N-terminal to C-terminal direction: a segment of the amino acid sequence of SEQ ID NO: 3 (CDR-L1), A segment of the amino acid sequence of SEQ ID NO: 4 (CDR-L2), and a segment (CDR-L3) of the amino acid sequence of SEQ ID NO: 5 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO: 5; The heavy chain (HC) variable region preferably comprises, in CDRs H1 to H3, the following amino acid sequence segments from the N-terminus to the C-terminus: a segment of the amino acid sequence of SEQ ID NO: 6 (CDR-H1), A segment of the amino acid sequence of SEQ ID NO: 7 (CDR-H2), and 6. The protein according to claim 4 or 5, comprising a segment (CDR-H3) of the amino acid sequence of SEQ ID NO: 8 or of an amino acid sequence having one amino acid residue substitution compared to the amino acid sequence of SEQ ID NO:

8.

7. The protein may be a single chain antibody (scFv), a Fab fragment, a F(ab) ２ a fragment thereof, or an immunoglobulin (Ig); and / or The protein may comprise a single chain antibody (scFv), a Fab fragment, a F(ab) fragment, or a fusion protein as the first and second fusion protein segments. ２ The protein of any one of claims 1 to 6, which is a fragment or a fusion protein comprising an immunoglobulin (Ig).

8. the protein comprises an antibody light chain (subunit) and an antibody heavy chain (subunit), the light chain comprises the light chain variable region and a light chain constant region; The protein according to any one of claims 1 to 6, wherein the heavy chain comprises the heavy chain variable region and at least one heavy chain constant region, preferably at least the constant region CH1.

9. the protein is an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM, or the protein is a fusion protein comprising said Ig and an additional fusion protein segment; and / or The protein according to any one of claims 1 to 8, wherein the protein is a fully human Ig produced in human immune cells.

10. The protein is a light chain, such as: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9, 10, or 11; or (b) a light chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10, or 11; and / or A heavy chain, such as: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15; or (d) the protein of any one of claims 2 to 9, comprising a heavy chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

11. The protein comprises a binding domain capable of binding to human transferrin receptor 1 (hTfR1) to enable the protein to cross the blood-brain barrier, and preferably the protein comprises a modified C Ｈ a heavy chain having three domains or capable of binding to hTfR1; Ｈ 11. The protein of any one of claims 1 to 10, comprising a three-domain C-terminal extension.

12. 12. The protein of any one of claims 1 to 11, wherein the protein is capable of binding to human TREM2 via its variable region, preferably to the stalk region of hTREM2, and / or wherein the protein activates human TREM2-dependent human pSYK signaling and / or is an hTREM2 agonist.

13. An antibody capable of binding to human TREM2, comprising an Ig light chain variable region as defined in claim 2 and an Ig heavy chain variable region as defined in claim 2, wherein the antibody is capable of activating human TREM2-dependent pSYK signaling.

14. A light chain, (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 9, 10, or 11; or (b) a light chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 9, 10, or 11; and a heavy chain comprising: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15; or (d) the antibody of claim 13, comprising a heavy chain, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

15. Two light chains, such as: (a) the amino acid sequence of the light chain is or comprises the amino acid sequence of SEQ ID NO: 10 or 11; or (b) two light chains, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 10 or 11; and Two heavy chains, such as: (c) the amino acid sequence of the heavy chain is or comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15; or (d) The antibody of claim 13 or 14, comprising two heavy chains, the amino acid sequence of which is or comprises an amino acid sequence having one or two amino acid residue substitutions compared to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15.

16. A pharmaceutical composition comprising the protein or antibody according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. The protein or antibody according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 for use in therapy or prevention.

18. The protein or antibody according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16, for use in a method for treating or preventing a neurodegenerative disease such as Alzheimer's disease.

19. 19. A protein or antibody for use according to claim 17 or 18, wherein said use is in a method for the treatment or prevention of a neurodegenerative disease, such as Alzheimer's disease, in a patient at an early stage of said neurodegenerative disease, such as Alzheimer's disease.

20. 20. The protein or antibody for use according to any one of claims 17 to 19, wherein the use is in a method for the treatment or prevention of a neurodegenerative disease, such as Alzheimer's disease, in patients with the following stages of the neurodegenerative disease, such as Alzheimer's disease: - cognitive impairment in said patient with a score of 23 or less, preferably a score between 10 and 23, more preferably a score between 19 and 23, on the Mini-Mental State Examination (MMSE) test, or - cognitive impairment of said patient with a score of 0.5 or more and 2 or less on the Clinical Dementia Rating Scale (CDR global score).

21. 21. The protein or antibody or pharmaceutical composition for use according to any one of claims 17 to 20, wherein said use comprises parenteral administration, preferably intravenous, subcutaneous or intraperitoneal administration, of said protein or antibody to a mammal.

22. A nucleic acid molecule encoding a protein, antibody, light chain and / or heavy chain as defined in any one of claims 1 to 15 or comprising or consisting of the nucleotide sequence of SEQ ID NO: 25 or 26.

23. A eukaryotic cell comprising a protein according to any one of claims 1 to 15 or a nucleic acid molecule according to claim 22.

24. 17. A method for treating or preventing a neurodegenerative disease such as Alzheimer's disease, comprising administering a protein or antibody as defined in any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 to a mammal in need of said treatment or prevention.

25. 25. The method of claim 24, comprising administering a protein or antibody as defined in any one of claims 1 to 15 or a pharmaceutical composition as defined in claim 16 to a human patient at an early stage of the disease.

26. 25. The method according to claim 24, comprising administering a protein or antibody as defined in any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 to a human patient in an early stage of said disease, as follows: - cognitive impairment in said patient with a score of 23 or less, preferably a score between 10 and 23, more preferably a score between 19 and 23, on the Mini-Mental State Examination (MMSE) test, or - cognitive impairment of said patient with a score of 0.5 or more and 2 or less on the Clinical Dementia Rating Scale (CDR global score).

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